Reprogramming cells into type 1 conventional dendritic cells or antigen-presenting cells

Abstract

The present invention relates to a composition comprising a transcription factor under the control of a promoter region, which composition can be used to reprogram a cell into a conventional dendritic cell or antigen presenting cell of type 1. The present invention further relates to a method for reprogramming a cell into a conventional dendritic cell or antigen presenting cell of type 1.

Description

[Technical field]

[0001] The present invention relates to compositions and methods for reprogramming cells into type 1 conventional dendritic cells or antigen presenting cells. [Background technology]

[0002] Cellular reprogramming relies on rewiring the epigenetic and transcriptional networks of one cell state to that of another cell type. Transcription factor (TF) overexpression experiments have highlighted the plasticity of adult somatic or differentiated cells, providing new techniques to generate any desired cell type. Forced expression of TFs can reprogram somatic or differentiated cells into induced pluripotent stem cells (iPSCs), which bear a striking resemblance to embryonic stem cells (Takahashi et al., 2007; Takahashi & Yamanaka, 2006). Alternatively, somatic cells can be directly converted into another specialized cell type (Pereira, Lemischka, & Moore, 2012). Direct lineage conversion has proven successful in reprogramming mouse and human fibroblasts into several cell types, such as neurons, cardiomyocytes, and hepatocytes, using TFs that specify target cell identity (Xu, Du, & Deng, 2015). Direct cell conversion has also been demonstrated in the hematopoietic system, where forced expression of TFs induces macrophage fates in B cells and fibroblasts (Xie, Ye, Feng, & Graf, 2004), and direct reprogramming of mouse fibroblasts into clonogenic hematopoietic progenitors was achieved by Gata2, Gfi1b, cFos, and Etv6 (Pereira et al., 2013). These four TFs induce a dynamic multistep hematopoietic process that progresses through an endothelial-like intermediate and recapitulates developmental hematopoiesis occurring in vitro (Pereira et al., 2016).

[0003] Reprogrammed cells are very promising therapeutic tools for regenerative medicine, and cells obtained by differentiation of iPSCs are already being tested in clinical studies (Pires et al., 2019). Recently, it has been demonstrated that antigen-presenting dendritic cells (DCs) can be reprogrammed from unrelated cell types by small combinations of TFs (Rosa et al., 2018), thereby opening up the opportunity to apply cell reprogramming to modulate immune responses and develop new immunotherapies.

[0004] Classically, the DC compartment can be divided into two functionally distinct DC subsets: conventional DCs (cDCs), which are professional antigen-presenting cells (APCs), and plasmacytoid DCs (pDCs). cDCs drive antigen-specific immune responses, whereas pDCs are professional producers of type I interferon during viral infection. However, the timing and precise mechanisms controlling the divergence of different subsets during DC development are still to be established.

[0005] DCs are a class of bone-marrow derived cells arising from lympho-myeloid hematopoiesis that scan the organism for pathogens, forming an essential interface between the activation of the innate and adaptive immune systems. DCs act as professional APCs that can activate T cell responses by presenting peptide antigens complexed with major histocompatibility complexes (MHC) on their surface, together with all necessary soluble and membrane-bound costimulatory molecules. DCs induce primary immune responses by priming naive T lymphocytes, enhance the effector functions of pre-primed T lymphocytes, and coordinate communication between innate and adaptive immunity. DCs are found in most tissues and use several types of receptors to continuously sample the antigen environment and monitor for pathogen invasion. At steady state, and at elevated rates in response to pathogen detection, sentinel DCs in non-lymphoid tissues migrate to lymphoid organs where they present collected and processed antigens to T cells. The phenotype that T cells acquire depends on the context of antigen presentation. When antigens are derived from pathogens or damaged self, DCs receive danger signals, become activated, and subsequently stimulate effector T cells necessary to confer protective immunity.

[0006] An important aspect of the control of immune responses is the existence of different types of DCs, each specialized to respond to a specific pathogen and to interact with a specific subset of T cells. In this context, cDCs can be further divided into myeloid / conventional DC type 1 (cDC1 or cDC1s) and myeloid / conventional DC type 2 (cDC2). This extends the flexibility of the immune system to respond appropriately to a wide range of different pathogens and danger signals.

[0007] Human cDC1s, characterized by surface expression of CD141, CLEC9A, XCR1, and CD226 (Wculek et al., 2019; Heidkamp et al., 2016; Dutertre et al., 2019), mediate antigen expression by secreting immunomodulatory cytokines, including IL-12 and interferons (IFNs), as well as chemokines, such as CXCL10, and mediate antigen expression by CD8+ / -CD8+ cells. +They are functionally defined by their ability to cross-present to T cells (Lauterbach et al., 2010; Poulin et al., 2010). In the context of antitumor immunity, Batf3- / - animals lacking cDC1s are unable to reject immunogenic tumors (Hildner et al., 2008). This effect has been shown to be dependent on tumor-resident cDC1s, highlighting the importance of this DC subset at the tumor site for immune rejection of established tumors (Bottcher et al., 2018) and mediating responses to therapy (Salmon et al., 2016; Spranger et al., 2017). Accordingly, cDC1 abundance in human tumors has been associated with patient survival and responsiveness to checkpoint inhibitors (Barry et al., 2018; Broz et al., 2014; Hubert et al., 2020; Mayoux et al. 2020; Spranger et al. 2017). Human primary cDC1s are extremely rare in vivo, so their research and translational applications require methods to generate functional cDC1s in vitro. Human CD34 + Bone marrow (BM) progenitor cells express CD141 in vitro in the presence of FLT3L with SCF, GM-CSF, and IL-4. + It has been used to induce cDC1 (Poulin et al., 2010). More recently, FLT3L was combined with co-culture with Notch-expressing stromal cell lines to promote cDC1 differentiation (Kirkling et al., 2018; Balan et al., 2018). The generation of cDC1-, cDC2-, and pDC-like cells from induced pluripotent stem cell (iPSC) cultures was also demonstrated (Sontag et al., 2017). However, these protocols are complex, require feeder layers, and result in low yields and a mixture of different DC subsets with competing functions.

[0008] Therefore, new strategies for generating homogenous populations of differentiated human cDC1 in vitro are needed. Summary of the Invention

[0009] Provided herein are compositions and methods for reprogramming cells into dendritic cells or antigen-presenting cells. The inventors have found that the expression of transcription factors BATF3, IRF8, PU.1 under specific promoters can significantly improve cell reprogramming. The inventors have also found additional transcription factors (i.e., IRF7 and BATF) that increase reprogramming efficiency when co-expressed with PU.1, IRF8, and BATF3.

[0010] Thus, as used herein, when expressed, the transcription factor: a) BATF3 or a biologically active variant thereof which is at least 70% identical to SEQ ID NO: 10 (BATF3), such as at least 75%, for example at least 80%, such as at least 85%, for example at least 90%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99%, for example 100% identical to SEQ ID NO: 10 (BATF3); b) IRF8 or a biologically active variant thereof which is at least 70% identical to SEQ ID NO: 11 (IRF8), such as at least 75%, for example at least 80%, such as at least 85%, for example at least 90%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99%, for example 100% identical to SEQ ID NO: 11 (IRF8), and c) PU.1 or a biologically active variant thereof which is at least 70% identical to SEQ ID NO: 12 (PU.1), such as at least 75%, for example at least 80%, such as at least 85%, for example at least 90%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99%, for example 100% identical to SEQ ID NO: 12 (PU.1); The one or more constructs or vectors encoding a transcription factor are provided, wherein the one or more constructs or vectors comprise a promoter region capable of controlling transcription of a transcription factor, and the promoter region comprises a spleen focus forming virus (SFFV) promoter.

[0011] Further provided herein is a cell comprising one or more constructs or vectors according to the compositions described herein.

[0012] Also provided herein is a method for reprogramming or inducing a cell into a dendritic cell or an antigen presenting cell, comprising: a) transducing a cell with a composition comprising a construct or vector according to the compositions described herein; b) expressing a transcription factor; thereby obtaining a reprogrammed or derived cell; A method is provided that includes:

[0013] Further provided herein are reprogrammed or derived cells obtained according to the methods disclosed herein.

[0014] Also provided herein is a method of treating cancer comprising administering to an individual in need thereof a composition, cell, pharmaceutical composition, and / or reprogrammed or derived cell according to the present invention.

