Methods and Compositions Involving Fusion Proteins for Improved Immunotherapy - Patent application

Abstract

Provided herein are nucleic acids, expression cassettes, modified lymphocytes, and compositions comprising thereof, that contain sequences encoding fusion proteins, TCRs, or CARs that contain domains of LTBR. In certain embodiments, the lymphocytes are T cells. Also described are methods of treatment using the provided compositions.

Description

[Technical Field]

[0001] Government Support Statement This invention was made with government support under grants R00HG008171, DP2HG010099, and R01CA218668 awarded by the National Institutes of Health, and D18AP00053 awarded by the Defense Advanced Research Projects Agency. The government has certain rights in this invention.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (NYG-LIPP-158PCT.xml, size: 304,622 bytes, and creation date: March 15, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0003] Cellular immunotherapy with engineered autologous patient T cells redirected against selected tumor antigens has shown great efficacy against hematologic cancers and has led to five U.S. Food and Drug Administration (FDA) approvals of chimeric antigen receptors (CARs) to date. 6 In contrast, CAR therapy for solid tumors has shown much lower efficacy overall due to the suppression of T cell effector function in the tumor microenvironment. Even for hematologic malignancies, with the exception of B-acute lymphoblastic leukemia, most patients do not experience a durable response, and resistance is primarily due to impaired T cell function rather than antigen loss. 7 Considerable efforts are being made to identify genes and pathways that contribute to T cell dysfunction. 8、9 However, to date, comprehensive, genome-wide screens for modulators of T cell function have been limited to loss-of-function screens. 2~4 .

[0004] Advances in CRISPR genome engineering have made it possible to easily knock out entire genes in the genome in a scalable and customizable manner. Its large size makes it difficult, if not impossible, to deliver Cas9 to primary T cells via lentivirus. 10 ) makes it difficult, but an alternative approach has been developed, which involves the use of the Cas9 protein 2 or mRNA 11 Transient delivery of HIV-1, HIV-2, or engineered isogenic mouse strains 3 However, these approaches are not amenable to gain-of-function screening in human cells, which requires continuous expression of a transcriptional activator that drives target gene expression.

[0005] What is needed are improved compositions and methods for more effective immunotherapy. Summary of the Invention

[0006] In a first aspect, provided herein are lymphocytes genetically modified to express a chimeric antigen receptor (CAR). The CAR comprises an antigen-binding domain, a transmembrane domain, and a signaling domain. In certain embodiments, at least one domain comprises an LTBR domain. In other embodiments, at least one domain comprises a domain from a gene in Table 3. In certain embodiments, the LTBR domain is the LTBR intracellular domain, or a fragment or variant thereof. In certain embodiments, the LTBR intracellular domain comprises amino acids 249-435 of SEQ ID NO: 2, or a fragment, deletion, or variant thereof.

[0007] In certain embodiments, the LTBR intracellular domain has a deletion at least at amino acids 393-435.

[0008] In another embodiment, the CAR comprises an antigen-binding domain, a transmembrane domain, a costimulatory signaling domain, and a signaling domain. In certain embodiments, at least one domain comprises an LTBR domain. In other embodiments, at least one domain comprises a domain from a gene in Table 3. In certain embodiments, the LTBR domain is the LTBR intracellular domain, or a fragment or variant thereof. In certain embodiments, the LTBR intracellular domain comprises amino acids 249-435 of SEQ ID NO: 2, or a fragment, deletion, or variant thereof. In certain embodiments, the LTBR intracellular domain has a deletion of at least amino acids 393-435.

[0009] In another aspect, a nucleic acid molecule is provided. The molecule comprises a sequence encoding a chimeric antigen receptor (CAR). The CAR comprises an antigen-binding domain, a transmembrane domain, and a signaling domain. In certain embodiments, at least one domain comprises an LTBR domain. In other embodiments, at least one domain comprises a domain from a gene in Table 3. In certain embodiments, the LTBR domain is the LTBR intracellular domain, or a fragment or variant thereof. In certain embodiments, the LTBR intracellular domain comprises amino acids 249-435 of SEQ ID NO: 2, or a fragment, deletion, or variant thereof. In certain embodiments, the LTBR intracellular domain has a deletion of at least amino acids 393-435. In another embodiment, the CAR comprises an antigen-binding domain, a transmembrane domain, a costimulatory signaling domain, and a signaling domain. In certain embodiments, at least one domain comprises an LTBR domain. In other embodiments, at least one domain comprises a domain from a gene in Table 3. In certain embodiments, the LTBR domain is the LTBR intracellular domain, or a fragment or variant thereof. In certain embodiments, the LTBR intracellular domain comprises amino acids 249-435 of SEQ ID NO: 2, or a fragment, deletion, or variant thereof. In certain embodiments, the LTBR intracellular domain has a deletion of at least amino acids 393-435.

[0010] In another aspect, an expression cassette is provided comprising a nucleic acid molecule comprising a sequence encoding a chimeric antigen receptor (CAR).

[0011] In another aspect, there is provided a method of treating cancer in a subject in need thereof, the method comprising administering a composition comprising the modified lymphocytes described herein.

[0012] In another aspect, a method of treating a viral disease in a subject in need thereof is provided, the method comprising administering a composition comprising modified lymphocytes.

[0013] In another aspect, a method of treating autoimmunity in a subject in need thereof is provided, the method comprising administering a composition comprising modified lymphocytes.

[0014] In another embodiment, a fusion protein is provided that includes an LTBR domain and at least one domain from a second protein that is not an LTBR.

[0015] In another aspect, a host cell is provided that comprises a nucleic acid molecule or expression cassette described herein.

[0016] Other aspects and advantages of the present invention will become readily apparent from the following detailed description of the invention. [Brief explanation of the drawings]

[0017] [Figure 1A]We demonstrate a genome-scale overexpression screen to identify genes that promote proliferation of primary human T cells. (Figure 1A) Overview of the pooled ORF screen. CD4+ and CD8+ T cells were isolated separately from peripheral blood from three healthy donors. The barcoded genome-scale ORF library was then introduced into CD3 / CD28-stimulated T cells, followed by selection of transduced cells. After 14 days of culture, T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and restimulated to induce proliferation. ORFs enriched in the CFSE-low population were identified by comparing the number of specific ORF barcodes before and after cell sorting. (Figure 1B) Normalized enrichment of individual barcodes for the indicated genes in the CD4+ screen. (Figure 1C) Robust rank integration of genes in both CFSE-low CD4+ and CFSE-low CD8+ T cells based on consistent enrichment of individual barcodes for each gene. (Figure 1D) Enrichment in individual donors and T cell populations of top-ranked genes (grouped by function and relevance to T cell proliferation) selected for further study. Neutral genes (MHC-I complex and cell type-specific differentiation markers) are included for comparison. Gene names are colored based on differential expression in CD3 / CD28 stimulated and resting T cells (green, upregulated; red, downregulated; gray, unchanged; black, no expression). [Figure 1B]We demonstrate a genome-scale overexpression screen to identify genes that promote proliferation of primary human T cells. (Figure 1A) Overview of the pooled ORF screen. CD4+ and CD8+ T cells were isolated separately from peripheral blood from three healthy donors. The barcoded genome-scale ORF library was then introduced into CD3 / CD28-stimulated T cells, followed by selection of transduced cells. After 14 days of culture, T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and restimulated to induce proliferation. ORFs enriched in the CFSE-low population were identified by comparing the number of specific ORF barcodes before and after cell sorting. (Figure 1B) Normalized enrichment of individual barcodes for the indicated genes in the CD4+ screen. (Figure 1C) Robust rank integration of genes in both CFSE-low CD4+ and CFSE-low CD8+ T cells based on consistent enrichment of individual barcodes for each gene. (Figure 1D) Enrichment in individual donors and T cell populations of top-ranked genes (grouped by function and relevance to T cell proliferation) selected for further study. Neutral genes (MHC-I complex and cell type-specific differentiation markers) are included for comparison. Gene names are colored based on differential expression in CD3 / CD28 stimulated and resting T cells (green, upregulated; red, downregulated; gray, unchanged; black, no expression). [Figure 1C]We demonstrate a genome-scale overexpression screen to identify genes that promote proliferation of primary human T cells. (Figure 1A) Overview of the pooled ORF screen. CD4+ and CD8+ T cells were isolated separately from peripheral blood from three healthy donors. The barcoded genome-scale ORF library was then introduced into CD3 / CD28-stimulated T cells, followed by selection of transduced cells. After 14 days of culture, T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and restimulated to induce proliferation. ORFs enriched in the CFSE-low population were identified by comparing the number of specific ORF barcodes before and after cell sorting. (Figure 1B) Normalized enrichment of individual barcodes for the indicated genes in the CD4+ screen. (Figure 1C) Robust rank integration of genes in both CFSE-low CD4+ and CFSE-low CD8+ T cells based on consistent enrichment of individual barcodes for each gene. (Figure 1D) Enrichment in individual donors and T cell populations of top-ranked genes (grouped by function and relevance to T cell proliferation) selected for further study. Neutral genes (MHC-I complex and cell type-specific differentiation markers) are included for comparison. Gene names are colored based on differential expression in CD3 / CD28 stimulated and resting T cells (green, upregulated; red, downregulated; gray, unchanged; black, no expression). [Figure 1D]We demonstrate a genome-scale overexpression screen to identify genes that promote proliferation of primary human T cells. (Figure 1A) Overview of the pooled ORF screen. CD4+ and CD8+ T cells were isolated separately from peripheral blood from three healthy donors. The barcoded genome-scale ORF library was then introduced into CD3 / CD28-stimulated T cells, followed by selection of transduced cells. After 14 days of culture, T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and restimulated to induce proliferation. ORFs enriched in the CFSE-low population were identified by comparing the number of specific ORF barcodes before and after cell sorting. (Figure 1B) Normalized enrichment of individual barcodes for the indicated genes in the CD4+ screen. (Figure 1C) Robust rank integration of genes in both CFSE-low CD4+ and CFSE-low CD8+ T cells based on consistent enrichment of individual barcodes for each gene. (Figure 1D) Enrichment in individual donors and T cell populations of top-ranked genes (grouped by function and relevance to T cell proliferation) selected for further study. Neutral genes (MHC-I complex and cell type-specific differentiation markers) are included for comparison. Gene names are colored based on differential expression in CD3 / CD28 stimulated and resting T cells (green, upregulated; red, downregulated; gray, unchanged; black, no expression). [Figure 2A]Overexpression of top-ranked ORFs increases proliferation, activation, and cytokine secretion in CD4+ and CD8+ T cells. (Figure 2A) CD4+ and CD8+ T cells from a donor uninvolved in screening were isolated separately and then transduced with lentivirus encoding the top-ranked ORFs along with a selection marker. After transduction and selection, T cells were restimulated before measuring proliferation, activation marker expression, and cytokine secretion. (Figure 2B) Proliferation of T cells normalized to tNGFR was measured as relative proliferation, defined as the ratio of stimulated cells to the corresponding unstimulated control. A minimum of two donors were tested in biological triplicates for each overexpressed gene. Boxes indicate the 25th to 75th percentiles with mean lines, and extensions extend to the maximum and minimum values. DUPD1 is also known as DUSP29. (Figure 2C) Average relative proliferation of ORF-transduced T cells in CD4+ and CD8+ T cells, normalized to tNGFR. Significant genes in both T cell subsets or either of them are marked (two-tailed Student's t-test P < 0.05 and false discovery rate < 0.1). (Figure 2D) Representative expression of CD25 or CD154 after restimulation. Numbers on the histograms correspond to the percentage of gated cells (CD8+CD154+) or mean fluorescence intensity (MFI). Dashed lines indicate the gate used to enumerate the MFI of CD154+ cells (CD8+) or control (tNGFR) cells. (Figure 2E) IL-2 and IFNγ secretion after restimulation normalized to tNGFR. Only genes that significantly increase T cell proliferation in CD4+, CD8+, or both T cell subsets are shown. A minimum of two donors were tested in triplicate per gene. Boxes indicate the 25th to 75th percentiles with mean lines, and extension lines extend to the maximum and minimum values. (FIG. 2F) Crossover between different T cell activation phenotypes significantly (P<0.05) improved by a given ORF in CD8+ or CD4+ T cells. [Figure 2B]Overexpression of top-ranked ORFs increases proliferation, activation, and cytokine secretion in CD4+ and CD8+ T cells. (Figure 2A) CD4+ and CD8+ T cells from a donor uninvolved in screening were isolated separately and then transduced with lentivirus encoding the top-ranked ORFs along with a selection marker. After transduction and selection, T cells were restimulated before measuring proliferation, activation marker expression, and cytokine secretion. (Figure 2B) Proliferation of T cells normalized to tNGFR was measured as relative proliferation, defined as the ratio of stimulated cells to the corresponding unstimulated control. A minimum of two donors were tested in biological triplicates for each overexpressed gene. Boxes indicate the 25th to 75th percentiles with mean lines, and extensions extend to the maximum and minimum values. DUPD1 is also known as DUSP29. (Figure 2C) Average relative proliferation of ORF-transduced T cells in CD4+ and CD8+ T cells, normalized to tNGFR. Significant genes in both T cell subsets or either of them are marked (two-tailed Student's t-test P < 0.05 and false discovery rate < 0.1). (Figure 2D) Representative expression of CD25 or CD154 after restimulation. Numbers on the histograms correspond to the percentage of gated cells (CD8+CD154+) or mean fluorescence intensity (MFI). Dashed lines indicate the gate used to enumerate the MFI of CD154+ cells (CD8+) or control (tNGFR) cells. (Figure 2E) IL-2 and IFNγ secretion after restimulation normalized to tNGFR. Only genes that significantly increase T cell proliferation in CD4+, CD8+, or both T cell subsets are shown. A minimum of two donors were tested in triplicate per gene. Boxes indicate the 25th to 75th percentiles with mean lines, and extension lines extend to the maximum and minimum values. (FIG. 2F) Crossover between different T cell activation phenotypes significantly (P<0.05) improved by a given ORF in CD8+ or CD4+ T cells. [Figure 2C]Overexpression of top-ranked ORFs increases proliferation, activation, and cytokine secretion in CD4+ and CD8+ T cells. (Figure 2A) CD4+ and CD8+ T cells from a donor uninvolved in screening were isolated separately and then transduced with lentivirus encoding the top-ranked ORFs along with a selection marker. After transduction and selection, T cells were restimulated before measuring proliferation, activation marker expression, and cytokine secretion. (Figure 2B) Proliferation of T cells normalized to tNGFR was measured as relative proliferation, defined as the ratio of stimulated cells to the corresponding unstimulated control. A minimum of two donors were tested in biological triplicates for each overexpressed gene. Boxes indicate the 25th to 75th percentiles with mean lines, and extensions extend to the maximum and minimum values. DUPD1 is also known as DUSP29. (Figure 2C) Average relative proliferation of ORF-transduced T cells in CD4+ and CD8+ T cells, normalized to tNGFR. Significant genes in both T cell subsets or either of them are marked (two-tailed Student's t-test P < 0.05 and false discovery rate < 0.1). (Figure 2D) Representative expression of CD25 or CD154 after restimulation. Numbers on the histograms correspond to the percentage of gated cells (CD8+CD154+) or mean fluorescence intensity (MFI). Dashed lines indicate the gate used to enumerate the MFI of CD154+ cells (CD8+) or control (tNGFR) cells. (Figure 2E) IL-2 and IFNγ secretion after restimulation normalized to tNGFR. Only genes that significantly increase T cell proliferation in CD4+, CD8+, or both T cell subsets are shown. A minimum of two donors were tested in triplicate per gene. Boxes indicate the 25th to 75th percentiles with mean lines, and extension lines extend to the maximum and minimum values. (FIG. 2F) Crossover between different T cell activation phenotypes significantly (P<0.05) improved by a given ORF in CD8+ or CD4+ T cells. [Figure 2D]Overexpression of top-ranked ORFs increases proliferation, activation, and cytokine secretion in CD4+ and CD8+ T cells. (Figure 2A) CD4+ and CD8+ T cells from a donor uninvolved in screening were isolated separately and then transduced with lentivirus encoding the top-ranked ORFs along with a selection marker. After transduction and selection, T cells were restimulated before measuring proliferation, activation marker expression, and cytokine secretion. (Figure 2B) Proliferation of T cells normalized to tNGFR was measured as relative proliferation, defined as the ratio of stimulated cells to the corresponding unstimulated control. A minimum of two donors were tested in biological triplicates for each overexpressed gene. Boxes indicate the 25th to 75th percentiles with mean lines, and extensions extend to the maximum and minimum values. DUPD1 is also known as DUSP29. (Figure 2C) Average relative proliferation of ORF-transduced T cells in CD4+ and CD8+ T cells, normalized to tNGFR. Significant genes in both T cell subsets or either of them are marked (two-tailed Student's t-test P < 0.05 and false discovery rate < 0.1). (Figure 2D) Representative expression of CD25 or CD154 after restimulation. Numbers on the histograms correspond to the percentage of gated cells (CD8+CD154+) or mean fluorescence intensity (MFI). Dashed lines indicate the gate used to enumerate the MFI of CD154+ cells (CD8+) or control (tNGFR) cells. (Figure 2E) IL-2 and IFNγ secretion after restimulation normalized to tNGFR. Only genes that significantly increase T cell proliferation in CD4+, CD8+, or both T cell subsets are shown. A minimum of two donors were tested in triplicate per gene. Boxes indicate the 25th to 75th percentiles with mean lines, and extension lines extend to the maximum and minimum values. (FIG. 2F) Crossover between different T cell activation phenotypes significantly (P<0.05) improved by a given ORF in CD8+ or CD4+ T cells. [Figure 2E]Overexpression of top-ranked ORFs increases proliferation, activation, and cytokine secretion in CD4+ and CD8+ T cells. (Figure 2A) CD4+ and CD8+ T cells from a donor uninvolved in screening were isolated separately and then transduced with lentivirus encoding the top-ranked ORFs along with a selection marker. After transduction and selection, T cells were restimulated before measuring proliferation, activation marker expression, and cytokine secretion. (Figure 2B) Proliferation of T cells normalized to tNGFR was measured as relative proliferation, defined as the ratio of stimulated cells to the corresponding unstimulated control. A minimum of two donors were tested in biological triplicates for each overexpressed gene. Boxes indicate the 25th to 75th percentiles with mean lines, and extensions extend to the maximum and minimum values. DUPD1 is also known as DUSP29. (Figure 2C) Average relative proliferation of ORF-transduced T cells in CD4+ and CD8+ T cells, normalized to tNGFR. Significant genes in both T cell subsets or either of them are marked (two-tailed Student's t-test P < 0.05 and false discovery rate < 0.1). (Figure 2D) Representative expression of CD25 or CD154 after restimulation. Numbers on the histograms correspond to the percentage of gated cells (CD8+CD154+) or mean fluorescence intensity (MFI). Dashed lines indicate the gate used to enumerate the MFI of CD154+ cells (CD8+) or control (tNGFR) cells. (Figure 2E) IL-2 and IFNγ secretion after restimulation normalized to tNGFR. Only genes that significantly increase T cell proliferation in CD4+, CD8+, or both T cell subsets are shown. A minimum of two donors were tested in triplicate per gene. Boxes indicate the 25th to 75th percentiles with mean lines, and extension lines extend to the maximum and minimum values. (FIG. 2F) Crossover between different T cell activation phenotypes significantly (P<0.05) improved by a given ORF in CD8+ or CD4+ T cells. [Figure 2F]Overexpression of top-ranked ORFs increases proliferation, activation, and cytokine secretion in CD4+ and CD8+ T cells. (Figure 2A) CD4+ and CD8+ T cells from a donor uninvolved in screening were isolated separately and then transduced with lentivirus encoding the top-ranked ORFs along with a selection marker. After transduction and selection, T cells were restimulated before measuring proliferation, activation marker expression, and cytokine secretion. (Figure 2B) Proliferation of T cells normalized to tNGFR was measured as relative proliferation, defined as the ratio of stimulated cells to the corresponding unstimulated control. A minimum of two donors were tested in biological triplicates for each overexpressed gene. Boxes indicate the 25th to 75th percentiles with mean lines, and extensions extend to the maximum and minimum values. DUPD1 is also known as DUSP29. (Figure 2C) Average relative proliferation of ORF-transduced T cells in CD4+ and CD8+ T cells, normalized to tNGFR. Significant genes in both T cell subsets or either of them are marked (two-tailed Student's t-test P < 0.05 and false discovery rate < 0.1). (Figure 2D) Representative expression of CD25 or CD154 after restimulation. Numbers on the histograms correspond to the percentage of gated cells (CD8+CD154+) or mean fluorescence intensity (MFI). Dashed lines indicate the gate used to enumerate the MFI of CD154+ cells (CD8+) or control (tNGFR) cells. (Figure 2E) IL-2 and IFNγ secretion after restimulation normalized to tNGFR. Only genes that significantly increase T cell proliferation in CD4+, CD8+, or both T cell subsets are shown. A minimum of two donors were tested in triplicate per gene. Boxes indicate the 25th to 75th percentiles with mean lines, and extension lines extend to the maximum and minimum values. (FIG. 2F) Crossover between different T cell activation phenotypes significantly (P<0.05) improved by a given ORF in CD8+ or CD4+ T cells. [Figure 3A]Single-cell OverCITE-seq identifies shared and distinct transcriptional programs induced by gene overexpression in T cells. (Figure 3A) OverCITE-seq captures overexpressed (ORF) constructs, transcriptomes, TCR clonotypes, cell surface proteins, and treatment hashtags in single cells. (Figure 3B) ORF assignment ratios in resting and CD3 / CD28-stimulated T cells. (Figure 3C) Antibody-derived tag sequencing (ADT, right) yields NGFR expression in tNGFR-transduced T cells similar to flow cytometry (left) using tNGFR-transduced T cells. Non-transduced cells (left) or cells assigned with a non-tNGFR ORF (right) are shown in gray. (Figure 3D) Uniform Variety Approximation and Projection (UMAP) view of single-cell transcriptomes after unsupervised clustering of OverCITE-seq-captured ORF singlets. The top left insert identifies stimulated and resting T cells by treatment hashtag. For each cluster, a subset of the top 20 differentially expressed genes is shown. HIST1H1B is also known as H1-5, and HIST1H3C is also known as H3C3. (Figure 3E) ORF occurrence in two representative clusters. Normalized residual values ​​are from chi-squared tests. ORFs of interest are indicated. [Figure 3B]Single-cell OverCITE-seq identifies shared and distinct transcriptional programs induced by gene overexpression in T cells. (Figure 3A) OverCITE-seq captures overexpressed (ORF) constructs, transcriptomes, TCR clonotypes, cell surface proteins, and treatment hashtags in single cells. (Figure 3B) ORF assignment ratios in resting and CD3 / CD28-stimulated T cells. (Figure 3C) Antibody-derived tag sequencing (ADT, right) yields NGFR expression in tNGFR-transduced T cells similar to flow cytometry (left) using tNGFR-transduced T cells. Non-transduced cells (left) or cells assigned with a non-tNGFR ORF (right) are shown in gray. (Figure 3D) Uniform Variety Approximation and Projection (UMAP) view of single-cell transcriptomes after unsupervised clustering of OverCITE-seq-captured ORF singlets. The top left insert identifies stimulated and resting T cells by treatment hashtag. For each cluster, a subset of the top 20 differentially expressed genes is shown. HIST1H1B is also known as H1-5, and HIST1H3C is also known as H3C3. (Figure 3E) ORF occurrence in two representative clusters. Normalized residual values ​​are from chi-squared tests. ORFs of interest are indicated. [Figure 3C]Single-cell OverCITE-seq identifies shared and distinct transcriptional programs induced by gene overexpression in T cells. (Figure 3A) OverCITE-seq captures overexpressed (ORF) constructs, transcriptomes, TCR clonotypes, cell surface proteins, and treatment hashtags in single cells. (Figure 3B) ORF assignment ratios in resting and CD3 / CD28-stimulated T cells. (Figure 3C) Antibody-derived tag sequencing (ADT, right) yields NGFR expression in tNGFR-transduced T cells similar to flow cytometry (left) using tNGFR-transduced T cells. Non-transduced cells (left) or cells assigned with a non-tNGFR ORF (right) are shown in gray. (Figure 3D) Uniform Variety Approximation and Projection (UMAP) view of single-cell transcriptomes after unsupervised clustering of OverCITE-seq-captured ORF singlets. The top left insert identifies stimulated and resting T cells by treatment hashtag. For each cluster, a subset of the top 20 differentially expressed genes is shown. HIST1H1B is also known as H1-5, and HIST1H3C is also known as H3C3. (Figure 3E) ORF occurrence in two representative clusters. Normalized residual values ​​are from chi-squared tests. ORFs of interest are indicated. [Figure 3D]Single-cell OverCITE-seq identifies shared and distinct transcriptional programs induced by gene overexpression in T cells. (Figure 3A) OverCITE-seq captures overexpressed (ORF) constructs, transcriptomes, TCR clonotypes, cell surface proteins, and treatment hashtags in single cells. (Figure 3B) ORF assignment ratios in resting and CD3 / CD28-stimulated T cells. (Figure 3C) Antibody-derived tag sequencing (ADT, right) yields NGFR expression in tNGFR-transduced T cells similar to flow cytometry (left) using tNGFR-transduced T cells. Non-transduced cells (left) or cells assigned with a non-tNGFR ORF (right) are shown in gray. (Figure 3D) Uniform Variety Approximation and Projection (UMAP) view of single-cell transcriptomes after unsupervised clustering of OverCITE-seq-captured ORF singlets. The top left insert identifies stimulated and resting T cells by treatment hashtag. For each cluster, a subset of the top 20 differentially expressed genes is shown. HIST1H1B is also known as H1-5, and HIST1H3C is also known as H3C3. (Figure 3E) ORF occurrence in two representative clusters. Normalized residual values ​​are from chi-squared tests. ORFs of interest are indicated. [Figure 3E]Single-cell OverCITE-seq identifies shared and distinct transcriptional programs induced by gene overexpression in T cells. (Figure 3A) OverCITE-seq captures overexpressed (ORF) constructs, transcriptomes, TCR clonotypes, cell surface proteins, and treatment hashtags in single cells. (Figure 3B) ORF assignment ratios in resting and CD3 / CD28-stimulated T cells. (Figure 3C) Antibody-derived tag sequencing (ADT, right) yields NGFR expression in tNGFR-transduced T cells similar to flow cytometry (left) using tNGFR-transduced T cells. Non-transduced cells (left) or cells assigned with a non-tNGFR ORF (right) are shown in gray. (Figure 3D) Uniform Variety Approximation and Projection (UMAP) view of single-cell transcriptomes after unsupervised clustering of OverCITE-seq-captured ORF singlets. The top left insert identifies stimulated and resting T cells by treatment hashtag. For each cluster, a subset of the top 20 differentially expressed genes is shown. HIST1H1B is also known as H1-5, and HIST1H3C is also known as H3C3. (Figure 3E) ORF occurrence in two representative clusters. Normalized residual values ​​are from chi-squared tests. ORFs of interest are indicated. [Figure 4A]We show that LTBR overexpression improves T cell function through activation of the canonical NF-κB pathway. (Figure 4A) Differential expression of genes in resting LTBR and tNGFR (negative control) T cells. Genes highlighted in red are those with a ≥2-fold change in expression and an adjusted P<0.05. (Figure 4B) Significantly enriched GO biological processes (p<0.05) in LTBR-overexpressing T cells. (Figure 4C) Cell viability of CD8+ T cells transduced with LTBR or tNGFR lentiviruses that were either restimulated with CD3 / CD28 for 4 days or left unstimulated (n=2 donors with 3 biological replicates each). (Figure 4D) PD-1 expression in resting LTBR or tNGFR T cells stimulated with a 3:1 excess of CD3 / CD28 beads every 3 days for up to 3 consecutive rounds of stimulation. (Figure 4E) ICAM-1 expression (resting) and IL-2 secretion (activated) by T cells transduced with Flag-tagged LTBR mutants, normalized to wild-type LTBR (n = 6 replicates across two experiments). (Figure 4F) Enrichment of transcription factor motifs in differentially accessible chromatin (top 10 motifs from each comparison). (Figure 4G) Quantification of phosphorylated RELA (phospho-RELA) in LTBR or tNGFR T cells stimulated with CD3 / CD28 antibodies for the indicated times. (Figures 4H, 4I) Quantification of phosphorylated IκBα (Figure 4H) or mature NF-κB2 (Figure 4I) in resting or CD3 / CD28-stimulated (15 min) LTBR or tNGFR cells. (Figure 4J) IFNγ secretion by LTBR or tNGFR cells stimulated after CRISPR knockout of the indicated genes (n = 18, three sgRNAs from two donors in three biological replicates). IFNγ levels were normalized to the corresponding non-targeting (NT) control (either LTBR or tNGFR) to allow for comparison of the relative effects of gene knockouts on T cell activation. (Figure 4K) Expression levels of core LTBR genes (n = 274 genes) in LTBR and tNGFR cells after CRISPR knockout of RELA or RELB (normalized to the non-targeting control in LTBR cells). Boxes indicate the 25th to 75th percentiles with median lines, and extended lines extend 1.5-fold between quartiles.Independent two-tailed t-test P values ​​(Figure 4C, 4G-K): not significant (NS) P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Error bars, s.e.m., n = 3 biological replicates unless otherwise stated. [Figure 4B]We show that LTBR overexpression improves T cell function through activation of the canonical NF-κB pathway. (Figure 4A) Differential expression of genes in resting LTBR and tNGFR (negative control) T cells. Genes highlighted in red are those with a ≥2-fold change in expression and an adjusted P<0.05. (Figure 4B) Significantly enriched GO biological processes (p<0.05) in LTBR-overexpressing T cells. (Figure 4C) Cell viability of CD8+ T cells transduced with LTBR or tNGFR lentiviruses that were either restimulated with CD3 / CD28 for 4 days or left unstimulated (n=2 donors with 3 biological replicates each). (Figure 4D) PD-1 expression in resting LTBR or tNGFR T cells stimulated with a 3:1 excess of CD3 / CD28 beads every 3 days for up to 3 consecutive rounds of stimulation. (Figure 4E) ICAM-1 expression (resting) and IL-2 secretion (activated) by T cells transduced with Flag-tagged LTBR mutants, normalized to wild-type LTBR (n = 6 replicates across two experiments). (Figure 4F) Enrichment of transcription factor motifs in differentially accessible chromatin (top 10 motifs from each comparison). (Figure 4G) Quantification of phosphorylated RELA (phospho-RELA) in LTBR or tNGFR T cells stimulated with CD3 / CD28 antibodies for the indicated times. (Figures 4H, 4I) Quantification of phosphorylated IκBα (Figure 4H) or mature NF-κB2 (Figure 4I) in resting or CD3 / CD28-stimulated (15 min) LTBR or tNGFR cells. (Figure 4J) IFNγ secretion by LTBR or tNGFR cells stimulated after CRISPR knockout of the indicated genes (n = 18, three sgRNAs from two donors in three biological replicates). IFNγ levels were normalized to the corresponding non-targeting (NT) control (either LTBR or tNGFR) to allow for comparison of the relative effects of gene knockouts on T cell activation. (Figure 4K) Expression levels of core LTBR genes (n = 274 genes) in LTBR and tNGFR cells after CRISPR knockout of RELA or RELB (normalized to the non-targeting control in LTBR cells). Boxes indicate the 25th to 75th percentiles with median lines, and extended lines extend 1.5-fold between quartiles.Independent two-tailed t-test P values ​​(Figure 4C, 4G-K): not significant (NS) P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Error bars, s.e.m., n = 3 biological replicates unless otherwise stated. [Figure 4C]We show that LTBR overexpression improves T cell function through activation of the canonical NF-κB pathway. (Figure 4A) Differential expression of genes in resting LTBR and tNGFR (negative control) T cells. Genes highlighted in red are those with a ≥2-fold change in expression and an adjusted P<0.05. (Figure 4B) Significantly enriched GO biological processes (p<0.05) in LTBR-overexpressing T cells. (Figure 4C) Cell viability of CD8+ T cells transduced with LTBR or tNGFR lentiviruses that were either restimulated with CD3 / CD28 for 4 days or left unstimulated (n=2 donors with 3 biological replicates each). (Figure 4D) PD-1 expression in resting LTBR or tNGFR T cells stimulated with a 3:1 excess of CD3 / CD28 beads every 3 days for up to 3 consecutive rounds of stimulation. (Figure 4E) ICAM-1 expression (resting) and IL-2 secretion (activated) by T cells transduced with Flag-tagged LTBR mutants, normalized to wild-type LTBR (n = 6 replicates across two experiments). (Figure 4F) Enrichment of transcription factor motifs in differentially accessible chromatin (top 10 motifs from each comparison). (Figure 4G) Quantification of phosphorylated RELA (phospho-RELA) in LTBR or tNGFR T cells stimulated with CD3 / CD28 antibodies for the indicated times. (Figures 4H, 4I) Quantification of phosphorylated IκBα (Figure 4H) or mature NF-κB2 (Figure 4I) in resting or CD3 / CD28-stimulated (15 min) LTBR or tNGFR cells. (Figure 4J) IFNγ secretion by LTBR or tNGFR cells stimulated after CRISPR knockout of the indicated genes (n = 18, three sgRNAs from two donors in three biological replicates). IFNγ levels were normalized to the corresponding non-targeting (NT) control (either LTBR or tNGFR) to allow for comparison of the relative effects of gene knockouts on T cell activation. (Figure 4K) Expression levels of core LTBR genes (n = 274 genes) in LTBR and tNGFR cells after CRISPR knockout of RELA or RELB (normalized to the non-targeting control in LTBR cells). Boxes indicate the 25th to 75th percentiles with median lines, and extended lines extend 1.5-fold between quartiles.Independent two-tailed t-test P values ​​(Figure 4C, 4G-K): not significant (NS) P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Error bars, s.e.m., n = 3 biological replicates unless otherwise stated. [Figure 4D]We show that LTBR overexpression improves T cell function through activation of the canonical NF-κB pathway. (Figure 4A) Differential expression of genes in resting LTBR and tNGFR (negative control) T cells. Genes highlighted in red are those with a ≥2-fold change in expression and an adjusted P<0.05. (Figure 4B) Significantly enriched GO biological processes (p<0.05) in LTBR-overexpressing T cells. (Figure 4C) Cell viability of CD8+ T cells transduced with LTBR or tNGFR lentiviruses that were either restimulated with CD3 / CD28 for 4 days or left unstimulated (n=2 donors with 3 biological replicates each). (Figure 4D) PD-1 expression in resting LTBR or tNGFR T cells stimulated with a 3:1 excess of CD3 / CD28 beads every 3 days for up to 3 consecutive rounds of stimulation. (Figure 4E) ICAM-1 expression (resting) and IL-2 secretion (activated) by T cells transduced with Flag-tagged LTBR mutants, normalized to wild-type LTBR (n = 6 replicates across two experiments). (Figure 4F) Enrichment of transcription factor motifs in differentially accessible chromatin (top 10 motifs from each comparison). (Figure 4G) Quantification of phosphorylated RELA (phospho-RELA) in LTBR or tNGFR T cells stimulated with CD3 / CD28 antibodies for the indicated times. (Figures 4H, 4I) Quantification of phosphorylated IκBα (Figure 4H) or mature NF-κB2 (Figure 4I) in resting or CD3 / CD28-stimulated (15 min) LTBR or tNGFR cells. (Figure 4J) IFNγ secretion by LTBR or tNGFR cells stimulated after CRISPR knockout of the indicated genes (n = 18, three sgRNAs from two donors in three biological replicates). IFNγ levels were normalized to the corresponding non-targeting (NT) control (either LTBR or tNGFR) to allow for comparison of the relative effects of gene knockouts on T cell activation. (Figure 4K) Expression levels of core LTBR genes (n = 274 genes) in LTBR and tNGFR cells after CRISPR knockout of RELA or RELB (normalized to the non-targeting control in LTBR cells). Boxes indicate the 25th to 75th percentiles with median lines, and extended lines extend 1.5-fold between quartiles.Independent two-tailed t-test P values ​​(Figure 4C, 4G-K): not significant (NS) P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Error bars, s.e.m., n = 3 biological replicates unless otherwise stated. [Figure 4E]We show that LTBR overexpression improves T cell function through activation of the canonical NF-κB pathway. (Figure 4A) Differential expression of genes in resting LTBR and tNGFR (negative control) T cells. Genes highlighted in red are those with a ≥2-fold change in expression and an adjusted P<0.05. (Figure 4B) Significantly enriched GO biological processes (p<0.05) in LTBR-overexpressing T cells. (Figure 4C) Cell viability of CD8+ T cells transduced with LTBR or tNGFR lentiviruses that were either restimulated with CD3 / CD28 for 4 days or left unstimulated (n=2 donors with 3 biological replicates each). (Figure 4D) PD-1 expression in resting LTBR or tNGFR T cells stimulated with a 3:1 excess of CD3 / CD28 beads every 3 days for up to 3 consecutive rounds of stimulation. (Figure 4E) ICAM-1 expression (resting) and IL-2 secretion (activated) by T cells transduced with Flag-tagged LTBR mutants, normalized to wild-type LTBR (n = 6 replicates across two experiments). (Figure 4F) Enrichment of transcription factor motifs in differentially accessible chromatin (top 10 motifs from each comparison). (Figure 4G) Quantification of phosphorylated RELA (phospho-RELA) in LTBR or tNGFR T cells stimulated with CD3 / CD28 antibodies for the indicated times. (Figures 4H, 4I) Quantification of phosphorylated IκBα (Figure 4H) or mature NF-κB2 (Figure 4I) in resting or CD3 / CD28-stimulated (15 min) LTBR or tNGFR cells. (Figure 4J) IFNγ secretion by LTBR or tNGFR cells stimulated after CRISPR knockout of the indicated genes (n = 18, three sgRNAs from two donors in three biological replicates). IFNγ levels were normalized to the corresponding non-targeting (NT) control (either LTBR or tNGFR) to allow for comparison of the relative effects of gene knockouts on T cell activation. (Figure 4K) Expression levels of core LTBR genes (n = 274 genes) in LTBR and tNGFR cells after CRISPR knockout of RELA or RELB (normalized to the non-targeting control in LTBR cells). Boxes indicate the 25th to 75th percentiles with median lines, and extended lines extend 1.5-fold between quartiles.Independent two-tailed t-test P values ​​(Figure 4C, 4G-K): not significant (NS) P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Error bars, s.e.m., n = 3 biological replicates unless otherwise stated. [Figure 4F]We show that LTBR overexpression improves T cell function through activation of the canonical NF-κB pathway. (Figure 4A) Differential expression of genes in resting LTBR and tNGFR (negative control) T cells. Genes highlighted in red are those with a ≥2-fold change in expression and an adjusted P<0.05. (Figure 4B) Significantly enriched GO biological processes (p<0.05) in LTBR-overexpressing T cells. (Figure 4C) Cell viability of CD8+ T cells transduced with LTBR or tNGFR lentiviruses that were either restimulated with CD3 / CD28 for 4 days or left unstimulated (n=2 donors with 3 biological replicates each). (Figure 4D) PD-1 expression in resting LTBR or tNGFR T cells stimulated with a 3:1 excess of CD3 / CD28 beads every 3 days for up to 3 consecutive rounds of stimulation. (Figure 4E) ICAM-1 expression (resting) and IL-2 secretion (activated) by T cells transduced with Flag-tagged LTBR mutants, normalized to wild-type LTBR (n = 6 replicates across two experiments). (Figure 4F) Enrichment of transcription factor motifs in differentially accessible chromatin (top 10 motifs from each comparison). (Figure 4G) Quantification of phosphorylated RELA (phospho-RELA) in LTBR or tNGFR T cells stimulated with CD3 / CD28 antibodies for the indicated times. (Figures 4H, 4I) Quantification of phosphorylated IκBα (Figure 4H) or mature NF-κB2 (Figure 4I) in resting or CD3 / CD28-stimulated (15 min) LTBR or tNGFR cells. (Figure 4J) IFNγ secretion by LTBR or tNGFR cells stimulated after CRISPR knockout of the indicated genes (n = 18, three sgRNAs from two donors in three biological replicates). IFNγ levels were normalized to the corresponding non-targeting (NT) control (either LTBR or tNGFR) to allow for comparison of the relative effects of gene knockouts on T cell activation. (Figure 4K) Expression levels of core LTBR genes (n = 274 genes) in LTBR and tNGFR cells after CRISPR knockout of RELA or RELB (normalized to the non-targeting control in LTBR cells). Boxes indicate the 25th to 75th percentiles with median lines, and extended lines extend 1.5-fold between quartiles.Independent two-tailed t-test P values ​​(Figure 4C, 4G-K): not significant (NS) P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Error bars, s.e.m., n = 3 biological replicates unless otherwise stated. [Figure 4G]We show that LTBR overexpression improves T cell function through activation of the canonical NF-κB pathway. (Figure 4A) Differential expression of genes in resting LTBR and tNGFR (negative control) T cells. Genes highlighted in red are those with a ≥2-fold change in expression and an adjusted P<0.05. (Figure 4B) Significantly enriched GO biological processes (p<0.05) in LTBR-overexpressing T cells. (Figure 4C) Cell viability of CD8+ T cells transduced with LTBR or tNGFR lentiviruses that were either restimulated with CD3 / CD28 for 4 days or left unstimulated (n=2 donors with 3 biological replicates each). (Figure 4D) PD-1 expression in resting LTBR or tNGFR T cells stimulated with a 3:1 excess of CD3 / CD28 beads every 3 days for up to 3 consecutive rounds of stimulation. (Figure 4E) ICAM-1 expression (resting) and IL-2 secretion (activated) by T cells transduced with Flag-tagged LTBR mutants, normalized to wild-type LTBR (n = 6 replicates across two experiments). (Figure 4F) Enrichment of transcription factor motifs in differentially accessible chromatin (top 10 motifs from each comparison). (Figure 4G) Quantification of phosphorylated RELA (phospho-RELA) in LTBR or tNGFR T cells stimulated with CD3 / CD28 antibodies for the indicated times. (Figures 4H, 4I) Quantification of phosphorylated IκBα (Figure 4H) or mature NF-κB2 (Figure 4I) in resting or CD3 / CD28-stimulated (15 min) LTBR or tNGFR cells. (Figure 4J) IFNγ secretion by LTBR or tNGFR cells stimulated after CRISPR knockout of the indicated genes (n = 18, three sgRNAs from two donors in three biological replicates). IFNγ levels were normalized to the corresponding non-targeting (NT) control (either LTBR or tNGFR) to allow for comparison of the relative effects of gene knockouts on T cell activation. (Figure 4K) Expression levels of core LTBR genes (n = 274 genes) in LTBR and tNGFR cells after CRISPR knockout of RELA or RELB (normalized to the non-targeting control in LTBR cells). Boxes indicate the 25th to 75th percentiles with median lines, and extended lines extend 1.5-fold between quartiles.Independent two-tailed t-test P values ​​(Figure 4C, 4G-K): not significant (NS) P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Error bars, s.e.m., n = 3 biological replicates unless otherwise stated. [Figure 4H]We show that LTBR overexpression improves T cell function through activation of the canonical NF-κB pathway. (Figure 4A) Differential expression of genes in resting LTBR and tNGFR (negative control) T cells. Genes highlighted in red are those with a ≥2-fold change in expression and an adjusted P<0.05. (Figure 4B) Significantly enriched GO biological processes (p<0.05) in LTBR-overexpressing T cells. (Figure 4C) Cell viability of CD8+ T cells transduced with LTBR or tNGFR lentiviruses that were either restimulated with CD3 / CD28 for 4 days or left unstimulated (n=2 donors with 3 biological replicates each). (Figure 4D) PD-1 expression in resting LTBR or tNGFR T cells stimulated with a 3:1 excess of CD3 / CD28 beads every 3 days for up to 3 consecutive rounds of stimulation. (Figure 4E) ICAM-1 expression (resting) and IL-2 secretion (activated) by T cells transduced with Flag-tagged LTBR mutants, normalized to wild-type LTBR (n = 6 replicates across two experiments). (Figure 4F) Enrichment of transcription factor motifs in differentially accessible chromatin (top 10 motifs from each comparison). (Figure 4G) Quantification of phosphorylated RELA (phospho-RELA) in LTBR or tNGFR T cells stimulated with CD3 / CD28 antibodies for the indicated times. (Figures 4H, 4I) Quantification of phosphorylated IκBα (Figure 4H) or mature NF-κB2 (Figure 4I) in resting or CD3 / CD28-stimulated (15 min) LTBR or tNGFR cells. (Figure 4J) IFNγ secretion by LTBR or tNGFR cells stimulated after CRISPR knockout of the indicated genes (n = 18, three sgRNAs from two donors in three biological replicates). IFNγ levels were normalized to the corresponding non-targeting (NT) control (either LTBR or tNGFR) to allow for comparison of the relative effects of gene knockouts on T cell activation. (Figure 4K) Expression levels of core LTBR genes (n = 274 genes) in LTBR and tNGFR cells after CRISPR knockout of RELA or RELB (normalized to the non-targeting control in LTBR cells). Boxes indicate the 25th to 75th percentiles with median lines, and extended lines extend 1.5-fold between quartiles.Independent two-tailed t-test P values ​​(Figure 4C, 4G-K): not significant (NS) P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Error bars, s.e.m., n = 3 biological replicates unless otherwise stated. [Figure 4I]We show that LTBR overexpression improves T cell function through activation of the canonical NF-κB pathway. (Figure 4A) Differential expression of genes in resting LTBR and tNGFR (negative control) T cells. Genes highlighted in red are those with a ≥2-fold change in expression and an adjusted P<0.05. (Figure 4B) Significantly enriched GO biological processes (p<0.05) in LTBR-overexpressing T cells. (Figure 4C) Cell viability of CD8+ T cells transduced with LTBR or tNGFR lentiviruses that were either restimulated with CD3 / CD28 for 4 days or left unstimulated (n=2 donors with 3 biological replicates each). (Figure 4D) PD-1 expression in resting LTBR or tNGFR T cells stimulated with a 3:1 excess of CD3 / CD28 beads every 3 days for up to 3 consecutive rounds of stimulation. (Figure 4E) ICAM-1 expression (resting) and IL-2 secretion (activated) by T cells transduced with Flag-tagged LTBR mutants, normalized to wild-type LTBR (n = 6 replicates across two experiments). (Figure 4F) Enrichment of transcription factor motifs in differentially accessible chromatin (top 10 motifs from each comparison). (Figure 4G) Quantification of phosphorylated RELA (phospho-RELA) in LTBR or tNGFR T cells stimulated with CD3 / CD28 antibodies for the indicated times. (Figures 4H, 4I) Quantification of phosphorylated IκBα (Figure 4H) or mature NF-κB2 (Figure 4I) in resting or CD3 / CD28-stimulated (15 min) LTBR or tNGFR cells. (Figure 4J) IFNγ secretion by LTBR or tNGFR cells stimulated after CRISPR knockout of the indicated genes (n = 18, three sgRNAs from two donors in three biological replicates). IFNγ levels were normalized to the corresponding non-targeting (NT) control (either LTBR or tNGFR) to allow for comparison of the relative effects of gene knockouts on T cell activation. (Figure 4K) Expression levels of core LTBR genes (n = 274 genes) in LTBR and tNGFR cells after CRISPR knockout of RELA or RELB (normalized to the non-targeting control in LTBR cells). Boxes indicate the 25th to 75th percentiles with median lines, and extended lines extend 1.5-fold between quartiles.Independent two-tailed t-test P values ​​(Figure 4C, 4G-K): not significant (NS) P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Error bars, s.e.m., n = 3 biological replicates unless otherwise stated. [Figure 4J]We show that LTBR overexpression improves T cell function through activation of the canonical NF-κB pathway. (Figure 4A) Differential expression of genes in resting LTBR and tNGFR (negative control) T cells. Genes highlighted in red are those with a ≥2-fold change in expression and an adjusted P<0.05. (Figure 4B) Significantly enriched GO biological processes (p<0.05) in LTBR-overexpressing T cells. (Figure 4C) Cell viability of CD8+ T cells transduced with LTBR or tNGFR lentiviruses that were either restimulated with CD3 / CD28 for 4 days or left unstimulated (n=2 donors with 3 biological replicates each). (Figure 4D) PD-1 expression in resting LTBR or tNGFR T cells stimulated with a 3:1 excess of CD3 / CD28 beads every 3 days for up to 3 consecutive rounds of stimulation. (Figure 4E) ICAM-1 expression (resting) and IL-2 secretion (activated) by T cells transduced with Flag-tagged LTBR mutants, normalized to wild-type LTBR (n = 6 replicates across two experiments). (Figure 4F) Enrichment of transcription factor motifs in differentially accessible chromatin (top 10 motifs from each comparison). (Figure 4G) Quantification of phosphorylated RELA (phospho-RELA) in LTBR or tNGFR T cells stimulated with CD3 / CD28 antibodies for the indicated times. (Figures 4H, 4I) Quantification of phosphorylated IκBα (Figure 4H) or mature NF-κB2 (Figure 4I) in resting or CD3 / CD28-stimulated (15 min) LTBR or tNGFR cells. (Figure 4J) IFNγ secretion by LTBR or tNGFR cells stimulated after CRISPR knockout of the indicated genes (n = 18, three sgRNAs from two donors in three biological replicates). IFNγ levels were normalized to the corresponding non-targeting (NT) control (either LTBR or tNGFR) to allow for comparison of the relative effects of gene knockouts on T cell activation. (Figure 4K) Expression levels of core LTBR genes (n = 274 genes) in LTBR and tNGFR cells after CRISPR knockout of RELA or RELB (normalized to the non-targeting control in LTBR cells). Boxes indicate the 25th to 75th percentiles with median lines, and extended lines extend 1.5-fold between quartiles.Independent two-tailed t-test P values ​​(Figure 4C, 4G-K): not significant (NS) P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Error bars, s.e.m., n = 3 biological replicates unless otherwise stated. [Figure 4K]We show that LTBR overexpression improves T cell function through activation of the canonical NF-κB pathway. (Figure 4A) Differential expression of genes in resting LTBR and tNGFR (negative control) T cells. Genes highlighted in red are those with a ≥2-fold change in expression and an adjusted P<0.05. (Figure 4B) Significantly enriched GO biological processes (p<0.05) in LTBR-overexpressing T cells. (Figure 4C) Cell viability of CD8+ T cells transduced with LTBR or tNGFR lentiviruses that were either restimulated with CD3 / CD28 for 4 days or left unstimulated (n=2 donors with 3 biological replicates each). (Figure 4D) PD-1 expression in resting LTBR or tNGFR T cells stimulated with a 3:1 excess of CD3 / CD28 beads every 3 days for up to 3 consecutive rounds of stimulation. (Figure 4E) ICAM-1 expression (resting) and IL-2 secretion (activated) by T cells transduced with Flag-tagged LTBR mutants, normalized to wild-type LTBR (n = 6 replicates across two experiments). (Figure 4F) Enrichment of transcription factor motifs in differentially accessible chromatin (top 10 motifs from each comparison). (Figure 4G) Quantification of phosphorylated RELA (phospho-RELA) in LTBR or tNGFR T cells stimulated with CD3 / CD28 antibodies for the indicated times. (Figures 4H, 4I) Quantification of phosphorylated IκBα (Figure 4H) or mature NF-κB2 (Figure 4I) in resting or CD3 / CD28-stimulated (15 min) LTBR or tNGFR cells. (Figure 4J) IFNγ secretion by LTBR or tNGFR cells stimulated after CRISPR knockout of the indicated genes (n = 18, three sgRNAs from two donors in three biological replicates). IFNγ levels were normalized to the corresponding non-targeting (NT) control (either LTBR or tNGFR) to allow for comparison of the relative effects of gene knockouts on T cell activation. (Figure 4K) Expression levels of core LTBR genes (n = 274 genes) in LTBR and tNGFR cells after CRISPR knockout of RELA or RELB (normalized to the non-targeting control in LTBR cells). Boxes indicate the 25th to 75th percentiles with median lines, and extended lines extend 1.5-fold between quartiles.Independent two-tailed t-test P values ​​(Figure 4C, 4G-K): not significant (NS) P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Error bars, s.e.m., n = 3 biological replicates unless otherwise stated. [Figure 5A]Top-ranked genes are shown to improve antigen-specific T