[0015] Further provided herein is the use of the compositions, cells, pharmaceutical compositions and / or reprogrammed or induced cells according to the invention for the manufacture of a medicament for treating cancer. [Brief description of the drawings]

[0016] [Figure 1A]PU.1, IRF8, and BATF3 induce a general cDC1 gene expression program in human fibroblasts. Human embryonic fibroblasts (HEFs) were co-transduced with DOX-inducible lentiviral particles encoding PU.1, IRF8, and BATF3 (PIB, TetO-PIB) and M2rtTA (UbC-M2rtTA). Purified PIB-transduced HEFs (hiDCs) were profiled by single-cell RNA-seq on day 3 (d3, CD45+, day 6 (d6, CD45+), and day 9 (CD45+HLA-DR-, d9 DR-, CD45+HLA-DR+, d9 DR+). HEFs and peripheral blood cDC1s, cDC2s, and pDCs were included as controls. [Figure 1B] PU.1, IRF8, and BATF3 induce a general cDC1 gene expression program in human fibroblasts. Flow cytometric analysis of hiDCs at days 3 and 9 after addition of Dox. [Figure 1C] PU.1, IRF8, and BATF3 induce a global cDC1 gene expression program in human fibroblasts. Kinetics of DR- (top) and DR+ (bottom) cell emergence (n=2-8, mean ± SD). [Figure 1D] PU.1, IRF8, and BATF3 induce a global cDC1 gene expression program in human fibroblasts. Scanning electron microscopy at day 9. Scale bar, 10 μm. [Figure 1E] PU.1, IRF8, and BATF3 induce a global cDC1 gene expression program in human fibroblasts. t-SNE plot showing 45,870 single cells. [Figure 1F] PU.1, IRF8, and BATF3 induce a general cDC1 gene expression program in human fibroblasts. Integration with published DC subset data (Villani et al. 2017) using scPred (Alquicira-Hernandez et al. 2019). Heatmap shows the percentage of single cells belonging to cDC1–DC6 subsets. [Figure 1G]PU.1, IRF8, and BATF3 induce a global cDC1 gene expression program in human fibroblasts. t-SNE plot of single cells assigned to cDC1. [Figure 1H] PU.1, IRF8, and BATF3 induce a general cDC1 gene expression program in human fibroblasts. Violin plot showing gene expression distribution of cDC1-specific genes. Log values ​​of gene counts are shown. [Figure 1I] PU.1, IRF8, and BATF3 induce a general cDC1 gene expression program in human fibroblasts. Heatmap showing differentially expressed genes across profiled populations in the five clusters. [Figure 1J] PU.1, IRF8, and BATF3 induce a general cDC1 gene expression program in human fibroblasts. Violin plot of selected genes from cluster 3. [Figure 1K] PU.1, IRF8, and BATF3 induce the global cDC1 gene expression program in human fibroblasts. Top 5 Reactome pathways enriched in each gene cluster. [Figure 1L] PU.1, IRF8, and BATF3 induce a general cDC1 gene expression program in human fibroblasts. Heatmap showing expression of genes associated with antigen cross-presentation. [Figure 1M] PU.1, IRF8, and BATF3 induce a general cDC1 gene expression program in human fibroblasts. Violin plot showing expression of genes associated with antigen cross-presentation. [Figure 2A] Pseudotemporal sequencing of single cells highlights pathways associated with successful and unsuccessful cDC1 reprogramming. Monocle3 reconstruction of single cell trajectories of hiDCs (DR- and DR+) non-attributed and attributed to cDC1s, and filtered cDC1s at HEF, day 3, day 6, and day 9 scPred (Alquicira-Hernandez et al. 2019). [Figure 2B] Pseudotime sequencing of single cells highlights pathways associated with successful and unsuccessful cDC1 reprogramming. cDC1 reprogramming trajectories colored by relative trajectory position (pseudotime, left). Box plot showing pseudotime distribution per cell type (right). [Figure 2C] Pseudotime sequencing of single cells highlights pathways associated with successful and unsuccessful cDC1 reprogramming. tSNE plot showing single cell velocities generated by scVelo (Bergen et al. 2020). Arrows indicate direction and thickness velocity along the trajectory. [Figure 2D] Pseudotime sequencing of single cells highlights pathways associated with successful vs. unsuccessful cDC1 reprogramming. Heatmap highlighting six gene clusters (A-F) showing dynamic expression along scVelo latency time. [Figure 2E] Pseudotime sequencing of single cells highlights pathways associated with successful and unsuccessful cDC1 reprogramming. Top 5 Reactome pathways enriched in each cluster. [Figure 2F] Pseudotime sequencing of single cells highlights pathways associated with successful and unsuccessful cDC1 reprogramming. Heatmap showing average expression values ​​of gene modules by differentially expressed cell type along the trajectory. [Figure 2G] Pseudotime sequencing of single cells highlights pathways associated with successful and unsuccessful cDC1 reprogramming. Violin plots showing the expression distribution of genes associated with unsuccessful and successful DC reprogramming. Log values ​​of gene counts are shown. [Figure 2H]Pseudotime sequencing of single cells highlights pathways associated with successful and unsuccessful cDC1 reprogramming. Unsuccessful (left) and successful (right) cDC1 reprogramming transcription factors with Chea3. SPI1, IRF8, and BATF are highlighted in bold. [Figure 2I] Pseudotime sequencing of single cells highlights pathways associated with successful and unsuccessful cDC1 reprogramming. Flow cytometric analysis of CD226 expression in hiDCs. [Figure 2J] Pseudotemporal sequencing of single cells highlights pathways associated with successful versus unsuccessful cDC1 reprogramming. Classification according to DC subset data (Villani et al. 2017). [Figure 2K] Pseudotime sequencing of single cells highlights pathways associated with successful and unsuccessful cDC1 reprogramming. Dead cell phagocytosis by CD45+HLA-DR+CD226+ and CD45+HLA-DR+CD226- hiDCs (n=6–7, mean ± SD). [Figure 2L] Pseudotime sequencing of single cells highlights pathways associated with successful and unsuccessful cDC1 reprogramming. Reprogramming efficiency at day 9 resulting from co-transduction of PIB with the indicated transcription factors (n=4, mean±SD). Cells transduced with M2rtTA and PIB were included as controls. **p<0.005, ****p<0.00005 [Diagram 3] Inflammatory cytokine signaling allows highly efficient reprogramming of human cDC1. Quantification of day 9 hiDC (CD45+HLA-DR+) obtained in the presence of (A) individual cytokines and (B) combinations of 2-3 cytokines. Non-transduced HEFs were included as controls (n=2-10, mean ± SD). [Figure 4A]Forced expression of transcription factors allows highly efficient reprogramming of human cDC1. Quantification of reprogrammed cells (tdT+MHC-II+) obtained by transducing Clec9a-tdTomato (tdT) reporter mouse embryonic fibroblasts with PIB-IRES-GFP driven by Dox-inducible (TetO) or constitutive promoters (UbC, SFFV, PGK, EF1S, EF1, and EF1i). Expression of GFP (tetO-GFP) was used as a control (n=2-6, mean ± SD). [Figure 4B] Forced expression of transcription factors enables highly efficient reprogramming of human cDC1. Quantification of day 9 hiDCs generated with TetO-PIB or SFFV-PIB in the presence or absence of IFN-γ, IFN-β, and TNF-α (n=4–19, mean ± SD). [Figure 4C] Forced expression of transcription factors enables highly efficient reprogramming of human cDC1. hiDC yield per input fibroblast (n=10-12, mean ± SD). [Figure 4D] Forced expression of transcription factors enables highly efficient reprogramming of human cDC1. Day 9 hiDCs generated in the four conditions were purified and profiled by scRNA-seq. Heatmaps show the percentage of single cells belonging to cDC1–DC6 subsets. [Diagram 5] Anti-inflammatory cytokine signaling does not inhibit cDC1 reprogramming. Flow cytometry quantification of CD45+HLA-DR+ cells (left) and CD40+ cells gated on CD45+HLA-DR+ cells (right) at day 9 generated by transducing HEFs with SFFV-PIB in the presence of anti-inflammatory cytokines (n=3, mean ± SD). [Figure 6A]The optimized reprogramming protocol allows the generation of functional human cDC1-like cells. Median fluorescence intensity (MFI) of CD40 and CD80 on day 9 in hiDCs (CD45+HLA-DR+) and peripheral blood CD141+CLEC9A+cDC1s generated by SFFV-PIB in the absence or presence of IFN-γ, IFN-β, and TNF-α (hiDC+cyt). Cells were stimulated overnight with individual TLR agonists LPS, Poly I:C (polyinosinic:polycytidylic acid), R848 or in combination (all) (n=2–14, mean ± SD). [Figure 6B] The optimized reprogramming protocol allows the generation of functional human cDC1-like cells. Quantification of dead cell phagocytosis by hiDCs on day 9 after 2 h incubation. HEFs and CD141+CLEC9A+cDC1s were included as controls (n=3–12, mean ± SD). [Figure 6C] The optimized reprogramming protocol allows the generation of functional human cDC1-like cells. Cytokine secretion of purified hiDCs on day 9 after overnight incubation with TLR agonists. HEFs, monocyte-derived DCs (moDCs), and CD141+CLEC9A+XCR1+cDC1s were included as controls (n=2–11, mean ± SD). [Figure 6D] The optimized reprogramming protocol allows the generation of functional human cDC1-like cells. Cells were incubated overnight with LPS, poly I:C, and R848, pulsed with CMV proteins for 3 h, washed, and co-cultured with CMV+CD8+ T cells. Antigen cross-presentation was quantified by measuring IFN-γ after 24 h (n=2–4, mean ± SD) *p<0.05, **p<0.005, ***p<0.0005, ****p<0.00005. [Figure 7A]Efficient cDC1 reprogramming of adult