cell responses and tumor killing. (Figures 5A-G) Co-delivery of anti-CD19 CAR and ORF to T cells from a healthy donor. (Figure 5A) Schematic of the tricistronic vector and CAR T cell experiment. (Figures 5B, 5C) Secretion of IFNγ (Figure 5B) and IL-2 (Figure 5C) after overnight co-incubation of CD8+ T cells with Nalm6 cells at a 1:1 ratio (n = 3 biological replicates, representing two donors). (Figure 5D) Representative images of Nalm6 GFP+ cells co-incubated with CAR T cells or untransduced control T cells for 48 hours. Scale bar, 200 μm. (Figure 5E) Nalm6 GFP+ cell proliferation (normalized total GFP per well) after co-incubation with T cells co-expressing the 19-28z CAR and LTBR or tNGFR (negative control) at the indicated effector-to-target ratios. (Figure 5F) Quantification of Nalm6 GFP+ clearance for T cells co-expressing the 19-28z or 18-BBz CAR and the top-ranked genes (n = 3 biological replicates, representing two donors) at an effector-to-target ratio of 0.25 and normalized to tNGFR after 48 hours of co-incubation. (Figure 5G) 19-BBz CAR T cells co-expressing LTBR or tNGFR were co-incubated with Nalm6 cells at a 1:1 ratio every 3 days for up to three rounds of stimulation (n = 3 biological replicates). Seven days after repeated antigen stimulation, CAR T cells were re-exposed to Nalm6 cells. IFNγ secretion was measured after overnight incubation. (Figure 5G) Co-delivery of anti-CD19 CAR and ORF to total PBMCs from a patient with diffuse large B-cell lymphoma. Transduced T cells were incubated alone or co-incubated with CD19+ Nalm6 or CD19- Jurkat cell lines at a 1:1 ratio (n=3 biological replicates, representing two patients). IFNγ and IL2 secretion was measured after overnight incubation. For the Nalm6 condition, the numbers above the indicated column pairs represent the fold increase in cytokine secretion by LTBR cells relative to tNGFR (negative control) cells. (Figure 5I) Delivery of ORFs to Vγ9Vδ2 T cells.IFNγ and IL-2 secretion after overnight co-incubation with the pancreatic ductal adenocarcinoma (PDAC) line Capan-2, pretreated with zoledronate to promote phosphoantigen accumulation (n=3 biological replicates). Data are mean ± sem, where applicable. [Figure 5B]Top-ranked genes are shown to improve antigen-specific T cell responses and tumor killing. (Figures 5A-G) Co-delivery of anti-CD19 CAR and ORF to T cells from a healthy donor. (Figure 5A) Schematic of the tricistronic vector and CAR T cell experiment. (Figures 5B, 5C) Secretion of IFNγ (Figure 5B) and IL-2 (Figure 5C) after overnight co-incubation of CD8+ T cells with Nalm6 cells at a 1:1 ratio (n = 3 biological replicates, representing two donors). (Figure 5D) Representative images of Nalm6 GFP+ cells co-incubated with CAR T cells or untransduced control T cells for 48 hours. Scale bar, 200 μm. (Figure 5E) Nalm6 GFP+ cell proliferation (normalized total GFP per well) after co-incubation with T cells co-expressing the 19-28z CAR and LTBR or tNGFR (negative control) at the indicated effector-to-target ratios. (Figure 5F) Quantification of Nalm6 GFP+ clearance for T cells co-expressing the 19-28z or 18-BBz CAR and the top-ranked genes (n = 3 biological replicates, representing two donors) at an effector-to-target ratio of 0.25 and normalized to tNGFR after 48 hours of co-incubation. (Figure 5G) 19-BBz CAR T cells co-expressing LTBR or tNGFR were co-incubated with Nalm6 cells at a 1:1 ratio every 3 days for up to three rounds of stimulation (n = 3 biological replicates). Seven days after repeated antigen stimulation, CAR T cells were re-exposed to Nalm6 cells. IFNγ secretion was measured after overnight incubation. (Figure 5G) Co-delivery of anti-CD19 CAR and ORF to total PBMCs from a patient with diffuse large B-cell lymphoma. Transduced T cells were incubated alone or co-incubated with CD19+ Nalm6 or CD19- Jurkat cell lines at a 1:1 ratio (n=3 biological replicates, representing two patients). IFNγ and IL2 secretion was measured after overnight incubation. For the Nalm6 condition, the numbers above the indicated column pairs represent the fold increase in cytokine secretion by LTBR cells relative to tNGFR (negative control) cells. (Figure 5I) Delivery of ORFs to Vγ9Vδ2 T cells.IFNγ and IL-2 secretion after overnight co-incubation with the pancreatic ductal adenocarcinoma (PDAC) line Capan-2, pretreated with zoledronate to promote phosphoantigen accumulation (n=3 biological replicates). Data are mean ± sem, where applicable. [Figure 5C]Top-ranked genes are shown to improve antigen-specific T cell responses and tumor killing. (Figures 5A-G) Co-delivery of anti-CD19 CAR and ORF to T cells from a healthy donor. (Figure 5A) Schematic of the tricistronic vector and CAR T cell experiment. (Figures 5B, 5C) Secretion of IFNγ (Figure 5B) and IL-2 (Figure 5C) after overnight co-incubation of CD8+ T cells with Nalm6 cells at a 1:1 ratio (n = 3 biological replicates, representing two donors). (Figure 5D) Representative images of Nalm6 GFP+ cells co-incubated with CAR T cells or untransduced control T cells for 48 hours. Scale bar, 200 μm. (Figure 5E) Nalm6 GFP+ cell proliferation (normalized total GFP per well) after co-incubation with T cells co-expressing the 19-28z CAR and LTBR or tNGFR (negative control) at the indicated effector-to-target ratios. (Figure 5F) Quantification of Nalm6 GFP+ clearance for T cells co-expressing the 19-28z or 18-BBz CAR and the top-ranked genes (n = 3 biological replicates, representing two donors) at an effector-to-target ratio of 0.25 and normalized to tNGFR after 48 hours of co-incubation. (Figure 5G) 19-BBz CAR T cells co-expressing LTBR or tNGFR were co-incubated with Nalm6 cells at a 1:1 ratio every 3 days for up to three rounds of stimulation (n = 3 biological replicates). Seven days after repeated antigen stimulation, CAR T cells were re-exposed to Nalm6 cells. IFNγ secretion was measured after overnight incubation. (Figure 5G) Co-delivery of anti-CD19 CAR and ORF to total PBMCs from a patient with diffuse large B-cell lymphoma. Transduced T cells were incubated alone or co-incubated with CD19+ Nalm6 or CD19- Jurkat cell lines at a 1:1 ratio (n=3 biological replicates, representing two patients). IFNγ and IL2 secretion was measured after overnight incubation. For the Nalm6 condition, the numbers above the indicated column pairs represent the fold increase in cytokine secretion by LTBR cells relative to tNGFR (negative control) cells. (Figure 5I) Delivery of ORFs to Vγ9Vδ2 T cells.IFNγ and IL-2 secretion after overnight co-incubation with the pancreatic ductal adenocarcinoma (PDAC) line Capan-2, pretreated with zoledronate to promote phosphoantigen accumulation (n=3 biological replicates). Data are mean ± sem, where applicable. [Figure 5D]Top-ranked genes are shown to improve antigen-specific T cell responses and tumor killing. (Figures 5A-G) Co-delivery of anti-CD19 CAR and ORF to T cells from a healthy donor. (Figure 5A) Schematic of the tricistronic vector and CAR T cell experiment. (Figures 5B, 5C) Secretion of IFNγ (Figure 5B) and IL-2 (Figure 5C) after overnight co-incubation of CD8+ T cells with Nalm6 cells at a 1:1 ratio (n = 3 biological replicates, representing two donors). (Figure 5D) Representative images of Nalm6 GFP+ cells co-incubated with CAR T cells or untransduced control T cells for 48 hours. Scale bar, 200 μm. (Figure 5E) Nalm6 GFP+ cell proliferation (normalized total GFP per well) after co-incubation with T cells co-expressing the 19-28z CAR and LTBR or tNGFR (negative control) at the indicated effector-to-target ratios. (Figure 5F) Quantification of Nalm6 GFP+ clearance for T cells co-expressing the 19-28z or 18-BBz CAR and the top-ranked genes (n = 3 biological replicates, representing two donors) at an effector-to-target ratio of 0.25 and normalized to tNGFR after 48 hours of co-incubation. (Figure 5G) 19-BBz CAR T cells co-expressing LTBR or tNGFR were co-incubated with Nalm6 cells at a 1:1 ratio every 3 days for up to three rounds of stimulation (n = 3 biological replicates). Seven days after repeated antigen stimulation, CAR T cells were re-exposed to Nalm6 cells. IFNγ secretion was measured after overnight incubation. (Figure 5G) Co-delivery of anti-CD19 CAR and ORF to total PBMCs from a patient with diffuse large B-cell lymphoma. Transduced T cells were incubated alone or co-incubated with CD19+ Nalm6 or CD19- Jurkat cell lines at a 1:1 ratio (n=3 biological replicates, representing two patients). IFNγ and IL2 secretion was measured after overnight incubation. For the Nalm6 condition, the numbers above the indicated column pairs represent the fold increase in cytokine secretion by LTBR cells relative to tNGFR (negative control) cells. (Figure 5I) Delivery of ORFs to Vγ9Vδ2 T cells.IFNγ and IL-2 secretion after overnight co-incubation with the pancreatic ductal adenocarcinoma (PDAC) line Capan-2, pretreated with zoledronate to promote phosphoantigen accumulation (n=3 biological replicates). Data are mean ± sem, where applicable. [Figure 5E]Top-ranked genes are shown to improve antigen-specific T cell responses and tumor killing. (Figures 5A-G) Co-delivery of anti-CD19 CAR and ORF to T cells from a healthy donor. (Figure 5A) Schematic of the tricistronic vector and CAR T cell experiment. (Figures 5B, 5C) Secretion of IFNγ (Figure 5B) and IL-2 (Figure 5C) after overnight co-incubation of CD8+ T cells with Nalm6 cells at a 1:1 ratio (n = 3 biological replicates, representing two donors). (Figure 5D) Representative images of Nalm6 GFP+ cells co-incubated with CAR T cells or untransduced control T cells for 48 hours. Scale bar, 200 μm. (Figure 5E) Nalm6 GFP+ cell proliferation (normalized total GFP per well) after co-incubation with T cells co-expressing the 19-28z CAR and LTBR or tNGFR (negative control) at the indicated effector-to-target ratios. (Figure 5F) Quantification of Nalm6 GFP+ clearance for T cells co-expressing the 19-28z or 18-BBz CAR and the top-ranked genes (n = 3 biological replicates, representing two donors) at an effector-to-target ratio of 0.25 and normalized to tNGFR after 48 hours of co-incubation. (Figure 5G) 19-BBz CAR T cells co-expressing LTBR or tNGFR were co-incubated with Nalm6 cells at a 1:1 ratio every 3 days for up to three rounds of stimulation (n = 3 biological replicates). Seven days after repeated antigen stimulation, CAR T cells were re-exposed to Nalm6 cells. IFNγ secretion was measured after overnight incubation. (Figure 5G) Co-delivery of anti-CD19 CAR and ORF to total PBMCs from a patient with diffuse large B-cell lymphoma. Transduced T cells were incubated alone or co-incubated with CD19+ Nalm6 or CD19- Jurkat cell lines at a 1:1 ratio (n=3 biological replicates, representing two patients). IFNγ and IL2 secretion was measured after overnight incubation. For the Nalm6 condition, the numbers above the indicated column pairs represent the fold increase in cytokine secretion by LTBR cells relative to tNGFR (negative control) cells. (Figure 5I) Delivery of ORFs to Vγ9Vδ2 T cells.IFNγ and IL-2 secretion after overnight co-incubation with the pancreatic ductal adenocarcinoma (PDAC) line Capan-2, pretreated with zoledronate to promote phosphoantigen accumulation (n=3 biological replicates). Data are mean ± sem, where applicable. [Figure 5F]Top-ranked genes are shown to improve antigen-specific T cell responses and tumor killing. (Figures 5A-G) Co-delivery of anti-CD19 CAR and ORF to T cells from a healthy donor. (Figure 5A) Schematic of the tricistronic vector and CAR T cell experiment. (Figures 5B, 5C) Secretion of IFNγ (Figure 5B) and IL-2 (Figure 5C) after overnight co-incubation of CD8+ T cells with Nalm6 cells at a 1:1 ratio (n = 3 biological replicates, representing two donors). (Figure 5D) Representative images of Nalm6 GFP+ cells co-incubated with CAR T cells or untransduced control T cells for 48 hours. Scale bar, 200 μm. (Figure 5E) Nalm6 GFP+ cell proliferation (normalized total GFP per well) after co-incubation with T cells co-expressing the 19-28z CAR and LTBR or tNGFR (negative control) at the indicated effector-to-target ratios. (Figure 5F) Quantification of Nalm6 GFP+ clearance for T cells co-expressing the 19-28z or 18-BBz CAR and the top-ranked genes (n = 3 biological replicates, representing two donors) at an effector-to-target ratio of 0.25 and normalized to tNGFR after 48 hours of co-incubation. (Figure 5G) 19-BBz CAR T cells co-expressing LTBR or tNGFR were co-incubated with Nalm6 cells at a 1:1 ratio every 3 days for up to three rounds of stimulation (n = 3 biological replicates). Seven days after repeated antigen stimulation, CAR T cells were re-exposed to Nalm6 cells. IFNγ secretion was measured after overnight incubation. (Figure 5G) Co-delivery of anti-CD19 CAR and ORF to total PBMCs from a patient with diffuse large B-cell lymphoma. Transduced T cells were incubated alone or co-incubated with CD19+ Nalm6 or CD19- Jurkat cell lines at a 1:1 ratio (n=3 biological replicates, representing two patients). IFNγ and IL2 secretion was measured after overnight incubation. For the Nalm6 condition, the numbers above the indicated column pairs represent the fold increase in cytokine secretion by LTBR cells relative to tNGFR (negative control) cells. (Figure 5I) Delivery of ORFs to Vγ9Vδ2 T cells.IFNγ and IL-2 secretion after overnight co-incubation with the pancreatic ductal adenocarcinoma (PDAC) line Capan-2, pretreated with zoledronate to promote phosphoantigen accumulation (n=3 biological replicates). Data are mean ± sem, where applicable. [Figure 5G]Top-ranked genes are shown to improve antigen-specific T cell responses and tumor killing. (Figures 5A-G) Co-delivery of anti-CD19 CAR and ORF to T cells from a healthy donor. (Figure 5A) Schematic of the tricistronic vector and CAR T cell experiment. (Figures 5B, 5C) Secretion of IFNγ (Figure 5B) and IL-2 (Figure 5C) after overnight co-incubation of CD8+ T cells with Nalm6 cells at a 1:1 ratio (n = 3 biological replicates, representing two donors). (Figure 5D) Representative images of Nalm6 GFP+ cells co-incubated with CAR T cells or untransduced control T cells for 48 hours. Scale bar, 200 μm. (Figure 5E) Nalm6 GFP+ cell proliferation (normalized total GFP per well) after co-incubation with T cells co-expressing the 19-28z CAR and LTBR or tNGFR (negative control) at the indicated effector-to-target ratios. (Figure 5F) Quantification of Nalm6 GFP+ clearance for T cells co-expressing the 19-28z or 18-BBz CAR and the top-ranked genes (n = 3 biological replicates, representing two donors) at an effector-to-target ratio of 0.25 and normalized to tNGFR after 48 hours of co-incubation. (Figure 5G) 19-BBz CAR T cells co-expressing LTBR or tNGFR were co-incubated with Nalm6 cells at a 1:1 ratio every 3 days for up to three rounds of stimulation (n = 3 biological replicates). Seven days after repeated antigen stimulation, CAR T cells were re-exposed to Nalm6 cells. IFNγ secretion was measured after overnight incubation. (Figure 5G) Co-delivery of anti-CD19 CAR and ORF to total PBMCs from a patient with diffuse large B-cell lymphoma. Transduced T cells were incubated alone or co-incubated with CD19+ Nalm6 or CD19- Jurkat cell lines at a 1:1 ratio (n=3 biological replicates, representing two patients). IFNγ and IL2 secretion was measured after overnight incubation. For the Nalm6 condition, the numbers above the indicated column pairs represent the fold increase in cytokine secretion by LTBR cells relative to tNGFR (negative control) cells. (Figure 5I) Delivery of ORFs to Vγ9Vδ2 T cells.IFNγ and IL-2 secretion after overnight co-incubation with the pancreatic ductal adenocarcinoma (PDAC) line Capan-2, pretreated with zoledronate to promote phosphoantigen accumulation (n=3 biological replicates). Data are mean ± sem, where applicable. [Figure 5H]Top-ranked genes are shown to improve antigen-specific T cell responses and tumor killing. (Figures 5A-G) Co-delivery of anti-CD19 CAR and ORF to T cells from a healthy donor. (Figure 5A) Schematic of the tricistronic vector and CAR T cell experiment. (Figures 5B, 5C) Secretion of IFNγ (Figure 5B) and IL-2 (Figure 5C) after overnight co-incubation of CD8+ T cells with Nalm6 cells at a 1:1 ratio (n = 3 biological replicates, representing two donors). (Figure 5D) Representative images of Nalm6 GFP+ cells co-incubated with CAR T cells or untransduced control T cells for 48 hours. Scale bar, 200 μm. (Figure 5E) Nalm6 GFP+ cell proliferation (normalized total GFP per well) after co-incubation with T cells co-expressing the 19-28z CAR and LTBR or tNGFR (negative control) at the indicated effector-to-target ratios. (Figure 5F) Quantification of Nalm6 GFP+ clearance for T cells co-expressing the 19-28z or 18-BBz CAR and the top-ranked genes (n = 3 biological replicates, representing two donors) at an effector-to-target ratio of 0.25 and normalized to tNGFR after 48 hours of co-incubation. (Figure 5G) 19-BBz CAR T cells co-expressing LTBR or tNGFR were co-incubated with Nalm6 cells at a 1:1 ratio every 3 days for up to three rounds of stimulation (n = 3 biological replicates). Seven days after repeated antigen stimulation, CAR T cells were re-exposed to Nalm6 cells. IFNγ secretion was measured after overnight incubation. (Figure 5G) Co-delivery of anti-CD19 CAR and ORF to total PBMCs from a patient with diffuse large B-cell lymphoma. Transduced T cells were incubated alone or co-incubated with CD19+ Nalm6 or CD19- Jurkat cell lines at a 1:1 ratio (n=3 biological replicates, representing two patients). IFNγ and IL2 secretion was measured after overnight incubation. For the Nalm6 condition, the numbers above the indicated column pairs represent the fold increase in cytokine secretion by LTBR cells relative to tNGFR (negative control) cells. (Figure 5I) Delivery of ORFs to Vγ9Vδ2 T cells.IFNγ and IL-2 secretion after overnight co-incubation with the pancreatic ductal adenocarcinoma (PDAC) line Capan-2, pretreated with zoledronate to promote phosphoantigen accumulation (n=3 biological replicates). Data are mean ± sem, where applicable. [Figure 5I]Top-ranked genes are shown to improve antigen-specific T cell responses and tumor killing. (Figures 5A-G) Co-delivery of anti-CD19 CAR and ORF to T cells from a healthy donor. (Figure 5A) Schematic of the tricistronic vector and CAR T cell experiment. (Figures 5B, 5C) Secretion of IFNγ (Figure 5B) and IL-2 (Figure 5C) after overnight co-incubation of CD8+ T cells with Nalm6 cells at a 1:1 ratio (n = 3 biological replicates, representing two donors). (Figure 5D) Representative images of Nalm6 GFP+ cells co-incubated with CAR T cells or untransduced control T cells for 48 hours. Scale bar, 200 μm. (Figure 5E) Nalm6 GFP+ cell proliferation (normalized total GFP per well) after co-incubation with T cells co-expressing the 19-28z CAR and LTBR or tNGFR (negative control) at the indicated effector-to-target ratios. (Figure 5F) Quantification of Nalm6 GFP+ clearance for T cells co-expressing the 19-28z or 18-BBz CAR and the top-ranked genes (n = 3 biological replicates, representing two donors) at an effector-to-target ratio of 0.25 and normalized to tNGFR after 48 hours of co-incubation. (Figure 5G) 19-BBz CAR T cells co-expressing LTBR or tNGFR were co-incubated with Nalm6 cells at a 1:1 ratio every 3 days for up to three rounds of stimulation (n = 3 biological replicates). Seven days after repeated antigen stimulation, CAR T cells were re-exposed to Nalm6 cells. IFNγ secretion was measured after overnight incubation. (Figure 5G) Co-delivery of anti-CD19 CAR and ORF to total PBMCs from a patient with diffuse large B-cell lymphoma. Transduced T cells were incubated alone or co-incubated with CD19+ Nalm6 or CD19- Jurkat cell lines at a 1:1 ratio (n=3 biological replicates, representing two patients). IFNγ and IL2 secretion was measured after overnight incubation. For the Nalm6 condition, the numbers above the indicated column pairs represent the fold increase in cytokine secretion by LTBR cells relative to tNGFR (negative control) cells. (Figure 5I) Delivery of ORFs to Vγ9Vδ2 T cells.IFNγ and IL-2 secretion after overnight co-incubation with the pancreatic ductal adenocarcinoma (PDAC) line Capan-2, pretreated with zoledronate to promote phosphoantigen accumulation (n=3 biological replicates). Data are mean ± sem, where applicable. [Figure 6A]Figure 6A shows the design of a human ORF library screening in primary T cells. (Figure 6A) Barcoded vector design for ORF overexpression. (Figure 6B) Distribution of the number of barcodes per ORF in the library. (Figure 6C) Vector design for quantifying the effect of different promoters and ORF insert sizes on lentiviral transduction efficiency. EFS - elongation factor-1α short promoter, CMV - cytomegalovirus promoter, PGK - phosphoglycerate kinase-1 promoter. (Figure 6D) Sequential gating strategy and representative histograms of cells transduced with the marker gene rat CD2 under different promoters. (Figure 6E) Percentage of positive cells and (Figure 6F) Mean Fluorescence Intensity (MFI) of rat CD2 (rCD2) expressed from the EFS and CMV promoters after puromycin selection of transduced primary CD4+ T cells. Each data point represents an individual transduction (n = 3 biological replicates). Error bars represent SEM. (Figure 6G) Distribution of ORF sizes in the genome-scale library. The sizes of the TCR-rCD2 constructs tested in panels 6E and 6F are shown. (Figure 6H) Titering of CD3 / CD28 antibodies. T cells were labeled with CFSE, stimulated, and incubated for 4 days. Gating for proliferating T cells was set at least twice to include expanded cells (third CFSE peak). (Figure 6I) Expansion of T cells from three healthy donors transduced with the ORF library. (Figure 6J) Representative CFSE profiles of restimulated CD8+ and CD4+ T cells before sorting. The CFSE low sorting gate is marked. (Figure 6K) Recovery of individual barcodes or corresponding ORFs in transduced T cells and plasmids used for lentivirus production. Samples from three donors were computationally pooled together with equal numbers of reads, and then the number of barcodes or ORFs present was counted with a minimum of one read. (Figure 6L) Distribution of reads corresponding to ORFs of different sizes. ORFs were assigned to deciles based on their size, with Q1 being the smallest size and Q10 being the largest (n = 1,161 ORFs per quantile). Boxes show the 25th–75th percentiles with a median line and extension lines extending to 1.5 × interquartile range.(Figure 6M) Gene enrichment in both CFSE-low CD4+ and CD8+ T cells calculated by collapsing individual barcodes into corresponding genes. Significantly enriched genes (log2 fold change >0.5 and adjusted p-value <0.05) are shown in red. Immune response genes of interest are indicated. (Figure 6N) Overlap of significantly enriched genes from Figure 6M in individual screening populations (CD4+, CD8+) analyzed separately. (Figure 6O) Normalized enrichment of individual barcodes for the indicated genes in the CD8+ screen. (Figure 6P) GO biological process for significantly enriched genes in Figure 6M. (Figure 6Q) Overlap of significantly enriched genes with genes differentially expressed between CD3 / CD28-stimulated and naive T cells. [Figure 6B]Figure 6A shows the design of a human ORF library screening in primary T cells. (Figure 6A) Barcoded vector design for ORF overexpression. (Figure 6B) Distribution of the number of barcodes per ORF in the library. (Figure 6C) Vector design for quantifying the effect of different promoters and ORF insert sizes on lentiviral transduction efficiency. EFS - elongation factor-1α short promoter, CMV - cytomegalovirus promoter, PGK - phosphoglycerate kinase-1 promoter. (Figure 6D) Sequential gating strategy and representative histograms of cells transduced with the marker gene rat CD2 under different promoters. (Figure 6E) Percentage of positive cells and (Figure 6F) Mean Fluorescence Intensity (MFI) of rat CD2 (rCD2) expressed from the EFS and CMV promoters after puromycin selection of transduced primary CD4+ T cells. Each data point represents an individual transduction (n = 3 biological replicates). Error bars represent SEM. (Figure 6G) Distribution of ORF sizes in the genome-scale library. The sizes of the TCR-rCD2 constructs tested in panels 6E and 6F are shown. (Figure 6H) Titering of CD3 / CD28 antibodies. T cells were labeled with CFSE, stimulated, and incubated for 4 days. Gating for proliferating T cells was set at least twice to include expanded cells (third CFSE peak). (Figure 6I) Expansion of T cells from three healthy donors transduced with the ORF library. (Figure 6J) Representative CFSE profiles of restimulated CD8+ and CD4+ T cells before sorting. The CFSE low sorting gate is marked. (Figure 6K) Recovery of individual barcodes or corresponding ORFs in transduced T cells and plasmids used for lentivirus production. Samples from three donors were computationally pooled together with equal numbers of reads, and then the number of barcodes or ORFs present was counted with a minimum of one read. (Figure 6L) Distribution of reads corresponding to ORFs of different sizes. ORFs were assigned to deciles based on their size, with Q1 being the smallest size and Q10 being the largest (n = 1,161 ORFs per quantile). Boxes show the 25th–75th percentiles with a median line and extension lines extending to 1.5 × interquartile range.(Figure 6M) Gene enrichment in both CFSE-low CD4+ and CD8+ T cells calculated by collapsing individual barcodes into corresponding genes. Significantly enriched genes (log2 fold change >0.5 and adjusted p-value <0.05) are shown in red. Immune response genes of interest are indicated. (Figure 6N) Overlap of significantly enriched genes from Figure 6M in individual screening populations (CD4+, CD8+) analyzed separately. (Figure 6O) Normalized enrichment of individual barcodes for the indicated genes in the CD8+ screen. (Figure 6P) GO biological process for significantly enriched genes in Figure 6M. (Figure 6Q) Overlap of significantly enriched genes with genes differentially expressed between CD3 / CD28-stimulated and naive T cells. [Figure 6C]Figure 6A shows the design of a human ORF library screening in primary T cells. (Figure 6A) Barcoded vector design for ORF overexpression. (Figure 6B) Distribution of the number of barcodes per ORF in the library. (Figure 6C) Vector design for quantifying the effect of different promoters and ORF insert sizes on lentiviral transduction efficiency. EFS - elongation factor-1α short promoter, CMV - cytomegalovirus promoter, PGK - phosphoglycerate kinase-1 promoter. (Figure 6D) Sequential gating strategy and representative histograms of cells transduced with the marker gene rat CD2 under different promoters. (Figure 6E) Percentage of positive cells and (Figure 6F) Mean Fluorescence Intensity (MFI) of rat CD2 (rCD2) expressed from the EFS and CMV promoters after puromycin selection of transduced primary CD4+ T cells. Each data point represents an individual transduction (n = 3 biological replicates). Error bars represent SEM. (Figure 6G) Distribution of ORF sizes in the genome-scale library. The sizes of the TCR-rCD2 constructs tested in panels 6E and 6F are shown. (Figure 6H) Titering of CD3 / CD28 antibodies. T cells were labeled with CFSE, stimulated, and incubated for 4 days. Gating for proliferating T cells was set at least twice to include expanded cells (third CFSE peak). (Figure 6I) Expansion of T cells from three healthy donors transduced with the ORF library. (Figure 6J) Representative CFSE profiles of restimulated CD8+ and CD4+ T cells before sorting. The CFSE low sorting gate is marked. (Figure 6K) Recovery of individual barcodes or corresponding ORFs in transduced T cells and plasmids used for lentivirus production. Samples from three donors were computationally pooled together with equal numbers of reads, and then the number of barcodes or ORFs present was counted with a minimum of one read. (Figure 6L) Distribution of reads corresponding to ORFs of different sizes. ORFs were assigned to deciles based on their size, with Q1 being the smallest size and Q10 being the largest (n = 1,161 ORFs per quantile). Boxes show the 25th–75th percentiles with a median line and extension lines extending to 1.5 × interquartile range.(Figure 6M) Gene enrichment in both CFSE-low CD4+ and CD8+ T cells calculated by collapsing individual barcodes into corresponding genes. Significantly enriched genes (log2 fold change >0.5 and adjusted p-value <0.05) are shown in red. Immune response genes of interest are indicated. (Figure 6N) Overlap of significantly enriched genes from Figure 6M in individual screening populations (CD4+, CD8+) analyzed separately. (Figure 6O) Normalized enrichment of individual barcodes for the indicated genes in the CD8+ screen. (Figure 6P) GO biological process for significantly enriched genes in Figure 6M. (Figure 6Q) Overlap of significantly enriched genes with genes differentially expressed between CD3 / CD28-stimulated and naive T cells. [Figure 6D]Figure 6A shows the design of a human ORF library screening in primary T cells. (Figure 6A) Barcoded vector design for ORF overexpression. (Figure 6B) Distribution of the number of barcodes per ORF in the library. (Figure 6C) Vector design for quantifying the effect of different promoters and ORF insert sizes on lentiviral transduction efficiency. EFS - elongation factor-1α short promoter, CMV - cytomegalovirus promoter, PGK - phosphoglycerate kinase-1 promoter. (Figure 6D) Sequential gating strategy and representative histograms of cells transduced with the marker gene rat CD2 under different promoters. (Figure 6E) Percentage of positive cells and (Figure 6F) Mean Fluorescence Intensity (MFI) of rat CD2 (rCD2) expressed from the EFS and CMV promoters after puromycin selection of transduced primary CD4+ T cells. Each data point represents an individual transduction (n = 3 biological replicates). Error bars represent SEM. (Figure 6G) Distribution of ORF sizes in the genome-scale library. The sizes of the TCR-rCD2 constructs tested in panels 6E and 6F are shown. (Figure 6H) Titering of CD3 / CD28 antibodies. T cells were labeled with CFSE, stimulated, and incubated for 4 days. Gating for proliferating T cells was set at least twice to include expanded cells (third CFSE peak). (Figure 6I) Expansion of T cells from three healthy donors transduced with the ORF library. (Figure 6J) Representative CFSE profiles of restimulated CD8+ and CD4+ T cells before sorting. The CFSE low sorting gate is marked. (Figure 6K) Recovery of individual barcodes or corresponding ORFs in transduced T cells and plasmids used for lentivirus production. Samples from three donors were computationally pooled together with equal numbers of reads, and then the number of barcodes or ORFs present was counted with a minimum of one read. (Figure 6L) Distribution of reads corresponding to ORFs of different sizes. ORFs were assigned to deciles based on their size, with Q1 being the smallest size and Q10 being the largest (n = 1,161 ORFs per quantile). Boxes show the 25th–75th percentiles with a median line and extension lines extending to 1.5 × interquartile range.(Figure 6M) Gene enrichment in both CFSE-low CD4+ and CD8+ T cells calculated by collapsing individual barcodes into corresponding genes. Significantly enriched genes (log2 fold change >0.5 and adjusted p-value <0.05) are shown in red. Immune response genes of interest are indicated. (Figure 6N) Overlap of significantly enriched genes from Figure 6M in individual screening populations (CD4+, CD8+) analyzed separately. (Figure 6O) Normalized enrichment of individual barcodes for the indicated genes in the CD8+ screen. (Figure 6P) GO biological process for significantly enriched genes in Figure 6M. (Figure 6Q) Overlap of significantly enriched genes with genes differentially expressed between CD3 / CD28-stimulated and naive T cells. [Figure 6E]Figure 6A shows the design of a human ORF library screening in primary T cells. (Figure 6A) Barcoded vector design for ORF overexpression. (Figure 6B) Distribution of the number of barcodes per ORF in the library. (Figure 6C) Vector design for quantifying the effect of different promoters and ORF insert sizes on lentiviral transduction efficiency. EFS - elongation factor-1α short promoter, CMV - cytomegalovirus promoter, PGK - phosphoglycerate kinase-1 promoter. (Figure 6D) Sequential gating strategy and representative histograms of cells transduced with the marker gene rat CD2 under different promoters. (Figure 6E) Percentage of positive cells and (Figure 6F) Mean Fluorescence Intensity (MFI) of rat CD2 (rCD2) expressed from the EFS and CMV promoters after puromycin selection of transduced primary CD4+ T cells. Each data point represents an individual transduction (n = 3 biological replicates). Error bars represent SEM. (Figure 6G) Distribution of ORF sizes in the genome-scale library. The sizes of the TCR-rCD2 constructs tested in panels 6E and 6F are shown. (Figure 6H) Titering of CD3 / CD28 antibodies. T cells were labeled with CFSE, stimulated, and incubated for 4 days. Gating for proliferating T cells was set at least twice to include expanded cells (third CFSE peak). (Figure 6I) Expansion of T cells from three healthy donors transduced with the ORF library. (Figure 6J) Representative CFSE profiles of restimulated CD8+ and CD4+ T cells before sorting. The CFSE low sorting gate is marked. (Figure 6K) Recovery of individual barcodes or corresponding ORFs in transduced T cells and plasmids used for lentivirus production. Samples from three donors were computationally pooled together with equal numbers of reads, and then the number of barcodes or ORFs present was counted with a minimum of one read. (Figure 6L) Distribution of reads corresponding to ORFs of different sizes. ORFs were assigned to deciles based on their size, with Q1 being the smallest size and Q10 being the largest (n = 1,161 ORFs per quantile). Boxes show the 25th–75th percentiles with a median line and extension lines extending to 1.5 × interquartile range.(Figure 6M) Gene enrichment in both CFSE-low CD4+ and CD8+ T cells calculated by collapsing individual barcodes into corresponding genes. Significantly enriched genes (log2 fold change >0.5 and adjusted p-value <0.05) are shown in red. Immune response genes of interest are indicated. (Figure 6N) Overlap of significantly enriched genes from Figure 6M in individual screening populations (CD4+, CD8+) analyzed separately. (Figure 6O) Normalized enrichment of individual barcodes for the indicated genes in the CD8+ screen. (Figure 6P) GO biological process for significantly enriched genes in Figure 6M. (Figure 6Q) Overlap of significantly enriched genes with genes differentially expressed between CD3 / CD28-stimulated and naive T cells. [Figure 6F]Figure 6A shows the design of a human ORF library screening in primary T cells. (Figure 6A) Barcoded vector design for ORF overexpression. (Figure 6B) Distribution of the number of barcodes per ORF in the library. (Figure 6C) Vector design for quantifying the effect of different promoters and ORF insert sizes on lentiviral transduction efficiency. EFS - elongation factor-1α short promoter, CMV - cytomegalovirus promoter, PGK - phosphoglycerate kinase-1 promoter. (Figure 6D) Sequential gating strategy and representative histograms of cells transduced with the marker gene rat CD2 under different promoters. (Figure 6E) Percentage of positive cells and (Figure 6F) Mean Fluorescence Intensity (MFI) of rat CD2 (rCD2) expressed from the EFS and CMV promoters after puromycin selection of transduced primary CD4+ T cells. Each data point represents an individual transduction (n = 3 biological replicates). Error bars represent SEM. (Figure 6G) Distribution of ORF sizes in the genome-scale library. The sizes of the TCR-rCD2 constructs tested in panels 6E and 6F are shown. (Figure 6H) Titering of CD3 / CD28 antibodies. T cells were labeled with CFSE, stimulated, and incubated for 4 days. Gating for proliferating T cells was set at least twice to include expanded cells (third CFSE peak). (Figure 6I) Expansion of T cells from three healthy donors transduced with the ORF library. (Figure 6J) Representative CFSE profiles of restimulated CD8+ and CD4+ T cells before sorting. The CFSE low sorting gate is marked. (Figure 6K) Recovery of individual barcodes or corresponding ORFs in transduced T cells and plasmids used for lentivirus production. Samples from three donors were computationally pooled together with equal numbers of reads, and then the number of barcodes or ORFs present was counted with a minimum of one read. (Figure 6L) Distribution of reads corresponding to ORFs of different sizes. ORFs were assigned to deciles based on their size, with Q1 being the smallest size and Q10 being the largest (n = 1,161 ORFs per quantile). Boxes show the 25th–75th percentiles with a median line and extension lines extending to 1.5 × interquartile range.(Figure 6M) Gene enrichment in both CFSE-low CD4+ and CD8+ T cells calculated by collapsing individual barcodes into corresponding genes. Significantly enriched genes (log2 fold change >0.5 and adjusted p-value <0.05) are shown in red. Immune response genes of interest are indicated. (Figure 6N) Overlap of significantly enriched genes from Figure 6M in individual screening populations (CD4+, CD8+) analyzed separately. (Figure 6O) Normalized enrichment of individual barcodes for the indicated genes in the CD8+ screen. (Figure 6P) GO biological process for significantly enriched genes in Figure 6M. (Figure 6Q) Overlap of significantly enriched genes with genes differentially expressed between CD3 / CD28-stimulated and naive T cells. [Figure 6G]Figure 6A shows the design of a human ORF library screening in primary T cells. (Figure 6A) Barcoded vector design for ORF overexpression. (Figure 6B) Distribution of the number of barcodes per ORF in the library. (Figure 6C) Vector design for quantifying the effect of different promoters and ORF insert sizes on lentiviral transduction efficiency. EFS - elongation factor-1α short promoter, CMV - cytomegalovirus promoter, PGK - phosphoglycerate kinase-1 promoter. (Figure 6D) Sequential gating strategy and representative histograms of cells transduced with the marker gene rat CD2 under different promoters. (Figure 6E) Percentage of positive cells and (Figure 6F) Mean Fluorescence Intensity (MFI) of rat CD2 (rCD2) expressed from the EFS and CMV promoters after puromycin selection of transduced primary CD4+ T cells. Each data point represents an individual transduction (n = 3 biological replicates). Error bars represent SEM. (Figure 6G) Distribution of ORF sizes in the genome-scale library. The sizes of the TCR-rCD2 constructs tested in panels 6E and 6F are shown. (Figure 6H) Titering of CD3 / CD28 antibodies. T cells were labeled with CFSE, stimulated, and incubated for 4 days. Gating for proliferating T cells was set at least twice to include expanded cells (third CFSE peak). (Figure 6I) Expansion of T cells from three healthy donors transduced with the ORF library. (Figure 6J) Representative CFSE profiles of restimulated CD8+ and CD4+ T cells before sorting. The CFSE low sorting gate is marked. (Figure 6K) Recovery of individual barcodes or corresponding ORFs in transduced T cells and plasmids used for lentivirus production. Samples from three donors were computationally pooled together with equal numbers of reads, and then the number of barcodes or ORFs present was counted with a minimum of one read. (Figure 6L) Distribution of reads corresponding to ORFs of different sizes. ORFs were assigned to deciles based on their size, with Q1 being the smallest size and Q10 being the largest (n = 1,161 ORFs per quantile). Boxes show the 25th–75th percentiles with a median line and extension lines extending to 1.5 × interquartile range.(Figure 6M) Gene enrichment in both CFSE-low CD4+ and CD8+ T cells calculated by collapsing individual barcodes into corresponding genes. Significantly enriched genes (log2 fold change >0.5 and adjusted p-value <0.05) are shown in red. Immune response genes of interest are indicated. (Figure 6N) Overlap of significantly enriched genes from Figure 6M in individual screening populations (CD4+, CD8+) analyzed separately. (Figure 6O) Normalized enrichment of individual barcodes for the indicated genes in the CD8+ screen. (Figure 6P) GO biological process for significantly enriched genes in Figure 6M. (Figure 6Q) Overlap of significantly enriched genes with genes differentially expressed between CD3 / CD28-stimulated and naive T cells. [Figure 6H]Figure 6A shows the design of a human ORF library screening in primary T cells. (Figure 6A) Barcoded vector design for ORF overexpression. (Figure 6B) Distribution of the number of barcodes per ORF in the library. (Figure 6C) Vector design for quantifying the effect of different promoters and ORF insert sizes on lentiviral transduction efficiency. EFS - elongation factor-1α short promoter, CMV - cytomegalovirus promoter, PGK - phosphoglycerate kinase-1 promoter. (Figure 6D) Sequential gating strategy and representative histograms of cells transduced with the marker gene rat CD2 under different promoters. (Figure 6E) Percentage of positive cells and (Figure 6F) Mean Fluorescence Intensity (MFI) of rat CD2 (rCD2) expressed from the EFS and CMV promoters after puromycin selection of transduced primary CD4+ T cells. Each data point represents an individual transduction (n = 3 biological replicates). Error bars represent SEM. (Figure 6G) Distribution of ORF sizes in the genome-scale library. The sizes of the TCR-rCD2 constructs tested in panels 6E and 6F are shown. (Figure 6H) Titering of CD3 / CD28 antibodies. T cells were labeled with CFSE, stimulated, and incubated for 4 days. Gating for proliferating T cells was set at least twice to include expanded cells (third CFSE peak). (Figure 6I) Expansion of T cells from three healthy donors transduced with the ORF library. (Figure 6J) Representative CFSE profiles of restimulated CD8+ and CD4+ T cells before sorting. The CFSE low sorting gate is marked. (Figure 6K) Recovery of individual barcodes or corresponding ORFs in transduced T cells and plasmids used for lentivirus production. Samples from three donors were computationally pooled together with equal numbers of reads, and then the number of barcodes or ORFs present was counted with a minimum of one read. (Figure 6L) Distribution of reads corresponding to ORFs of different sizes. ORFs were assigned to deciles based on their size, with Q1 being the smallest size and Q10 being the largest (n = 1,161 ORFs per quantile). Boxes show the 25th–75th percentiles with a median line and extension lines extending to 1.5 × interquartile range.(Figure 6M) Gene enrichment in both CFSE-low CD4+ and CD8+ T cells calculated by collapsing individual barcodes into corresponding genes. Significantly enriched genes (log2 fold change >0.5 and adjusted p-value <0.05) are shown in red. Immune response genes of interest are indicated. (Figure 6N) Overlap of significantly enriched genes from Figure 6M in individual screening populations (CD4+, CD8+) analyzed separately. (Figure 6O) Normalized enrichment of individual barcodes for the indicated genes in the CD8+ screen. (Figure 6P) GO biological process for significantly enriched genes in Figure 6M. (Figure 6Q) Overlap of significantly enriched genes with genes differentially expressed between CD3 / CD28-stimulated and naive T cells. [Figure 6I]Figure 6A shows the design of a human ORF library screening in primary T cells. (Figure 6A) Barcoded vector design for ORF overexpression. (Figure 6B) Distribution of the number of barcodes per ORF in the library. (Figure 6C) Vector design for quantifying the effect of different promoters and ORF insert sizes on lentiviral transduction efficiency. EFS - elongation factor-1α short promoter, CMV - cytomegalovirus promoter, PGK - phosphoglycerate kinase-1 promoter. (Figure 6D) Sequential gating strategy and representative histograms of cells transduced with the marker gene rat CD2 under different promoters. (Figure 6E) Percentage of positive cells and (Figure 6F) Mean Fluorescence Intensity (MFI) of rat CD2 (rCD2) expressed from the EFS and CMV promoters after puromycin selection of transduced primary CD4+ T cells. Each data point represents an individual transduction (n = 3 biological replicates). Error bars represent SEM. (Figure 6G) Distribution of ORF sizes in the genome-scale library. The sizes of the TCR-rCD2 constructs tested in panels 6E and 6F are shown. (Figure 6H) Titering of CD3 / CD28 antibodies. T cells were labeled with CFSE, stimulated, and incubated for 4 days. Gating for proliferating T cells was set at least twice to include expanded cells (third CFSE peak). (Figure 6I) Expansion of T cells from three healthy donors transduced with the ORF library. (Figure 6J) Representative CFSE profiles of restimulated CD8+ and CD4+ T cells before sorting. The CFSE low sorting gate is marked. (Figure 6K) Recovery of individual barcodes or corresponding ORFs in transduced T cells and plasmids used for lentivirus production. Samples from three donors were computationally pooled together with equal numbers of reads, and then the number of barcodes or ORFs present was counted with a minimum of one read. (Figure 6L) Distribution of reads corresponding to ORFs of different sizes. ORFs were assigned to deciles based on their size, with Q1 being the smallest size and Q10 being the largest (n = 1,161 ORFs per quantile). Boxes show the 25th–75th percentiles with a median line and extension lines extending to 1.5 × interquartile range.(Figure 6M) Gene enrichment in both CFSE-low CD4+ and CD8+ T cells calculated by collapsing individual barcodes into corresponding genes. Significantly enriched genes (log2 fold change >0.5 and adjusted p-value <0.05) are shown in red. Immune response genes of interest are indicated. (Figure 6N) Overlap of significantly enriched genes from Figure 6M in individual screening populations (CD4+, CD8+) analyzed separately. (Figure 6O) Normalized enrichment of individual barcodes for the indicated genes in the CD8+ screen. (Figure 6P) GO biological process for significantly enriched genes in Figure 6M. (Figure 6Q) Overlap of significantly enriched genes with genes differentially expressed between CD3 / CD28-stimulated and naive T cells. [Figure 6J]Figure 6A shows the design of a human ORF library screening in primary T cells. (Figure 6A) Barcoded vector design for ORF overexpression. (Figure 6B) Distribution of the number of barcodes per ORF in the library. (Figure 6C) Vector design for quantifying the effect of different promoters and ORF insert sizes on lentiviral transduction efficiency. EFS - elongation factor-1α short promoter, CMV - cytomegalovirus promoter, PGK - phosphoglycerate kinase-1 promoter. (Figure 6D) Sequential gating strategy and representative histograms of cells transduced with the marker gene rat CD2 under different promoters. (Figure 6E) Percentage of positive cells and (Figure 6F) Mean Fluorescence Intensity (MFI) of rat CD2 (rCD2) expressed from the EFS and CMV promoters after puromycin selection of transduced primary CD4+ T cells. Each data point represents an individual transduction (n = 3 biological replicates). Error bars represent SEM. (Figure 6G) Distribution of ORF sizes in the genome-scale library. The sizes of the TCR-rCD2 constructs tested in panels 6E and 6F are shown. (Figure 6H) Titering of CD3 / CD28 antibodies. T cells were labeled with CFSE, stimulated, and incubated for 4 days. Gating for proliferating T cells was set at least twice to include expanded cells (third CFSE peak). (Figure 6I) Expansion of T cells from three healthy donors transduced with the ORF library. (Figure 6J) Representative CFSE profiles of restimulated CD8+ and CD4+ T cells before sorting. The CFSE low sorting gate is marked. (Figure 6K) Recovery of individual barcodes or corresponding ORFs in transduced T cells and plasmids used for lentivirus production. Samples from three donors were computationally pooled together with equal numbers of reads, and then the number of barcodes or ORFs present was counted with a minimum of one read. (Figure 6L) Distribution of reads corresponding to ORFs of different sizes. ORFs were assigned to deciles based on their size, with Q1 being the smallest size and Q10 being the largest (n = 1,161 ORFs per quantile). Boxes show the 25th–75th percentiles with a median line and extension lines extending to 1.5 × interquartile range.(Figure 6M) Gene enrichment in both CFSE-low CD4+ and CD8+ T cells calculated by collapsing individual barcodes into corresponding genes. Significantly enriched genes (log2 fold change >0.5 and adjusted p-value <0.05) are shown in red. Immune response genes of interest are indicated. (Figure 6N) Overlap of significantly enriched genes from Figure 6M in individual screening populations (CD4+, CD8+) analyzed separately. (Figure 6O) Normalized enrichment of individual barcodes for the indicated genes in the CD8+ screen. (Figure 6P) GO biological process for significantly enriched genes in Figure 6M. (Figure 6Q) Overlap of significantly enriched genes with genes differentially expressed between CD3 / CD28-stimulated and naive T cells. [Figure 6K]Figure 6A shows the design of a human ORF library screening in primary T cells. (Figure 6A) Barcoded vector design for ORF overexpression. (Figure 6B) Distribution of the number of barcodes per ORF in the library. (Figure 6C) Vector design for quantifying the effect of different promoters and ORF insert sizes on lentiviral transduction efficiency. EFS - elongation factor-1α short promoter, CMV - cytomegalovirus promoter, PGK - phosphoglycerate kinase-1 promoter. (Figure 6D) Sequential gating strategy and representative histograms of cells transduced with the marker gene rat CD2 under different promoters. (Figure 6E) Percentage of positive cells and (Figure 6F) Mean Fluorescence Intensity (MFI) of rat CD2 (rCD2) expressed from the EFS and CMV promoters after puromycin selection of transduced primary CD4+ T cells. Each data point represents an individual transduction (n = 3 biological replicates). Error bars represent SEM. (Figure 6G) Distribution of ORF sizes in the genome-scale library. The sizes of the TCR-rCD2 constructs tested in panels 6E and 6F are shown. (Figure 6H) Titering of CD3 / CD28 antibodies. T cells were labeled with CFSE, stimulated, and incubated for 4 days. Gating for proliferating T cells was set at least twice to include expanded cells (third CFSE peak). (Figure 6I) Expansion of T cells from three healthy donors transduced with the ORF library. (Figure 6J) Representative CFSE profiles of restimulated CD8+ and CD4+ T cells before sorting. The CFSE low sorting gate is marked. (Figure 6K) Recovery of individual barcodes or corresponding ORFs in transduced T cells and plasmids used for lentivirus production. Samples from three donors were computationally pooled together with equal numbers of reads, and then the number of barcodes or ORFs present was counted with a minimum of one read. (Figure 6L) Distribution of reads corresponding to ORFs of different sizes. ORFs were assigned to deciles based on their size, with Q1 being the smallest size and Q10 being the largest (n = 1,161 ORFs per quantile). Boxes show the 25th–75th percentiles with a median line and extension lines extending to 1.5 × interquartile range.(Figure 6M) Gene enrichment in both CFSE-low CD4+ and CD8+ T cells calculated by collapsing individual barcodes into corresponding genes. Significantly enriched genes (log2 fold change >0.5 and adjusted p-value <0.05) are shown in red. Immune response genes of interest are indicated. (Figure 6N) Overlap of significantly enriched genes from Figure 6M in individual screening populations (CD4+, CD8+) analyzed separately. (Figure 6O) Normalized enrichment of individual barcodes for the indicated genes in the CD8+ screen. (Figure 6P) GO biological process for significantly enriched genes in Figure 6M. (Figure 6Q) Overlap of significantly enriched genes with genes differentially expressed between CD3 / CD28-stimulated and naive T cells. [Figure 6L]Figure 6A shows the design of a human ORF library screening in primary T cells. (Figure 6A) Barcoded vector design for ORF overexpression. (Figure 6B) Distribution of the number of barcodes per ORF in the library. (Figure 6C) Vector design for quantifying the effect of different promoters and ORF insert sizes on lentiviral transduction efficiency. EFS - elongation factor-1α short promoter, CMV - cytomegalovirus promoter, PGK - phosphoglycerate kinase-1 promoter. (Figure 6D) Sequential gating strategy and representative histograms of cells transduced with the marker gene rat CD2 under different promoters. (Figure 6E) Percentage of positive cells and (Figure 6F) Mean Fluorescence Intensity (MFI) of rat CD2 (rCD2) expressed from the EFS and CMV promoters after puromycin selection of transduced primary CD4+ T cells. Each data point represents an individual transduction (n = 3 biological replicates). Error bars represent SEM. (Figure 6G) Distribution of ORF sizes in the genome-scale library. The sizes of the TCR-rCD2 constructs tested in panels 6E and 6F are shown. (Figure 6H) Titering of CD3 / CD28 antibodies. T cells were labeled with CFSE, stimulated, and incubated for 4 days. Gating for proliferating T cells was set at least twice to include expanded cells (third CFSE peak). (Figure 6I) Expansion of T cells from three healthy donors transduced with the ORF library. (Figure 6J) Representative CFSE profiles of restimulated CD8+ and CD4+ T cells before sorting. The CFSE low sorting gate is marked. (Figure 6K) Recovery of individual barcodes or corresponding ORFs in transduced T cells and plasmids used for lentivirus production. Samples from three donors were computationally pooled together with equal numbers of reads, and then the number of barcodes or ORFs present was counted with a minimum of one read. (Figure 6L) Distribution of reads corresponding to ORFs of different sizes. ORFs were assigned to deciles based on their size, with Q1 being the smallest size and Q10 being the largest (n = 1,161 ORFs per quantile). Boxes show the 25th–75th percentiles with a median line and extension lines extending to 1.5 × interquartile range.(Figure 6M) Gene enrichment in both CFSE-low CD4+ and CD8+ T cells calculated by collapsing individual barcodes into corresponding genes. Significantly enriched genes (log2 fold change >0.5 and adjusted p-value <0.05) are shown in red. Immune response genes of interest are indicated. (Figure 6N) Overlap of significantly enriched genes from Figure 6M in individual screening populations (CD4+, CD8+) analyzed separately. (Figure 6O) Normalized enrichment of individual barcodes for the indicated genes in the CD8+ screen. (Figure 6P) GO biological process for significantly enriched genes in Figure 6M. (Figure 6Q) Overlap of significantly enriched genes with genes differentially expressed between CD3 / CD28-stimulated and naive T cells. [Figure 6M]Figure 6A shows the design of a human ORF library screening in primary T cells. (Figure 6A) Barcoded vector design for ORF overexpression. (Figure 6B) Distribution of the number of barcodes per ORF in the library. (Figure 6C) Vector design for quantifying the effect of different promoters and ORF insert sizes on lentiviral transduction efficiency. EFS - elongation factor-1α short promoter, CMV - cytomegalovirus promoter, PGK - phosphoglycerate kinase-1 promoter. (Figure 6D) Sequential gating strategy and representative histograms of cells transduced with the marker gene rat CD2 under different promoters. (Figure 6E) Percentage of positive cells and (Figure 6F) Mean Fluorescence Intensity (MFI) of rat CD2 (rCD2) expressed from the EFS and CMV promoters after puromycin selection of transduced primary CD4+ T cells. Each data point represents an individual transduction (n = 3 biological replicates). Error bars represent SEM. (Figure 6G) Distribution of ORF sizes in the genome-scale library. The sizes of the TCR-rCD2 constructs tested in panels 6E and 6F are shown. (Figure 6H) Titering of CD3 / CD28 antibodies. T cells were labeled with CFSE, stimulated, and incubated for 4 days. Gating for proliferating T cells was set at least twice to include expanded cells (third CFSE peak). (Figure 6I) Expansion of T cells from three healthy donors transduced with the ORF library. (Figure 6J) Representative CFSE profiles of restimulated CD8+ and CD4+ T cells before sorting. The CFSE low sorting gate is marked. (Figure 6K) Recovery of individual barcodes or corresponding ORFs in transduced T cells and plasmids used for lentivirus production. Samples from three donors were computationally pooled together with equal numbers of reads, and then the number of barcodes or ORFs present was counted with a minimum of one read. (Figure 6L) Distribution of reads corresponding to ORFs of different sizes. ORFs were assigned to deciles based on their size, with Q1 being the smallest size and Q10 being the largest (n = 1,161 ORFs per quantile). Boxes show the 25th–75th percentiles with a median line and extension lines extending to 1.5 × interquartile range.(Figure 6M) Gene enrichment in both CFSE-low CD4+ and CD8+ T cells calculated by collapsing individual barcodes into corresponding genes. Significantly enriched genes (log2 fold change >0.5 and adjusted p-value <0.05) are shown in red. Immune response genes of interest are indicated. (Figure 6N) Overlap of significantly enriched genes from Figure 6M in individual screening populations (CD4+, CD8+) analyzed separately. (Figure 6O) Normalized enrichment of individual barcodes for the indicated genes in the CD8+ screen. (Figure 6P) GO biological process for significantly enriched genes in Figure 6M. (Figure 6Q) Overlap of significantly enriched genes with genes differentially expressed between CD3 / CD28-stimulated and naive T cells. [Figure 6N]Figure 6A shows the design of a human ORF library screening in primary T cells. (Figure 6A) Barcoded vector design for ORF overexpression. (Figure 6B) Distribution of the number of barcodes per ORF in the library. (Figure 6C) Vector design for quantifying the effect of different promoters and ORF insert sizes on lentiviral transduction efficiency. EFS - elongation factor-1α short promoter, CMV - cytomegalovirus promoter, PGK - phosphoglycerate kinase-1 promoter. (Figure 6D) Sequential gating strategy and representative histograms of cells transduced with the marker gene rat CD2 under different promoters. (Figure 6E) Percentage of positive cells and (Figure 6F) Mean Fluorescence Intensity (MFI) of rat CD2 (rCD2) expressed from the EFS and CMV promoters after puromycin selection of transduced primary CD4+ T cells. Each data point represents an individual transduction (n = 3 biological replicates). Error bars represent SEM. (Figure 6G) Distribution of ORF sizes in the genome-scale library. The sizes of the TCR-rCD2 constructs tested in panels 6E and 6F are shown. (Figure 6H) Titering of CD3 / CD28 antibodies. T cells were labeled with CFSE, stimulated, and incubated for 4 days. Gating for proliferating T cells was set at least twice to include expanded cells (third CFSE peak). (Figure 6I) Expansion of T cells from three healthy donors transduced with the ORF library. (Figure 6J) Representative CFSE profiles of restimulated CD8+ and CD4+ T cells before sorting. The CFSE low sorting gate is marked. (Figure 6K) Recovery of individual barcodes or corresponding ORFs in transduced T cells and plasmids used for lentivirus production. Samples from three donors were computationally pooled together with equal numbers of reads, and then the number of barcodes or ORFs present was counted with a minimum of one read. (Figure 6L) Distribution of reads corresponding to ORFs of different sizes. ORFs were assigned to deciles based on their size, with Q1 being the smallest size and Q10 being the largest (n = 1,161 ORFs per quantile). Boxes show the 25th–75th percentiles with a median line and extension lines extending to 1.5 × interquartile range.(Figure 6M) Gene enrichment in both CFSE-low CD4+ and CD8+ T cells calculated by collapsing individual barcodes into corresponding genes. Significantly enriched genes (log2 fold change >0.5 and adjusted p-value <0.05) are shown in red. Immune response genes of interest are indicated. (Figure 6N) Overlap of significantly enriched genes from Figure 6M in individual screening populations (CD4+, CD8+) analyzed separately. (Figure 6O) Normalized enrichment of individual barcodes for the indicated genes in the CD8+ screen. (Figure 6P) GO biological process for significantly enriched genes in Figure 6M. (Figure 6Q) Overlap of significantly enriched genes with genes differentially expressed between CD3 / CD28-stimulated and naive T cells. [Figure 6O]Figure 6A shows the design of a human ORF library screening in primary T cells. (Figure 6A) Barcoded vector design for ORF overexpression. (Figure 6B) Distribution of the number of barcodes per ORF in the library. (Figure 6C) Vector design for quantifying the effect of different promoters and ORF insert sizes on lentiviral transduction efficiency. EFS - elongation factor-1α short promoter, CMV - cytomegalovirus promoter, PGK - phosphoglycerate kinase-1 promoter. (Figure 6D) Sequential gating strategy and representative histograms of cells transduced with the marker gene rat CD2 under different promoters. (Figure 6E) Percentage of positive cells and (Figure 6F) Mean Fluorescence Intensity (MFI) of rat CD2 (rCD2) expressed from the EFS and CMV promoters after puromycin selection of transduced primary CD4+ T cells. Each data point represents an individual transduction (n = 3 biological replicates). Error bars represent SEM. (Figure 6G) Distribution of ORF sizes in the genome-scale library. The sizes of the TCR-rCD2 constructs tested in panels 6E and 6F are shown. (Figure 6H) Titering of CD3 / CD28 antibodies. T cells were labeled with CFSE, stimulated, and incubated for 4 days. Gating for proliferating T cells was set at least twice to include expanded cells (third CFSE peak). (Figure 6I) Expansion of T cells from three healthy donors transduced with the ORF library. (Figure 6J) Representative CFSE profiles of restimulated CD8+ and CD4+ T cells before sorting. The CFSE low sorting gate is marked. (Figure 6K) Recovery of individual barcodes or corresponding ORFs in transduced T cells and plasmids used for lentivirus production. Samples from three donors were computationally pooled together with equal numbers of reads, and then the number of barcodes or ORFs present was counted with a minimum of one read. (Figure 6L) Distribution of reads corresponding to ORFs of different sizes. ORFs were assigned to deciles based on their size, with Q1 being the smallest size and Q10 being the largest (n = 1,161 ORFs per quantile). Boxes show the 25th–75th percentiles with a median line and extension lines extending to 1.5 × interquartile range.(Figure 6M) Gene enrichment in both CFSE-low CD4+ and CD8+ T cells calculated by collapsing individual barcodes into corresponding genes. Significantly enriched genes (log2 fold change >0.5 and adjusted p-value <0.05) are shown in red. Immune response genes of interest are indicated. (Figure 6N) Overlap of significantly enriched genes from Figure 6M in individual screening populations (CD4+, CD8+) analyzed separately. (Figure 6O) Normalized enrichment of individual barcodes for the indicated genes in the CD8+ screen. (Figure 6P) GO biological process for significantly enriched genes in Figure 6M. (Figure 6Q) Overlap of significantly enriched genes with genes differentially expressed between CD3 / CD28-stimulated and naive T cells. [Figure 6P]Figure 6A shows the design of a human ORF library screening in primary T cells. (Figure 6A) Barcoded vector design for ORF overexpression. (Figure 6B) Distribution of the number of barcodes per ORF in the library. (Figure 6C) Vector design for quantifying the effect of different promoters and ORF insert sizes on lentiviral transduction efficiency. EFS - elongation factor-1α short promoter, CMV - cytomegalovirus promoter, PGK - phosphoglycerate kinase-1 promoter. (Figure 6D) Sequential gating strategy and representative histograms of cells transduced with the marker gene rat CD2 under different promoters. (Figure 6E) Percentage of positive cells and (Figure 6F) Mean Fluorescence Intensity (MFI) of rat CD2 (rCD2) expressed from the EFS and CMV promoters after puromycin selection of transduced primary CD4+ T cells. Each data point represents an individual transduction (n = 3 biological replicates). Error bars represent SEM. (Figure 6G) Distribution of ORF sizes in the genome-scale library. The sizes of the TCR-rCD2 constructs tested in panels 6E and 6F are shown. (Figure 6H) Titering of CD3 / CD28 antibodies. T cells were labeled with CFSE, stimulated, and incubated for 4 days. Gating for proliferating T cells was set at least twice to include expanded cells (third CFSE peak). (Figure 6I) Expansion of T cells from three healthy donors transduced with the ORF library. (Figure 6J) Representative CFSE profiles of restimulated CD8+ and CD4+ T cells before sorting. The CFSE low sorting gate is marked. (Figure 6K) Recovery of individual barcodes or corresponding ORFs in transduced T cells and plasmids used for lentivirus production. Samples from three donors were computationally pooled together with equal numbers of reads, and then the number of barcodes or ORFs present was counted with a minimum of one read. (Figure 6L) Distribution of reads corresponding to ORFs of different sizes. ORFs were assigned to deciles based on their size, with Q1 being the smallest size and Q10 being the largest (n = 1,161 ORFs per quantile). Boxes show the 25th–75th percentiles with a median line and extension lines extending to 1.5 × interquartile range.(Figure 6M) Gene enrichment in both CFSE-low CD4+ and CD8+ T cells calculated by collapsing individual barcodes into corresponding genes. Significantly enriched genes (log2 fold change >0.5 and adjusted p-value <0.05) are shown in red. Immune response genes of interest are indicated. (Figure 6N) Overlap of significantly enriched genes from Figure 6M in individual screening populations (CD4+, CD8+) analyzed separately. (Figure 6O) Normalized enrichment of individual barcodes for the indicated genes in the CD8+ screen. (Figure 6P) GO biological process for significantly enriched genes in Figure 6M. (Figure 6Q) Overlap of significantly enriched genes with genes differentially expressed between CD3 / CD28-stimulated and naive T cells. [Figure 6Q]Figure 6A shows the design of a human ORF library screening in primary T cells. (Figure 6A) Barcoded vector design for ORF overexpression. (Figure 6B) Distribution of the number of barcodes per ORF in the library. (Figure 6C) Vector design for quantifying the effect of different promoters and ORF insert sizes on lentiviral transduction efficiency. EFS - elongation factor-1α short promoter, CMV - cytomegalovirus promoter, PGK - phosphoglycerate kinase-1 promoter. (Figure 6D) Sequential gating strategy and representative histograms of cells transduced with the marker gene rat CD2 under different promoters. (Figure 6E) Percentage of positive cells and (Figure 6F) Mean Fluorescence Intensity (MFI) of rat CD2 (rCD2) expressed from the EFS and CMV promoters after puromycin selection of transduced primary CD4+ T cells. Each data point represents an individual transduction (n = 3 biological replicates). Error bars represent SEM. (Figure 6G) Distribution of ORF sizes in the genome-scale library. The sizes of the TCR-rCD2 constructs tested in panels 6E and 6F are shown. (Figure 6H) Titering of CD3 / CD28 antibodies. T cells were labeled with CFSE, stimulated, and incubated for 4 days. Gating for proliferating T cells was set at least twice to include expanded cells (third CFSE peak). (Figure 6I) Expansion of T cells from three healthy donors transduced with the ORF library. (Figure 6J) Representative CFSE profiles of restimulated CD8+ and CD4+ T cells before sorting. The CFSE low sorting gate is marked. (Figure 6K) Recovery of individual barcodes or corresponding ORFs in transduced T cells and plasmids used for lentivirus production. Samples from three donors were computationally pooled together with equal numbers of reads, and then the number of barcodes or ORFs present was counted with a minimum of one read. (Figure 6L) Distribution of reads corresponding to ORFs of different sizes. ORFs were assigned to deciles based on their size, with Q1 being the smallest size and Q10 being the largest (n = 1,161 ORFs per quantile). Boxes show the 25th–75th percentiles with a median line and extension lines extending to 1.5 × interquartile range.(Figure 6M) Gene enrichment in both CFSE-low CD4+ and CD8+ T cells calculated by collapsing individual barcodes into corresponding genes. Significantly enriched genes (log2 fold change >0.5 and adjusted p-value <0.05) are shown in red. Immune response genes of interest are indicated. (Figure 6N) Overlap of significantly enriched genes from Figure 6M in individual screening populations (CD4+, CD8+) analyzed separately. (Figure 6O) Normalized enrichment of individual barcodes for the indicated genes in the CD8+ screen. (Figure 6P) GO biological process for significantly enriched genes in Figure 6M. (Figure 6Q) Overlap of significantly enriched genes with genes differentially expressed between CD3 / CD28-stimulated and naive T cells. [Figure 7A]Overexpression of selected ORFs in donors unrelated to the screen is shown. (Figure 7A) Histogram of selected ORF expression in T cells after puromycin selection. (Figure 7B) Quantification of tNGFR expression in transduced CD4+ and CD8+ T cells. Puromycin selection was completed 7 days after transduction. To maintain T cells in culture, they were restimulated with CD3 / CD28 on days 21 and 42. (Figure 7C) Correlation between ORF size and change in proliferation compared to tNGFR. Mean log2 fold change is shown. (Figure 7D) Proliferation of restimulated CD8+ or (Figure 7E) CD4+ T cells compared to tNGFR in individual donors (n=3 biological replicates). Mean and SEM are shown. (Figure 7F, Figure 7G) Proliferation of T cells transduced with ORFs that significantly improved T cell proliferation (see Figure 2C), as measured by CellTrace Yellow dilution. Representative CellTrace Yellow histograms and fitted distributions (Figure 7F) and quantification of the proliferation index (Figure 7G) are shown (n = 3 biological replicates). P values: <0.0001, 0.0008, <0.0001, 0.011, 0.0031, 0.0007, <0.0001, 0.28, 0.004, <0.0001, 0.58, 0.01, 0.0003, <0.0001, 0.036, 0.0049 (from left to right). (Figure 7H) Viability of ORF-transduced T cells 4 days after CD3 / CD28 restimulation. Representative data from one donor (out of four donors tested) are shown (n = 3 biological replicates). (Figure 7I, Figure 7J) Cell cycle analysis of T cells stimulated with CD3 / CD28 for 24 hours. Gating was based on isotype and fluorescence minus one controls. Representative gating (Figure 7I) and quantification (Figure 7J) of cells in S-G2-M phases (for stimulated T cells) are shown (n = 6 biological replicates from two donors). P values: 1, 0.29, 0.0065, 0.17, 0.0051, 1, 0.13, 0.55, 0.0004, 0.98, 0.0088, 0.68, 0.91, 0.7, 1 (left to right).Statistical significance in panels Figure 7G and Figure 7I: One-way ANOVA with Darnett's multiple comparison test *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Error bars indicate SEM. [Figure 7B]Overexpression of selected ORFs in donors unrelated to the screen is shown. (Figure 7A) Histogram of selected ORF expression in T cells after puromycin selection. (Figure 7B) Quantification of tNGFR expression in transduced CD4+ and CD8+ T cells. Puromycin selection was completed 7 days after transduction. To maintain T cells in culture, they were restimulated with CD3 / CD28 on days 21 and 42. (Figure 7C) Correlation between ORF size and change in proliferation compared to tNGFR. Mean log2 fold change is shown. (Figure 7D) Proliferation of restimulated CD8+ or (Figure 7E) CD4+ T cells compared to tNGFR in individual donors (n=3 biological replicates). Mean and SEM are shown. (Figure 7F, Figure 7G) Proliferation of T cells transduced with ORFs that significantly improved T cell proliferation (see Figure 2C), as measured by CellTrace Yellow dilution. Representative CellTrace Yellow histograms and fitted distributions (Figure 7F) and quantification of the proliferation index (Figure 7G) are shown (n = 3 biological replicates). P values: <0.0001, 0.0008, <0.0001, 0.011, 0.0031, 0.0007, <0.0001, 0.28, 0.004, <0.0001, 0.58, 0.01, 0.0003, <0.0001, 0.036, 0.0049 (from left to right). (Figure 7H) Viability of ORF-transduced T cells 4 days after CD3 / CD28 restimulation. Representative data from one donor (out of four donors tested) are shown (n = 3 biological replicates). (Figure 7I, Figure 7J) Cell cycle analysis of T cells stimulated with CD3 / CD28 for 24 hours. Gating was based on isotype and fluorescence minus one controls. Representative gating (Figure 7I) and quantification (Figure 7J) of cells in S-G2-M phases (for stimulated T cells) are shown (n = 6 biological replicates from two donors). P values: 1, 0.29, 0.0065, 0.17, 0.0051, 1, 0.13, 0.55, 0.0004, 0.98, 0.0088, 0.68, 0.91, 0.7, 1 (left to right).Statistical significance in panels Figure 7G and Figure 7I: One-way ANOVA with Darnett's multiple comparison test *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Error bars indicate SEM. [Figure 7C]Overexpression of selected ORFs in donors unrelated to the screen is shown. (Figure 7A) Histogram of selected ORF expression in T cells after puromycin selection. (Figure 7B) Quantification of tNGFR expression in transduced CD4+ and CD8+ T cells. Puromycin selection was completed 7 days after transduction. To maintain T cells in culture, they were restimulated with CD3 / CD28 on days 21 and 42. (Figure 7C) Correlation between ORF size and change in proliferation compared to tNGFR. Mean log2 fold change is shown. (Figure 7D) Proliferation of restimulated CD8+ or (Figure 7E) CD4+ T cells compared to tNGFR in individual donors (n=3 biological replicates). Mean and SEM are shown. (Figure 7F, Figure 7G) Proliferation of T cells transduced with ORFs that significantly improved T cell proliferation (see Figure 2C), as measured by CellTrace Yellow dilution. Representative CellTrace Yellow histograms and fitted distributions (Figure 7F) and quantification of the proliferation index (Figure 7G) are shown (n = 3 biological replicates). P values: <0.0001, 0.0008, <0.0001, 0.011, 0.0031, 0.0007, <0.0001, 0.28, 0.004, <0.0001, 0.58, 0.01, 0.0003, <0.0001, 0.036, 0.0049 (from left to right). (Figure 7H) Viability of ORF-transduced T cells 4 days after CD3 / CD28 restimulation. Representative data from one donor (out of four donors tested) are shown (n = 3 biological replicates). (Figure 7I, Figure 7J) Cell cycle analysis of T cells stimulated with CD3 / CD28 for 24 hours. Gating was based on isotype and fluorescence minus one controls. Representative gating (Figure 7I) and quantification (Figure 7J) of cells in S-G2-M phases (for stimulated T cells) are shown (n = 6 biological replicates from two donors). P values: 1, 0.29, 0.0065, 0.17, 0.0051, 1, 0.13, 0.55, 0.0004, 0.98, 0.0088, 0.68, 0.91, 0.7, 1 (left to right).Statistical significance in panels Figure 7G and Figure 7I: One-way ANOVA with Darnett's multiple comparison test *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Error bars indicate SEM. [Figure 7D]Overexpression of selected ORFs in donors unrelated to the screen is shown. (Figure 7A) Histogram of selected ORF expression in T cells after puromycin selection. (Figure 7B) Quantification of tNGFR expression in transduced CD4+ and CD8+ T cells. Puromycin selection was completed 7 days after transduction. To maintain T cells in culture, they were restimulated with CD3 / CD28 on days 21 and 42. (Figure 7C) Correlation between ORF size and change in proliferation compared to tNGFR. Mean log2 fold change is shown. (Figure 7D) Proliferation of restimulated CD8+ or (Figure 7E) CD4+ T cells compared to tNGFR in individual donors (n=3 biological replicates). Mean and SEM are shown. (Figure 7F, Figure 7G) Proliferation of T cells transduced with ORFs that significantly improved T cell proliferation (see Figure 2C), as measured by CellTrace Yellow dilution. Representative CellTrace Yellow histograms and fitted distributions (Figure 7F) and quantification of the proliferation index (Figure 7G) are shown (n = 3 biological replicates). P values: <0.0001, 0.0008, <0.0001, 0.011, 0.0031, 0.0007, <0.0001, 0.28, 0.004, <0.0001, 0.58, 0.01, 0.0003, <0.0001, 0.036, 0.0049 (from left to right). (Figure 7H) Viability of ORF-transduced T cells 4 days after CD3 / CD28 restimulation. Representative data from one donor (out of four donors tested) are shown (n = 3 biological replicates). (Figure 7I, Figure 7J) Cell cycle analysis of T cells stimulated with CD3 / CD28 for 24 hours. Gating was based on isotype and fluorescence minus one controls. Representative gating (Figure 7I) and quantification (Figure 7J) of cells in S-G2-M phases (for stimulated T cells) are shown (n = 6 biological replicates from two donors). P values: 1, 0.29, 0.0065, 0.17, 0.0051, 1, 0.13, 0.55, 0.0004, 0.98, 0.0088, 0.68, 0.91, 0.7, 1 (left to right).Statistical significance in panels Figure 7G and Figure 7I: One-way ANOVA with Darnett's multiple comparison test *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Error bars indicate SEM. [Figure 7E]Overexpression of selected ORFs in donors unrelated to the screen is shown. (Figure 7A) Histogram of selected ORF expression in T cells after puromycin selection. (Figure 7B) Quantification of tNGFR expression in transduced CD4+ and CD8+ T cells. Puromycin selection was completed 7 days after transduction. To maintain T cells in culture, they were restimulated with CD3 / CD28 on days 21 and 42. (Figure 7C) Correlation between ORF size and change in proliferation compared to tNGFR. Mean log2 fold change is shown. (Figure 7D) Proliferation of restimulated CD8+ or (Figure 7E) CD4+ T cells compared to tNGFR in individual donors (n=3 biological replicates). Mean and SEM are shown. (Figure 7F, Figure 7G) Proliferation of T cells transduced with ORFs that significantly improved T cell proliferation (see Figure 2C), as measured by CellTrace Yellow dilution. Representative CellTrace Yellow histograms and fitted distributions (Figure 7F) and quantification of the proliferation index (Figure 7G) are shown (n = 3 biological replicates). P values: <0.0001, 0.0008, <0.0001, 0.011, 0.0031, 0.0007, <0.0001, 0.28, 0.004, <0.0001, 0.58, 0.01, 0.0003, <0.0001, 0.036, 0.0049 (from left to right). (Figure 7H) Viability of ORF-transduced T cells 4 days after CD3 / CD28 restimulation. Representative data from one donor (out of four donors tested) are shown (n = 3 biological replicates). (Figure 7I, Figure 7J) Cell cycle analysis of T cells stimulated with CD3 / CD28 for 24 hours. Gating was based on isotype and fluorescence minus one controls. Representative gating (Figure 7I) and quantification (Figure 7J) of cells in S-G2-M phases (for stimulated T cells) are shown (n = 6 biological replicates from two donors). P values: 1, 0.29, 0.0065, 0.17, 0.0051, 1, 0.13, 0.55, 0.0004, 0.98, 0.0088, 0.68, 0.91, 0.7, 1 (left to right).Statistical significance in panels Figure 7G and Figure 7I: One-way ANOVA with Darnett's multiple comparison test *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Error bars indicate SEM. [Figure 7F]Overexpression of selected ORFs in donors unrelated to the screen is shown. (Figure 7A) Histogram of selected ORF expression in T cells after puromycin selection. (Figure 7B) Quantification of tNGFR expression in transduced CD4+ and CD8+ T cells. Puromycin selection was completed 7 days after transduction. To maintain T cells in culture, they were restimulated with CD3 / CD28 on days 21 and 42. (Figure 7C) Correlation between ORF size and change in proliferation compared to tNGFR. Mean log2 fold change is shown. (Figure 7D) Proliferation of restimulated CD8+ or (Figure 7E) CD4+ T cells compared to tNGFR in individual donors (n=3 biological replicates). Mean and SEM are shown. (Figure 7F, Figure 7G) Proliferation of T cells transduced with ORFs that significantly improved T cell proliferation (see Figure 2C), as measured by CellTrace Yellow dilution. Representative CellTrace Yellow histograms and fitted distributions (Figure 7F) and quantification of the proliferation index (Figure 7G) are shown (n = 3 biological replicates). P values: <0.0001, 0.0008, <0.0001, 0.011, 0.0031, 0.0007, <0.0001, 0.28, 0.004, <0.0001, 0.58, 0.01, 0.0003, <0.0001, 0.036, 0.0049 (from left to right). (Figure 7H) Viability of ORF-transduced T cells 4 days after CD3 / CD28 restimulation. Representative data from one donor (out of four donors tested) are shown (n = 3 biological replicates). (Figure 7I, Figure 7J) Cell cycle analysis of T cells stimulated with CD3 / CD28 for 24 hours. Gating was based on isotype and fluorescence minus one controls. Representative gating (Figure 7I) and quantification (Figure 7J) of cells in S-G2-M phases (for stimulated T cells) are shown (n = 6 biological replicates from two donors). P values: 1, 0.29, 0.0065, 0.17, 0.0051, 1, 0.13, 0.55, 0.0004, 0.98, 0.0088, 0.68, 0.91, 0.7, 1 (left to right).Statistical significance in panels Figure 7G and Figure 7I: One-way ANOVA with Darnett's multiple comparison test *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Error bars indicate SEM. [Figure 7G]Overexpression of selected ORFs in donors unrelated to the screen is shown. (Figure 7A) Histogram of selected ORF expression in T cells after puromycin selection. (Figure 7B) Quantification of tNGFR expression in transduced CD4+ and CD8+ T cells. Puromycin selection was completed 7 days after transduction. To maintain T cells in culture, they were restimulated with CD3 / CD28 on days 21 and 42. (Figure 7C) Correlation between ORF size and change in proliferation compared to tNGFR. Mean log2 fold change is shown. (Figure 7D) Proliferation of restimulated CD8+ or (Figure 7E) CD4+ T cells compared to tNGFR in individual donors (n=3 biological replicates). Mean and SEM are shown. (Figure 7F, Figure 7G) Proliferation of T cells transduced with ORFs that significantly improved T cell proliferation (see Figure 2C), as measured by CellTrace Yellow dilution. Representative CellTrace Yellow histograms and fitted distributions (Figure 7F) and quantification of the proliferation index (Figure 7G) are shown (n = 3 biological replicates). P values: <0.0001, 0.0008, <0.0001, 0.011, 0.0031, 0.0007, <0.0001, 0.28, 0.004, <0.0001, 0.58, 0.01, 0.0003, <0.0001, 0.036, 0.0049 (from left to right). (Figure 7H) Viability of ORF-transduced T cells 4 days after CD3 / CD28 restimulation. Representative data from one donor (out of four donors tested) are shown (n = 3 biological replicates). (Figure 7I, Figure 7J) Cell cycle analysis of T cells stimulated with CD3 / CD28 for 24 hours. Gating was based on isotype and fluorescence minus one controls. Representative gating (Figure 7I) and quantification (Figure 7J) of cells in S-G2-M phases (for stimulated T cells) are shown (n = 6 biological replicates from two donors). P values: 1, 0.29, 0.0065, 0.17, 0.0051, 1, 0.13, 0.55, 0.0004, 0.98, 0.0088, 0.68, 0.91, 0.7, 1 (left to right).Statistical significance in panels Figure 7G and Figure 7I: One-way ANOVA with Darnett's multiple comparison test *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Error bars indicate SEM. [Figure 7H]Overexpression of selected ORFs in donors unrelated to the screen is shown. (Figure 7A) Histogram of selected ORF expression in T cells after puromycin selection. (Figure 7B) Quantification of tNGFR expression in transduced CD4+ and CD8+ T cells. Puromycin selection was completed 7 days after transduction. To maintain T cells in culture, they were restimulated with CD3 / CD28 on days 21 and 42. (Figure 7C) Correlation between ORF size and change in proliferation compared to tNGFR. Mean log2 fold change is shown. (Figure 7D) Proliferation of restimulated CD8+ or (Figure 7E) CD4+ T cells compared to tNGFR in individual donors (n=3 biological replicates). Mean and SEM are shown. (Figure 7F, Figure 7G) Proliferation of T cells transduced with ORFs that significantly improved T cell proliferation (see Figure 2C), as measured by CellTrace Yellow dilution. Representative CellTrace Yellow histograms and fitted distributions (Figure 7F) and quantification of the proliferation index (Figure 7G) are shown (n = 3 biological replicates). P values: <0.0001, 0.0008, <0.0001, 0.011, 0.0031, 0.0007, <0.0001, 0.28, 0.004, <0.0001, 0.58, 0.01, 0.0003, <0.0001, 0.036, 0.0049 (from left to right). (Figure 7H) Viability of ORF-transduced T cells 4 days after CD3 / CD28 restimulation. Representative data from one donor (out of four donors tested) are shown (n = 3 biological replicates). (Figure 7I, Figure 7J) Cell cycle analysis of T cells stimulated with CD3 / CD28 for 24 hours. Gating was based on isotype and fluorescence minus one controls. Representative gating (Figure 7I) and quantification (Figure 7J) of cells in S-G2-M phases (for stimulated T cells) are shown (n = 6 biological replicates from two donors). P values: 1, 0.29, 0.0065, 0.17, 0.0051, 1, 0.13, 0.55, 0.0004, 0.98, 0.0088, 0.68, 0.91, 0.7, 1 (left to right).Statistical significance in panels Figure 7G and Figure 7I: One-way ANOVA with Darnett's multiple comparison test *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Error bars indicate SEM. [Figure 7I]Overexpression of selected ORFs in donors unrelated to the screen is shown. (Figure 7A) Histogram of selected ORF expression in T cells after puromycin selection. (Figure 7B) Quantification of tNGFR expression in transduced CD4+ and CD8+ T cells. Puromycin selection was completed 7 days after transduction. To maintain T cells in culture, they were restimulated with CD3 / CD28 on days 21 and 42. (Figure 7C) Correlation between ORF size and change in proliferation compared to tNGFR. Mean log2 fold change is shown. (Figure 7D) Proliferation of restimulated CD8+ or (Figure 7E) CD4+ T cells compared to tNGFR in individual donors (n=3 biological replicates). Mean and SEM are shown. (Figure 7F, Figure 7G) Proliferation of T cells transduced with ORFs that significantly improved T cell proliferation (see Figure 2C), as measured by CellTrace Yellow dilution. Representative CellTrace Yellow histograms and fitted distributions (Figure 7F) and quantification of the proliferation index (Figure 7G) are shown (n = 3 biological replicates). P values: <0.0001, 0.0008, <0.0001, 0.011, 0.0031, 0.0007, <0.0001, 0.28, 0.004, <0.0001, 0.58, 0.01, 0.0003, <0.0001, 0.036, 0.0049 (from left to right). (Figure 7H) Viability of ORF-transduced T cells 4 days after CD3 / CD28 restimulation. Representative data from one donor (out of four donors tested) are shown (n = 3 biological replicates). (Figure 7I, Figure 7J) Cell cycle analysis of T cells stimulated with CD3 / CD28 for 24 hours. Gating was based on isotype and fluorescence minus one controls. Representative gating (Figure 7I) and quantification (Figure 7J) of cells in S-G2-M phases (for stimulated T cells) are shown (n = 6 biological replicates from two donors). P values: 1, 0.29, 0.0065, 0.17, 0.0051, 1, 0.13, 0.55, 0.0004, 0.98, 0.0088, 0.68, 0.91, 0.7, 1 (left to right).Statistical significance in panels Figure 7G and Figure 7I: One-way ANOVA with Darnett's multiple comparison test *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Error bars indicate SEM. [Figure 7J]Overexpression of selected ORFs in donors unrelated to the screen is shown. (Figure 7A) Histogram of selected ORF expression in T cells after puromycin selection. (Figure 7B) Quantification of tNGFR expression in transduced CD4+ and CD8+ T cells. Puromycin selection was completed 7 days after transduction. To maintain T cells in culture, they were restimulated with CD3 / CD28 on days 21 and 42. (Figure 7C) Correlation between ORF size and change in proliferation compared to tNGFR. Mean log2 fold change is shown. (Figure 7D) Proliferation of restimulated CD8+ or (Figure 7E) CD4+ T cells compared to tNGFR in individual donors (n=3 biological replicates). Mean and SEM are shown. (Figure 7F, Figure 7G) Proliferation of T cells transduced with ORFs that significantly improved T cell proliferation (see Figure 2C), as measured by CellTrace Yellow dilution. Representative CellTrace Yellow histograms and fitted distributions (Figure 7F) and quantification of the proliferation index (Figure 7G) are shown (n = 3 biological replicates). P values: <0.0001, 0.0008, <0.0001, 0.011, 0.0031, 0.0007, <0.0001, 0.28, 0.004, <0.0001, 0.58, 0.01, 0.0003, <0.0001, 0.036, 0.0049 (from left to right). (Figure 7H) Viability of ORF-transduced T cells 4 days after CD3 / CD28 restimulation. Representative data from one donor (out of four donors tested) are shown (n = 3 biological replicates). (Figure 7I, Figure 7J) Cell cycle analysis of T cells stimulated with CD3 / CD28 for 24 hours. Gating was based on isotype and fluorescence minus one controls. Representative gating (Figure 7I) and quantification (Figure 7J) of cells in S-G2-M phases (for stimulated T cells) are shown (n = 6 biological replicates from two donors). P values: 1, 0.29, 0.0065, 0.17, 0.0051, 1, 0.13, 0.55, 0.0004, 0.98, 0.0088, 0.68, 0.91, 0.7, 1 (left to right).Statistical significance in panels Figure 7G and Figure 7I: One-way ANOVA with Darnett's multiple comparison test *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Error bars indicate SEM. [Figure 8A] Functional responses of ORF-overexpressing T cells are shown. (Figure 8A) Quantitative expression of CD25 or CD154 after restimulation. A minimum of two donors were tested in triplicate per gene. Only genes that significantly increased T cell proliferation in CD4+, CD8+, or both T cell subsets are shown. Means and SEMs are shown. (Figures 8B, 8C) Sensitivity to antigen dose. T cells were incubated with the indicated anti-CD3 antibody concentrations for 24 hours, and the amount of secreted IFNγ was quantified. Representative dose-response curve fitting (Figure 8B) and IC50 determination (Figure 8C) are shown (n = 2 biological replicates). (Figure 8D) Quantification of secreted IL-2 and IFNγ in T cells incubated alone or with CD3 / CD28 antibodies for 24 hours. Representative data from one of four donors (n = 3 biological replicates) are shown. (FIG. 8E) Multiplex quantification of selected secreted cytokines and chemokines by ORF-transduced T cells 24 hours after CD3 / CD28 stimulation. Mean z-scores (from independent samples) of duplicate determinations normalized to tNGFR are shown. [Figure 8B]Functional responses of ORF-overexpressing T cells are shown. (Figure 8A) Quantitative expression of CD25 or CD154 after restimulation. A minimum of two donors were tested in triplicate per gene. Only genes that significantly increased T cell proliferation in CD4+, CD8+, or both T cell subsets are shown. Means and SEMs are shown. (Figures 8B, 8C) Sensitivity to antigen dose. T cells were incubated with the indicated anti-CD3 antibody concentrations for 24 hours, and the amount of secreted IFNγ was quantified. Representative dose-response curve fitting (Figure 8B) and IC50 determination (Figure 8C) are shown (n = 2 biological replicates). (Figure 8D) Quantification of secreted IL-2 and IFNγ in T cells incubated alone or with CD3 / CD28 antibodies for 24 hours. Representative data from one of four donors (n = 3 biological replicates) are shown. (FIG. 8E) Multiplex quantification of selected secreted cytokines and chemokines by ORF-transduced T cells 24 hours after CD3 / CD28 stimulation. Mean z-scores (from independent samples) of duplicate determinations normalized to tNGFR are shown. [Figure 8C] Functional responses of ORF-overexpressing T cells are shown. (Figure 8A) Quantitative expression of CD25 or CD154 after restimulation. A minimum of two donors were tested in triplicate per gene. Only genes that significantly increased T cell proliferation in CD4+, CD8+, or both T cell subsets are shown. Means and SEMs are shown. (Figures 8B, 8C) Sensitivity to antigen dose. T cells were incubated with the indicated anti-CD3 antibody concentrations for 24 hours, and the amount of secreted IFNγ was quantified. Representative dose-response curve fitting (Figure 8B) and IC50 determination (Figure 8C) are shown (n = 2 biological replicates). (Figure 8D) Quantification of secreted IL-2 and IFNγ in T cells incubated alone or with CD3 / CD28 antibodies for 24 hours. Representative data from one of four donors (n = 3 biological replicates) are shown. (FIG. 8E) Multiplex quantification of selected secreted cytokines and chemokines by ORF-transduced T cells 24 hours after CD3 / CD28 stimulation. Mean z-scores (from independent samples) of duplicate determinations normalized to tNGFR are shown. [Figure 8D]Functional responses of ORF-overexpressing T cells are shown. (Figure 8A) Quantitative expression of CD25 or CD154 after restimulation. A minimum of two donors were tested in triplicate per gene. Only genes that significantly increased T cell proliferation in CD4+, CD8+, or both T cell subsets are shown. Means and SEMs are shown. (Figures 8B, 8C) Sensitivity to antigen dose. T cells were incubated with the indicated anti-CD3 antibody concentrations for 24 hours, and the amount of secreted IFNγ was quantified. Representative dose-response curve fitting (Figure 8B) and IC50 determination (Figure 8C) are shown (n = 2 biological replicates). (Figure 8D) Quantification of secreted IL-2 and IFNγ in T cells incubated alone or with CD3 / CD28 antibodies for 24 hours. Representative data from one of four donors (n = 3 biological replicates) are shown. (FIG. 8E) Multiplex quantification of selected secreted cytokines and chemokines by ORF-transduced T cells 24 hours after CD3 / CD28 stimulation. Mean z-scores (from independent samples) of duplicate determinations normalized to tNGFR are shown. [Figure 8E] Functional responses of ORF-overexpressing T cells are shown. (Figure 8A) Quantitative expression of CD25 or CD154 after restimulation. A minimum of two donors were tested in triplicate per gene. Only genes that significantly increased T cell proliferation in CD4+, CD8+, or both T cell subsets are shown. Means and SEMs are shown. (Figures 8B, 8C) Sensitivity to antigen dose. T cells were incubated with the indicated anti-CD3 antibody concentrations for 24 hours, and the amount of secreted IFNγ was quantified. Representative dose-response curve fitting (Figure 8B) and IC50 determination (Figure 8C) are shown (n = 2 biological replicates). (Figure 8D) Quantification of secreted IL-2 and IFNγ in T cells incubated alone or with CD3 / CD28 antibodies for 24 hours. Representative data from one of four donors (n = 3 biological replicates) are shown. (FIG. 8E) Multiplex quantification of selected secreted cytokines and chemokines by ORF-transduced T cells 24 hours after CD3 / CD28 stimulation. Mean z-scores (from independent samples) of duplicate determinations normalized to tNGFR are shown. [Figure 9A]OverCITE-seq analysis identifies ORFs and their transcriptional effects. (Figure 9A) Cell quality parameters identified by gel bead barcodes. Negatives, singlets, and doublets are assigned based on cell hashing. (Figure 9B) Percentage of stimulated and resting T cells among cells assigned to each ORF. Chi-squared p-values ​​are shown for ORFs with significantly shifted (uneven) distributions of stimulated and resting cells. (Figure 9C) Cell cycle-corrected, scaled expression of overexpressed genes in cells transduced with each ORF and the negative control (tNGFR). Two-tailed Wilcoxon p-values ​​shown above the violin plot indicate statistical significance of gene expression levels between specific ORF-transduced and tNGFR-transduced T cells. The boxes indicate the 25th to 75th percentiles along with the median line, and extensions extend to the maximum and minimum values. N=71(ADA), 147(AHCY), 190(AHNAK), 119(AKR1C4), 124(ATF6B), 179(BATF), 137(CALML3), 189(CDK1), 129(CDK2), 236(CLIC1), 84(CRLF2), 91(CXCL12), 88(CYP27A1), 129(DBI), 26(DCLRE1B), 261(DUPD1), Figure 9D: Expression of all ORF genes by cell assigned to each ORF. Each row is z-score normalized. Figure 9E: Distribution of individual ORF frequencies within clusters. The number of ORF cells and chi-squared residuals are displayed. The chi-squared p-value indicating whether the ORF distribution in each cluster is significantly different from the overall ORF distribution is displayed above the plot. The percentage of stimulated and resting T cells in each cluster is shown below the cluster label.