fibroblasts. Flow cytometry analysis on day 9 of hiDCs (CD45+HLA-DR+) generated from human dermal fibroblasts (HDFs) from three independent donors in the absence (SFFV-PIB) or presence (SFFV-PIB+cyt) of IFN-γ, IFN-β, and TNF-α. [Figure 7B] Efficient cDC1 reprogramming of adult fibroblasts. Quantification of hiDCs (CD45+HLA-DR+) generated from human dermal fibroblasts (HDFs) from three independent donors in the absence (SFFV-PIB) or presence (SFFV-PIB+cyt) of IFN-γ, IFN-β, and TNF-α on day 9. HDFs were included as controls (n=3–13, mean ± SD). [Figure 7C] Efficient cDC1 reprogramming of adult fibroblasts. Expression of CD40 and CD80. [Figure 7D] Efficient cDC1 reprogramming of adult fibroblasts. Day 9 HDF-derived hiDCs were purified and profiled by scRNA-seq. Heatmaps show the percentage of single cells belonging to cDC1–6 subsets. [Figure 7E] Efficient cDC1 reprogramming of adult fibroblasts. Heatmap shows expression of genes upregulated during reprogramming and expressed in cDC1. Genes involved in cDC1 and antigen presentation are highlighted in bold. [Figure 7F] Efficient cDC1 reprogramming of adult fibroblasts. Heatmap shows expression of genes upregulated during reprogramming and expressed in cDC1. Genes of cDC1 and antigen presentation are shown as violin plots. Log values ​​of gene counts are shown. [Figure 8A] Efficient cDC1 reprogramming of mesenchymal stromal cells. Strategy to obtain hiDCs from human mesenchymal stromal cells (MSCs) under xeno-free conditions. MSCs were isolated from three healthy donors, FACS purified (Lin-CD45-CD271+), expanded in pHPL medium, transduced and cultured in X-VIVO 15. dX=day X. [Figure 8B]Efficient cDC1 reprogramming of mesenchymal stromal cells. Quantification of MSC-derived hiDCs generated with or without cytokines at day 9 (n=3–14, mean ± SD). [Figure 8C] Efficient cDC1 reprogramming of mesenchymal stromal cells. Quantification of MSC-derived hiDCs generated with or without cytokines at day 9 (n=3–14, mean ± SD). [Figure 8D] Efficient cDC1 reprogramming of mesenchymal stromal cells. Flow cytometry analysis of CD40 and CD80. ns-not significant, ****p<0.00005. [Figure 9A] Induced DCs induce antitumor immunity in vivo. Kinetics of acquisition of cross-presentation capability during DC reprogramming. Representative flow cytometry plots show CTV labeling of TCR+CD8+CD44+ T cells co-cultured with 50,000 tdT+ cells on d4, d7, and d9 of reprogramming. MEFs were included as controls. [Figure 9B] Induced DCs induce antitumor immunity in vivo. Quantification of proliferating T cells after co-culture with tdT+ cells sorted at three different ratios at different time points (n=4, mean±SD). [Figure 9C] Induced DCs induce antitumor immunity in vivo. Cytokine secretion of sorted tdtT+ cells after stimulation with LPS or poly I:C (n=2, mean ± SD). MEFs and CD103+ bone marrow-derived DCs (BM-DCs) were included as controls. [Figure 9D] Induced DCs induce antitumor immunity in vivo. Purified tdT+iDCs were mixed with 0.5 M B16OVA cells, and then tumors were subcutaneously implanted into C57BL / 6 mice. Tumor volumes were assessed over 14 days (n=5-6, mean ± SEM). [Figure 9E] Induced DCs induce antitumor immunity in vivo. Purified tdT+iDCs were injected intratumorally into B16OVA tumors 8 days after establishment. Tumor volumes were assessed up to day 20 (n=4-9, mean ± SEM, two independent experiments). Animals injected with PBS, MEFs, and CD103+BM-DCs were included as controls. [Figure 9F] Induced DCs induce antitumor immunity in vivo. CTV-labeled OT-I CD8+ T cells were injected intravenously on the same day as iDCs, and tumors and tumor-draining lymph nodes were analyzed 4 days later. Tumor infiltration (left) and expression of IFN-γ and granzyme B (GzmB) (right) of in vitro restimulation of OVA-restricted CD8+ T cells were quantified (n = 2–4, mean ± SD). [Figure 10A] PU.1 has independent chromatin targeting ability and recruits IRF8 and BATF3 to the same binding site. Strategy to profile the chromatin binding sites of PU.1, IRF8, and BATF3 (PIB) at early stages of reprogramming. HDFs were transduced with PIB (left) or individual factors (right) and analyzed by ChIP-seq after 48 hours. [Figure 10B] PU.1 has independent chromatin targeting capabilities and recruits IRF8 and BATF3 to the same binding site. Heatmap showing genome-wide distribution of PU.1, IRF8, and BATF3 when expressed in combination (left) or individually (right). Signals are displayed within 8 kb windows centered on individual peaks. The number of peaks in each condition is indicated. The average signal intensity of the peaks is shown (lower panel). [Figure 10C] PU.1 has independent chromatin targeting capabilities and recruits IRF8 and BATF3 to the same binding site. De novo motif prediction analysis of target sites for PU.1, IRF8, and BATF3 when expressed in combination or individually. The motifs of PU.1 are highlighted in bold. [Figure 11A] PU.1, IRF8, and BATF3 bind in open chromatin to inhibit fibroblast genes and impose the cDC1 transcriptional program. Venn diagram shows genome-wide peak overlap between PU.1, IRF8, and BATF3 (PIB). [Figure 11B]PU.1, IRF8, and BATF3 bind in open chromatin to inhibit fibroblast genes and impose the cDC1 transcriptional program. De novo motif prediction analysis for co-binding sites of PU.1, IRF8, and BATF3 when expressed in combination. PU.1-IRF and BATF motifs are highlighted in bold. [Figure 11C] PU.1, IRF8, and BATF3 bind in open chromatin to inhibit fibroblast genes and impose the cDC1 transcriptional program. Motif comparison of PU.1-IRF and BATF. Jaccard's similarity coefficient = 0.02. [Figure 11D] PU.1, IRF8, and BATF3 bind in open chromatin to inhibit fibroblast genes and impose the cDC1 transcriptional program. Immunoblot (left) showing immunoprecipitation (IP) of PU.1 (top), IRF8 (middle), and BATF3 (bottom) in HEK293T cells 24 hours after transfection with PIB. Co-immunoprecipitation (Co-IP) was performed with one million, two million, and five million (M) cells (right). Input (10%) and IgG isotype were used as controls. [Figure 11E] PU.1, IRF8, and BATF3 bind in open chromatin to inhibit fibroblast genes and impose the cDC1 transcriptional program. Heatmap showing differentially expressed genes between day 9 HDFs and hiDCs that are bound by either PU.1, IRF8, and BATF3 or by all three factors (crossover). [Figure 11F] PU.1, IRF8, and BATF3 bind in open chromatin to inhibit fibroblast genes and impose the cDC1 transcriptional program. Heatmap of normalized read coverage of chromatin marks in HDFs relative to co-binding sites. Signals are displayed within 8 kb windows and centered on transcription factor binding sites. Average signal intensity is shown (upper panel). [Figure 11G]PU.1, IRF8, and BATF3 bind in open chromatin to inhibit fibroblast genes and impose the cDC1 transcriptional program. A model for the mechanisms that set in motion cDC1 reprogramming. [Figure 12A] PU.1, IRF8, and BATF3 reprogram mouse cancer cells into cDC1-like cells. Flow cytometry analysis of mouse Lewis lung carcinoma (3LL) and melanoma (B16) cells (tumor antigen presenting cells, Tumour-APC) 9 days after transduction with SFFV-PIB-GFP lentiviral particles. Parental cell lines transduced with SFFV-GFP were included as controls. [Figure 12B] PU.1, IRF8, and BATF3 reprogram mouse cancer cells into cDC1-like cells. Reprogrammed 2 cells (GFP+CD45+MHC-II+) from Lewis lung carcinoma (LLC) and melanoma B16 were purified by FACS on day 9 (d9). GFP vector-transduced cancer cells 3 were included as controls (d0). Heatmaps show expressed 4 genes related to IFN-γ (left) and STING (right) pathways in reprogrammed LLC and 5 induced dendritic cells (iDCs). Type 1 (cDC1) splenic dendritic cells were included as reference 6 (GSE103618). [Figure 12C] PU.1, IRF8, and BATF3 reprogram mouse cancer cells into cDC1-like cells. Where indicated, flow cytometry analysis (left) and quantification (right) of endogenous antigen presentation measured as CD8+ T cell proliferation (CTV dilution) and activation (CD44+) after co-culture with FACS-purified B16-OVA cells transduced with PIB or eGFP lentivirus on day 3 of reprogramming after overnight stimulation with poly(I:C) (P(I:C)) (n=3-4). [Figure 12D] PU.1, IRF8, and BATF3 reprogram mouse cancer cells into cDC1-like cells. Flow cytometric analysis (left) and quantification (right) of T cell killing of PIB-transduced or IFN-treated B16-OVA target cells (mOrange+) after 72 h of coculture (n=6–9). [Figure 12E] PU.1, IRF8, and BATF3 reprogram mouse cancer cells into cDC1-like cells. Flow cytometry quantification of antigen cross-presentation capacity measured as the percentage of CD44+ proliferative OT-I CD8+9 T cells after coculture with B16 cells transduced with SFFV-PIB-GFP lentiviral particles, incubated overnight with OVA protein in the presence of P(I:C) and / or interferon gamma (IFN-g) where indicated (n=4–8). [Figure 12F] PU.1, IRF8, and BATF3 reprogram mouse cancer cells into cDC1-like cells. Tumor-APCs derived from B16 on day 5 of reprogramming were pulsed with OVA protein and P(I:C) and injected intratumorally into pre-established B16-OVA tumors on days 7, 10, and 13. [Figure 12G] PU.1, IRF8, and BATF3 reprogram mouse cancer cells into cDC1-like cells. Tumor growth (n=6) in mice