(Figure 9F, Figure 9G) Spearman correlation between transcriptional profiles of selected ORF cells in resting (Figure 9F) and stimulated (Figure 9G) populations. (Figure 9H) Fold change of the top differentially expressed genes between cells with the indicated ORFs in resting and stimulated T cells. For each condition, the ORF with the strongest transcriptional change (compared to tNGFR cells) is shown. (Figure 9I) Differential gene expression in stimulated ORF T cells compared to resting T cells. Genes with significant expression changes in at least one ORF are shown (DESeq2 adjusted p<0.05). For all genes, the log2 fold change of each ORF (stimulated) relative to tNGFR (resting) is shown, normalized to the log2 fold change of tNGFR (stimulated) relative to tNGFR (resting). Genes of interest in each cluster are labeled. (Figure 9J) Average TCR clonotype diversity in ORF cells. [Figure 9B]OverCITE-seq analysis identifies ORFs and their transcriptional effects. (Figure 9A) Cell quality parameters identified by gel bead barcodes. Negatives, singlets, and doublets are assigned based on cell hashing. (Figure 9B) Percentage of stimulated and resting T cells among cells assigned to each ORF. Chi-squared p-values ​​are shown for ORFs with significantly shifted (uneven) distributions of stimulated and resting cells. (Figure 9C) Cell cycle-corrected, scaled expression of overexpressed genes in cells transduced with each ORF and the negative control (tNGFR). Two-tailed Wilcoxon p-values ​​shown above the violin plot indicate statistical significance of gene expression levels between specific ORF-transduced and tNGFR-transduced T cells. The boxes indicate the 25th to 75th percentiles along with the median line, and extensions extend to the maximum and minimum values. N=71(ADA), 147(AHCY), 190(AHNAK), 119(AKR1C4), 124(ATF6B), 179(BATF), 137(CALML3), 189(CDK1), 129(CDK2), 236(CLIC1), 84(CRLF2), 91(CXCL12), 88(CYP27A1), 129(DBI), 26(DCLRE1B), 261(DUPD1), Figure 9D: Expression of all ORF genes by cell assigned to each ORF. Each row is z-score normalized. Figure 9E: Distribution of individual ORF frequencies within clusters. The number of ORF cells and chi-squared residuals are displayed. The chi-squared p-value indicating whether the ORF distribution in each cluster is significantly different from the overall ORF distribution is displayed above the plot. The percentage of stimulated and resting T cells in each cluster is shown below the cluster label.(Figure 9F, Figure 9G) Spearman correlation between transcriptional profiles of selected ORF cells in resting (Figure 9F) and stimulated (Figure 9G) populations. (Figure 9H) Fold change of the top differentially expressed genes between cells with the indicated ORFs in resting and stimulated T cells. For each condition, the ORF with the strongest transcriptional change (compared to tNGFR cells) is shown. (Figure 9I) Differential gene expression in stimulated ORF T cells compared to resting T cells. Genes with significant expression changes in at least one ORF are shown (DESeq2 adjusted p<0.05). For all genes, the log2 fold change of each ORF (stimulated) relative to tNGFR (resting) is shown, normalized to the log2 fold change of tNGFR (stimulated) relative to tNGFR (resting). Genes of interest in each cluster are labeled. (Figure 9J) Average TCR clonotype diversity in ORF cells. [Figure 9C]OverCITE-seq analysis identifies ORFs and their transcriptional effects. (Figure 9A) Cell quality parameters identified by gel bead barcodes. Negatives, singlets, and doublets are assigned based on cell hashing. (Figure 9B) Percentage of stimulated and resting T cells among cells assigned to each ORF. Chi-squared p-values ​​are shown for ORFs with significantly shifted (uneven) distributions of stimulated and resting cells. (Figure 9C) Cell cycle-corrected, scaled expression of overexpressed genes in cells transduced with each ORF and the negative control (tNGFR). Two-tailed Wilcoxon p-values ​​shown above the violin plot indicate statistical significance of gene expression levels between specific ORF-transduced and tNGFR-transduced T cells. The boxes indicate the 25th to 75th percentiles along with the median line, and extensions extend to the maximum and minimum values. N=71(ADA), 147(AHCY), 190(AHNAK), 119(AKR1C4), 124(ATF6B), 179(BATF), 137(CALML3), 189(CDK1), 129(CDK2), 236(CLIC1), 84(CRLF2), 91(CXCL12), 88(CYP27A1), 129(DBI), 26(DCLRE1B), 261(DUPD1), Figure 9D: Expression of all ORF genes by cell assigned to each ORF. Each row is z-score normalized. Figure 9E: Distribution of individual ORF frequencies within clusters. The number of ORF cells and chi-squared residuals are displayed. The chi-squared p-value indicating whether the ORF distribution in each cluster is significantly different from the overall ORF distribution is displayed above the plot. The percentage of stimulated and resting T cells in each cluster is shown below the cluster label.(Figure 9F, Figure 9G) Spearman correlation between transcriptional profiles of selected ORF cells in resting (Figure 9F) and stimulated (Figure 9G) populations. (Figure 9H) Fold change of the top differentially expressed genes between cells with the indicated ORFs in resting and stimulated T cells. For each condition, the ORF with the strongest transcriptional change (compared to tNGFR cells) is shown. (Figure 9I) Differential gene expression in stimulated ORF T cells compared to resting T cells. Genes with significant expression changes in at least one ORF are shown (DESeq2 adjusted p<0.05). For all genes, the log2 fold change of each ORF (stimulated) relative to tNGFR (resting) is shown, normalized to the log2 fold change of tNGFR (stimulated) relative to tNGFR (resting). Genes of interest in each cluster are labeled. (Figure 9J) Average TCR clonotype diversity in ORF cells. [Figure 9D]OverCITE-seq analysis identifies ORFs and their transcriptional effects. (Figure 9A) Cell quality parameters identified by gel bead barcodes. Negatives, singlets, and doublets are assigned based on cell hashing. (Figure 9B) Percentage of stimulated and resting T cells among cells assigned to each ORF. Chi-squared p-values ​​are shown for ORFs with significantly shifted (uneven) distributions of stimulated and resting cells. (Figure 9C) Cell cycle-corrected, scaled expression of overexpressed genes in cells transduced with each ORF and the negative control (tNGFR). Two-tailed Wilcoxon p-values ​​shown above the violin plot indicate statistical significance of gene expression levels between specific ORF-transduced and tNGFR-transduced T cells. The boxes indicate the 25th to 75th percentiles along with the median line, and extensions extend to the maximum and minimum values. N=71(ADA), 147(AHCY), 190(AHNAK), 119(AKR1C4), 124(ATF6B), 179(BATF), 137(CALML3), 189(CDK1), 129(CDK2), 236(CLIC1), 84(CRLF2), 91(CXCL12), 88(CYP27A1), 129(DBI), 26(DCLRE1B), 261(DUPD1), Figure 9D: Expression of all ORF genes by cell assigned to each ORF. Each row is z-score normalized. Figure 9E: Distribution of individual ORF frequencies within clusters. The number of ORF cells and chi-squared residuals are displayed. The chi-squared p-value indicating whether the ORF distribution in each cluster is significantly different from the overall ORF distribution is displayed above the plot. The percentage of stimulated and resting T cells in each cluster is shown below the cluster label.(Figure 9F, Figure 9G) Spearman correlation between transcriptional profiles of selected ORF cells in resting (Figure 9F) and stimulated (Figure 9G) populations. (Figure 9H) Fold change of the top differentially expressed genes between cells with the indicated ORFs in resting and stimulated T cells. For each condition, the ORF with the strongest transcriptional change (compared to tNGFR cells) is shown. (Figure 9I) Differential gene expression in stimulated ORF T cells compared to resting T cells. Genes with significant expression changes in at least one ORF are shown (DESeq2 adjusted p<0.05). For all genes, the log2 fold change of each ORF (stimulated) relative to tNGFR (resting) is shown, normalized to the log2 fold change of tNGFR (stimulated) relative to tNGFR (resting). Genes of interest in each cluster are labeled. (Figure 9J) Average TCR clonotype diversity in ORF cells. [Figure 9E]OverCITE-seq analysis identifies ORFs and their transcriptional effects. (Figure 9A) Cell quality parameters identified by gel bead barcodes. Negatives, singlets, and doublets are assigned based on cell hashing. (Figure 9B) Percentage of stimulated and resting T cells among cells assigned to each ORF. Chi-squared p-values ​​are shown for ORFs with significantly shifted (uneven) distributions of stimulated and resting cells. (Figure 9C) Cell cycle-corrected, scaled expression of overexpressed genes in cells transduced with each ORF and the negative control (tNGFR). Two-tailed Wilcoxon p-values ​​shown above the violin plot indicate statistical significance of gene expression levels between specific ORF-transduced and tNGFR-transduced T cells. The boxes indicate the 25th to 75th percentiles along with the median line, and extensions extend to the maximum and minimum values. N=71(ADA), 147(AHCY), 190(AHNAK), 119(AKR1C4), 124(ATF6B), 179(BATF), 137(CALML3), 189(CDK1), 129(CDK2), 236(CLIC1), 84(CRLF2), 91(CXCL12), 88(CYP27A1), 129(DBI), 26(DCLRE1B), 261(DUPD1), Figure 9D: Expression of all ORF genes by cell assigned to each ORF. Each row is z-score normalized. Figure 9E: Distribution of individual ORF frequencies within clusters. The number of ORF cells and chi-squared residuals are displayed. The chi-squared p-value indicating whether the ORF distribution in each cluster is significantly different from the overall ORF distribution is displayed above the plot. The percentage of stimulated and resting T cells in each cluster is shown below the cluster label.(Figure 9F, Figure 9G) Spearman correlation between transcriptional profiles of selected ORF cells in resting (Figure 9F) and stimulated (Figure 9G) populations. (Figure 9H) Fold change of the top differentially expressed genes between cells with the indicated ORFs in resting and stimulated T cells. For each condition, the ORF with the strongest transcriptional change (compared to tNGFR cells) is shown. (Figure 9I) Differential gene expression in stimulated ORF T cells compared to resting T cells. Genes with significant expression changes in at least one ORF are shown (DESeq2 adjusted p<0.05). For all genes, the log2 fold change of each ORF (stimulated) relative to tNGFR (resting) is shown, normalized to the log2 fold change of tNGFR (stimulated) relative to tNGFR (resting). Genes of interest in each cluster are labeled. (Figure 9J) Average TCR clonotype diversity in ORF cells. [Figure 9F]OverCITE-seq analysis identifies ORFs and their transcriptional effects. (Figure 9A) Cell quality parameters identified by gel bead barcodes. Negatives, singlets, and doublets are assigned based on cell hashing. (Figure 9B) Percentage of stimulated and resting T cells among cells assigned to each ORF. Chi-squared p-values ​​are shown for ORFs with significantly shifted (uneven) distributions of stimulated and resting cells. (Figure 9C) Cell cycle-corrected, scaled expression of overexpressed genes in cells transduced with each ORF and the negative control (tNGFR). Two-tailed Wilcoxon p-values ​​shown above the violin plot indicate statistical significance of gene expression levels between specific ORF-transduced and tNGFR-transduced T cells. The boxes indicate the 25th to 75th percentiles along with the median line, and extensions extend to the maximum and minimum values. N=71(ADA), 147(AHCY), 190(AHNAK), 119(AKR1C4), 124(ATF6B), 179(BATF), 137(CALML3), 189(CDK1), 129(CDK2), 236(CLIC1), 84(CRLF2), 91(CXCL12), 88(CYP27A1), 129(DBI), 26(DCLRE1B), 261(DUPD1), Figure 9D: Expression of all ORF genes by cell assigned to each ORF. Each row is z-score normalized. Figure 9E: Distribution of individual ORF frequencies within clusters. The number of ORF cells and chi-squared residuals are displayed. The chi-squared p-value indicating whether the ORF distribution in each cluster is significantly different from the overall ORF distribution is displayed above the plot. The percentage of stimulated and resting T cells in each cluster is shown below the cluster label.(Figure 9F, Figure 9G) Spearman correlation between transcriptional profiles of selected ORF cells in resting (Figure 9F) and stimulated (Figure 9G) populations. (Figure 9H) Fold change of the top differentially expressed genes between cells with the indicated ORFs in resting and stimulated T cells. For each condition, the ORF with the strongest transcriptional change (compared to tNGFR cells) is shown. (Figure 9I) Differential gene expression in stimulated ORF T cells compared to resting T cells. Genes with significant expression changes in at least one ORF are shown (DESeq2 adjusted p<0.05). For all genes, the log2 fold change of each ORF (stimulated) relative to tNGFR (resting) is shown, normalized to the log2 fold change of tNGFR (stimulated) relative to tNGFR (resting). Genes of interest in each cluster are labeled. (Figure 9J) Average TCR clonotype diversity in ORF cells. [Figure 9G]OverCITE-seq analysis identifies ORFs and their transcriptional effects. (Figure 9A) Cell quality parameters identified by gel bead barcodes. Negatives, singlets, and doublets are assigned based on cell hashing. (Figure 9B) Percentage of stimulated and resting T cells among cells assigned to each ORF. Chi-squared p-values ​​are shown for ORFs with significantly shifted (uneven) distributions of stimulated and resting cells. (Figure 9C) Cell cycle-corrected, scaled expression of overexpressed genes in cells transduced with each ORF and the negative control (tNGFR). Two-tailed Wilcoxon p-values ​​shown above the violin plot indicate statistical significance of gene expression levels between specific ORF-transduced and tNGFR-transduced T cells. The boxes indicate the 25th to 75th percentiles along with the median line, and extensions extend to the maximum and minimum values. N=71(ADA), 147(AHCY), 190(AHNAK), 119(AKR1C4), 124(ATF6B), 179(BATF), 137(CALML3), 189(CDK1), 129(CDK2), 236(CLIC1), 84(CRLF2), 91(CXCL12), 88(CYP27A1), 129(DBI), 26(DCLRE1B), 261(DUPD1), Figure 9D: Expression of all ORF genes by cell assigned to each ORF. Each row is z-score normalized. Figure 9E: Distribution of individual ORF frequencies within clusters. The number of ORF cells and chi-squared residuals are displayed. The chi-squared p-value indicating whether the ORF distribution in each cluster is significantly different from the overall ORF distribution is displayed above the plot. The percentage of stimulated and resting T cells in each cluster is shown below the cluster label.(Figure 9F, Figure 9G) Spearman correlation between transcriptional profiles of selected ORF cells in resting (Figure 9F) and stimulated (Figure 9G) populations. (Figure 9H) Fold change of the top differentially expressed genes between cells with the indicated ORFs in resting and stimulated T cells. For each condition, the ORF with the strongest transcriptional change (compared to tNGFR cells) is shown. (Figure 9I) Differential gene expression in stimulated ORF T cells compared to resting T cells. Genes with significant expression changes in at least one ORF are shown (DESeq2 adjusted p<0.05). For all genes, the log2 fold change of each ORF (stimulated) relative to tNGFR (resting) is shown, normalized to the log2 fold change of tNGFR (stimulated) relative to tNGFR (resting). Genes of interest in each cluster are labeled. (Figure 9J) Average TCR clonotype diversity in ORF cells. [Figure 9H]OverCITE-seq analysis identifies ORFs and their transcriptional effects. (Figure 9A) Cell quality parameters identified by gel bead barcodes. Negatives, singlets, and doublets are assigned based on cell hashing. (Figure 9B) Percentage of stimulated and resting T cells among cells assigned to each ORF. Chi-squared p-values ​​are shown for ORFs with significantly shifted (uneven) distributions of stimulated and resting cells. (Figure 9C) Cell cycle-corrected, scaled expression of overexpressed genes in cells transduced with each ORF and the negative control (tNGFR). Two-tailed Wilcoxon p-values ​​shown above the violin plot indicate statistical significance of gene expression levels between specific ORF-transduced and tNGFR-transduced T cells. The boxes indicate the 25th to 75th percentiles along with the median line, and extensions extend to the maximum and minimum values. N=71(ADA), 147(AHCY), 190(AHNAK), 119(AKR1C4), 124(ATF6B), 179(BATF), 137(CALML3), 189(CDK1), 129(CDK2), 236(CLIC1), 84(CRLF2), 91(CXCL12), 88(CYP27A1), 129(DBI), 26(DCLRE1B), 261(DUPD1), Figure 9D: Expression of all ORF genes by cell assigned to each ORF. Each row is z-score normalized. Figure 9E: Distribution of individual ORF frequencies within clusters. The number of ORF cells and chi-squared residuals are displayed. The chi-squared p-value indicating whether the ORF distribution in each cluster is significantly different from the overall ORF distribution is displayed above the plot. The percentage of stimulated and resting T cells in each cluster is shown below the cluster label.(Figure 9F, Figure 9G) Spearman correlation between transcriptional profiles of selected ORF cells in resting (Figure 9F) and stimulated (Figure 9G) populations. (Figure 9H) Fold change of the top differentially expressed genes between cells with the indicated ORFs in resting and stimulated T cells. For each condition, the ORF with the strongest transcriptional change (compared to tNGFR cells) is shown. (Figure 9I) Differential gene expression in stimulated ORF T cells compared to resting T cells. Genes with significant expression changes in at least one ORF are shown (DESeq2 adjusted p<0.05). For all genes, the log2 fold change of each ORF (stimulated) relative to tNGFR (resting) is shown, normalized to the log2 fold change of tNGFR (stimulated) relative to tNGFR (resting). Genes of interest in each cluster are labeled. (Figure 9J) Average TCR clonotype diversity in ORF cells. [Figure 9I]OverCITE-seq analysis identifies ORFs and their transcriptional effects. (Figure 9A) Cell quality parameters identified by gel bead barcodes. Negatives, singlets, and doublets are assigned based on cell hashing. (Figure 9B) Percentage of stimulated and resting T cells among cells assigned to each ORF. Chi-squared p-values ​​are shown for ORFs with significantly shifted (uneven) distributions of stimulated and resting cells. (Figure 9C) Cell cycle-corrected, scaled expression of overexpressed genes in cells transduced with each ORF and the negative control (tNGFR). Two-tailed Wilcoxon p-values ​​shown above the violin plot indicate statistical significance of gene expression levels between specific ORF-transduced and tNGFR-transduced T cells. The boxes indicate the 25th to 75th percentiles along with the median line, and extensions extend to the maximum and minimum values. N=71(ADA), 147(AHCY), 190(AHNAK), 119(AKR1C4), 124(ATF6B), 179(BATF), 137(CALML3), 189(CDK1), 129(CDK2), 236(CLIC1), 84(CRLF2), 91(CXCL12), 88(CYP27A1), 129(DBI), 26(DCLRE1B), 261(DUPD1), Figure 9D: Expression of all ORF genes by cell assigned to each ORF. Each row is z-score normalized. Figure 9E: Distribution of individual ORF frequencies within clusters. The number of ORF cells and chi-squared residuals are displayed. The chi-squared p-value indicating whether the ORF distribution in each cluster is significantly different from the overall ORF distribution is displayed above the plot. The percentage of stimulated and resting T cells in each cluster is shown below the cluster label.(Figure 9F, Figure 9G) Spearman correlation between transcriptional profiles of selected ORF cells in resting (Figure 9F) and stimulated (Figure 9G) populations. (Figure 9H) Fold change of the top differentially expressed genes between cells with the indicated ORFs in resting and stimulated T cells. For each condition, the ORF with the strongest transcriptional change (compared to tNGFR cells) is shown. (Figure 9I) Differential gene expression in stimulated ORF T cells compared to resting T cells. Genes with significant expression changes in at least one ORF are shown (DESeq2 adjusted p<0.05). For all genes, the log2 fold change of each ORF (stimulated) relative to tNGFR (resting) is shown, normalized to the log2 fold change of tNGFR (stimulated) relative to tNGFR (resting). Genes of interest in each cluster are labeled. (Figure 9J) Average TCR clonotype diversity in ORF cells. [Figure 9J]OverCITE-seq analysis identifies ORFs and their transcriptional effects. (Figure 9A) Cell quality parameters identified by gel bead barcodes. Negatives, singlets, and doublets are assigned based on cell hashing. (Figure 9B) Percentage of stimulated and resting T cells among cells assigned to each ORF. Chi-squared p-values ​​are shown for ORFs with significantly shifted (uneven) distributions of stimulated and resting cells. (Figure 9C) Cell cycle-corrected, scaled expression of overexpressed genes in cells transduced with each ORF and the negative control (tNGFR). Two-tailed Wilcoxon p-values ​​shown above the violin plot indicate statistical significance of gene expression levels between specific ORF-transduced and tNGFR-transduced T cells. The boxes indicate the 25th to 75th percentiles along with the median line, and extensions extend to the maximum and minimum values. N=71(ADA), 147(AHCY), 190(AHNAK), 119(AKR1C4), 124(ATF6B), 179(BATF), 137(CALML3), 189(CDK1), 129(CDK2), 236(CLIC1), 84(CRLF2), 91(CXCL12), 88(CYP27A1), 129(DBI), 26(DCLRE1B), 261(DUPD1), Figure 9D: Expression of all ORF genes by cell assigned to each ORF. Each row is z-score normalized. Figure 9E: Distribution of individual ORF frequencies within clusters. The number of ORF cells and chi-squared residuals are displayed. The chi-squared p-value indicating whether the ORF distribution in each cluster is significantly different from the overall ORF distribution is displayed above the plot. The percentage of stimulated and resting T cells in each cluster is shown below the cluster label.(Figure 9F, Figure 9G) Spearman correlation between transcriptional profiles of selected ORF cells in resting (Figure 9F) and stimulated (Figure 9G) populations. (Figure 9H) Fold change of the top differentially expressed genes between cells with the indicated ORFs in resting and stimulated T cells. For each condition, the ORF with the strongest transcriptional change (compared to tNGFR cells) is shown. (Figure 9I) Differential gene expression in stimulated ORF T cells compared to resting T cells. Genes with significant expression changes in at least one ORF are shown (DESeq2 adjusted p<0.05). For all genes, the log2 fold change of each ORF (stimulated) relative to tNGFR (resting) is shown, normalized to the log2 fold change of tNGFR (stimulated) relative to tNGFR (resting). Genes of interest in each cluster are labeled. (Figure 9J) Average TCR clonotype diversity in ORF cells. [Figure 10A]Functional analysis of LTBR overexpression in T cells is shown. (Figure 10A) LTBR expression in the indicated human primary tissues from the Genotype-Tissue Expression (GTEx) project v875 (n=948 donors). Boxes indicate the 25th-75th percentiles with a median line. (Figure 10B) LTBR expression in peripheral blood mononuclear cells (PBMCs) from 31,021 cells from two donors. 76 Cell types shown are derived from Harmony tSNE clustering of single-cell transcriptomes. (Figure 10C) Overlap between significantly upregulated genes in LTBR cells compared to tNGFR cells identified by single-cell or bulk RNA-seq. (Figure 10D, Figure 10E) TCF1 expression in LTBR- or tNGFR-transduced T cells. (Figure 10D) Representative histogram of TCF1 expression and the gate (dashed line) for TCF1+ cells, and (Figure 10E) quantification of TCF1+ cells are shown (n = 3 biological replicates). (Figures 10F-10H) ICAM-1, CD70, CD74, and MHC-II expression in LTBR and tNGFR T cells. Representative histogram (Figure 10F), quantification (Figure 10G), and time course (Figure 10H) of expression in LTBR and tNGFR cells after CD3 / CD28 stimulation in n = 3 donors (CD8+) or n = 4 donors (CD4+) (n = 3 biological replicates). (Figure 10I) Differentiation phenotype of NGFR- and LTBR-transduced T cells (n = 4 donors, CD4+ and CD8+ separately). CM: central memory. EM: effector memory. Differentiation was defined based on CD45RO and CCR7 expression (naive: CD45ROneg CCR7+, CM: CD45RO+ CCR7+, EM: CD45RO+ CCR7neg, effector: CD45ROneg CCR7neg). (Figure 10J) Representative dot plot of T cell viability after CD3 / CD28 stimulation. Viable cells are in the lower left quadrant. (Figure 10K) Cell viability of LTBR or tNGFR lentivirally transduced CD4+ T cells that were either restimulated with CD3 / CD28 for 4 days or left unstimulated (n = 2 donors with 3 biological replicates each).(Figures 10L and 10M) LTBR and tNGFR cells were stimulated with a 3:1 excess of CD3 / CD28 beads every 3 days for up to three rounds of stimulation. After repeated stimulation, TIM-3 and LAG-3 expression (Figure 10L) was measured in resting cells, and IFNγ and IL2 secretion (Figure 10M) was measured in restimulated cells (n = 3 biological replicates). Statistical significance in panels 10E, 10I, and 10K: two-tailed unpaired t-test; panel 10G: two-tailed paired t-test. Error bars indicate SEM. [Figure 10B]Functional analysis of LTBR overexpression in T cells is shown. (Figure 10A) LTBR expression in the indicated human primary tissues from the Genotype-Tissue Expression (GTEx) project v875 (n=948 donors). Boxes indicate the 25th-75th percentiles with a median line. (Figure 10B) LTBR expression in peripheral blood mononuclear cells (PBMCs) from 31,021 cells from two donors. 76 Cell types shown are derived from Harmony tSNE clustering of single-cell transcriptomes. (Figure 10C) Overlap between significantly upregulated genes in LTBR cells compared to tNGFR cells identified by single-cell or bulk RNA-seq. (Figure 10D, Figure 10E) TCF1 expression in LTBR- or tNGFR-transduced T cells. (Figure 10D) Representative histogram of TCF1 expression and the gate (dashed line) for TCF1+ cells, and (Figure 10E) quantification of TCF1+ cells are shown (n = 3 biological replicates). (Figures 10F-10H) ICAM-1, CD70, CD74, and MHC-II expression in LTBR and tNGFR T cells. Representative histogram (Figure 10F), quantification (Figure 10G), and time course (Figure 10H) of expression in LTBR and tNGFR cells after CD3 / CD28 stimulation in n = 3 donors (CD8+) or n = 4 donors (CD4+) (n = 3 biological replicates). (Figure 10I) Differentiation phenotype of NGFR- and LTBR-transduced T cells (n = 4 donors, CD4+ and CD8+ separately). CM: central memory. EM: effector memory. Differentiation was defined based on CD45RO and CCR7 expression (naive: CD45ROneg CCR7+, CM: CD45RO+ CCR7+, EM: CD45RO+ CCR7neg, effector: CD45ROneg CCR7neg). (Figure 10J) Representative dot plot of T cell viability after CD3 / CD28 stimulation. Viable cells are in the lower left quadrant. (Figure 10K) Cell viability of LTBR or tNGFR lentivirally transduced CD4+ T cells that were either restimulated with CD3 / CD28 for 4 days or left unstimulated (n = 2 donors with 3 biological replicates each).(Figures 10L and 10M) LTBR and tNGFR cells were stimulated with a 3:1 excess of CD3 / CD28 beads every 3 days for up to three rounds of stimulation. After repeated stimulation, TIM-3 and LAG-3 expression (Figure 10L) was measured in resting cells, and IFNγ and IL2 secretion (Figure 10M) was measured in restimulated cells (n = 3 biological replicates). Statistical significance in panels 10E, 10I, and 10K: two-tailed unpaired t-test; panel 10G: two-tailed paired t-test. Error bars indicate SEM. [Figure 10C]Functional analysis of LTBR overexpression in T cells is shown. (Figure 10A) LTBR expression in the indicated human primary tissues from the Genotype-Tissue Expression (GTEx) project v875 (n=948 donors). Boxes indicate the 25th-75th percentiles with a median line. (Figure 10B) LTBR expression in peripheral blood mononuclear cells (PBMCs) from 31,021 cells from two donors. 76 Cell types shown are derived from Harmony tSNE clustering of single-cell transcriptomes. (Figure 10C) Overlap between significantly upregulated genes in LTBR cells compared to tNGFR cells identified by single-cell or bulk RNA-seq. (Figure 10D, Figure 10E) TCF1 expression in LTBR- or tNGFR-transduced T cells. (Figure 10D) Representative histogram of TCF1 expression and the gate (dashed line) for TCF1+ cells, and (Figure 10E) quantification of TCF1+ cells are shown (n = 3 biological replicates). (Figures 10F-10H) ICAM-1, CD70, CD74, and MHC-II expression in LTBR and tNGFR T cells. Representative histogram (Figure 10F), quantification (Figure 10G), and time course (Figure 10H) of expression in LTBR and tNGFR cells after CD3 / CD28 stimulation in n = 3 donors (CD8+) or n = 4 donors (CD4+) (n = 3 biological replicates). (Figure 10I) Differentiation phenotype of NGFR- and LTBR-transduced T cells (n = 4 donors, CD4+ and CD8+ separately). CM: central memory. EM: effector memory. Differentiation was defined based on CD45RO and CCR7 expression (naive: CD45ROneg CCR7+, CM: CD45RO+ CCR7+, EM: CD45RO+ CCR7neg, effector: CD45ROneg CCR7neg). (Figure 10J) Representative dot plot of T cell viability after CD3 / CD28 stimulation. Viable cells are in the lower left quadrant. (Figure 10K) Cell viability of LTBR or tNGFR lentivirally transduced CD4+ T cells that were either restimulated with CD3 / CD28 for 4 days or left unstimulated (n = 2 donors with 3 biological replicates each).(Figures 10L and 10M) LTBR and tNGFR cells were stimulated with a 3:1 excess of CD3 / CD28 beads every 3 days for up to three rounds of stimulation. After repeated stimulation, TIM-3 and LAG-3 expression (Figure 10L) was measured in resting cells, and IFNγ and IL2 secretion (Figure 10M) was measured in restimulated cells (n = 3 biological replicates). Statistical significance in panels 10E, 10I, and 10K: two-tailed unpaired t-test; panel 10G: two-tailed paired t-test. Error bars indicate SEM. [Figure 10D]Functional analysis of LTBR overexpression in T cells is shown. (Figure 10A) LTBR expression in the indicated human primary tissues from the Genotype-Tissue Expression (GTEx) project v875 (n=948 donors). Boxes indicate the 25th-75th percentiles with a median line. (Figure 10B) LTBR expression in peripheral blood mononuclear cells (PBMCs) from 31,021 cells from two donors. 76 Cell types shown are derived from Harmony tSNE clustering of single-cell transcriptomes. (Figure 10C) Overlap between significantly upregulated genes in LTBR cells compared to tNGFR cells identified by single-cell or bulk RNA-seq. (Figure 10D, Figure 10E) TCF1 expression in LTBR- or tNGFR-transduced T cells. (Figure 10D) Representative histogram of TCF1 expression and the gate (dashed line) for TCF1+ cells, and (Figure 10E) quantification of TCF1+ cells are shown (n = 3 biological replicates). (Figures 10F-10H) ICAM-1, CD70, CD74, and MHC-II expression in LTBR and tNGFR T cells. Representative histogram (Figure 10F), quantification (Figure 10G), and time course (Figure 10H) of expression in LTBR and tNGFR cells after CD3 / CD28 stimulation in n = 3 donors (CD8+) or n = 4 donors (CD4+) (n = 3 biological replicates). (Figure 10I) Differentiation phenotype of NGFR- and LTBR-transduced T cells (n = 4 donors, CD4+ and CD8+ separately). CM: central memory. EM: effector memory. Differentiation was defined based on CD45RO and CCR7 expression (naive: CD45ROneg CCR7+, CM: CD45RO+ CCR7+, EM: CD45RO+ CCR7neg, effector: CD45ROneg CCR7neg). (Figure 10J) Representative dot plot of T cell viability after CD3 / CD28 stimulation. Viable cells are in the lower left quadrant. (Figure 10K) Cell viability of LTBR or tNGFR lentivirally transduced CD4+ T cells that were either restimulated with CD3 / CD28 for 4 days or left unstimulated (n = 2 donors with 3 biological replicates each).(Figures 10L and 10M) LTBR and tNGFR cells were stimulated with a 3:1 excess of CD3 / CD28 beads every 3 days for up to three rounds of stimulation. After repeated stimulation, TIM-3 and LAG-3 expression (Figure 10L) was measured in resting cells, and IFNγ and IL2 secretion (Figure 10M) was measured in restimulated cells (n = 3 biological replicates). Statistical significance in panels 10E, 10I, and 10K: two-tailed unpaired t-test; panel 10G: two-tailed paired t-test. Error bars indicate SEM. [Figure 10E]Functional analysis of LTBR overexpression in T cells is shown. (Figure 10A) LTBR expression in the indicated human primary tissues from the Genotype-Tissue Expression (GTEx) project v875 (n=948 donors). Boxes indicate the 25th-75th percentiles with a median line. (Figure 10B) LTBR expression in peripheral blood mononuclear cells (PBMCs) from 31,021 cells from two donors. 76 Cell types shown are derived from Harmony tSNE clustering of single-cell transcriptomes. (Figure 10C) Overlap between significantly upregulated genes in LTBR cells compared to tNGFR cells identified by single-cell or bulk RNA-seq. (Figure 10D, Figure 10E) TCF1 expression in LTBR- or tNGFR-transduced T cells. (Figure 10D) Representative histogram of TCF1 expression and the gate (dashed line) for TCF1+ cells, and (Figure 10E) quantification of TCF1+ cells are shown (n = 3 biological replicates). (Figures 10F-10H) ICAM-1, CD70, CD74, and MHC-II expression in LTBR and tNGFR T cells. Representative histogram (Figure 10F), quantification (Figure 10G), and time course (Figure 10H) of expression in LTBR and tNGFR cells after CD3 / CD28 stimulation in n = 3 donors (CD8+) or n = 4 donors (CD4+) (n = 3 biological replicates). (Figure 10I) Differentiation phenotype of NGFR- and LTBR-transduced T cells (n = 4 donors, CD4+ and CD8+ separately). CM: central memory. EM: effector memory. Differentiation was defined based on CD45RO and CCR7 expression (naive: CD45ROneg CCR7+, CM: CD45RO+ CCR7+, EM: CD45RO+ CCR7neg, effector: CD45ROneg CCR7neg). (Figure 10J) Representative dot plot of T cell viability after CD3 / CD28 stimulation. Viable cells are in the lower left quadrant. (Figure 10K) Cell viability of LTBR or tNGFR lentivirally transduced CD4+ T cells that were either restimulated with CD3 / CD28 for 4 days or left unstimulated (n = 2 donors with 3 biological replicates each).(Figures 10L and 10M) LTBR and tNGFR cells were stimulated with a 3:1 excess of CD3 / CD28 beads every 3 days for up to three rounds of stimulation. After repeated stimulation, TIM-3 and LAG-3 expression (Figure 10L) was measured in resting cells, and IFNγ and IL2 secretion (Figure 10M) was measured in restimulated cells (n = 3 biological replicates). Statistical significance in panels 10E, 10I, and 10K: two-tailed unpaired t-test; panel 10G: two-tailed paired t-test. Error bars indicate SEM. [Figure 10F]Functional analysis of LTBR overexpression in T cells is shown. (Figure 10A) LTBR expression in the indicated human primary tissues from the Genotype-Tissue Expression (GTEx) project v875 (n=948 donors). Boxes indicate the 25th-75th percentiles with a median line. (Figure 10B) LTBR expression in peripheral blood mononuclear cells (PBMCs) from 31,021 cells from two donors. 76 Cell types shown are derived from Harmony tSNE clustering of single-cell transcriptomes. (Figure 10C) Overlap between significantly upregulated genes in LTBR cells compared to tNGFR cells identified by single-cell or bulk RNA-seq. (Figure 10D, Figure 10E) TCF1 expression in LTBR- or tNGFR-transduced T cells. (Figure 10D) Representative histogram of TCF1 expression and the gate (dashed line) for TCF1+ cells, and (Figure 10E) quantification of TCF1+ cells are shown (n = 3 biological replicates). (Figures 10F-10H) ICAM-1, CD70, CD74, and MHC-II expression in LTBR and tNGFR T cells. Representative histogram (Figure 10F), quantification (Figure 10G), and time course (Figure 10H) of expression in LTBR and tNGFR cells after CD3 / CD28 stimulation in n = 3 donors (CD8+) or n = 4 donors (CD4+) (n = 3 biological replicates). (Figure 10I) Differentiation phenotype of NGFR- and LTBR-transduced T cells (n = 4 donors, CD4+ and CD8+ separately). CM: central memory. EM: effector memory. Differentiation was defined based on CD45RO and CCR7 expression (naive: CD45ROneg CCR7+, CM: CD45RO+ CCR7+, EM: CD45RO+ CCR7neg, effector: CD45ROneg CCR7neg). (Figure 10J) Representative dot plot of T cell viability after CD3 / CD28 stimulation. Viable cells are in the lower left quadrant. (Figure 10K) Cell viability of LTBR or tNGFR lentivirally transduced CD4+ T cells that were either restimulated with CD3 / CD28 for 4 days or left unstimulated (n = 2 donors with 3 biological replicates each).(Figures 10L and 10M) LTBR and tNGFR cells were stimulated with a 3:1 excess of CD3 / CD28 beads every 3 days for up to three rounds of stimulation. After repeated stimulation, TIM-3 and LAG-3 expression (Figure 10L) was measured in resting cells, and IFNγ and IL2 secretion (Figure 10M) was measured in restimulated cells (n = 3 biological replicates). Statistical significance in panels 10E, 10I, and 10K: two-tailed unpaired t-test; panel 10G: two-tailed paired t-test. Error bars indicate SEM. [Figure 10G]Functional analysis of LTBR overexpression in T cells is shown. (Figure 10A) LTBR expression in the indicated human primary tissues from the Genotype-Tissue Expression (GTEx) project v875 (n=948 donors). Boxes indicate the 25th-75th percentiles with a median line. (Figure 10B) LTBR expression in peripheral blood mononuclear cells (PBMCs) from 31,021 cells from two donors. 76 Cell types shown are derived from Harmony tSNE clustering of single-cell transcriptomes. (Figure 10C) Overlap between significantly upregulated genes in LTBR cells compared to tNGFR cells identified by single-cell or bulk RNA-seq. (Figure 10D, Figure 10E) TCF1 expression in LTBR- or tNGFR-transduced T cells. (Figure 10D) Representative histogram of TCF1 expression and the gate (dashed line) for TCF1+ cells, and (Figure 10E) quantification of TCF1+ cells are shown (n = 3 biological replicates). (Figures 10F-10H) ICAM-1, CD70, CD74, and MHC-II expression in LTBR and tNGFR T cells. Representative histogram (Figure 10F), quantification (Figure 10G), and time course (Figure 10H) of expression in LTBR and tNGFR cells after CD3 / CD28 stimulation in n = 3 donors (CD8+) or n = 4 donors (CD4+) (n = 3 biological replicates). (Figure 10I) Differentiation phenotype of NGFR- and LTBR-transduced T cells (n = 4 donors, CD4+ and CD8+ separately). CM: central memory. EM: effector memory. Differentiation was defined based on CD45RO and CCR7 expression (naive: CD45ROneg CCR7+, CM: CD45RO+ CCR7+, EM: CD45RO+ CCR7neg, effector: CD45ROneg CCR7neg). (Figure 10J) Representative dot plot of T cell viability after CD3 / CD28 stimulation. Viable cells are in the lower left quadrant. (Figure 10K) Cell viability of LTBR or tNGFR lentivirally transduced CD4+ T cells that were either restimulated with CD3 / CD28 for 4 days or left unstimulated (n = 2 donors with 3 biological replicates each).(Figures 10L and 10M) LTBR and tNGFR cells were stimulated with a 3:1 excess of CD3 / CD28 beads every 3 days for up to three rounds of stimulation. After repeated stimulation, TIM-3 and LAG-3 expression (Figure 10L) was measured in resting cells, and IFNγ and IL2 secretion (Figure 10M) was measured in restimulated cells (n = 3 biological replicates). Statistical significance in panels 10E, 10I, and 10K: two-tailed unpaired t-test; panel 10G: two-tailed paired t-test. Error bars indicate SEM. [Figure 10H]Functional analysis of LTBR overexpression in T cells is shown. (Figure 10A) LTBR expression in the indicated human primary tissues from the Genotype-Tissue Expression (GTEx) project v875 (n=948 donors). Boxes indicate the 25th-75th percentiles with a median line. (Figure 10B) LTBR expression in peripheral blood mononuclear cells (PBMCs) from 31,021 cells from two donors. 76 Cell types shown are derived from Harmony tSNE clustering of single-cell transcriptomes. (Figure 10C) Overlap between significantly upregulated genes in LTBR cells compared to tNGFR cells identified by single-cell or bulk RNA-seq. (Figure 10D, Figure 10E) TCF1 expression in LTBR- or tNGFR-transduced T cells. (Figure 10D) Representative histogram of TCF1 expression and the gate (dashed line) for TCF1+ cells, and (Figure 10E) quantification of TCF1+ cells are shown (n = 3 biological replicates). (Figures 10F-10H) ICAM-1, CD70, CD74, and MHC-II expression in LTBR and tNGFR T cells. Representative histogram (Figure 10F), quantification (Figure 10G), and time course (Figure 10H) of expression in LTBR and tNGFR cells after CD3 / CD28 stimulation in n = 3 donors (CD8+) or n = 4 donors (CD4+) (n = 3 biological replicates). (Figure 10I) Differentiation phenotype of NGFR- and LTBR-transduced T cells (n = 4 donors, CD4+ and CD8+ separately). CM: central memory. EM: effector memory. Differentiation was defined based on CD45RO and CCR7 expression (naive: CD45ROneg CCR7+, CM: CD45RO+ CCR7+, EM: CD45RO+ CCR7neg, effector: CD45ROneg CCR7neg). (Figure 10J) Representative dot plot of T cell viability after CD3 / CD28 stimulation. Viable cells are in the lower left quadrant. (Figure 10K) Cell viability of LTBR or tNGFR lentivirally transduced CD4+ T cells that were either restimulated with CD3 / CD28 for 4 days or left unstimulated (n = 2 donors with 3 biological replicates each).(Figures 10L and 10M) LTBR and tNGFR cells were stimulated with a 3:1 excess of CD3 / CD28 beads every 3 days for up to three rounds of stimulation. After repeated stimulation, TIM-3 and LAG-3 expression (Figure 10L) was measured in resting cells, and IFNγ and IL2 secretion (Figure 10M) was measured in restimulated cells (n = 3 biological replicates). Statistical significance in panels 10E, 10I, and 10K: two-tailed unpaired t-test; panel 10G: two-tailed paired t-test. Error bars indicate SEM. [Figure 10I]Functional analysis of LTBR overexpression in T cells is shown. (Figure 10A) LTBR expression in the indicated human primary tissues from the Genotype-Tissue Expression (GTEx) project v875 (n=948 donors). Boxes indicate the 25th-75th percentiles with a median line. (Figure 10B) LTBR expression in peripheral blood mononuclear cells (PBMCs) from 31,021 cells from two donors. 76 Cell types shown are derived from Harmony tSNE clustering of single-cell transcriptomes. (Figure 10C) Overlap between significantly upregulated genes in LTBR cells compared to tNGFR cells identified by single-cell or bulk RNA-seq. (Figure 10D, Figure 10E) TCF1 expression in LTBR- or tNGFR-transduced T cells. (Figure 10D) Representative histogram of TCF1 expression and the gate (dashed line) for TCF1+ cells, and (Figure 10E) quantification of TCF1+ cells are shown (n = 3 biological replicates). (Figures 10F-10H) ICAM-1, CD70, CD74, and MHC-II expression in LTBR and tNGFR T cells. Representative histogram (Figure 10F), quantification (Figure 10G), and time course (Figure 10H) of expression in LTBR and tNGFR cells after CD3 / CD28 stimulation in n = 3 donors (CD8+) or n = 4 donors (CD4+) (n = 3 biological replicates). (Figure 10I) Differentiation phenotype of NGFR- and LTBR-transduced T cells (n = 4 donors, CD4+ and CD8+ separately). CM: central memory. EM: effector memory. Differentiation was defined based on CD45RO and CCR7 expression (naive: CD45ROneg CCR7+, CM: CD45RO+ CCR7+, EM: CD45RO+ CCR7neg, effector: CD45ROneg CCR7neg). (Figure 10J) Representative dot plot of T cell viability after CD3 / CD28 stimulation. Viable cells are in the lower left quadrant. (Figure 10K) Cell viability of LTBR or tNGFR lentivirally transduced CD4+ T cells that were either restimulated with CD3 / CD28 for 4 days or left unstimulated (n = 2 donors with 3 biological replicates each).(Figures 10L and 10M) LTBR and tNGFR cells were stimulated with a 3:1 excess of CD3 / CD28 beads every 3 days for up to three rounds of stimulation. After repeated stimulation, TIM-3 and LAG-3 expression (Figure 10L) was measured in resting cells, and IFNγ and IL2 secretion (Figure 10M) was measured in restimulated cells (n = 3 biological replicates). Statistical significance in panels 10E, 10I, and 10K: two-tailed unpaired t-test; panel 10G: two-tailed paired t-test. Error bars indicate SEM. [Figure 10J]Functional analysis of LTBR overexpression in T cells is shown. (Figure 10A) LTBR expression in the indicated human primary tissues from the Genotype-Tissue Expression (GTEx) project v875 (n=948 donors). Boxes indicate the 25th-75th percentiles with a median line. (Figure 10B) LTBR expression in peripheral blood mononuclear cells (PBMCs) from 31,021 cells from two donors. 76 Cell types shown are derived from Harmony tSNE clustering of single-cell transcriptomes. (Figure 10C) Overlap between significantly upregulated genes in LTBR cells compared to tNGFR cells identified by single-cell or bulk RNA-seq. (Figure 10D, Figure 10E) TCF1 expression in LTBR- or tNGFR-transduced T cells. (Figure 10D) Representative histogram of TCF1 expression and the gate (dashed line) for TCF1+ cells, and (Figure 10E) quantification of TCF1+ cells are shown (n = 3 biological replicates). (Figures 10F-10H) ICAM-1, CD70, CD74, and MHC-II expression in LTBR and tNGFR T cells. Representative histogram (Figure 10F), quantification (Figure 10G), and time course (Figure 10H) of expression in LTBR and tNGFR cells after CD3 / CD28 stimulation in n = 3 donors (CD8+) or n = 4 donors (CD4+) (n = 3 biological replicates). (Figure 10I) Differentiation phenotype of NGFR- and LTBR-transduced T cells (n = 4 donors, CD4+ and CD8+ separately). CM: central memory. EM: effector memory. Differentiation was defined based on CD45RO and CCR7 expression (naive: CD45ROneg CCR7+, CM: CD45RO+ CCR7+, EM: CD45RO+ CCR7neg, effector: CD45ROneg CCR7neg). (Figure 10J) Representative dot plot of T cell viability after CD3 / CD28 stimulation. Viable cells are in the lower left quadrant. (Figure 10K) Cell viability of LTBR or tNGFR lentivirally transduced CD4+ T cells that were either restimulated with CD3 / CD28 for 4 days or left unstimulated (n = 2 donors with 3 biological replicates each).(Figures 10L and 10M) LTBR and tNGFR cells were stimulated with a 3:1 excess of CD3 / CD28 beads every 3 days for up to three rounds of stimulation. After repeated stimulation, TIM-3 and LAG-3 expression (Figure 10L) was measured in resting cells, and IFNγ and IL2 secretion (Figure 10M) was measured in restimulated cells (n = 3 biological replicates). Statistical significance in panels 10E, 10I, and 10K: two-tailed unpaired t-test; panel 10G: two-tailed paired t-test. Error bars indicate SEM. [Figure 10K]Functional analysis of LTBR overexpression in T cells is shown. (Figure 10A) LTBR expression in the indicated human primary tissues from the Genotype-Tissue Expression (GTEx) project v875 (n=948 donors). Boxes indicate the 25th-75th percentiles with a median line. (Figure 10B) LTBR expression in peripheral blood mononuclear cells (PBMCs) from 31,021 cells from two donors. 76 Cell types shown are derived from Harmony tSNE clustering of single-cell transcriptomes. (Figure 10C) Overlap between significantly upregulated genes in LTBR cells compared to tNGFR cells identified by single-cell or bulk RNA-seq. (Figure 10D, Figure 10E) TCF1 expression in LTBR- or tNGFR-transduced T cells. (Figure 10D) Representative histogram of TCF1 expression and the gate (dashed line) for TCF1+ cells, and (Figure 10E) quantification of TCF1+ cells are shown (n = 3 biological replicates). (Figures 10F-10H) ICAM-1, CD70, CD74, and MHC-II expression in LTBR and tNGFR T cells. Representative histogram (Figure 10F), quantification (Figure 10G), and time course (Figure 10H) of expression in LTBR and tNGFR cells after CD3 / CD28 stimulation in n = 3 donors (CD8+) or n = 4 donors (CD4+) (n = 3 biological replicates). (Figure 10I) Differentiation phenotype of NGFR- and LTBR-transduced T cells (n = 4 donors, CD4+ and CD8+ separately). CM: central memory. EM: effector memory. Differentiation was defined based on CD45RO and CCR7 expression (naive: CD45ROneg CCR7+, CM: CD45RO+ CCR7+, EM: CD45RO+ CCR7neg, effector: CD45ROneg CCR7neg). (Figure 10J) Representative dot plot of T cell viability after CD3 / CD28 stimulation. Viable cells are in the lower left quadrant. (Figure 10K) Cell viability of LTBR or tNGFR lentivirally transduced CD4+ T cells that were either restimulated with CD3 / CD28 for 4 days or left unstimulated (n = 2 donors with 3 biological replicates each).