injected with tumor APC (PIB), PBS, or control lentivirus (MCS)-transduced B16 cells. [Figure 12H] PU.1, IRF8, and BATF3 reprogram mouse cancer cells into cDC1-like cells. Survival in mice injected with tumor APC (PIB), PBS, or control lentivirus (MCS)-transduced B16 cells (n=6). Mean ± SD is represented. **p<0.01, ****p<0.0001. [Figure 13A] PU.1, IRF8, and BATF3 reprogram human cancer cells into cDC1-like cells. Reprogramming efficiency of glioblastoma (T98G), rectal carcinoma (ECC4), and mesothelioma (ACC-Meso-1, ACCM1) cell lines analyzed by flow cytometry as the percentage of cells co-expressing CD45 and HLA-DR gated on transduced EGFP+ cells (red) when transduced with SFFV-PIB-GFP or control SFFV-GFP lentivirus. [Figure 13B]PU.1, IRF8, and BATF3 reprogram human cancer cells into cDC1-like cells. Reprogramming efficiency of cDC1 across 28 solid tumor cell lines. Reprogrammed populations expressing (CD45+HLA-DR+) and intermediate populations (CD45+HLA-DR- or CD45-HLA-DR+) are shown (n=2-8). Mean ± SD is represented. [Figure 13C] PU.1, IRF8, and BATF3 reprogram human cancer cells into cDC1-like cells. Flow cytometry quantification of cDC1 surface markers (CLEC9A, CD141, CD11c) in human glioblastoma (T98G) cells 9 days after transduction with SFFV-PIB-GFP lentiviral particles. The parental cell line transduced with SFFV-GFP was included as a control. [Figure 13D] PU.1, IRF8, and BATF3 reprogram human cancer cells into cDC1-like cells. Kinetics of surface expression of costimulatory molecules CD40, CD80, and CD86 in CD45+HLA-DR+ cancer cells 9 days after transduction with SFFV-PIB-GFP lentiviral particles stimulated overnight with poly I:C and LPS. SFFV-GFP-transduced parental cell lines and unstimulated SFFV-PIB-GFP-transduced parental cell lines were included as controls. [Figure 13E] PU.1, IRF8, and BATF3 reprogram human cancer cells into cDC1-like cells. Exemplary plots at day 9 are shown. [Figure 13F] PU.1, IRF8, and BATF3 reprogram human cancer cells into cDC1-like cells. Human primary tonsillar cancer tissue (JCA10) and patient-xenograft derived bladder cancer cells (U3P2E2) were transduced with hPIB-IRES-EGFP or EGFP control vector (day 0) and analyzed by flow cytometry on day 9 to determine the percentage of reprogrammed CD45+HLA-DR+ cells (black) and partially reprogrammed cells expressing either CD45 or HLA-DR. [Figure 13G]PU.1, IRF8, and BATF3 reprogram human cancer cells into cDC1-like cells. Reprogramming efficiency shown in primary human tumor cells derived from melanoma (n=2), lung cancer (n=2), head and neck cancer (tonsil, n=2, tongue, n=3), pancreatic cancer (n=2), breast cancer (n=2), and bladder cancer (n=2) and cancer-associated fibroblasts (CAF, n=2). Partially reprogrammed cells are shown (CD45+HLA-DR-, CD45-HLA-DR+). [Figure 14A] PU.1, IRF8, and BATF3 induce rapid global transcriptional and epigenetic reprogramming. Experimental design for evaluating the kinetics of transcriptomics and epigenetic reprogramming. Human glioblastoma cell line (T98G) was transduced with SFFV-hPIB-IRES-EGFP. Reprogrammed (CD45+HLA-DR+,++) and partially reprogrammed (CD45-HLA-DR+,+) cells were profiled by FACS sorting and mRNA and ATAC sequencing on days 3 (d3), 5 (d5), 7 (d7), and 9 (d9). Control cells transduced with empty EGFP vector are represented as day 0 (d0). cDC1 donor cells were used as reference. [Figure 14B] PU.1, IRF8, and BATF3 induce rapid global transcriptional and epigenetic reprogramming. Principal component analysis (PCA) of the time course of cancer cell reprogramming based on differentially expressed genes (left panel). Human embryonic fibroblast (HEF) reprogramming was also included as a reference for the dynamics of this process. Arrows highlight the reprogramming trajectories. PCA based on differentially accessible chromatin regions (right panel). Donor peripheral blood cDC1 was used as a reference. [Figure 14C]PU.1, IRF8, and BATF3 induce rapid global transcriptional and epigenetic reprogramming. Establishment of the tumor-APC transcriptomic signature in reprogrammed and partially reprogrammed T98G cells (left). Chromatin accessibility in the tumor-APC gene set is shown on the right. [Figure 15A] Histone deacetylase inhibition enhances tumor-APC reprogramming efficiency. Lewis lung carcinoma (LLC) and B16 cancer cells were transduced with PU.1, IRF8, and BATF3 (SFFV-PIB-eGFP), cultured in the presence or absence of valproic acid (VPA), and analyzed for CD45 and MHC-II expression by flow cytometry on day 9. [Figure 15B] Histone deacetylase inhibition enhances tumor-APC reprogramming efficiency. Quantification of reprogramming efficiency in the presence of VPA, gated on eGFP+ transduced cells (%CD45+MHC-II+ cells) (n=6–16). Cancer cells transduced with eGFP vector (green, striped) were included as a control. [Figure 15C] Histone deacetylase inhibition enhances tumor-APC reprogramming efficiency. Quantification of the percentage of MHC-I+ cells gated on eGFP+ transduced cells (n=6–11). [Figure 15D] Histone deacetylase inhibition enhances tumor-APC reprogramming efficiency. Quantification of CD44+ proliferative OT-I CD8+ T cells after co-culture with reprogrammed LLC-OVA and B16-OVA cells (n=4-11). [Figure 15E] Histone deacetylase inhibition enhances tumor-APC reprogramming efficiency. Quantification of T cell-mediated killing by flow cytometry of reprogrammed B16-OVA target cells (mOrange+) and non-targeting B16-OVA cells at 0 and 72 hours in a 1:1 ratio of activated OT-I T cells to cancer cells (n=5-7). [Figure 15F]Histone deacetylase inhibition enhances tumor-APC reprogramming efficiency. Quantification of CD44+ proliferative OT-I CD8+ T cells after co-culture with reprogrammed LLC or B16 cells pre-incubated with OVA peptide (SIINFKL) (n=4–12). Mean ± SD is represented. **p<0.01, ****p<0.0001. [Figure 15G] Histone deacetylase inhibition enhances tumor-APC reprogramming efficiency. Flow cytometry analysis (upper panel) and quantification (lower panel) of cDC1 reprogramming efficiency in the presence and absence of valproic acid (VPA) in six human cancer cell lines on day 9. Cancer cell lines were transduced with SFFV-hPIB-IRES-EGFP lentiviral particles or EGFP as a control and cultured in the presence or absence of VPA from days 1 to 4 of reprogramming. [Figure 16] SPIB and SPIC compensate for the role of PU.1 in cDC1 reprogramming. (A) Flow cytometric quantification of Clec9a reporter activation in mouse embryonic fibroblasts (MEFs) 5 days after transduction with PU.1 homologs alone or in combination with IRF8 and BATF3. (B) Flow cytometric quantification of CD45 and MHC-II expression levels (gated on tdTomato+ cells). Graph bars represent mean ± SEM (N=4). **p<0.01, ****p<0.0001. [Figure 17A] Adenovirus- and adeno-associated virus-delivered PU.1, IRF8, and BATF3 enable cDC1 reprogramming in mouse and human cells. Flow cytometry analysis of Clec9a reporter activation in mouse embryonic fibroblasts (MEFs) 9 days after transduction with lentivirus (Lenti), adenovirus (Ad5 and Ad5 / F35), and adeno-associated virus (AAV-DJ and AAV2-qYF) encoding PU.1, IRF8, and BATF3 (PIB), and GFP (PIB-GFP). [Figure 17B]Adenovirus- and adeno-associated virus-delivered PU.1, IRF8, and BATF3 enable cDC1 reprogramming in mouse and human cells. Quantification of CD45 and MHC-II expression in mouse embryonic fibroblasts (MEFs) 9 days after transduction with lentivirus (Lenti), adenovirus (Ad5 and Ad5 / F35), and adeno-associated virus (AAV-DJ and AAV2-qYF) encoding PU.1, IRF8, and BATF3 (PIB), and GFP (PIB-GFP). Viruses encoding GFP only were included as controls and did not induce tdTomato expression in transduced cells. Graph bars show mean ± SEM (N=4). [Figure 17C] Adenovirus- and adeno-associated virus-delivered PU.1, IRF8, and BATF3 enable cDC1 reprogramming in mouse and human cells. Flow cytometry quantification of cDC1 reprogramming efficiency in B2905 mouse melanoma cell line measured as CD45 and MHC-II expression 9 days after transduction with virus encoding PIB-GFP. Lentivirus encoding GFP only was included as a control. Graph bars show mean ± SEM (N=4). [Figure 17D] Adenovirus- and adeno-associated virus-delivered PU.1, IRF8, and BATF3 enable cDC1 reprogramming in mouse and human cells. Flow cytometric quantification of cDC1 reprogramming efficiency in two human cancer cell lines (IGR-39 and T98G) and one primary melanoma sample (2778) measured as CD45 and HLA-DR expression 9 days after transduction with virus encoding PIB-GFP. Lentivirus encoding GFP only was included as a control. Graph bars show mean ± SEM (N=4). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] definition "Biologically active variant" refers herein to a biologically active variant of a transcription factor (TF) that retains at least some of the activity of the parent TF. For example, biologically active variants of basic leucine zipper ATF-like transcription factor 3 (BATF3), interferon regulatory factor 8 (IRF8), and PU.1 can act as the respective TFs mentioned above, inducing or inhibiting the expression of the same genes, respectively, in cells, as BATF3, IRF8, and PU.1 do, respectively, but the efficiency of induction may be different, e.g., the efficiency of gene induction or inhibition is reduced or increased compared to the parent TF.

[0018] "Identity and homology" with respect to polynucleotides or polypeptides is defined herein as the percentage of nucleic acids or amino acids in a candidate sequence that are identical or homologous to the corresponding naturally occurring nucleic acid or amino acid residues, respectively, after aligning the sequences, introducing gaps, if necessary, to achieve the maximum identity / similarity / homology percentage, and taking into account any conservative substitutions according to the NCIUB rules (hftp: / / www.chem.qmul.ac.uk / iubmb / misc / naseq.html; NC-IUB, Eur J Biochem (1985)) as part of the sequence identity. Neither 5' or 3' extensions, nor insertions (in the case of nucleic acids), nor N' or C' extensions, nor insertions (in the case of polypeptides) result in a loss of identity, similarity or homology. Methods and computer programs for alignment are well known in the art. In general, a given homology between two sequences means that the identity between these sequences is at least equal to the homology. For example, if two sequences are 70% homologous to each other, they may not be less than 70% identical to each other, but may share 80% identity.

[0019] As used herein, "mesenchymal stem cells" or "mesenchymal stromal cells" (both referred to as "MSCs") are used interchangeably and are referred to herein as multipotent stromal cells that can differentiate into a variety of cell types, including, but not limited to, osteoblasts (bone cells), chondrocytes (cartilage cells), and adipocytes (fat cells).

[0020] "Murine," as used herein, refers to any and all members of the Muridae family, including rats and mice.

[0021] "Reprogramming" as used herein refers to the process of converting a cell from one cell type to differentiate into another cell type. In particular, reprogramming as used herein refers to the conversion or transdifferentiation of any type of cell into a type of conventional dendritic cell or antigen-presenting cell.

[0022] "Treating" or "treatment," as used herein, refers to any administration or application of a therapeutic agent to the disclosed diseases, disorders, and conditions in a subject, including inhibiting the progression of a disease, slowing the disease or its progression, arresting the onset of a disease, partially or completely alleviating a disease, or partially or completely alleviating one or more symptoms of a disease.

[0023] As used herein, the term "adenovirus" is used to refer to any and all viruses that can be categorized as adenoviruses, including any adenovirus that infects humans or non-human animals, including all groups, subgroups, and serotypes, unless otherwise required. Thus, as used herein, "adenovirus" refers to the virus itself or its derivatives, and includes all serotypes and subtypes, naturally occurring (wild type), modified forms used as adenovirus vectors, e.g., gene delivery vehicles, modified forms known in the art, such as capsid mutants, recombinant forms, replication-competent, conditionally replication-competent, or replication-deficient forms, unless otherwise specified.

[0024] As used herein, the term "adeno-associated virus" can be used to refer to the naturally occurring wild-type virus itself or its derivatives. This term is used to refer to any and all viruses that can be categorized as adeno-associated viruses, including any adeno-associated virus that infects humans or non-human animals, including all subtypes, serotypes, and pseudotypes, as well as both naturally occurring forms and modified and recombinant forms, such as those used as adeno-associated virus vectors, e.g., gene delivery vehicles, unless otherwise required.

[0025] As used herein, the abbreviation "Ad" in relation to viral vectors refers to adenovirus, typically followed by a number indicating the serotype of the adenovirus. For example, "Ad5" refers to serotype 5 of adenovirus.

[0026] Any Ad suitable for the purpose may be used herein, including, but not limited to, an Ad from any serotype from any of the A, B, C, D, E, F, G Ad subgroups, such as Ad2, Ad5 or Ad35, avian Ad, bovine Ad, canine Ad, caprine Ad, equine Ad, primate Ad, non-primate Ad, and ovine Ad. "Primate Ad" refers to an Ad that infects primates, "non-primate Ad" refers to an Ad that infects non-primate mammals, and "bovine Ad" refers to an Ad that infects bovine mammals.

[0027] The genomic sequences of the various serotypes of Ad, as well as the sequences of the native terminal repeats (TRs) and capsid subunits, are known in the art.

[0028] As used herein, the abbreviation "AAV" in relation to viral vectors refers to adeno-associated virus, typically followed by a number indicating the serotype of the adeno-associated virus. For example, "AAV2" refers to adeno-associated virus serotype 2.