(Figures 10L and 10M) LTBR and tNGFR cells were stimulated with a 3:1 excess of CD3 / CD28 beads every 3 days for up to three rounds of stimulation. After repeated stimulation, TIM-3 and LAG-3 expression (Figure 10L) was measured in resting cells, and IFNγ and IL2 secretion (Figure 10M) was measured in restimulated cells (n = 3 biological replicates). Statistical significance in panels 10E, 10I, and 10K: two-tailed unpaired t-test; panel 10G: two-tailed paired t-test. Error bars indicate SEM. [Figure 10L]Functional analysis of LTBR overexpression in T cells is shown. (Figure 10A) LTBR expression in the indicated human primary tissues from the Genotype-Tissue Expression (GTEx) project v875 (n=948 donors). Boxes indicate the 25th-75th percentiles with a median line. (Figure 10B) LTBR expression in peripheral blood mononuclear cells (PBMCs) from 31,021 cells from two donors. 76 Cell types shown are derived from Harmony tSNE clustering of single-cell transcriptomes. (Figure 10C) Overlap between significantly upregulated genes in LTBR cells compared to tNGFR cells identified by single-cell or bulk RNA-seq. (Figure 10D, Figure 10E) TCF1 expression in LTBR- or tNGFR-transduced T cells. (Figure 10D) Representative histogram of TCF1 expression and the gate (dashed line) for TCF1+ cells, and (Figure 10E) quantification of TCF1+ cells are shown (n = 3 biological replicates). (Figures 10F-10H) ICAM-1, CD70, CD74, and MHC-II expression in LTBR and tNGFR T cells. Representative histogram (Figure 10F), quantification (Figure 10G), and time course (Figure 10H) of expression in LTBR and tNGFR cells after CD3 / CD28 stimulation in n = 3 donors (CD8+) or n = 4 donors (CD4+) (n = 3 biological replicates). (Figure 10I) Differentiation phenotype of NGFR- and LTBR-transduced T cells (n = 4 donors, CD4+ and CD8+ separately). CM: central memory. EM: effector memory. Differentiation was defined based on CD45RO and CCR7 expression (naive: CD45ROneg CCR7+, CM: CD45RO+ CCR7+, EM: CD45RO+ CCR7neg, effector: CD45ROneg CCR7neg). (Figure 10J) Representative dot plot of T cell viability after CD3 / CD28 stimulation. Viable cells are in the lower left quadrant. (Figure 10K) Cell viability of LTBR or tNGFR lentivirally transduced CD4+ T cells that were either restimulated with CD3 / CD28 for 4 days or left unstimulated (n = 2 donors with 3 biological replicates each).(Figures 10L and 10M) LTBR and tNGFR cells were stimulated with a 3:1 excess of CD3 / CD28 beads every 3 days for up to three rounds of stimulation. After repeated stimulation, TIM-3 and LAG-3 expression (Figure 10L) was measured in resting cells, and IFNγ and IL2 secretion (Figure 10M) was measured in restimulated cells (n = 3 biological replicates). Statistical significance in panels 10E, 10I, and 10K: two-tailed unpaired t-test; panel 10G: two-tailed paired t-test. Error bars indicate SEM. [Figure 10M]Functional analysis of LTBR overexpression in T cells is shown. (Figure 10A) LTBR expression in the indicated human primary tissues from the Genotype-Tissue Expression (GTEx) project v875 (n=948 donors). Boxes indicate the 25th-75th percentiles with a median line. (Figure 10B) LTBR expression in peripheral blood mononuclear cells (PBMCs) from 31,021 cells from two donors. 76 Cell types shown are derived from Harmony tSNE clustering of single-cell transcriptomes. (Figure 10C) Overlap between significantly upregulated genes in LTBR cells compared to tNGFR cells identified by single-cell or bulk RNA-seq. (Figure 10D, Figure 10E) TCF1 expression in LTBR- or tNGFR-transduced T cells. (Figure 10D) Representative histogram of TCF1 expression and the gate (dashed line) for TCF1+ cells, and (Figure 10E) quantification of TCF1+ cells are shown (n = 3 biological replicates). (Figures 10F-10H) ICAM-1, CD70, CD74, and MHC-II expression in LTBR and tNGFR T cells. Representative histogram (Figure 10F), quantification (Figure 10G), and time course (Figure 10H) of expression in LTBR and tNGFR cells after CD3 / CD28 stimulation in n = 3 donors (CD8+) or n = 4 donors (CD4+) (n = 3 biological replicates). (Figure 10I) Differentiation phenotype of NGFR- and LTBR-transduced T cells (n = 4 donors, CD4+ and CD8+ separately). CM: central memory. EM: effector memory. Differentiation was defined based on CD45RO and CCR7 expression (naive: CD45ROneg CCR7+, CM: CD45RO+ CCR7+, EM: CD45RO+ CCR7neg, effector: CD45ROneg CCR7neg). (Figure 10J) Representative dot plot of T cell viability after CD3 / CD28 stimulation. Viable cells are in the lower left quadrant. (Figure 10K) Cell viability of LTBR or tNGFR lentivirally transduced CD4+ T cells that were either restimulated with CD3 / CD28 for 4 days or left unstimulated (n = 2 donors with 3 biological replicates each).(Figures 10L and 10M) LTBR and tNGFR cells were stimulated with a 3:1 excess of CD3 / CD28 beads every 3 days for up to three rounds of stimulation. After repeated stimulation, TIM-3 and LAG-3 expression (Figure 10L) was measured in resting cells, and IFNγ and IL2 secretion (Figure 10M) was measured in restimulated cells (n = 3 biological replicates). Statistical significance in panels 10E, 10I, and 10K: two-tailed unpaired t-test; panel 10G: two-tailed paired t-test. Error bars indicate SEM. [Figure 11A] Expression of LTBR ligands and LTBR, ​​either via mRNA or with deletions and point mutations, is shown. (Figure 11A) IL2 secretion after 24 hours of stimulation with CD3 / CD28 antibodies. Where indicated, recombinant soluble LTA (1 ng / mL) or LIGHT (10 ng / mL) was added together with CD3 / CD28 antibodies. CD4+ T cells from one donor were tested in triplicate. (Figures 11B and 11C) CD4+ and CD8+ T cells from two donors were co-incubated with CD3 / CD28 antibodies or recombinant soluble LTA or LIGHT for 24 hours, followed by measurement of IL2 (Figure 11B) and IFNγ (Figure 11C). (n = 3 biological replicates) (Figures 10D, 10E) Differentiation phenotype (Figure 10D) or proliferation (Figure 10E) after restimulation of tNGFR- and LTBR-transduced T cells incubated with either IL2 alone or LTA (1 ng / mL) or LIGHT (10 ng / mL) during the culture period (n = 3 biological replicates). CM: central memory. EM: effector memory. Independent two-tailed t-test p-values ​​are shown. (Figures 11F, 11I) Transient LTBR or tNGFR expression via mRNA nucleofection (Figure 101F). T cells were nucleofected with LTBR or tNGFR mRNA (n = 3 biological replicates), and surface expression of LTBR (Figure 11G), tNGFR (Figure 11H), or four genes upregulated in LTBR cells (Figure 11I) was monitored over 21 days. At each time point, expression of the target genes was normalized to the appropriate tNGFR control. (FIG. 11J) Schematic of FLAG-tagged LTBR mutants. (FIG. 11K) LTBR and FLAG expression in T cells transduced with LTBR mutants. Error bars indicate SEM. [Figure 11B] Expression of LTBR ligands and LTBR, ​​either via mRNA or with deletions and point mutations, is shown. (Figure 11A) IL2 secretion after 24 hours of stimulation with CD3 / CD28 antibodies. Where indicated, recombinant soluble LTA (1 ng / mL) or LIGHT (10 ng / mL) was added together with CD3 / CD28 antibodies. CD4+ T cells from one donor were tested in triplicate. (Figures 11B and 11C) CD4+ and CD8+ T cells from two donors were co-incubated with CD3 / CD28 antibodies or recombinant soluble LTA or LIGHT for 24 hours, followed by measurement of IL2 (Figure 11B) and IFNγ (Figure 11C). (n = 3 biological replicates) (Figures 10D, 10E) Differentiation phenotype (Figure 10D) or proliferation (Figure 10E) after restimulation of tNGFR- and LTBR-transduced T cells incubated with either IL2 alone or LTA (1 ng / mL) or LIGHT (10 ng / mL) during the culture period (n = 3 biological replicates). CM: central memory. EM: effector memory. Independent two-tailed t-test p-values ​​are shown. (Figures 11F, 11I) Transient LTBR or tNGFR expression via mRNA nucleofection (Figure 101F). T cells were nucleofected with LTBR or tNGFR mRNA (n = 3 biological replicates), and surface expression of LTBR (Figure 11G), tNGFR (Figure 11H), or four genes upregulated in LTBR cells (Figure 11I) was monitored over 21 days. At each time point, expression of the target genes was normalized to the appropriate tNGFR control. (FIG. 11J) Schematic of FLAG-tagged LTBR mutants. (FIG. 11K) LTBR and FLAG expression in T cells transduced with LTBR mutants. Error bars indicate SEM. [Figure 11C]Expression of LTBR ligands and LTBR, ​​either via mRNA or with deletions and point mutations, is shown. (Figure 11A) IL2 secretion after 24 hours of stimulation with CD3 / CD28 antibodies. Where indicated, recombinant soluble LTA (1 ng / mL) or LIGHT (10 ng / mL) was added together with CD3 / CD28 antibodies. CD4+ T cells from one donor were tested in triplicate. (Figures 11B and 11C) CD4+ and CD8+ T cells from two donors were co-incubated with CD3 / CD28 antibodies or recombinant soluble LTA or LIGHT for 24 hours, followed by measurement of IL2 (Figure 11B) and IFNγ (Figure 11C). (n = 3 biological replicates) (Figures 10D, 10E) Differentiation phenotype (Figure 10D) or proliferation (Figure 10E) after restimulation of tNGFR- and LTBR-transduced T cells incubated with either IL2 alone or LTA (1 ng / mL) or LIGHT (10 ng / mL) during the culture period (n = 3 biological replicates). CM: central memory. EM: effector memory. Independent two-tailed t-test p-values ​​are shown. (Figures 11F, 11I) Transient LTBR or tNGFR expression via mRNA nucleofection (Figure 101F). T cells were nucleofected with LTBR or tNGFR mRNA (n = 3 biological replicates), and surface expression of LTBR (Figure 11G), tNGFR (Figure 11H), or four genes upregulated in LTBR cells (Figure 11I) was monitored over 21 days. At each time point, expression of the target genes was normalized to the appropriate tNGFR control. (FIG. 11J) Schematic of FLAG-tagged LTBR mutants. (FIG. 11K) LTBR and FLAG expression in T cells transduced with LTBR mutants. Error bars indicate SEM. [Figure 11D]Expression of LTBR ligands and LTBR, ​​either via mRNA or with deletions and point mutations, is shown. (Figure 11A) IL2 secretion after 24 hours of stimulation with CD3 / CD28 antibodies. Where indicated, recombinant soluble LTA (1 ng / mL) or LIGHT (10 ng / mL) was added together with CD3 / CD28 antibodies. CD4+ T cells from one donor were tested in triplicate. (Figures 11B and 11C) CD4+ and CD8+ T cells from two donors were co-incubated with CD3 / CD28 antibodies or recombinant soluble LTA or LIGHT for 24 hours, followed by measurement of IL2 (Figure 11B) and IFNγ (Figure 11C). (n = 3 biological replicates) (Figures 10D, 10E) Differentiation phenotype (Figure 10D) or proliferation (Figure 10E) after restimulation of tNGFR- and LTBR-transduced T cells incubated with either IL2 alone or LTA (1 ng / mL) or LIGHT (10 ng / mL) during the culture period (n = 3 biological replicates). CM: central memory. EM: effector memory. Independent two-tailed t-test p-values ​​are shown. (Figures 11F, 11I) Transient LTBR or tNGFR expression via mRNA nucleofection (Figure 101F). T cells were nucleofected with LTBR or tNGFR mRNA (n = 3 biological replicates), and surface expression of LTBR (Figure 11G), tNGFR (Figure 11H), or four genes upregulated in LTBR cells (Figure 11I) was monitored over 21 days. At each time point, expression of the target genes was normalized to the appropriate tNGFR control. (FIG. 11J) Schematic of FLAG-tagged LTBR mutants. (FIG. 11K) LTBR and FLAG expression in T cells transduced with LTBR mutants. Error bars indicate SEM. [Figure 11E]Expression of LTBR ligands and LTBR, ​​either via mRNA or with deletions and point mutations, is shown. (Figure 11A) IL2 secretion after 24 hours of stimulation with CD3 / CD28 antibodies. Where indicated, recombinant soluble LTA (1 ng / mL) or LIGHT (10 ng / mL) was added together with CD3 / CD28 antibodies. CD4+ T cells from one donor were tested in triplicate. (Figures 11B and 11C) CD4+ and CD8+ T cells from two donors were co-incubated with CD3 / CD28 antibodies or recombinant soluble LTA or LIGHT for 24 hours, followed by measurement of IL2 (Figure 11B) and IFNγ (Figure 11C). (n = 3 biological replicates) (Figures 10D, 10E) Differentiation phenotype (Figure 10D) or proliferation (Figure 10E) after restimulation of tNGFR- and LTBR-transduced T cells incubated with either IL2 alone or LTA (1 ng / mL) or LIGHT (10 ng / mL) during the culture period (n = 3 biological replicates). CM: central memory. EM: effector memory. Independent two-tailed t-test p-values ​​are shown. (Figures 11F, 11I) Transient LTBR or tNGFR expression via mRNA nucleofection (Figure 101F). T cells were nucleofected with LTBR or tNGFR mRNA (n = 3 biological replicates), and surface expression of LTBR (Figure 11G), tNGFR (Figure 11H), or four genes upregulated in LTBR cells (Figure 11I) was monitored over 21 days. At each time point, expression of the target genes was normalized to the appropriate tNGFR control. (FIG. 11J) Schematic of FLAG-tagged LTBR mutants. (FIG. 11K) LTBR and FLAG expression in T cells transduced with LTBR mutants. Error bars indicate SEM. [Figure 11F]Expression of LTBR ligands and LTBR, ​​either via mRNA or with deletions and point mutations, is shown. (Figure 11A) IL2 secretion after 24 hours of stimulation with CD3 / CD28 antibodies. Where indicated, recombinant soluble LTA (1 ng / mL) or LIGHT (10 ng / mL) was added together with CD3 / CD28 antibodies. CD4+ T cells from one donor were tested in triplicate. (Figures 11B and 11C) CD4+ and CD8+ T cells from two donors were co-incubated with CD3 / CD28 antibodies or recombinant soluble LTA or LIGHT for 24 hours, followed by measurement of IL2 (Figure 11B) and IFNγ (Figure 11C). (n = 3 biological replicates) (Figures 10D, 10E) Differentiation phenotype (Figure 10D) or proliferation (Figure 10E) after restimulation of tNGFR- and LTBR-transduced T cells incubated with either IL2 alone or LTA (1 ng / mL) or LIGHT (10 ng / mL) during the culture period (n = 3 biological replicates). CM: central memory. EM: effector memory. Independent two-tailed t-test p-values ​​are shown. (Figures 11F, 11I) Transient LTBR or tNGFR expression via mRNA nucleofection (Figure 101F). T cells were nucleofected with LTBR or tNGFR mRNA (n = 3 biological replicates), and surface expression of LTBR (Figure 11G), tNGFR (Figure 11H), or four genes upregulated in LTBR cells (Figure 11I) was monitored over 21 days. At each time point, expression of the target genes was normalized to the appropriate tNGFR control. (FIG. 11J) Schematic of FLAG-tagged LTBR mutants. (FIG. 11K) LTBR and FLAG expression in T cells transduced with LTBR mutants. Error bars indicate SEM. [Figure 11G]Expression of LTBR ligands and LTBR, ​​either via mRNA or with deletions and point mutations, is shown. (Figure 11A) IL2 secretion after 24 hours of stimulation with CD3 / CD28 antibodies. Where indicated, recombinant soluble LTA (1 ng / mL) or LIGHT (10 ng / mL) was added together with CD3 / CD28 antibodies. CD4+ T cells from one donor were tested in triplicate. (Figures 11B and 11C) CD4+ and CD8+ T cells from two donors were co-incubated with CD3 / CD28 antibodies or recombinant soluble LTA or LIGHT for 24 hours, followed by measurement of IL2 (Figure 11B) and IFNγ (Figure 11C). (n = 3 biological replicates) (Figures 10D, 10E) Differentiation phenotype (Figure 10D) or proliferation (Figure 10E) after restimulation of tNGFR- and LTBR-transduced T cells incubated with either IL2 alone or LTA (1 ng / mL) or LIGHT (10 ng / mL) during the culture period (n = 3 biological replicates). CM: central memory. EM: effector memory. Independent two-tailed t-test p-values ​​are shown. (Figures 11F, 11I) Transient LTBR or tNGFR expression via mRNA nucleofection (Figure 101F). T cells were nucleofected with LTBR or tNGFR mRNA (n = 3 biological replicates), and surface expression of LTBR (Figure 11G), tNGFR (Figure 11H), or four genes upregulated in LTBR cells (Figure 11I) was monitored over 21 days. At each time point, expression of the target genes was normalized to the appropriate tNGFR control. (FIG. 11J) Schematic of FLAG-tagged LTBR mutants. (FIG. 11K) LTBR and FLAG expression in T cells transduced with LTBR mutants. Error bars indicate SEM. [Figure 11H]Expression of LTBR ligands and LTBR, ​​either via mRNA or with deletions and point mutations, is shown. (Figure 11A) IL2 secretion after 24 hours of stimulation with CD3 / CD28 antibodies. Where indicated, recombinant soluble LTA (1 ng / mL) or LIGHT (10 ng / mL) was added together with CD3 / CD28 antibodies. CD4+ T cells from one donor were tested in triplicate. (Figures 11B and 11C) CD4+ and CD8+ T cells from two donors were co-incubated with CD3 / CD28 antibodies or recombinant soluble LTA or LIGHT for 24 hours, followed by measurement of IL2 (Figure 11B) and IFNγ (Figure 11C). (n = 3 biological replicates) (Figures 10D, 10E) Differentiation phenotype (Figure 10D) or proliferation (Figure 10E) after restimulation of tNGFR- and LTBR-transduced T cells incubated with either IL2 alone or LTA (1 ng / mL) or LIGHT (10 ng / mL) during the culture period (n = 3 biological replicates). CM: central memory. EM: effector memory. Independent two-tailed t-test p-values ​​are shown. (Figures 11F, 11I) Transient LTBR or tNGFR expression via mRNA nucleofection (Figure 101F). T cells were nucleofected with LTBR or tNGFR mRNA (n = 3 biological replicates), and surface expression of LTBR (Figure 11G), tNGFR (Figure 11H), or four genes upregulated in LTBR cells (Figure 11I) was monitored over 21 days. At each time point, expression of the target genes was normalized to the appropriate tNGFR control. (FIG. 11J) Schematic of FLAG-tagged LTBR mutants. (FIG. 11K) LTBR and FLAG expression in T cells transduced with LTBR mutants. Error bars indicate SEM. [Figure 11I]Expression of LTBR ligands and LTBR, ​​either via mRNA or with deletions and point mutations, is shown. (Figure 11A) IL2 secretion after 24 hours of stimulation with CD3 / CD28 antibodies. Where indicated, recombinant soluble LTA (1 ng / mL) or LIGHT (10 ng / mL) was added together with CD3 / CD28 antibodies. CD4+ T cells from one donor were tested in triplicate. (Figures 11B and 11C) CD4+ and CD8+ T cells from two donors were co-incubated with CD3 / CD28 antibodies or recombinant soluble LTA or LIGHT for 24 hours, followed by measurement of IL2 (Figure 11B) and IFNγ (Figure 11C). (n = 3 biological replicates) (Figures 10D, 10E) Differentiation phenotype (Figure 10D) or proliferation (Figure 10E) after restimulation of tNGFR- and LTBR-transduced T cells incubated with either IL2 alone or LTA (1 ng / mL) or LIGHT (10 ng / mL) during the culture period (n = 3 biological replicates). CM: central memory. EM: effector memory. Independent two-tailed t-test p-values ​​are shown. (Figures 11F, 11I) Transient LTBR or tNGFR expression via mRNA nucleofection (Figure 101F). T cells were nucleofected with LTBR or tNGFR mRNA (n = 3 biological replicates), and surface expression of LTBR (Figure 11G), tNGFR (Figure 11H), or four genes upregulated in LTBR cells (Figure 11I) was monitored over 21 days. At each time point, expression of the target genes was normalized to the appropriate tNGFR control. (FIG. 11J) Schematic of FLAG-tagged LTBR mutants. (FIG. 11K) LTBR and FLAG expression in T cells transduced with LTBR mutants. Error bars indicate SEM. [Figure 11J]Expression of LTBR ligands and LTBR, ​​either via mRNA or with deletions and point mutations, is shown. (Figure 11A) IL2 secretion after 24 hours of stimulation with CD3 / CD28 antibodies. Where indicated, recombinant soluble LTA (1 ng / mL) or LIGHT (10 ng / mL) was added together with CD3 / CD28 antibodies. CD4+ T cells from one donor were tested in triplicate. (Figures 11B and 11C) CD4+ and CD8+ T cells from two donors were co-incubated with CD3 / CD28 antibodies or recombinant soluble LTA or LIGHT for 24 hours, followed by measurement of IL2 (Figure 11B) and IFNγ (Figure 11C). (n = 3 biological replicates) (Figures 10D, 10E) Differentiation phenotype (Figure 10D) or proliferation (Figure 10E) after restimulation of tNGFR- and LTBR-transduced T cells incubated with either IL2 alone or LTA (1 ng / mL) or LIGHT (10 ng / mL) during the culture period (n = 3 biological replicates). CM: central memory. EM: effector memory. Independent two-tailed t-test p-values ​​are shown. (Figures 11F, 11I) Transient LTBR or tNGFR expression via mRNA nucleofection (Figure 101F). T cells were nucleofected with LTBR or tNGFR mRNA (n = 3 biological replicates), and surface expression of LTBR (Figure 11G), tNGFR (Figure 11H), or four genes upregulated in LTBR cells (Figure 11I) was monitored over 21 days. At each time point, expression of the target genes was normalized to the appropriate tNGFR control. (FIG. 11J) Schematic of FLAG-tagged LTBR mutants. (FIG. 11K) LTBR and FLAG expression in T cells transduced with LTBR mutants. Error bars indicate SEM. [Figure 11K]Expression of LTBR ligands and LTBR, ​​either via mRNA or with deletions and point mutations, is shown. (Figure 11A) IL2 secretion after 24 hours of stimulation with CD3 / CD28 antibodies. Where indicated, recombinant soluble LTA (1 ng / mL) or LIGHT (10 ng / mL) was added together with CD3 / CD28 antibodies. CD4+ T cells from one donor were tested in triplicate. (Figures 11B and 11C) CD4+ and CD8+ T cells from two donors were co-incubated with CD3 / CD28 antibodies or recombinant soluble LTA or LIGHT for 24 hours, followed by measurement of IL2 (Figure 11B) and IFNγ (Figure 11C). (n = 3 biological replicates) (Figures 10D, 10E) Differentiation phenotype (Figure 10D) or proliferation (Figure 10E) after restimulation of tNGFR- and LTBR-transduced T cells incubated with either IL2 alone or LTA (1 ng / mL) or LIGHT (10 ng / mL) during the culture period (n = 3 biological replicates). CM: central memory. EM: effector memory. Independent two-tailed t-test p-values ​​are shown. (Figures 11F, 11I) Transient LTBR or tNGFR expression via mRNA nucleofection (Figure 101F). T cells were nucleofected with LTBR or tNGFR mRNA (n = 3 biological replicates), and surface expression of LTBR (Figure 11G), tNGFR (Figure 11H), or four genes upregulated in LTBR cells (Figure 11I) was monitored over 21 days. At each time point, expression of the target genes was normalized to the appropriate tNGFR control. (FIG. 11J) Schematic of FLAG-tagged LTBR mutants. (FIG. 11K) LTBR and FLAG expression in T cells transduced with LTBR mutants. Error bars indicate SEM. [Figure 12A]Chromatin accessibility in LTBR T cells is shown. (Figure 12A) Principal component (PC) analysis of globally accessible chromatin regions in LTBR and tNGFR T cells either resting or stimulated with CD3 / CD28 for 24 hours. (Figure 12B) Differentially accessible chromatin regions between stimulated tNGFR and resting tNGFR, between stimulated LTBR and resting LTBR, ​​between resting LTBR and resting tNGFR, and between stimulated LTBR and stimulated tNGFR. The number of peaks gained / lost is shown (using an absolute log2 fold change of 1 and an adjusted p-value <0.1 as the cutoff). (Figures 12C, 12D) Changes in chromatin accessibility for differentially expressed (adjusted p<0.05) genes (Figure 12C) or changes in gene expression for differentially accessible (adjusted p<0.05) regions (Figure 12D). Two-tailed t-test p-values ​​are shown. Boxes indicate the 25th–75th percentiles with a median line, and extensions extend to the 1.5× interquartile range. N = 614 genes (Figure 12C) or genomic regions (Figure 12D). (Figures 12E, 12F) Chromatin accessibility profiles at loci with more (Figure 12E) or less (Figure 12F) openness in LTBR compared with tNGFR cells, either resting or stimulated for 24 hours. The y-axis represents normalized reads (scale: 0–860 for BATF3, 0–1950 for IL13, 0–1230 for TRAF1, 0–1000 for TNFSF4, 0–300 for PDCD1, and 0–2350 for LAG3). (Figure 12G) Chromatin accessibility in resting or stimulated LTBR and tNGFR cells. Each row represents a peak that was significantly enriched in LTBR compared to the corresponding tNGFR control (log2 fold change >1, DESeq2 adjusted p-value <0.05). Peaks were clustered using k-means clustering, and selected genes at or near the peak from each cluster are shown. (Figure 12H) Correlation of each ATAC sample (biological replicate) based on bias-corrected deviation. (Figure 12I) Top transcription factor (TF) motifs enriched at differentially accessible chromatin regions in resting LTBR cells compared to resting tNGFR cells. [Figure 12B] Chromatin accessibility in LTBR T cells is shown. (Figure 12A) Principal component (PC) analysis of globally accessible chromatin regions in LTBR and tNGFR T cells either resting or stimulated with CD3 / CD28 for 24 hours. (Figure 12B) Differentially accessible chromatin regions between stimulated tNGFR and resting tNGFR, between stimulated LTBR and resting LTBR, ​​between resting LTBR and resting tNGFR, and between stimulated LTBR and stimulated tNGFR. The number of peaks gained / lost is shown (using an absolute log2 fold change of 1 and an adjusted p-value <0.1 as the cutoff). (Figures 12C, 12D) Changes in chromatin accessibility for differentially expressed (adjusted p<0.05) genes (Figure 12C) or changes in gene expression for differentially accessible (adjusted p<0.05) regions (Figure 12D). Two-tailed t-test p-values ​​are shown. Boxes indicate the 25th–75th percentiles with a median line, and extensions extend to the 1.5× interquartile range. N = 614 genes (Figure 12C) or genomic regions (Figure 12D). (Figures 12E, 12F) Chromatin accessibility profiles at loci with more (Figure 12E) or less (Figure 12F) openness in LTBR compared with tNGFR cells, either resting or stimulated for 24 hours. The y-axis represents normalized reads (scale: 0–860 for BATF3, 0–1950 for IL13, 0–1230 for TRAF1, 0–1000 for TNFSF4, 0–300 for PDCD1, and 0–2350 for LAG3). (Figure 12G) Chromatin accessibility in resting or stimulated LTBR and tNGFR cells. Each row represents a peak that was significantly enriched in LTBR compared to the corresponding tNGFR control (log2 fold change >1, DESeq2 adjusted p-value <0.05). Peaks were clustered using k-means clustering, and selected genes at or near the peak from each cluster are shown. (Figure 12H) Correlation of each ATAC sample (biological replicate) based on bias-corrected deviation. (Figure 12I) Top transcription factor (TF) motifs enriched at differentially accessible chromatin regions in resting LTBR cells compared to resting tNGFR cells. [Figure 12C]Chromatin accessibility in LTBR T cells is shown. (Figure 12A) Principal component (PC) analysis of globally accessible chromatin regions in LTBR and tNGFR T cells either resting or stimulated with CD3 / CD28 for 24 hours. (Figure 12B) Differentially accessible chromatin regions between stimulated tNGFR and resting tNGFR, between stimulated LTBR and resting LTBR, ​​between resting LTBR and resting tNGFR, and between stimulated LTBR and stimulated tNGFR. The number of peaks gained / lost is shown (using an absolute log2 fold change of 1 and an adjusted p-value <0.1 as the cutoff). (Figures 12C, 12D) Changes in chromatin accessibility for differentially expressed (adjusted p<0.05) genes (Figure 12C) or changes in gene expression for differentially accessible (adjusted p<0.05) regions (Figure 12D). Two-tailed t-test p-values ​​are shown. Boxes indicate the 25th–75th percentiles with a median line, and extensions extend to the 1.5× interquartile range. N = 614 genes (Figure 12C) or genomic regions (Figure 12D). (Figures 12E, 12F) Chromatin accessibility profiles at loci with more (Figure 12E) or less (Figure 12F) openness in LTBR compared with tNGFR cells, either resting or stimulated for 24 hours. The y-axis represents normalized reads (scale: 0–860 for BATF3, 0–1950 for IL13, 0–1230 for TRAF1, 0–1000 for TNFSF4, 0–300 for PDCD1, and 0–2350 for LAG3). (Figure 12G) Chromatin accessibility in resting or stimulated LTBR and tNGFR cells. Each row represents a peak that was significantly enriched in LTBR compared to the corresponding tNGFR control (log2 fold change >1, DESeq2 adjusted p-value <0.05). Peaks were clustered using k-means clustering, and selected genes at or near the peak from each cluster are shown. (Figure 12H) Correlation of each ATAC sample (biological replicate) based on bias-corrected deviation. (Figure 12I) Top transcription factor (TF) motifs enriched at differentially accessible chromatin regions in resting LTBR cells compared to resting tNGFR cells. [Figure 12D] Chromatin accessibility in LTBR T cells is shown. (Figure 12A) Principal component (PC) analysis of globally accessible chromatin regions in LTBR and tNGFR T cells either resting or stimulated with CD3 / CD28 for 24 hours. (Figure 12B) Differentially accessible chromatin regions between stimulated tNGFR and resting tNGFR, between stimulated LTBR and resting LTBR, ​​between resting LTBR and resting tNGFR, and between stimulated LTBR and stimulated tNGFR. The number of peaks gained / lost is shown (using an absolute log2 fold change of 1 and an adjusted p-value <0.1 as the cutoff). (Figures 12C, 12D) Changes in chromatin accessibility for differentially expressed (adjusted p<0.05) genes (Figure 12C) or changes in gene expression for differentially accessible (adjusted p<0.05) regions (Figure 12D). Two-tailed t-test p-values ​​are shown. Boxes indicate the 25th–75th percentiles with a median line, and extensions extend to the 1.5× interquartile range. N = 614 genes (Figure 12C) or genomic regions (Figure 12D). (Figures 12E, 12F) Chromatin accessibility profiles at loci with more (Figure 12E) or less (Figure 12F) openness in LTBR compared with tNGFR cells, either resting or stimulated for 24 hours. The y-axis represents normalized reads (scale: 0–860 for BATF3, 0–1950 for IL13, 0–1230 for TRAF1, 0–1000 for TNFSF4, 0–300 for PDCD1, and 0–2350 for LAG3). (Figure 12G) Chromatin accessibility in resting or stimulated LTBR and tNGFR cells. Each row represents a peak that was significantly enriched in LTBR compared to the corresponding tNGFR control (log2 fold change >1, DESeq2 adjusted p-value <0.05). Peaks were clustered using k-means clustering, and selected genes at or near the peak from each cluster are shown. (Figure 12H) Correlation of each ATAC sample (biological replicate) based on bias-corrected deviation. (Figure 12I) Top transcription factor (TF) motifs enriched at differentially accessible chromatin regions in resting LTBR cells compared to resting tNGFR cells. [Figure 12E]Chromatin accessibility in LTBR T cells is shown. (Figure 12A) Principal component (PC) analysis of globally accessible chromatin regions in LTBR and tNGFR T cells either resting or stimulated with CD3 / CD28 for 24 hours. (Figure 12B) Differentially accessible chromatin regions between stimulated tNGFR and resting tNGFR, between stimulated LTBR and resting LTBR, ​​between resting LTBR and resting tNGFR, and between stimulated LTBR and stimulated tNGFR. The number of peaks gained / lost is shown (using an absolute log2 fold change of 1 and an adjusted p-value <0.1 as the cutoff). (Figures 12C, 12D) Changes in chromatin accessibility for differentially expressed (adjusted p<0.05) genes (Figure 12C) or changes in gene expression for differentially accessible (adjusted p<0.05) regions (Figure 12D). Two-tailed t-test p-values ​​are shown. Boxes indicate the 25th–75th percentiles with a median line, and extensions extend to the 1.5× interquartile range. N = 614 genes (Figure 12C) or genomic regions (Figure 12D). (Figures 12E, 12F) Chromatin accessibility profiles at loci with more (Figure 12E) or less (Figure 12F) openness in LTBR compared with tNGFR cells, either resting or stimulated for 24 hours. The y-axis represents normalized reads (scale: 0–860 for BATF3, 0–1950 for IL13, 0–1230 for TRAF1, 0–1000 for TNFSF4, 0–300 for PDCD1, and 0–2350 for LAG3). (Figure 12G) Chromatin accessibility in resting or stimulated LTBR and tNGFR cells. Each row represents a peak that was significantly enriched in LTBR compared to the corresponding tNGFR control (log2 fold change >1, DESeq2 adjusted p-value <0.05). Peaks were clustered using k-means clustering, and selected genes at or near the peak from each cluster are shown. (Figure 12H) Correlation of each ATAC sample (biological replicate) based on bias-corrected deviation. (Figure 12I) Top transcription factor (TF) motifs enriched at differentially accessible chromatin regions in resting LTBR cells compared to resting tNGFR cells. [Figure 12F] Chromatin accessibility in LTBR T cells is shown. (Figure 12A) Principal component (PC) analysis of globally accessible chromatin regions in LTBR and tNGFR T cells either resting or stimulated with CD3 / CD28 for 24 hours. (Figure 12B) Differentially accessible chromatin regions between stimulated tNGFR and resting tNGFR, between stimulated LTBR and resting LTBR, ​​between resting LTBR and resting tNGFR, and between stimulated LTBR and stimulated tNGFR. The number of peaks gained / lost is shown (using an absolute log2 fold change of 1 and an adjusted p-value <0.1 as the cutoff). (Figures 12C, 12D) Changes in chromatin accessibility for differentially expressed (adjusted p<0.05) genes (Figure 12C) or changes in gene expression for differentially accessible (adjusted p<0.05) regions (Figure 12D). Two-tailed t-test p-values ​​are shown. Boxes indicate the 25th–75th percentiles with a median line, and extensions extend to the 1.5× interquartile range. N = 614 genes (Figure 12C) or genomic regions (Figure 12D). (Figures 12E, 12F) Chromatin accessibility profiles at loci with more (Figure 12E) or less (Figure 12F) openness in LTBR compared with tNGFR cells, either resting or stimulated for 24 hours. The y-axis represents normalized reads (scale: 0–860 for BATF3, 0–1950 for IL13, 0–1230 for TRAF1, 0–1000 for TNFSF4, 0–300 for PDCD1, and 0–2350 for LAG3). (Figure 12G) Chromatin accessibility in resting or stimulated LTBR and tNGFR cells. Each row represents a peak that was significantly enriched in LTBR compared to the corresponding tNGFR control (log2 fold change >1, DESeq2 adjusted p-value <0.05). Peaks were clustered using k-means clustering, and selected genes at or near the peak from each cluster are shown. (Figure 12H) Correlation of each ATAC sample (biological replicate) based on bias-corrected deviation. (Figure 12I) Top transcription factor (TF) motifs enriched at differentially accessible chromatin regions in resting LTBR cells compared to resting tNGFR cells. [Figure 12G]Chromatin accessibility in LTBR T cells is shown. (Figure 12A) Principal component (PC) analysis of globally accessible chromatin regions in LTBR and tNGFR T cells either resting or stimulated with CD3 / CD28 for 24 hours. (Figure 12B) Differentially accessible chromatin regions between stimulated tNGFR and resting tNGFR, between stimulated LTBR and resting LTBR, ​​between resting LTBR and resting tNGFR, and between stimulated LTBR and stimulated tNGFR. The number of peaks gained / lost is shown (using an absolute log2 fold change of 1 and an adjusted p-value <0.1 as the cutoff). (Figures 12C, 12D) Changes in chromatin accessibility for differentially expressed (adjusted p<0.05) genes (Figure 12C) or changes in gene expression for differentially accessible (adjusted p<0.05) regions (Figure 12D). Two-tailed t-test p-values ​​are shown. Boxes indicate the 25th–75th percentiles with a median line, and extensions extend to the 1.5× interquartile range. N = 614 genes (Figure 12C) or genomic regions (Figure 12D). (Figures 12E, 12F) Chromatin accessibility profiles at loci with more (Figure 12E) or less (Figure 12F) openness in LTBR compared with tNGFR cells, either resting or stimulated for 24 hours. The y-axis represents normalized reads (scale: 0–860 for BATF3, 0–1950 for IL13, 0–1230 for TRAF1, 0–1000 for TNFSF4, 0–300 for PDCD1, and 0–2350 for LAG3). (Figure 12G) Chromatin accessibility in resting or stimulated LTBR and tNGFR cells. Each row represents a peak that was significantly enriched in LTBR compared to the corresponding tNGFR control (log2 fold change >1, DESeq2 adjusted p-value <0.05). Peaks were clustered using k-means clustering, and selected genes at or near the peak from each cluster are shown. (Figure 12H) Correlation of each ATAC sample (biological replicate) based on bias-corrected deviation. (Figure 12I) Top transcription factor (TF) motifs enriched at differentially accessible chromatin regions in resting LTBR cells compared to resting tNGFR cells. [Figure 12H] Chromatin accessibility in LTBR T cells is shown. (Figure 12A) Principal component (PC) analysis of globally accessible chromatin regions in LTBR and tNGFR T cells either resting or stimulated with CD3 / CD28 for 24 hours. (Figure 12B) Differentially accessible chromatin regions between stimulated tNGFR and resting tNGFR, between stimulated LTBR and resting LTBR, ​​between resting LTBR and resting tNGFR, and between stimulated LTBR and stimulated tNGFR. The number of peaks gained / lost is shown (using an absolute log2 fold change of 1 and an adjusted p-value <0.1 as the cutoff). (Figures 12C, 12D) Changes in chromatin accessibility for differentially expressed (adjusted p<0.05) genes (Figure 12C) or changes in gene expression for differentially accessible (adjusted p<0.05) regions (Figure 12D). Two-tailed t-test p-values ​​are shown. Boxes indicate the 25th–75th percentiles with a median line, and extensions extend to the 1.5× interquartile range. N = 614 genes (Figure 12C) or genomic regions (Figure 12D). (Figures 12E, 12F) Chromatin accessibility profiles at loci with more (Figure 12E) or less (Figure 12F) openness in LTBR compared with tNGFR cells, either resting or stimulated for 24 hours. The y-axis represents normalized reads (scale: 0–860 for BATF3, 0–1950 for IL13, 0–1230 for TRAF1, 0–1000 for TNFSF4, 0–300 for PDCD1, and 0–2350 for LAG3). (Figure 12G) Chromatin accessibility in resting or stimulated LTBR and tNGFR cells. Each row represents a peak that was significantly enriched in LTBR compared to the corresponding tNGFR control (log2 fold change >1, DESeq2 adjusted p-value <0.05). Peaks were clustered using k-means clustering, and selected genes at or near the peak from each cluster are shown. (Figure 12H) Correlation of each ATAC sample (biological replicate) based on bias-corrected deviation. (Figure 12I) Top transcription factor (TF) motifs enriched at differentially accessible chromatin regions in resting LTBR cells compared to resting tNGFR cells. [Figure 12I]Chromatin accessibility in LTBR T cells is shown. (Figure 12A) Principal component (PC) analysis of globally accessible chromatin regions in LTBR and tNGFR T cells either resting or stimulated with CD3 / CD28 for 24 hours. (Figure 12B) Differentially accessible chromatin regions between stimulated tNGFR and resting tNGFR, between stimulated LTBR and resting LTBR, ​​between resting LTBR and resting tNGFR, and between stimulated LTBR and stimulated tNGFR. The number of peaks gained / lost is shown (using an absolute log2 fold change of 1 and an adjusted p-value <0.1 as the cutoff). (Figures 12C, 12D) Changes in chromatin accessibility for differentially expressed (adjusted p<0.05) genes (Figure 12C) or changes in gene expression for differentially accessible (adjusted p<0.05) regions (Figure 12D). Two-tailed t-test p-values ​​are shown. Boxes indicate the 25th–75th percentiles with a median line, and extensions extend to the 1.5× interquartile range. N = 614 genes (Figure 12C) or genomic regions (Figure 12D). (Figures 12E, 12F) Chromatin accessibility profiles at loci with more (Figure 12E) or less (Figure 12F) openness in LTBR compared with tNGFR cells, either resting or stimulated for 24 hours. The y-axis represents normalized reads (scale: 0–860 for BATF3, 0–1950 for IL13, 0–1230 for TRAF1, 0–1000 for TNFSF4, 0–300 for PDCD1, and 0–2350 for LAG3). (Figure 12G) Chromatin accessibility in resting or stimulated LTBR and tNGFR cells. Each row represents a peak that was significantly enriched in LTBR compared to the corresponding tNGFR control (log2 fold change >1, DESeq2 adjusted p-value <0.05). Peaks were clustered using k-means clustering, and selected genes at or near the peak from each cluster are shown. (Figure 12H) Correlation of each ATAC sample (biological replicate) based on bias-corrected deviation. (Figure 12I) Top transcription factor (TF) motifs enriched at differentially accessible chromatin regions in resting LTBR cells compared to resting tNGFR cells. [Figure 13A] Proteomic and functional genomic assays of NF-κB activation are shown. (Figure 13A) Phospho-RELA staining by intracellular flow cytometry in LTBR and tNGFR cells. Gating for identifying phospho-RELA+ cells is shown. (Figures 13B, 13C) Western blot quantification of key proteins in the NF-κB pathway in LTBR and tNGFR cells resting or stimulated with CD3 / CD28 for 15 minutes. A representative gel (Figure 13B) or quantification of band intensity for GAPHD (Figure 13C) is shown (n=3 biological replicates). p-values ​​from an independent two-tailed t-test are indicated. (Figure 13D) Representation of the LTBR signaling pathway. Each gene is colored based on its differential expression in LTBR versus the corresponding tNGFR cells (CD4+ and CD8+ T cells, resting or stimulated for 24 hours). (Figures 13E-G) Simultaneous gene knockout via CRISPR and ORF overexpression. T cells were transduced with a lentiviral vector co-expressing a single-stranded guide RNA (sgRNA) and the LTBR ORF. After transduction, Cas9 protein was delivered via nucleofection. (Figure 13F) Representative target gene expression in LTBR cells co-expressing sgRNAs targeting B2M, an essential component of the MHC-I complex, or TRBC1 / 2, an essential component of the αβ TCR. (Figure 13G) IFNγ quantification after restimulation (n = 3 sgRNAs). (Figures 13H-13O) Representative protein-level-based quantification of gene knockout efficiency. Representative histograms (Figures 13H, 13J, 13L) and quantification of relative expression levels (Figures 13I, 13K, 13M) of LTA, LIGHT, and RELA are shown (n = 3 sgRNAs). Dashed lines represent the gate used to enumerate cells expressing a given protein. A representative gel (Figure 13N) and quantification of RELB expression (Figure 13O) are shown (n=3 sgRNAs for RELB and n=2 non-targeting control sgRNAs). (Figure 13P) Identification of 274 genes identified as enriched in both CD4+ and CD8+ T cells transduced with LTBR relative to the corresponding tNGFR control ("core LTBR" genes). Error bars indicate SEM. [Figure 13B]Proteomic and functional genomic assays of NF-κB activation are shown. (Figure 13A) Phospho-RELA staining by intracellular flow cytometry in LTBR and tNGFR cells. Gating for identifying phospho-RELA+ cells is shown. (Figures 13B, 13C) Western blot quantification of key proteins in the NF-κB pathway in LTBR and tNGFR cells resting or stimulated with CD3 / CD28 for 15 minutes. A representative gel (Figure 13B) or quantification of band intensity for GAPHD (Figure 13C) is shown (n=3 biological replicates). p-values ​​from an independent two-tailed t-test are indicated. (Figure 13D) Representation of the LTBR signaling pathway. Each gene is colored based on its differential expression in LTBR versus the corresponding tNGFR cells (CD4+ and CD8+ T cells, resting or stimulated for 24 hours). (Figures 13E-G) Simultaneous gene knockout via CRISPR and ORF overexpression. T cells were transduced with a lentiviral vector co-expressing a single-stranded guide RNA (sgRNA) and the LTBR ORF. After transduction, Cas9 protein was delivered via nucleofection. (Figure 13F) Representative target gene expression in LTBR cells co-expressing sgRNAs targeting B2M, an essential component of the MHC-I complex, or TRBC1 / 2, an essential component of the αβ TCR. (Figure 13G) IFNγ quantification after restimulation (n = 3 sgRNAs). (Figures 13H-13O) Representative protein-level-based quantification of gene knockout efficiency. Representative histograms (Figures 13H, 13J, 13L) and quantification of relative expression levels (Figures 13I, 13K, 13M) of LTA, LIGHT, and RELA are shown (n = 3 sgRNAs). Dashed lines represent the gate used to enumerate cells expressing a given protein. A representative gel (Figure 13N) and quantification of RELB expression (Figure 13O) are shown (n=3 sgRNAs for RELB and n=2 non-targeting control sgRNAs). (Figure 13P) Identification of 274 genes identified as enriched in both CD4+ and CD8+ T cells transduced with LTBR relative to the corresponding tNGFR control ("core LTBR" genes). Error bars indicate SEM. [Figure 13C]Proteomic and functional genomic assays of NF-κB activation are shown. (Figure 13A) Phospho-RELA staining by intracellular flow cytometry in LTBR and tNGFR cells. Gating for identifying phospho-RELA+ cells is shown. (Figures 13B, 13C) Western blot quantification of key proteins in the NF-κB pathway in LTBR and tNGFR cells resting or stimulated with CD3 / CD28 for 15 minutes. A representative gel (Figure 13B) or quantification of band intensity for GAPHD (Figure 13C) is shown (n=3 biological replicates). p-values ​​from an independent two-tailed t-test are indicated. (Figure 13D) Representation of the LTBR signaling pathway. Each gene is colored based on its differential expression in LTBR versus the corresponding tNGFR cells (CD4+ and CD8+ T cells, resting or stimulated for 24 hours). (Figures 13E-G) Simultaneous gene knockout via CRISPR and ORF overexpression. T cells were transduced with a lentiviral vector co-expressing a single-stranded guide RNA (sgRNA) and the LTBR ORF. After transduction, Cas9 protein was delivered via nucleofection. (Figure 13F) Representative target gene expression in LTBR cells co-expressing sgRNAs targeting B2M, an essential component of the MHC-I complex, or TRBC1 / 2, an essential component of the αβ TCR. (Figure 13G) IFNγ quantification after restimulation (n = 3 sgRNAs). (Figures 13H-13O) Representative protein-level-based quantification of gene knockout efficiency. Representative histograms (Figures 13H, 13J, 13L) and quantification of relative expression levels (Figures 13I, 13K, 13M) of LTA, LIGHT, and RELA are shown (n = 3 sgRNAs). Dashed lines represent the gate used to enumerate cells expressing a given protein. A representative gel (Figure 13N) and quantification of RELB expression (Figure 13O) are shown (n=3 sgRNAs for RELB and n=2 non-targeting control sgRNAs). (Figure 13P) Identification of 274 genes identified as enriched in both CD4+ and CD8+ T cells transduced with LTBR relative to the corresponding tNGFR control ("core LTBR" genes). Error bars indicate SEM. [Figure 13D]Proteomic and functional genomic assays of NF-κB activation are shown. (Figure 13A) Phospho-RELA staining by intracellular flow cytometry in LTBR and tNGFR cells. Gating for identifying phospho-RELA+ cells is shown. (Figures 13B, 13C) Western blot quantification of key proteins in the NF-κB pathway in LTBR and tNGFR cells resting or stimulated with CD3 / CD28 for 15 minutes. A representative gel (Figure 13B) or quantification of band intensity for GAPHD (Figure 13C) is shown (n=3 biological replicates). p-values ​​from an independent two-tailed t-test are indicated. (Figure 13D) Representation of the LTBR signaling pathway. Each gene is colored based on its differential expression in LTBR versus the corresponding tNGFR cells (CD4+ and CD8+ T cells, resting or stimulated for 24 hours). (Figures 13E-G) Simultaneous gene knockout via CRISPR and ORF overexpression. T cells were transduced with a lentiviral vector co-expressing a single-stranded guide RNA (sgRNA) and the LTBR ORF. After transduction, Cas9 protein was delivered via nucleofection. (Figure 13F) Representative target gene expression in LTBR cells co-expressing sgRNAs targeting B2M, an essential component of the MHC-I complex, or TRBC1 / 2, an essential component of the αβ TCR. (Figure 13G) IFNγ quantification after restimulation (n = 3 sgRNAs). (Figures 13H-13O) Representative protein-level-based quantification of gene knockout efficiency. Representative histograms (Figures 13H, 13J, 13L) and quantification of relative expression levels (Figures 13I, 13K, 13M) of LTA, LIGHT, and RELA are shown (n = 3 sgRNAs). Dashed lines represent the gate used to enumerate cells expressing a given protein. A representative gel (FIG. 13N) and quantification of RELB expression (FIG. 13O) are shown (n=3 sgRNAs for RELB and n=2 non-targeting control sgRNAs). (FIG. 13P) Identification of 274 genes identified as enriched in both CD4+ and CD8 T cells transduced with LTBR relative to the corresponding tNGFR control ("core LTBR" genes). Error bars indicate SEM. [Figure 13E]Proteomic and functional genomic assays of NF-κB activation