[0029] Any AAV suitable for this purpose may be used herein, including, but not limited to, AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 3A (AAV3A), AAV serotype 3B (AAV3B), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAV serotype 10 (AAV10), avian AAV, bovine AAV, canine AAV, caprine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. For example, "primate AAV" refers to AAV that infects primates, "non-primate AAV" refers to AAV that infects non-primate mammals, and "bovine AAV" refers to AAV that infects bovine mammals.

[0030] The genomic sequences of the various serotypes of AAV, as well as the sequences of the native terminal repeats (TRs), Rep proteins, and capsid subunits, are known in the art.

[0031] As used herein, a "hybrid" Ad or AAV vector refers to an Ad or AAV-based vector that has been engineered such that the Ad or AAV vector contains proteins from two or more different Ad or AAV serotypes.

[0032] As used herein, "AAV2-qYF" or "AAV2-QuadYF" refers to a quadruple tyrosine to phenylalanine mutant of AAV2.

[0033] As used herein, "AAV-DJ" refers to a hybrid capsid derived from DNA family shuffling of eight wild-type AAV serotypes, including AAV2, 4, 5, 8, 9, avian, bovine, and caprine AAV. AAV-DJ is a synthetic serotype 2 / 8 / 9 chimera that is distinct from its closest natural relative (AAV-2) by 60 capsid amino acids.

[0034] composition The present invention relates to compositions and their use in methods for reprogramming or inducing cells into dendritic cells or antigen-presenting cells.The inventors have surprisingly found that expressing TFs BATF3, IRF8, and PU.1 under specific promoters can significantly improve reprogramming.

[0035] Thus, as used herein, when expressed, the transcription factor: a) BATF3, or a biologically active variant thereof that is at least 70% identical to SEQ ID NO: 10 (BATF3); b) IRF8 or a biologically active variant thereof that is at least 70% identical to SEQ ID NO: 11 (IRF8), and c) PU.1 or a biologically active variant thereof which is at least 70% identical to SEQ ID NO: 12 (PU.1); and wherein the one or more constructs or vectors comprise a promoter region capable of controlling transcription of a transcription factor, the promoter region comprising a spleen focus forming virus (SFFV) promoter, an MND (myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer binding site substitution) promoter, a CAG (CMV early enhancer / chicken beta actin) promoter, a cytomegalovirus (CMV) promoter, a ubiquitin C (UbC) promoter, an EF-1 alpha (EF-1α) promoter, an EF-1 alpha short (EF1S) promoter, an EF-1 alpha with intron (EF1i) promoter, a phosphoglycerate kinase (PGK) promoter, or a promoter that exhibits substantially the same effect.

[0036] The TF may be as defined in the "Transcription Factor" section herein.

[0037] The promoter region may be as defined in the "Promoter" section herein.

[0038] TFs can be expressed from one or more vectors or constructs as polycistronic, dicistronic (or bicistronic), and / or monocistronic constructs. An mRNA molecule is said to be monocistronic if it contains genetic information for translating only a single protein chain. On the other hand, a polycistronic mRNA has multiple open reading frames (ORFs), each of which is translated into a polypeptide. A dicistronic mRNA encodes only two proteins. Polycistronic and dicistronic mRNAs are expressed from a single promoter or promoter region.

[0039] In one embodiment, the composition, upon expression, comprises: a) IRF7 or a biologically active variant thereof that is at least 70% identical to SEQ ID NO: 21 (IRF7); b) BATF or a biologically active variant thereof that is at least 70% identical to SEQ ID NO: 19 (BATF); c) SPIB or a biologically active variant thereof which is at least 70% identical to SEQ ID NO: 23 (SPIB); d) SPIC or a biologically active variant thereof which is at least 70% identical to SEQ ID NO: 25 (SPIC); e) CEBPα or a biologically active variant thereof which is at least 70% identical to SEQ ID NO: 13 (CEBPα); and wherein the one or more constructs or vectors comprise a promoter region capable of controlling transcription of the transcription factor, the promoter region comprising a spleen focus forming virus (SFFV) promoter, an MND (myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer binding site substitution) promoter, a CAG (CMV early enhancer / chicken beta actin) promoter, a cytomegalovirus (CMV) promoter, an ubiquitin C (UbC) promoter, an EF-1 alpha (EF-1α) promoter, an EF-1 alpha short (EF1S) promoter, an EF-1 alpha with intron (EF1i) promoter, a phosphoglycerate kinase (PGK) promoter, or a promoter exhibiting substantially the same effect.

[0040] In one embodiment, the composition comprises: a) one construct or vector that upon expression encodes the transcription factors BATF3, IRF8, and PU.1; b) one construct or vector that, upon expression, encodes the transcription factors BATF3, IRF8, and SPIB; c) a first construct or vector that, upon expression, encodes the transcription factors BATF3 and IRF8, and a second construct or vector that, upon expression, encodes the transcription factor PU.1; d) a first construct or vector that, upon expression, encodes the transcription factors BATF3 and IRF8, and a second construct or vector that, upon expression, encodes the transcription factor SPIB; e) a first construct or vector that, upon expression, encodes the transcription factor BATF3 and a second construct or vector that, upon expression, encodes the transcription factors IRF8 and PU.1; f) a first construct or vector that, upon expression, encodes the transcription factor BATF3 and a second construct or vector that, upon expression, encodes the transcription factors IRF8 and SPIB; g) a first construct or vector that, upon expression, encodes the transcription factor IRF8 and a second construct or vector that, upon expression, encodes the transcription factors BATF3 and PU.1; h) a first construct or vector that, upon expression, encodes the transcription factor IRF8 and a second construct or vector that, upon expression, encodes the transcription factors BATF3 and SPIB; i) a first construct or vector that, when expressed, encodes the transcription factor BATF3; a second construct or vector that, when expressed, encodes the transcription factor IRF8; and a third construct or vector that, when expressed, encodes the transcription factor PU.1; and / or j) a first construct or vector that, when expressed, encodes the transcription factor BATF3; a second construct or vector that, when expressed, encodes the transcription factor IRF8; and a third construct or vector that, when expressed, encodes the transcription factor SPIB; Includes.

[0041] In one embodiment, one or more of the constructs or vectors, upon expression, further encodes the transcription factor CCAAT / enhancer-binding protein alpha (cΕΒΡα), or a biologically active variant thereof, which may be as defined in the section entitled "Transcription Factors" herein.

[0042] In another embodiment, one or more of the constructs or vectors further encodes, upon expression, the transcription factor interferon regulatory factor 7 (IRF7), or a biologically active variant thereof. IRF7 may be as defined in the "Transcription Factors" section herein.

[0043] In another embodiment, one or more of the constructs or vectors further encodes, upon expression, a transcription factor, basic leucine zipper ATF-like (BATF), or a biologically active variant thereof. BATF may be as defined in the "Transcription Factors" section herein.

[0044] In another embodiment, one or more of the constructs or vectors further encodes, upon expression, a transcription factor Spi-C (SPIC), or a biologically active variant thereof. SPIC may be as defined in the "Transcription Factors" section herein.

[0045] In one embodiment, the composition comprises: a) one construct or vector that upon expression encodes the transcription factors BATF3, IRF8, PU.1, and IRF7; b) a first construct or vector that, upon expression, encodes the transcription factors BATF3 and IRF8, and a second construct or vector that, upon expression, encodes the transcription factors PU.1 and IRF7; c) a first construct or vector that, upon expression, encodes the transcription factors BATF3 and PU.1, and a second construct or vector that, upon expression, encodes the transcription factors IRF8 and IRF7; d) a first construct or vector that, upon expression, encodes the transcription factors PU.1 and IRF8, and a second construct or vector that, upon expression, encodes the transcription factors BATF3 and IRF7; e) a first construct or vector that, upon expression, encodes the transcription factors BATF3, IRF8, and PU.1, and a second construct or vector that, upon expression, encodes the transcription factor IRF7; f) a first construct or vector that, upon expression, encodes the transcription factor BATF3 and a second construct or vector that, upon expression, encodes the transcription factors IRF8, PU.1, and IRF7; g) a first construct or vector that, upon expression, encodes the transcription factor IRF8 and a second construct or vector that, upon expression, encodes the transcription factors BATF3, PU.1, and IRF7; h) a first construct or vector that, upon expression, encodes the transcription factor PU.1 and a second construct or vector that, upon expression, encodes the transcription factors BATF3, IRF8, and IRF7; and / or i) a first construct or vector that, when expressed, encodes the transcription factor BATF3, a second construct or vector that, when expressed, encodes the transcription factor IRF8, a third construct or vector that, when expressed, encodes the transcription factor PU.1, and a fourth construct or vector that, when expressed, encodes the transcription factor IRF7; Includes.

[0046] In one embodiment, the composition comprises: a) one construct or vector that, upon expression, encodes the transcription factors BATF3, IRF8, PU.1, and BATF; b) a first construct or vector that, upon expression, encodes the transcription factors BATF3 and IRF8, and a second construct or vector that, upon expression, encodes the transcription factors PU.1 and BATF; c) a first construct or vector that, upon expression, encodes the transcription factors BATF3 and PU.1, and a second construct or vector that, upon expression, encodes the transcription factors IRF8 and BATF; d) a first construct or vector that, upon expression, encodes the transcription factors PU.1 and IRF8, and a second construct or vector that, upon expression, encodes the transcription factors BATF3 and BATF; e) a first construct or vector that upon expression encodes the transcription factors BATF3, IRF8, and PU.1, and a second construct or vector that upon expression encodes the transcription factor BATF; f) a first construct or vector that, upon expression, encodes the transcription factor BATF3 and a second construct or vector that, upon expression, encodes the transcription factors IRF8, PU.1, and BATF; g) a first construct or vector that, upon expression, encodes the transcription factor IRF8 and a second construct or vector that, upon expression, encodes the transcription factors BATF3, PU.1, and BATF; h) a first construct or vector that, upon expression, encodes the transcription factor PU.1 and a second construct or vector that, upon expression, encodes the transcription factors BATF3, IRF8, and BATF; and / or i) a first construct or vector that, when expressed, encodes the transcription factor BATF3, a second construct or vector that, when expressed, encodes the transcription factor IRF8, a third construct or vector that, when expressed, encodes the transcription factor PU.1, and a fourth construct or vector that, when expressed, encodes the transcription factor BATF; Includes.

[0047] In one embodiment, the composition comprises: a) one construct or vector that, upon expression, encodes the transcription factors BATF3, IRF8, SPIB, and IRF7; b) a first construct or vector that, upon expression, encodes the transcription factors BATF3 and IRF8, and a second construct or vector that, upon expression, encodes the transcription factors SPIB and IRF7; c) a first construct or vector that, upon expression, encodes the transcription factors BATF3 and SPIB, and a second construct or vector that, upon expression, encodes the transcription factors IRF8 and IRF7; d) a first construct or vector that, upon expression, encodes the transcription factors SPIB and IRF8, and a second construct or vector that, upon expression, encodes the transcription factors BATF3 and IRF7; e) a first construct or vector that, upon expression, encodes the transcription factors BATF3, IRF8, and SPIB, and a second construct or vector that, upon expression, encodes the transcription factor IRF7; f) a first construct or vector that, upon expression, encodes the transcription factor BATF3 and a second construct or vector that, upon expression, encodes the transcription factors IRF8, SPIB, and IRF7; g) a first construct or vector that, upon expression, encodes the transcription factor IRF8 and a second construct or vector that, upon expression, encodes the transcription factors BATF3, SPIB, and IRF7; h) a first construct or vector that, upon expression, encodes the transcription factor SPIB and a second construct or vector that, upon expression, encodes the transcription factors BATF3, IRF8, and IRF7; and / or i) a first construct or vector that encodes the transcription factor BATF3 when expressed, a second construct or vector that encodes the transcription factor IRF8 when expressed, a third construct or vector that encodes the transcription factor SPIB when expressed, and a fourth construct or vector that encodes the transcription factor IRF7 when expressed; Includes.

[0048] In one embodiment, the composition comprises: a) one construct or vector that, upon expression, encodes the transcription factors BATF3, IRF8, SPIB, and BATF; b) a first construct or vector that, upon expression, encodes the transcription factors BATF3 and IRF8, and a second construct or vector that, upon expression, encodes the transcription factors SPIB and BATF; c) a first construct or vector that, upon expression, encodes the transcription factors BATF3 and SPIB, and a second construct or vector that, upon expression, encodes the transcription factors IRF8 and BATF; d) a first construct or vector that, upon expression, encodes the transcription factors SPIB and IRF8, and a second construct or vector that, upon expression, encodes the transcription factors BATF3 and BATF; e) a first construct or vector that, upon expression, encodes the transcription factors BATF3, IRF8, and SPIB, and a second construct or vector that, upon expression, encodes the transcription factor BATF; f) a first construct or vector that, upon expression, encodes the transcription factor BATF3 and a second construct or vector that, upon expression, encodes the transcription factors IRF8, SPIB, and BATF; g) a first construct or vector that, upon expression, encodes the transcription factor IRF8 and a second construct or vector that, upon expression, encodes the transcription factors BATF3, SPIB, and BATF; h) a first construct or vector that, upon expression, encodes the transcription factor SPIB and a second construct or vector that, upon expression, encodes the transcription factors BATF3, IRF8, and BATF; and / or i) a first construct or vector that encodes the transcription factor BATF3 when expressed, a second construct or vector that encodes the transcription factor IRF8 when expressed, a third construct or vector that encodes the transcription factor SPIB when expressed, and a fourth construct or vector that encodes the transcription factor BATF when expressed; Includes.

[0049] One or more of the constructs and vectors disclosed herein can be any type of construct or vector, such as a plasmid.

[0050] In one embodiment, one or more constructs or vectors are one or more viral vectors.In another embodiment, the viral vector is selected from the group consisting of lentivirus vector, retrovirus vector, adenovirus vector, herpesvirus vector, poxvirus vector, adeno-associated virus vector, paramyxovirus vector, rhabdovirus vector, alphavirus vector, flavivirus vector, and adeno-associated virus vector.In another embodiment, the viral vector is a lentivirus vector.

[0051] Adenovirus (Ad) vectors and adeno-associated virus (AAV) vectors may be vectors derived from any Ad or AAV serotype known in the art, and may allow gene expression in specific cells (e.g., nerve cells, muscle cells, and liver cells), tissues, and organs, for example, by applying the specificity of the target cells infected for each serotype. Ad or AAV may be wild-type or may be deleted in whole or in part of one or more wild-type genes. Ad or AAV may be further engineered by any method known in the art, such as, for example, pseudotyping, resulting in hybrid (or chimeric) virus particles, such as hybrid virus capsids.

[0052] The AAV or Ad viral particles may also be mutated at one or more amino acid residues, such as, for example, one or more tyrosine residues.

[0053] In one embodiment, the adenoviral vector is selected from the group consisting of a wild-type Ad vector, a hybrid Ad vector, and a mutant Ad vector.

[0054] In another embodiment, the adeno-associated virus vector is selected from the group consisting of a wild-type AAV vector, a hybrid AAV vector, and a mutant AAV vector.

[0055] In a further embodiment, the wild-type Ad vector is Ad5 and the hybrid Ad vector is Ad5 / F35.

[0056] In yet another embodiment, the hybrid AAV vector is AAV-DJ and the mutant AAV vector is AAV2-QuadYF.

[0057] In one embodiment, the vector or construct is a synthetic mRNA, a naked alphavirus RNA replicon or a naked flavivirus RNA replicon.

[0058] In one embodiment, the lentiviral vector contains a chimeric 5' long terminal repeat (LTR) fused to a heterologous enhancer / promoter, such as a Rous Sarcoma Virus (RSV) or CMV promoter.

[0059] In one embodiment, the lentiviral vector contains a deletion within the U3 region of the 3'LTR, such that the vector is replication-incompetent and self-inactivates after integration.

[0060] In one embodiment, the one or more constructs or vectors are one or more plasmids.

[0061] In one embodiment, the backbone of one or more constructs or vectors is selected from the group consisting of FUW, pRRL-cPPT, pRLL, pCCL, pCLL, pHAGE2, pWPXL, pLKO, pHIV, pLL, pCDH, and pLenti.

[0062] pRRL, pRLL, pCCL, and pCLL are lentiviral transfer vectors that contain a chimeric Rous Sarcoma Virus (RSV)-HIV or CMV-HIV 5'LTR and a vector backbone in which the Simian Virus 40 origin of polyadenylation and (enhancer-less) replication sequences are included downstream of the HIV 3'LTR, replacing most of the human sequences remaining from the HIV integration site. In pRRL, the enhancer and promoter from the U3 region of RSV (nucleotides -233 to -1 relative to the transcription start site, GenBank Accession No. J02342) are joined to the R region of the HIV-1 LTR. In pRLL, the RSV enhancer (nucleotides -233 to -50) sequences are joined to the promoter region of HIV-1 (from position -78 relative to the transcription start site). In pCCL, the CMV enhancer and promoter (nucleotides -673 to -1 relative to the transcription start site, GenBank accession number K03104) are linked to the R region of HIV-1. In pCLL, the CMV enhancer (nucleotides -673 to -220) is linked to the HIV-1 promoter region (position -78).