are shown. (Figure 13A) Phospho-RELA staining by intracellular flow cytometry in LTBR and tNGFR cells. Gating for identifying phospho-RELA+ cells is shown. (Figures 13B, 13C) Western blot quantification of key proteins in the NF-κB pathway in LTBR and tNGFR cells resting or stimulated with CD3 / CD28 for 15 minutes. A representative gel (Figure 13B) or quantification of band intensity for GAPHD (Figure 13C) is shown (n=3 biological replicates). p-values ​​from an independent two-tailed t-test are indicated. (Figure 13D) Representation of the LTBR signaling pathway. Each gene is colored based on its differential expression in LTBR versus the corresponding tNGFR cells (CD4+ and CD8+ T cells, resting or stimulated for 24 hours). (Figures 13E-G) Simultaneous gene knockout via CRISPR and ORF overexpression. T cells were transduced with a lentiviral vector co-expressing a single-stranded guide RNA (sgRNA) and the LTBR ORF. After transduction, Cas9 protein was delivered via nucleofection. (Figure 13F) Representative target gene expression in LTBR cells co-expressing sgRNAs targeting B2M, an essential component of the MHC-I complex, or TRBC1 / 2, an essential component of the αβ TCR. (Figure 13G) IFNγ quantification after restimulation (n = 3 sgRNAs). (Figures 13H-13O) Representative protein-level-based quantification of gene knockout efficiency. Representative histograms (Figures 13H, 13J, 13L) and quantification of relative expression levels (Figures 13I, 13K, 13M) of LTA, LIGHT, and RELA are shown (n = 3 sgRNAs). Dashed lines represent the gate used to enumerate cells expressing a given protein. A representative gel (Figure 13N) and quantification of RELB expression (Figure 13O) are shown (n=3 sgRNAs for RELB and n=2 non-targeting control sgRNAs). (Figure 13P) Identification of 274 genes identified as enriched in both CD4+ and CD8+ T cells transduced with LTBR relative to the corresponding tNGFR control ("core LTBR" genes). Error bars indicate SEM. [Figure 13F]Proteomic and functional genomic assays of NF-κB activation are shown. (Figure 13A) Phospho-RELA staining by intracellular flow cytometry in LTBR and tNGFR cells. Gating for identifying phospho-RELA+ cells is shown. (Figures 13B, 13C) Western blot quantification of key proteins in the NF-κB pathway in LTBR and tNGFR cells resting or stimulated with CD3 / CD28 for 15 minutes. A representative gel (Figure 13B) or quantification of band intensity for GAPHD (Figure 13C) is shown (n=3 biological replicates). p-values ​​from an independent two-tailed t-test are indicated. (Figure 13D) Representation of the LTBR signaling pathway. Each gene is colored based on its differential expression in LTBR versus the corresponding tNGFR cells (CD4+ and CD8+ T cells, resting or stimulated for 24 hours). (Figures 13E-G) Simultaneous gene knockout via CRISPR and ORF overexpression. T cells were transduced with a lentiviral vector co-expressing a single-stranded guide RNA (sgRNA) and the LTBR ORF. After transduction, Cas9 protein was delivered via nucleofection. (Figure 13F) Representative target gene expression in LTBR cells co-expressing sgRNAs targeting B2M, an essential component of the MHC-I complex, or TRBC1 / 2, an essential component of the αβ TCR. (Figure 13G) IFNγ quantification after restimulation (n = 3 sgRNAs). (Figures 13H-13O) Representative protein-level-based quantification of gene knockout efficiency. Representative histograms (Figures 13H, 13J, 13L) and quantification of relative expression levels (Figures 13I, 13K, 13M) of LTA, LIGHT, and RELA are shown (n = 3 sgRNAs). Dashed lines represent the gate used to enumerate cells expressing a given protein. A representative gel (Figure 13N) and quantification of RELB expression (Figure 13O) are shown (n=3 sgRNAs for RELB and n=2 non-targeting control sgRNAs). (Figure 13P) Identification of 274 genes identified as enriched in both CD4+ and CD8+ T cells transduced with LTBR relative to the corresponding tNGFR control ("core LTBR" genes). Error bars indicate SEM. [Figure 13G]Proteomic and functional genomic assays of NF-κB activation are shown. (Figure 13A) Phospho-RELA staining by intracellular flow cytometry in LTBR and tNGFR cells. Gating for identifying phospho-RELA+ cells is shown. (Figures 13B, 13C) Western blot quantification of key proteins in the NF-κB pathway in LTBR and tNGFR cells resting or stimulated with CD3 / CD28 for 15 minutes. A representative gel (Figure 13B) or quantification of band intensity for GAPHD (Figure 13C) is shown (n=3 biological replicates). p-values ​​from an independent two-tailed t-test are indicated. (Figure 13D) Representation of the LTBR signaling pathway. Each gene is colored based on its differential expression in LTBR versus the corresponding tNGFR cells (CD4+ and CD8+ T cells, resting or stimulated for 24 hours). (Figures 13E-G) Simultaneous gene knockout via CRISPR and ORF overexpression. T cells were transduced with a lentiviral vector co-expressing a single-stranded guide RNA (sgRNA) and the LTBR ORF. After transduction, Cas9 protein was delivered via nucleofection. (Figure 13F) Representative target gene expression in LTBR cells co-expressing sgRNAs targeting B2M, an essential component of the MHC-I complex, or TRBC1 / 2, an essential component of the αβ TCR. (Figure 13G) IFNγ quantification after restimulation (n = 3 sgRNAs). (Figures 13H-13O) Representative protein-level-based quantification of gene knockout efficiency. Representative histograms (Figures 13H, 13J, 13L) and quantification of relative expression levels (Figures 13I, 13K, 13M) of LTA, LIGHT, and RELA are shown (n = 3 sgRNAs). Dashed lines represent the gate used to enumerate cells expressing a given protein. A representative gel (Figure 13N) and quantification of RELB expression (Figure 13O) are shown (n=3 sgRNAs for RELB and n=2 non-targeting control sgRNAs). (Figure 13P) Identification of 274 genes identified as enriched in both CD4+ and CD8+ T cells transduced with LTBR relative to the corresponding tNGFR control ("core LTBR" genes). Error bars indicate SEM. [Figure 13H]Proteomic and functional genomic assays of NF-κB activation are shown. (Figure 13A) Phospho-RELA staining by intracellular flow cytometry in LTBR and tNGFR cells. Gating for identifying phospho-RELA+ cells is shown. (Figures 13B, 13C) Western blot quantification of key proteins in the NF-κB pathway in LTBR and tNGFR cells resting or stimulated with CD3 / CD28 for 15 minutes. A representative gel (Figure 13B) or quantification of band intensity for GAPHD (Figure 13C) is shown (n=3 biological replicates). p-values ​​from an independent two-tailed t-test are indicated. (Figure 13D) Representation of the LTBR signaling pathway. Each gene is colored based on its differential expression in LTBR versus the corresponding tNGFR cells (CD4+ and CD8+ T cells, resting or stimulated for 24 hours). (Figures 13E-G) Simultaneous gene knockout via CRISPR and ORF overexpression. T cells were transduced with a lentiviral vector co-expressing a single-stranded guide RNA (sgRNA) and the LTBR ORF. After transduction, Cas9 protein was delivered via nucleofection. (Figure 13F) Representative target gene expression in LTBR cells co-expressing sgRNAs targeting B2M, an essential component of the MHC-I complex, or TRBC1 / 2, an essential component of the αβ TCR. (Figure 13G) IFNγ quantification after restimulation (n = 3 sgRNAs). (Figures 13H-13O) Representative protein-level-based quantification of gene knockout efficiency. Representative histograms (Figures 13H, 13J, 13L) and quantification of relative expression levels (Figures 13I, 13K, 13M) of LTA, LIGHT, and RELA are shown (n = 3 sgRNAs). Dashed lines represent the gate used to enumerate cells expressing a given protein. A representative gel (Figure 13N) and quantification of RELB expression (Figure 13O) are shown (n=3 sgRNAs for RELB and n=2 non-targeting control sgRNAs). (Figure 13P) Identification of 274 genes identified as enriched in both CD4+ and CD8+ T cells transduced with LTBR relative to the corresponding tNGFR control ("core LTBR" genes). Error bars indicate SEM. [Figure 13I]Proteomic and functional genomic assays of NF-κB activation are shown. (Figure 13A) Phospho-RELA staining by intracellular flow cytometry in LTBR and tNGFR cells. Gating for identifying phospho-RELA+ cells is shown. (Figures 13B, 13C) Western blot quantification of key proteins in the NF-κB pathway in LTBR and tNGFR cells resting or stimulated with CD3 / CD28 for 15 minutes. A representative gel (Figure 13B) or quantification of band intensity for GAPHD (Figure 13C) is shown (n=3 biological replicates). p-values ​​from an independent two-tailed t-test are indicated. (Figure 13D) Representation of the LTBR signaling pathway. Each gene is colored based on its differential expression in LTBR versus the corresponding tNGFR cells (CD4+ and CD8+ T cells, resting or stimulated for 24 hours). (Figures 13E-G) Simultaneous gene knockout via CRISPR and ORF overexpression. T cells were transduced with a lentiviral vector co-expressing a single-stranded guide RNA (sgRNA) and the LTBR ORF. After transduction, Cas9 protein was delivered via nucleofection. (Figure 13F) Representative target gene expression in LTBR cells co-expressing sgRNAs targeting B2M, an essential component of the MHC-I complex, or TRBC1 / 2, an essential component of the αβ TCR. (Figure 13G) IFNγ quantification after restimulation (n = 3 sgRNAs). (Figures 13H-13O) Representative protein-level-based quantification of gene knockout efficiency. Representative histograms (Figures 13H, 13J, 13L) and quantification of relative expression levels (Figures 13I, 13K, 13M) of LTA, LIGHT, and RELA are shown (n = 3 sgRNAs). Dashed lines represent the gate used to enumerate cells expressing a given protein. A representative gel (Figure 13N) and quantification of RELB expression (Figure 13O) are shown (n=3 sgRNAs for RELB and n=2 non-targeting control sgRNAs). (Figure 13P) Identification of 274 genes identified as enriched in both CD4+ and CD8+ T cells transduced with LTBR relative to the corresponding tNGFR control ("core LTBR" genes). Error bars indicate SEM. [Figure 13J]Proteomic and functional genomic assays of NF-κB activation are shown. (Figure 13A) Phospho-RELA staining by intracellular flow cytometry in LTBR and tNGFR cells. Gating for identifying phospho-RELA+ cells is shown. (Figures 13B, 13C) Western blot quantification of key proteins in the NF-κB pathway in LTBR and tNGFR cells resting or stimulated with CD3 / CD28 for 15 minutes. A representative gel (Figure 13B) or quantification of band intensity for GAPHD (Figure 13C) is shown (n=3 biological replicates). p-values ​​from an independent two-tailed t-test are indicated. (Figure 13D) Representation of the LTBR signaling pathway. Each gene is colored based on its differential expression in LTBR versus the corresponding tNGFR cells (CD4+ and CD8+ T cells, resting or stimulated for 24 hours). (Figures 13E-G) Simultaneous gene knockout via CRISPR and ORF overexpression. T cells were transduced with a lentiviral vector co-expressing a single-stranded guide RNA (sgRNA) and the LTBR ORF. After transduction, Cas9 protein was delivered via nucleofection. (Figure 13F) Representative target gene expression in LTBR cells co-expressing sgRNAs targeting B2M, an essential component of the MHC-I complex, or TRBC1 / 2, an essential component of the αβ TCR. (Figure 13G) IFNγ quantification after restimulation (n = 3 sgRNAs). (Figures 13H-13O) Representative protein-level-based quantification of gene knockout efficiency. Representative histograms (Figures 13H, 13J, 13L) and quantification of relative expression levels (Figures 13I, 13K, 13M) of LTA, LIGHT, and RELA are shown (n = 3 sgRNAs). Dashed lines represent the gate used to enumerate cells expressing a given protein. A representative gel (Figure 13N) and quantification of RELB expression (Figure 13O) are shown (n=3 sgRNAs for RELB and n=2 non-targeting control sgRNAs). (Figure 13P) Identification of 274 genes identified as enriched in both CD4+ and CD8+ T cells transduced with LTBR relative to the corresponding tNGFR control ("core LTBR" genes). Error bars indicate SEM. [Figure 13K]Proteomic and functional genomic assays of NF-κB activation are shown. (Figure 13A) Phospho-RELA staining by intracellular flow cytometry in LTBR and tNGFR cells. Gating for identifying phospho-RELA+ cells is shown. (Figures 13B, 13C) Western blot quantification of key proteins in the NF-κB pathway in LTBR and tNGFR cells resting or stimulated with CD3 / CD28 for 15 minutes. A representative gel (Figure 13B) or quantification of band intensity for GAPHD (Figure 13C) is shown (n=3 biological replicates). p-values ​​from an independent two-tailed t-test are indicated. (Figure 13D) Representation of the LTBR signaling pathway. Each gene is colored based on its differential expression in LTBR versus the corresponding tNGFR cells (CD4+ and CD8+ T cells, resting or stimulated for 24 hours). (Figures 13E-G) Simultaneous gene knockout via CRISPR and ORF overexpression. T cells were transduced with a lentiviral vector co-expressing a single-stranded guide RNA (sgRNA) and the LTBR ORF. After transduction, Cas9 protein was delivered via nucleofection. (Figure 13F) Representative target gene expression in LTBR cells co-expressing sgRNAs targeting B2M, an essential component of the MHC-I complex, or TRBC1 / 2, an essential component of the αβ TCR. (Figure 13G) IFNγ quantification after restimulation (n = 3 sgRNAs). (Figures 13H-13O) Representative protein-level-based quantification of gene knockout efficiency. Representative histograms (Figures 13H, 13J, 13L) and quantification of relative expression levels (Figures 13I, 13K, 13M) of LTA, LIGHT, and RELA are shown (n = 3 sgRNAs). Dashed lines represent the gate used to enumerate cells expressing a given protein. A representative gel (Figure 13N) and quantification of RELB expression (Figure 13O) are shown (n=3 sgRNAs for RELB and n=2 non-targeting control sgRNAs). (Figure 13P) Identification of 274 genes identified as enriched in both CD4+ and CD8+ T cells transduced with LTBR relative to the corresponding tNGFR control ("core LTBR" genes). Error bars indicate SEM. [Figure 13L]Proteomic and functional genomic assays of NF-κB activation are shown. (Figure 13A) Phospho-RELA staining by intracellular flow cytometry in LTBR and tNGFR cells. Gating for identifying phospho-RELA+ cells is shown. (Figures 13B, 13C) Western blot quantification of key proteins in the NF-κB pathway in LTBR and tNGFR cells resting or stimulated with CD3 / CD28 for 15 minutes. A representative gel (Figure 13B) or quantification of band intensity for GAPHD (Figure 13C) is shown (n=3 biological replicates). p-values ​​from an independent two-tailed t-test are indicated. (Figure 13D) Representation of the LTBR signaling pathway. Each gene is colored based on its differential expression in LTBR versus the corresponding tNGFR cells (CD4+ and CD8+ T cells, resting or stimulated for 24 hours). (Figures 13E-G) Simultaneous gene knockout via CRISPR and ORF overexpression. T cells were transduced with a lentiviral vector co-expressing a single-stranded guide RNA (sgRNA) and the LTBR ORF. After transduction, Cas9 protein was delivered via nucleofection. (Figure 13F) Representative target gene expression in LTBR cells co-expressing sgRNAs targeting B2M, an essential component of the MHC-I complex, or TRBC1 / 2, an essential component of the αβ TCR. (Figure 13G) IFNγ quantification after restimulation (n = 3 sgRNAs). (Figures 13H-13O) Representative protein-level-based quantification of gene knockout efficiency. Representative histograms (Figures 13H, 13J, 13L) and quantification of relative expression levels (Figures 13I, 13K, 13M) of LTA, LIGHT, and RELA are shown (n = 3 sgRNAs). Dashed lines represent the gate used to enumerate cells expressing a given protein. A representative gel (Figure 13N) and quantification of RELB expression (Figure 13O) are shown (n=3 sgRNAs for RELB and n=2 non-targeting control sgRNAs). (Figure 13P) Identification of 274 genes identified as enriched in both CD4+ and CD8+ T cells transduced with LTBR relative to the corresponding tNGFR control ("core LTBR" genes). Error bars indicate SEM. [Figure 13M]Proteomic and functional genomic assays of NF-κB activation are shown. (Figure 13A) Phospho-RELA staining by intracellular flow cytometry in LTBR and tNGFR cells. Gating for identifying phospho-RELA+ cells is shown. (Figures 13B, 13C) Western blot quantification of key proteins in the NF-κB pathway in LTBR and tNGFR cells resting or stimulated with CD3 / CD28 for 15 minutes. A representative gel (Figure 13B) or quantification of band intensity for GAPHD (Figure 13C) is shown (n=3 biological replicates). p-values ​​from an independent two-tailed t-test are indicated. (Figure 13D) Representation of the LTBR signaling pathway. Each gene is colored based on its differential expression in LTBR versus the corresponding tNGFR cells (CD4+ and CD8+ T cells, resting or stimulated for 24 hours). (Figures 13E-G) Simultaneous gene knockout via CRISPR and ORF overexpression. T cells were transduced with a lentiviral vector co-expressing a single-stranded guide RNA (sgRNA) and the LTBR ORF. After transduction, Cas9 protein was delivered via nucleofection. (Figure 13F) Representative target gene expression in LTBR cells co-expressing sgRNAs targeting B2M, an essential component of the MHC-I complex, or TRBC1 / 2, an essential component of the αβ TCR. (Figure 13G) IFNγ quantification after restimulation (n = 3 sgRNAs). (Figures 13H-13O) Representative protein-level-based quantification of gene knockout efficiency. Representative histograms (Figures 13H, 13J, 13L) and quantification of relative expression levels (Figures 13I, 13K, 13M) of LTA, LIGHT, and RELA are shown (n = 3 sgRNAs). Dashed lines represent the gate used to enumerate cells expressing a given protein. A representative gel (Figure 13N) and quantification of RELB expression (Figure 13O) are shown (n=3 sgRNAs for RELB and n=2 non-targeting control sgRNAs). (Figure 13P) Identification of 274 genes identified as enriched in both CD4+ and CD8+ T cells transduced with LTBR relative to the corresponding tNGFR control ("core LTBR" genes). Error bars indicate SEM. [Figure 13N]Proteomic and functional genomic assays of NF-κB activation are shown. (Figure 13A) Phospho-RELA staining by intracellular flow cytometry in LTBR and tNGFR cells. Gating for identifying phospho-RELA+ cells is shown. (Figures 13B, 13C) Western blot quantification of key proteins in the NF-κB pathway in LTBR and tNGFR cells resting or stimulated with CD3 / CD28 for 15 minutes. A representative gel (Figure 13B) or quantification of band intensity for GAPHD (Figure 13C) is shown (n=3 biological replicates). p-values ​​from an independent two-tailed t-test are indicated. (Figure 13D) Representation of the LTBR signaling pathway. Each gene is colored based on its differential expression in LTBR versus the corresponding tNGFR cells (CD4+ and CD8+ T cells, resting or stimulated for 24 hours). (Figures 13E-G) Simultaneous gene knockout via CRISPR and ORF overexpression. T cells were transduced with a lentiviral vector co-expressing a single-stranded guide RNA (sgRNA) and the LTBR ORF. After transduction, Cas9 protein was delivered via nucleofection. (Figure 13F) Representative target gene expression in LTBR cells co-expressing sgRNAs targeting B2M, an essential component of the MHC-I complex, or TRBC1 / 2, an essential component of the αβ TCR. (Figure 13G) IFNγ quantification after restimulation (n = 3 sgRNAs). (Figures 13H-13O) Representative protein-level-based quantification of gene knockout efficiency. Representative histograms (Figures 13H, 13J, 13L) and quantification of relative expression levels (Figures 13I, 13K, 13M) of LTA, LIGHT, and RELA are shown (n = 3 sgRNAs). Dashed lines represent the gate used to enumerate cells expressing a given protein. A representative gel (Figure 13N) and quantification of RELB expression (Figure 13O) are shown (n=3 sgRNAs for RELB and n=2 non-targeting control sgRNAs). (Figure 13P) Identification of 274 genes identified as enriched in both CD4+ and CD8+ T cells transduced with LTBR relative to the corresponding tNGFR control ("core LTBR" genes). Error bars indicate SEM. [Figure 13O]Proteomic and functional genomic assays of NF-κB activation are shown. (Figure 13A) Phospho-RELA staining by intracellular flow cytometry in LTBR and tNGFR cells. Gating for identifying phospho-RELA+ cells is shown. (Figures 13B, 13C) Western blot quantification of key proteins in the NF-κB pathway in LTBR and tNGFR cells resting or stimulated with CD3 / CD28 for 15 minutes. A representative gel (Figure 13B) or quantification of band intensity for GAPHD (Figure 13C) is shown (n=3 biological replicates). p-values ​​from an independent two-tailed t-test are indicated. (Figure 13D) Representation of the LTBR signaling pathway. Each gene is colored based on its differential expression in LTBR versus the corresponding tNGFR cells (CD4+ and CD8+ T cells, resting or stimulated for 24 hours). (Figures 13E-G) Simultaneous gene knockout via CRISPR and ORF overexpression. T cells were transduced with a lentiviral vector co-expressing a single-stranded guide RNA (sgRNA) and the LTBR ORF. After transduction, Cas9 protein was delivered via nucleofection. (Figure 13F) Representative target gene expression in LTBR cells co-expressing sgRNAs targeting B2M, an essential component of the MHC-I complex, or TRBC1 / 2, an essential component of the αβ TCR. (Figure 13G) IFNγ quantification after restimulation (n = 3 sgRNAs). (Figures 13H-13O) Representative protein-level-based quantification of gene knockout efficiency. Representative histograms (Figures 13H, 13J, 13L) and quantification of relative expression levels (Figures 13I, 13K, 13M) of LTA, LIGHT, and RELA are shown (n = 3 sgRNAs). Dashed lines represent the gate used to enumerate cells expressing a given protein. A representative gel (Figure 13N) and quantification of RELB expression (Figure 13O) are shown (n=3 sgRNAs for RELB and n=2 non-targeting control sgRNAs). (Figure 13P) Identification of 274 genes identified as enriched in both CD4+ and CD8+ T cells transduced with LTBR relative to the corresponding tNGFR control ("core LTBR" genes). Error bars indicate SEM. [Figure 13P]Proteomic and functional genomic assays of NF-κB activation are shown. (Figure 13A) Phospho-RELA staining by intracellular flow cytometry in LTBR and tNGFR cells. Gating for identifying phospho-RELA+ cells is shown. (Figures 13B, 13C) Western blot quantification of key proteins in the NF-κB pathway in LTBR and tNGFR cells resting or stimulated with CD3 / CD28 for 15 minutes. A representative gel (Figure 13B) or quantification of band intensity for GAPHD (Figure 13C) is shown (n=3 biological replicates). p-values ​​from an independent two-tailed t-test are indicated. (Figure 13D) Representation of the LTBR signaling pathway. Each gene is colored based on its differential expression in LTBR versus the corresponding tNGFR cells (CD4+ and CD8+ T cells, resting or stimulated for 24 hours). (Figures 13E-G) Simultaneous gene knockout via CRISPR and ORF overexpression. T cells were transduced with a lentiviral vector co-expressing a single-stranded guide RNA (sgRNA) and the LTBR ORF. After transduction, Cas9 protein was delivered via nucleofection. (Figure 13F) Representative target gene expression in LTBR cells co-expressing sgRNAs targeting B2M, an essential component of the MHC-I complex, or TRBC1 / 2, an essential component of the αβ TCR. (Figure 13G) IFNγ quantification after restimulation (n = 3 sgRNAs). (Figures 13H-13O) Representative protein-level-based quantification of gene knockout efficiency. Representative histograms (Figures 13H, 13J, 13L) and quantification of relative expression levels (Figures 13I, 13K, 13M) of LTA, LIGHT, and RELA are shown (n = 3 sgRNAs). Dashed lines represent the gate used to enumerate cells expressing a given protein. A representative gel (Figure 13N) and quantification of RELB expression (Figure 13O) are shown (n=3 sgRNAs for RELB and n=2 non-targeting control sgRNAs). (Figure 13P) Identification of 274 genes identified as enriched in both CD4+ and CD8+ T cells transduced with LTBR relative to the corresponding tNGFR control ("core LTBR" genes). Error bars indicate SEM. [Figure 14A]Co-delivery of ORF with CD19-targeted CAR is shown. (Figure 14A) Transduction efficiency of CAR+ORF lentiviral vector or ORF alone (n=4 biological replicates). (Figure 14B, Figure 14C) CAR expression levels determined by staining with anti-mouse Fab F(ab')2. Representative histograms (Figure 14B) and quantification of CAR expression relative to tNGFR (Figure 14C) are shown for two healthy donors and two patients with diffuse large B-cell lymphoma (DLBCL). (Figure 14D) Expansion curves of CAR+ORF-transduced T cells (n=4 biological replicates). (Figure 14E) LTBR expression in autologous CD14+ monocytes and T cells transduced with LTBR alone or CAR+LTBR. (Figures 14F-14I) Expression of ICAM-1 (Figure 14F), CD70 (Figure 14G), CD74 (Figure 14H), and MHC-II (Figure 14I) by T cells transduced with LTBR ORF alone, CAR+LTBR, ​​or CAR+tNGFR. All data are normalized to tNGFR alone (no CAR). p-values ​​from an independent two-tailed t-test are shown. (Figures 14J-14M) Expression of exhaustion markers PD-1 (Figure 14J), TIM-3 (Figure 14K), LAG-3 (Figure 14L), and CD39 (Figure 14M) in CAR+ORF T cells. (Figure 14N) Differentiation phenotype of CAR+ORF T cells. CM: central memory. EM: effector memory. Differentiation was defined based on CD45RO and CCR7 expression (naive: CD45ROneg CCR7+, CM: CD45RO+CCR7+, EM: CD45RO+CCR7neg, effector: CD45ROneg CCR7neg). (Figures 14O, 14P) Expression of activation markers CD25 (Figure 14O) and CD69 (Figure 14P) in CAR+ORF T cells incubated alone or with Nalm6 cells for 24 hours. Error bars indicate SEM. N=3 biological replicates unless otherwise indicated. [Figure 14B]Co-delivery of ORF with CD19-targeted CAR is shown. (Figure 14A) Transduction efficiency of CAR+ORF lentiviral vector or ORF alone (n=4 biological replicates). (Figure 14B, Figure 14C) CAR expression levels determined by staining with anti-mouse Fab F(ab')2. Representative histograms (Figure 14B) and quantification of CAR expression relative to tNGFR (Figure 14C) are shown for two healthy donors and two patients with diffuse large B-cell lymphoma (DLBCL). (Figure 14D) Expansion curves of CAR+ORF-transduced T cells (n=4 biological replicates). (Figure 14E) LTBR expression in autologous CD14+ monocytes and T cells transduced with LTBR alone or CAR+LTBR. (Figures 14F-14I) Expression of ICAM-1 (Figure 14F), CD70 (Figure 14G), CD74 (Figure 14H), and MHC-II (Figure 14I) by T cells transduced with LTBR ORF alone, CAR+LTBR, ​​or CAR+tNGFR. All data are normalized to tNGFR alone (no CAR). p-values ​​from an independent two-tailed t-test are shown. (Figures 14J-14M) Expression of exhaustion markers PD-1 (Figure 14J), TIM-3 (Figure 14K), LAG-3 (Figure 14L), and CD39 (Figure 14M) in CAR+ORF T cells. (Figure 14N) Differentiation phenotype of CAR+ORF T cells. CM: central memory. EM: effector memory. Differentiation was defined based on CD45RO and CCR7 expression (naive: CD45ROneg CCR7+, CM: CD45RO+CCR7+, EM: CD45RO+CCR7neg, effector: CD45ROneg CCR7neg). (Figures 14O, 14P) Expression of activation markers CD25 (Figure 14O) and CD69 (Figure 14P) in CAR+ORF T cells incubated alone or with Nalm6 cells for 24 hours. Error bars indicate SEM. N=3 biological replicates unless otherwise indicated. [Figure 14C]Co-delivery of ORF with CD19-targeted CAR is shown. (Figure 14A) Transduction efficiency of CAR+ORF lentiviral vector or ORF alone (n=4 biological replicates). (Figure 14B, Figure 14C) CAR expression levels determined by staining with anti-mouse Fab F(ab')2. Representative histograms (Figure 14B) and quantification of CAR expression relative to tNGFR (Figure 14C) are shown for two healthy donors and two patients with diffuse large B-cell lymphoma (DLBCL). (Figure 14D) Expansion curves of CAR+ORF-transduced T cells (n=4 biological replicates). (Figure 14E) LTBR expression in autologous CD14+ monocytes and T cells transduced with LTBR alone or CAR+LTBR. (Figures 14F-14I) Expression of ICAM-1 (Figure 14F), CD70 (Figure 14G), CD74 (Figure 14H), and MHC-II (Figure 14I) by T cells transduced with LTBR ORF alone, CAR+LTBR, ​​or CAR+tNGFR. All data are normalized to tNGFR alone (no CAR). p-values ​​from an independent two-tailed t-test are shown. (Figures 14J-14M) Expression of exhaustion markers PD-1 (Figure 14J), TIM-3 (Figure 14K), LAG-3 (Figure 14L), and CD39 (Figure 14M) in CAR+ORF T cells. (Figure 14N) Differentiation phenotype of CAR+ORF T cells. CM: central memory. EM: effector memory. Differentiation was defined based on CD45RO and CCR7 expression (naive: CD45ROneg CCR7+, CM: CD45RO+CCR7+, EM: CD45RO+CCR7neg, effector: CD45ROneg CCR7neg). (Figures 14O, 14P) Expression of activation markers CD25 (Figure 14O) and CD69 (Figure 14P) in CAR+ORF T cells incubated alone or with Nalm6 cells for 24 hours. Error bars indicate SEM. N=3 biological replicates unless otherwise indicated. [Figure 14D]Co-delivery of ORF with CD19-targeted CAR is shown. (Figure 14A) Transduction efficiency of CAR+ORF lentiviral vector or ORF alone (n=4 biological replicates). (Figure 14B, Figure 14C) CAR expression levels determined by staining with anti-mouse Fab F(ab')2. Representative histograms (Figure 14B) and quantification of CAR expression relative to tNGFR (Figure 14C) are shown for two healthy donors and two patients with diffuse large B-cell lymphoma (DLBCL). (Figure 14D) Expansion curves of CAR+ORF-transduced T cells (n=4 biological replicates). (Figure 14E) LTBR expression in autologous CD14+ monocytes and T cells transduced with LTBR alone or CAR+LTBR. (Figures 14F-14I) Expression of ICAM-1 (Figure 14F), CD70 (Figure 14G), CD74 (Figure 14H), and MHC-II (Figure 14I) by T cells transduced with LTBR ORF alone, CAR+LTBR, ​​or CAR+tNGFR. All data are normalized to tNGFR alone (no CAR). p-values ​​from an independent two-tailed t-test are shown. (Figures 14J-14M) Expression of exhaustion markers PD-1 (Figure 14J), TIM-3 (Figure 14K), LAG-3 (Figure 14L), and CD39 (Figure 14M) in CAR+ORF T cells. (Figure 14N) Differentiation phenotype of CAR+ORF T cells. CM: central memory. EM: effector memory. Differentiation was defined based on CD45RO and CCR7 expression (naive: CD45ROneg CCR7+, CM: CD45RO+CCR7+, EM: CD45RO+CCR7neg, effector: CD45ROneg CCR7neg). (Figures 14O, 14P) Expression of activation markers CD25 (Figure 14O) and CD69 (Figure 14P) in CAR+ORF T cells incubated alone or with Nalm6 cells for 24 hours. Error bars indicate SEM. N=3 biological replicates unless otherwise indicated. [Figure 14E]Co-delivery of ORF with CD19-targeted CAR is shown. (Figure 14A) Transduction efficiency of CAR+ORF lentiviral vector or ORF alone (n=4 biological replicates). (Figure 14B, Figure 14C) CAR expression levels determined by staining with anti-mouse Fab F(ab')2. Representative histograms (Figure 14B) and quantification of CAR expression relative to tNGFR (Figure 14C) are shown for two healthy donors and two patients with diffuse large B-cell lymphoma (DLBCL). (Figure 14D) Expansion curves of CAR+ORF-transduced T cells (n=4 biological replicates). (Figure 14E) LTBR expression in autologous CD14+ monocytes and T cells transduced with LTBR alone or CAR+LTBR. (Figures 14F-14I) Expression of ICAM-1 (Figure 14F), CD70 (Figure 14G), CD74 (Figure 14H), and MHC-II (Figure 14I) by T cells transduced with LTBR ORF alone, CAR+LTBR, ​​or CAR+tNGFR. All data are normalized to tNGFR alone (no CAR). p-values ​​from an independent two-tailed t-test are shown. (Figures 14J-14M) Expression of exhaustion markers PD-1 (Figure 14J), TIM-3 (Figure 14K), LAG-3 (Figure 14L), and CD39 (Figure 14M) in CAR+ORF T cells. (Figure 14N) Differentiation phenotype of CAR+ORF T cells. CM: central memory. EM: effector memory. Differentiation was defined based on CD45RO and CCR7 expression (naive: CD45ROneg CCR7+, CM: CD45RO+CCR7+, EM: CD45RO+CCR7neg, effector: CD45ROneg CCR7neg). (Figures 14O, 14P) Expression of activation markers CD25 (Figure 14O) and CD69 (Figure 14P) in CAR+ORF T cells incubated alone or with Nalm6 cells for 24 hours. Error bars indicate SEM. N=3 biological replicates unless otherwise indicated. [Figure 14F]Co-delivery of ORF with CD19-targeted CAR is shown. (Figure 14A) Transduction efficiency of CAR+ORF lentiviral vector or ORF alone (n=4 biological replicates). (Figure 14B, Figure 14C) CAR expression levels determined by staining with anti-mouse Fab F(ab')2. Representative histograms (Figure 14B) and quantification of CAR expression relative to tNGFR (Figure 14C) are shown for two healthy donors and two patients with diffuse large B-cell lymphoma (DLBCL). (Figure 14D) Expansion curves of CAR+ORF-transduced T cells (n=4 biological replicates). (Figure 14E) LTBR expression in autologous CD14+ monocytes and T cells transduced with LTBR alone or CAR+LTBR. (Figures 14F-14I) Expression of ICAM-1 (Figure 14F), CD70 (Figure 14G), CD74 (Figure 14H), and MHC-II (Figure 14I) by T cells transduced with LTBR ORF alone, CAR+LTBR, ​​or CAR+tNGFR. All data are normalized to tNGFR alone (no CAR). p-values ​​from an independent two-tailed t-test are shown. (Figures 14J-14M) Expression of exhaustion markers PD-1 (Figure 14J), TIM-3 (Figure 14K), LAG-3 (Figure 14L), and CD39 (Figure 14M) in CAR+ORF T cells. (Figure 14N) Differentiation phenotype of CAR+ORF T cells. CM: central memory. EM: effector memory. Differentiation was defined based on CD45RO and CCR7 expression (naive: CD45ROneg CCR7+, CM: CD45RO+CCR7+, EM: CD45RO+CCR7neg, effector: CD45ROneg CCR7neg). (Figures 14O, 14P) Expression of activation markers CD25 (Figure 14O) and CD69 (Figure 14P) in CAR+ORF T cells incubated alone or with Nalm6 cells for 24 hours. Error bars indicate SEM. N=3 biological replicates unless otherwise indicated. [Figure 14G]Co-delivery of ORF with CD19-targeted CAR is shown. (Figure 14A) Transduction efficiency of CAR+ORF lentiviral vector or ORF alone (n=4 biological replicates). (Figure 14B, Figure 14C) CAR expression levels determined by staining with anti-mouse Fab F(ab')2. Representative histograms (Figure 14B) and quantification of CAR expression relative to tNGFR (Figure 14C) are shown for two healthy donors and two patients with diffuse large B-cell lymphoma (DLBCL). (Figure 14D) Expansion curves of CAR+ORF-transduced T cells (n=4 biological replicates). (Figure 14E) LTBR expression in autologous CD14+ monocytes and T cells transduced with LTBR alone or CAR+LTBR. (Figures 14F-14I) Expression of ICAM-1 (Figure 14F), CD70 (Figure 14G), CD74 (Figure 14H), and MHC-II (Figure 14I) by T cells transduced with LTBR ORF alone, CAR+LTBR, ​​or CAR+tNGFR. All data are normalized to tNGFR alone (no CAR). p-values ​​from an independent two-tailed t-test are shown. (Figures 14J-14M) Expression of exhaustion markers PD-1 (Figure 14J), TIM-3 (Figure 14K), LAG-3 (Figure 14L), and CD39 (Figure 14M) in CAR+ORF T cells. (Figure 14N) Differentiation phenotype of CAR+ORF T cells. CM: central memory. EM: effector memory. Differentiation was defined based on CD45RO and CCR7 expression (naive: CD45ROneg CCR7+, CM: CD45RO+CCR7+, EM: CD45RO+CCR7neg, effector: CD45ROneg CCR7neg). (Figures 14O, 14P) Expression of activation markers CD25 (Figure 14O) and CD69 (Figure 14P) in CAR+ORF T cells incubated alone or with Nalm6 cells for 24 hours. Error bars indicate SEM. N=3 biological replicates unless otherwise indicated. [Figure 14H]Co-delivery of ORF with CD19-targeted CAR is shown. (Figure 14A) Transduction efficiency of CAR+ORF lentiviral vector or ORF alone (n=4 biological replicates). (Figure 14B, Figure 14C) CAR expression levels determined by staining with anti-mouse Fab F(ab')2. Representative histograms (Figure 14B) and quantification of CAR expression relative to tNGFR (Figure 14C) are shown for two healthy donors and two patients with diffuse large B-cell lymphoma (DLBCL). (Figure 14D) Expansion curves of CAR+ORF-transduced T cells (n=4 biological replicates). (Figure 14E) LTBR expression in autologous CD14+ monocytes and T cells transduced with LTBR alone or CAR+LTBR. (Figures 14F-14I) Expression of ICAM-1 (Figure 14F), CD70 (Figure 14G), CD74 (Figure 14H), and MHC-II (Figure 14I) by T cells transduced with LTBR ORF alone, CAR+LTBR, ​​or CAR+tNGFR. All data are normalized to tNGFR alone (no CAR). p-values ​​from an independent two-tailed t-test are shown. (Figures 14J-14M) Expression of exhaustion markers PD-1 (Figure 14J), TIM-3 (Figure 14K), LAG-3 (Figure 14L), and CD39 (Figure 14M) in CAR+ORF T cells. (Figure 14N) Differentiation phenotype of CAR+ORF T cells. CM: central memory. EM: effector memory. Differentiation was defined based on CD45RO and CCR7 expression (naive: CD45ROneg CCR7+, CM: CD45RO+CCR7+, EM: CD45RO+CCR7neg, effector: CD45ROneg CCR7neg). (Figures 14O, 14P) Expression of activation markers CD25 (Figure 14O) and CD69 (Figure 14P) in CAR+ORF T cells incubated alone or with Nalm6 cells for 24 hours. Error bars indicate SEM. N=3 biological replicates unless otherwise indicated. [Figure 14I]Co-delivery of ORF with CD19-targeted CAR is shown. (Figure 14A) Transduction efficiency of CAR+ORF lentiviral vector or ORF alone (n=4 biological replicates). (Figure 14B, Figure 14C) CAR expression levels determined by staining with anti-mouse Fab F(ab')2. Representative histograms (Figure 14B) and quantification of CAR expression relative to tNGFR (Figure 14C) are shown for two healthy donors and two patients with diffuse large B-cell lymphoma (DLBCL). (Figure 14D) Expansion curves of CAR+ORF-transduced T cells (n=4 biological replicates). (Figure 14E) LTBR expression in autologous CD14+ monocytes and T cells transduced with LTBR alone or CAR+LTBR. (Figures 14F-14I) Expression of ICAM-1 (Figure 14F), CD70 (Figure 14G), CD74 (Figure 14H), and MHC-II (Figure 14I) by T cells transduced with LTBR ORF alone, CAR+LTBR, ​​or CAR+tNGFR. All data are normalized to tNGFR alone (no CAR). p-values ​​from an independent two-tailed t-test are shown. (Figures 14J-14M) Expression of exhaustion markers PD-1 (Figure 14J), TIM-3 (Figure 14K), LAG-3 (Figure 14L), and CD39 (Figure 14M) in CAR+ORF T cells. (Figure 14N) Differentiation phenotype of CAR+ORF T cells. CM: central memory. EM: effector memory. Differentiation was defined based on CD45RO and CCR7 expression (naive: CD45ROneg CCR7+, CM: CD45RO+CCR7+, EM: CD45RO+CCR7neg, effector: CD45ROneg CCR7neg). (Figures 14O, 14P) Expression of activation markers CD25 (Figure 14O) and CD69 (Figure 14P) in CAR+ORF T cells incubated alone or with Nalm6 cells for 24 hours. Error bars indicate SEM. N=3 biological replicates unless otherwise indicated. [Figure 14J]Co-delivery of ORF with CD19-targeted CAR is shown. (Figure 14A) Transduction efficiency of CAR+ORF lentiviral vector or ORF alone (n=4 biological replicates). (Figure 14B, Figure 14C) CAR expression levels determined by staining with anti-mouse Fab F(ab')2. Representative histograms (Figure 14B) and quantification of CAR expression relative to tNGFR (Figure 14C) are shown for two healthy donors and two patients with diffuse large B-cell lymphoma (DLBCL). (Figure 14D) Expansion curves of CAR+ORF-transduced T cells (n=4 biological replicates). (Figure 14E) LTBR expression in autologous CD14+ monocytes and T cells transduced with LTBR alone or CAR+LTBR. (Figures 14F-14I) Expression of ICAM-1 (Figure 14F), CD70 (Figure 14G), CD74 (Figure 14H), and MHC-II (Figure 14I) by T cells transduced with LTBR ORF alone, CAR+LTBR, ​​or CAR+tNGFR. All data are normalized to tNGFR alone (no CAR). p-values ​​from an independent two-tailed t-test are shown. (Figures 14J-14M) Expression of exhaustion markers PD-1 (Figure 14J), TIM-3 (Figure 14K), LAG-3 (Figure 14L), and CD39 (Figure 14M) in CAR+ORF T cells. (Figure 14N) Differentiation phenotype of CAR+ORF T cells. CM: central memory. EM: effector memory. Differentiation was defined based on CD45RO and CCR7 expression (naive: CD45ROneg CCR7+, CM: CD45RO+CCR7+, EM: CD45RO+CCR7neg, effector: CD45ROneg CCR7neg). (Figures 14O, 14P) Expression of activation markers CD25 (Figure 14O) and CD69 (Figure 14P) in CAR+ORF T cells incubated alone or with Nalm6 cells for 24 hours. Error bars indicate SEM. N=3 biological replicates unless otherwise indicated. [Figure 14K]Co-delivery of ORF with CD19-targeted CAR is shown. (Figure 14A) Transduction efficiency of CAR+ORF lentiviral vector or ORF alone (n=4 biological replicates). (Figure 14B, Figure 14C) CAR expression levels determined by staining with anti-mouse Fab F(ab')2. Representative histograms (Figure 14B) and quantification of CAR expression relative to tNGFR (Figure 14C) are shown for two healthy donors and two patients with diffuse large B-cell lymphoma (DLBCL). (Figure 14D) Expansion curves of CAR+ORF-transduced T cells (n=4 biological replicates). (Figure 14E) LTBR expression in autologous CD14+ monocytes and T cells transduced with LTBR alone or CAR+LTBR. (Figures 14F-14I) Expression of ICAM-1 (Figure 14F), CD70 (Figure 14G), CD74 (Figure 14H), and MHC-II (Figure 14I) by T cells transduced with LTBR ORF alone, CAR+LTBR, ​​or CAR+tNGFR. All data are normalized to tNGFR alone (no CAR). p-values ​​from an independent two-tailed t-test are shown. (Figures 14J-14M) Expression of exhaustion markers PD-1 (Figure 14J), TIM-3 (Figure 14K), LAG-3 (Figure 14L), and CD39 (Figure 14M) in CAR+ORF T cells. (Figure 14N) Differentiation phenotype of CAR+ORF T cells. CM: central memory. EM: effector memory. Differentiation was defined based on CD45RO and CCR7 expression (naive: CD45ROneg CCR7+, CM: CD45RO+CCR7+, EM: CD45RO+CCR7neg, effector: CD45ROneg CCR7neg). (Figures 14O, 14P) Expression of activation markers CD25 (Figure 14O) and CD69 (Figure 14P) in CAR+ORF T cells incubated alone or with Nalm6 cells for 24 hours. Error bars indicate SEM. N=3 biological replicates unless otherwise indicated. [Figure 14L]Co-delivery of ORF with CD19-targeted CAR is shown. (Figure 14A) Transduction efficiency of CAR+ORF lentiviral vector or ORF alone (n=4 biological replicates). (Figure 14B, Figure 14C) CAR expression levels determined by staining with anti-mouse Fab F(ab')2. Representative histograms (Figure 14B) and quantification of CAR expression relative to tNGFR (Figure 14C) are shown for two healthy donors and two patients with diffuse large B-cell lymphoma (DLBCL). (Figure 14D) Expansion curves of CAR+ORF-transduced T cells (n=4 biological replicates). (Figure 14E) LTBR expression in autologous CD14+ monocytes and T cells transduced with LTBR alone or CAR+LTBR. (Figures 14F-14I) Expression of ICAM-1 (Figure 14F), CD70 (Figure 14G), CD74 (Figure 14H), and MHC-II (Figure 14I) by T cells transduced with LTBR ORF alone, CAR+LTBR, ​​or CAR+tNGFR. All data are normalized to tNGFR alone (no CAR). p-values ​​from an independent two-tailed t-test are shown. (Figures 14J-14M) Expression of exhaustion markers PD-1 (Figure 14J), TIM-3 (Figure 14K), LAG-3 (Figure 14L), and CD39 (Figure 14M) in CAR+ORF T cells. (Figure 14N) Differentiation phenotype of CAR+ORF T cells. CM: central memory. EM: effector memory. Differentiation was defined based on CD45RO and CCR7 expression (naive: CD45ROneg CCR7+, CM: CD45RO+CCR7+, EM: CD45RO+CCR7neg, effector: CD45ROneg CCR7neg). (Figures 14O, 14P) Expression of activation markers CD25 (Figure 14O) and CD69 (Figure 14P) in CAR+ORF T cells incubated alone or with Nalm6 cells for 24 hours. Error bars indicate SEM. N=3 biological replicates unless otherwise indicated. [Figure 14M]Co-delivery of ORF with CD19-targeted CAR is shown. (Figure 14A) Transduction efficiency of CAR+ORF lentiviral vector or ORF alone (n=4 biological replicates). (Figure 14B, Figure 14C) CAR expression levels determined by staining with anti-mouse Fab F(ab')2. Representative histograms (Figure 14B) and quantification of CAR expression relative to tNGFR (Figure 14C) are shown for two healthy donors and two patients with diffuse large B-cell lymphoma (DLBCL). (Figure 14D) Expansion curves of CAR+ORF-transduced T cells (n=4 biological replicates). (Figure 14E) LTBR expression in autologous CD14+ monocytes and T cells transduced with LTBR alone or CAR+LTBR. (Figures 14F-14I) Expression of ICAM-1 (Figure 14F), CD70 (Figure 14G), CD74 (Figure 14H), and MHC-II (Figure 14I) by T cells transduced with LTBR ORF alone, CAR+LTBR, ​​or CAR+tNGFR. All data are normalized to tNGFR alone (no CAR). p-values ​​from an independent two-tailed t-test are shown. (Figures 14J-14M) Expression of exhaustion markers PD-1 (Figure 14J), TIM-3 (Figure 14K), LAG-3 (Figure 14L), and CD39 (Figure 14M) in CAR+ORF T cells. (Figure 14N) Differentiation phenotype of CAR+ORF T cells. CM: central memory. EM: effector memory. Differentiation was defined based on CD45RO and CCR7 expression (naive: CD45ROneg CCR7+, CM: CD45RO+CCR7+, EM: CD45RO+CCR7neg, effector: CD45ROneg CCR7neg). (Figures 14O, 14P) Expression of activation markers CD25 (Figure 14O) and CD69 (Figure 14P) in CAR+ORF T cells incubated alone or with Nalm6 cells for 24 hours. Error bars indicate SEM. N=3 biological replicates unless otherwise indicated. [Figure 14N]Co-delivery of ORF with CD19-targeted CAR is shown. (Figure 14A) Transduction efficiency of CAR+ORF lentiviral vector or ORF alone (n=4 biological replicates). (Figure 14B, Figure 14C) CAR expression levels determined by staining with anti-mouse Fab F(ab')2. Representative histograms (Figure 14B) and quantification of CAR expression relative to tNGFR (Figure 14C) are shown for two healthy donors and two patients with diffuse large B-cell lymphoma (DLBCL). (Figure 14D) Expansion curves of CAR+ORF-transduced T cells (n=4 biological replicates). (Figure 14E) LTBR expression in autologous CD14+ monocytes and T cells transduced with LTBR alone or CAR+LTBR. (Figures 14F-14I) Expression of ICAM-1 (Figure 14F), CD70 (Figure 14G), CD74 (Figure 14H), and MHC-II (Figure 14I) by T cells transduced with LTBR ORF alone, CAR+LTBR, ​​or CAR+tNGFR. All data are normalized to tNGFR alone (no CAR). p-values ​​from an independent two-tailed t-test are shown. (Figures 14J-14M) Expression of exhaustion markers PD-1 (Figure 14J), TIM-3 (Figure 14K), LAG-3 (Figure 14L), and CD39 (Figure 14M) in CAR+ORF T cells. (Figure 14N) Differentiation phenotype of CAR+ORF T cells. CM: central memory. EM: effector memory. Differentiation was defined based on CD45RO and CCR7 expression (naive: CD45ROneg CCR7+, CM: CD45RO+CCR7+, EM: CD45RO+CCR7neg, effector: CD45ROneg CCR7neg). (Figures 14O, 14P) Expression of activation markers CD25 (Figure 14O) and CD69 (Figure 14P) in CAR+ORF T cells incubated alone or with Nalm6 cells for 24 hours. Error bars indicate SEM. N=3 biological replicates unless otherwise indicated. [Figure 14O]Co-delivery of ORF with CD19-targeted CAR is shown. (Figure 14A) Transduction efficiency of CAR+ORF lentiviral vector or ORF alone (n=4 biological replicates). (Figure 14B, Figure 14C) CAR expression levels determined by staining with anti-mouse Fab F(ab')2. Representative histograms (Figure 14B) and quantification of CAR expression relative to tNGFR (Figure 14C) are shown for two healthy donors and two patients with diffuse large B-cell lymphoma (DLBCL). (Figure 14D) Expansion curves of CAR+ORF-transduced T cells (n=4 biological replicates). (Figure 14E) LTBR expression in autologous CD14+ monocytes and T cells transduced with LTBR alone or CAR+LTBR. (Figures 14F-14I) Expression of ICAM-1 (Figure 14F), CD70 (Figure 14G), CD74 (Figure 14H), and MHC-II (Figure 14I) by T cells transduced with LTBR ORF alone, CAR+LTBR, ​​or CAR+tNGFR. All data are normalized to tNGFR alone (no CAR). p-values ​​from an independent two-tailed t-test are shown. (Figures 14J-14M) Expression of exhaustion markers PD-1 (Figure 14J), TIM-3 (Figure 14K), LAG-3 (Figure 14L), and CD39 (Figure 14M) in CAR+ORF T cells. (Figure 14N) Differentiation phenotype of CAR+ORF T cells. CM: central memory. EM: effector memory. Differentiation was defined based on CD45RO and CCR7 expression (naive: CD45ROneg CCR7+, CM: CD45RO+CCR7+, EM: CD45RO+CCR7neg, effector: CD45ROneg CCR7neg). (Figures 14O, 14P) Expression of activation markers CD25 (Figure 14O) and CD69 (Figure 14P) in CAR+ORF T cells incubated alone or with Nalm6 cells for 24 hours. Error bars indicate SEM. N=3 biological replicates unless otherwise indicated. [Figure 14P]Co-delivery of ORF with CD19-targeted...

Claims

1. Lymphocytes genetically modified to express a chimeric antigen receptor (CAR), wherein the CAR is a) Antigen-binding domain and b) Transmembrane domain and c) A signaling domain, including, Lymphocytes in which at least one domain contains an LTBR domain.

2. The modified lymphocyte according to claim 1, wherein the LTBR domain is an LTBR intracellular domain, or a fragment or variant thereof.

3. The LTBR intracellular domain contains amino acids 249-435 of SEQ ID NO: 2, or a fragment, deletion, or variant thereof, or The LTBR intracellular domain has a deletion in at least amino acids 393-435 of SEQ ID NO:

2. Modified lymphocytes according to claim 2.

4. The modified lymphocyte according to claim 1, wherein the CAR further comprises a co-stimulatory domain.

5. The modified lymphocyte according to claim 1, wherein the lymphocyte is a T cell, an NK cell, or an NKT cell.

6. The modified lymphocyte according to claim 1, wherein the lymphocyte is an alpha-beta T cell or a gamma-delta T cell, or optionally a Vγ9Vδ2 T cell.

7. (i) The LTBR intracellular domain is located between the co-stimulatory signaling domain and the signaling domain of (c), (ii) The LTBR domain is located after the signal transduction domain of (c), Modified lymphocytes according to claim 1.

8. The modified lymphocyte according to claim 1, wherein the CAR is axicabutagene ciloleucel (Yescarta®), brexucagene autoleucel (Tecartus®), idecabutagene vicleucel (Abecma®), lysocabtagene maraleucel (Breyanzi®), or tisagenlecleucel (Kyrmriah®), modified to include an LTBR domain.

9. A nucleic acid molecule comprising a sequence encoding a chimeric antigen receptor (CAR), wherein the CAR is a) Antigen-binding domain and b) Transmembrane domain and c) A signaling domain, including, A nucleic acid molecule in which at least one domain contains an LTBR domain.

10. The LTBR domain is an LTBR intracellular domain, or a fragment or variant thereof. (i) The LTBR intracellular domain contains amino acids 249-435 of SEQ ID NO: 2, or a fragment, deletion, or variant thereof, (ii) The LTBR intracellular domain has a deletion in at least amino acids 393 to 435 of SEQ ID NO: 2 The nucleic acid molecule according to claim 9.

11. The nucleic acid molecule according to claim 9, wherein the CAR further comprises a co-stimulus signaling domain.

12. An expression cassette comprising the nucleic acid molecule described in claim 9.

13. A modified lymphocyte comprising the expression cassette described in Claim 12.

14. A method for producing modified lymphocytes, comprising introducing the expression cassette described in claim 12 into lymphocytes.

15. A pharmaceutical composition comprising modified lymphocytes for use in the treatment of cancer in a subject.

16. A method for increasing T cell effector function, including proliferation or cytokine production and / or secretion, wherein the method comprises administering to the subject a composition comprising the modified lymphocytes described in claim 1.

17. A fusion protein comprising an LTBR domain and at least one domain from a second protein that is not an LTBR domain.

18. A nucleic acid molecule comprising a sequence encoding the fusion protein described in claim 17.

19. A host cell comprising the nucleic acid molecule described in claim 18.

20. Lymphocytes that have been genetically modified to express T cell receptors (TCRs), (i) The TCR includes an alpha chain fused to the intracellular domain of the LTBR, (ii) The TCR includes a beta chain fused to the intracellular domain of the LTBR, (iii) The TCR includes a gamma chain fused to the intracellular domain of the LTBR, (iv) The TCR includes a delta chain fused to the intracellular domain of the LTBR, Lymphocytes.

21. a. Antigen-binding domain and b. LTBR and, c. A fusion protein comprising at least one domain from a second protein that is not an LTBR.

22. A nucleic acid molecule comprising a sequence encoding the fusion protein described in claim 21.

23. Modified lymphocytes comprising the nucleic acid molecule described in claim 22.

24. A modified lymphocyte comprising the nucleic acid molecule described in claim 22, and further comprising the nucleic acid molecule encoding CAR.

25. A manipulated lentiviral vector comprising the sequence of Sequence ID No. 132, or a sequence that shares at least 90% identity with Sequence ID No. 132 and, optionally, the open reading frame (ORF) of the gene of interest and / or a barcode inserted within the sequence.

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