[0063] One or more of the constructs or vectors disclosed herein can include any type of element in addition to a polynucleotide encoding a TF and a promoter region(s) that facilitates expression of the TF, for example, one or more of the constructs or vectors can include regulatory, selectable, and / or structural elements and / or sequences.

[0064] In one embodiment, the one or more constructs or vectors comprise a self-cleaving peptide operably linked to at least two of the at least three coding regions, thus forming a single open reading frame. The self-cleaving peptide may be any type of self-cleaving peptide. In one embodiment, the self-cleaving peptide is a 2A peptide. In one embodiment, the 2A peptide is selected from the group consisting of Equine rhinitis A virus (E2A), foot and mouth disease virus (F2A), porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A) peptides.

[0065] In one embodiment, one or more of the constructs or vectors comprises a post-transcriptional regulatory element (PRE) sequence. In a preferred embodiment, the PRE sequence is a Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE).

[0066] In one embodiment, one or more of the constructs or vectors comprise 5' and 3' terminal repeats. In a preferred embodiment, at least one of the 5' and 3' terminal repeats is a self-inactivating (SIN) design with a partial deletion of U3 of the lentiviral long terminal repeat or the 3' long terminal repeat.

[0067] In one embodiment, one or more of the constructs or vectors comprises a central polypurine tract (cPPT).

[0068] In one embodiment, one or more of the constructs or vectors comprises a nucleocapsid protein packaging target site. In a preferred embodiment, the protein packaging target site comprises an HIV-1 psi sequence.

[0069] In one embodiment, one or more of the constructs or vectors comprises a REV protein response element (RRE).

[0070] The compositions disclosed herein may further comprise additional components, such as components that improve the efficiency of reprogramming cells according to the methods disclosed herein. The additional components may be macromolecules, such as proteins, for example cytokines.

[0071] Cytokines are small proteins (peptides) important in cell signaling. They cannot cross the lipid bilayer of a cell to enter the cytoplasm, but act through surface receptors that regulate intracellular signaling pathways. They have been shown to participate in autocrine, paracrine, and endocrine signaling as immunomodulatory agents. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors.

[0072] In one embodiment, the composition further comprises one or more pro-inflammatory cytokines. In one embodiment, the composition further comprises one or more hematopoietic cytokines. In one embodiment, the composition further comprises one or more cytokines selected from the group consisting of IFNβ, IFNγ, TNFα, IFNα, IL-1β, IL-6, CD40I, Flt3I, GM-CSF, IFN-λ1, IFN-ω, IL-2, IL-4, IL-15, prostaglandin 2, SCF, and oncostatin M (OM). In a preferred embodiment, the one or more cytokines are selected from the group consisting of IFNβ, IFNγ, and TNFα.

[0073] Additional components may include, for example, small molecules. Small molecules are low molecular weight molecules including lipids, monosaccharides, second messengers, other natural products, and metabolites, as well as other xenobiotics that are distinct from macromolecules such as drugs and proteins. Small molecules have a high level of cell permeability, are inexpensive to produce, are easy to synthesize, and are standardized.

[0074] In one embodiment, the composition further comprises one or more small molecules.

[0075] The small molecule may be, for example, a small molecule that functions as an epigenetic modulator. The small molecule may also be, for example, a small molecule that targets epigenetic control of gene expression, such as histone deacetylase inhibitors (HDACi), DNA methyltransferase inhibitors, histone methyltransferase (HMT) inhibitors, or histone demethylase inhibitors.

[0076] Thus, in one embodiment, the composition further comprises one or more histone deacetylase inhibitors.

[0077] In one embodiment, the composition further comprises valproic acid, suberoylanilide hydroxamic acid (SAHA), trichostatin A (TSA), sodium butyrate.

[0078] Thus, in one embodiment, the composition further comprises one or more DNA methyltransferase inhibitors, such as 5'-azacytidine (5'-azaC) or RG108.

[0079] In one embodiment, the composition further comprises one or more histone methyltransferase (HMT) inhibitors, for example, BIX-01294, an inhibitor of G9a-mediated inhibition of H3K9me2 methylation.

[0080] In one embodiment, the composition further comprises one or more histone demethylase inhibitors, such as parnate (an LSD1 inhibitor).

[0081] Such additional components may also include, for example, nucleic acids encoding additional TFs or genes associated with successful reprogramming.

[0082] Thus, in one embodiment, the composition further comprises one or more additional TFs and / or genes encoding one or more additional TFs, wherein the one or more TFs are associated with successful reprogramming. In one embodiment, the one or more TFs associated with successful reprogramming are selected from the TFs associated with successful reprogramming listed in Table 1.

[0083] In one embodiment, the composition further comprises one or more additional TFs associated with successful reprogramming and / or genes encoding additional TFs, wherein the one or more TFs associated with successful reprogramming are selected from the list in Table 1.

[0084] In one embodiment, the composition comprises cells expressing one or more additional surface markers, wherein the one or more additional surface markers are selected from the surface markers listed in Table 1.

[0085] [Table 1] TIFF2024522074000003.tif239159TIFF2024522074000004.tif242159TIFF2024522074000005.tif242159TIFF2024522074000006.tif213159

[0086] As described above, the composition may further comprise a gene encoding a protein associated with successful reprogramming, hi one embodiment, the gene encodes a protein other than a TF.

[0087] Expression of the genes can be examined using methods known in the art, such as, for example, transcriptomics or other methods described herein.

[0088] In one embodiment, the composition is a pharmaceutical composition.

[0089] cell As used herein, a transcription factor upon expression: a) BATF3, or a biologically active variant thereof that is at least 70% identical to SEQ ID NO: 10 (BATF3); b) IRF8 or a biologically active variant thereof that is at least 70% identical to SEQ ID NO: 11 (IRF8), and c) PU.1 or a biologically active variant thereof which is at least 70% identical to SEQ ID NO: 12 (PU.1); d) IRF7 or a biologically active variant thereof that is at least 70% identical to SEQ ID NO: 21 (IRF7); e) BATF or a biologically active variant thereof that is at least 70% identical to SEQ ID NO: 19 (BATF); f) SPIB or a biologically active variant thereof which is at least 70% identical to SEQ ID NO: 23 (SPIB); g) SPIC or a biologically active variant thereof which is at least 70% identical to SEQ ID NO: 25 (SPIC), and / or h) CEBPα or a biologically active variant thereof which is at least 70% identical to SEQ ID NO: 13 (CEBPα), or any combination thereof; and wherein the one or more constructs or vectors comprise a promoter region capable of controlling transcription of a transcription factor, the promoter region comprising a spleen focus forming virus (SFFV) promoter, an MND (myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer binding site substitution) promoter, a CAG (CMV early enhancer / chicken beta actin) promoter, a cytomegalovirus (CMV) promoter, a ubiquitin C (UbC) promoter, an EF-1 alpha (EF-1α) promoter, an EF-1 alpha short (EF1S) promoter, an EF-1 alpha with intron (EF1i) promoter, a phosphoglycerate kinase (PGK) promoter, or a promoter that exhibits substantially the same effect.

[0090] In one embodiment, the cell comprises: a) one construct or vector that upon expression encodes the transcription factors BATF3, IRF8, and PU.1; b) one construct or vector that, upon expression, encodes the transcription factors BATF3, IRF8, and SPIB; c) a first construct or vector that, upon expression, encodes the transcription factors BATF3 and IRF8, and a second construct or vector that, upon expression, encodes the transcription factor PU.1; d) a first construct or vector that, upon expression, encodes the transcription factors BATF3 and IRF8, and a second construct or vector that, upon expression, encodes the transcription factor SPIB; e) a first construct or vector that, upon expression, encodes the transcription factor BATF3 and a second construct or vector that, upon expression, encodes the transcription factors IRF8 and PU.1; f) a first construct or vector that, upon expression, encodes the transcription factor BATF3 and a second construct or vector that, upon expression, encodes the transcription factors IRF8 and SPIB; g) a first construct or vector that, upon expression, encodes the transcription factor IRF8 and a second construct or vector that, upon expression, encodes the transcription factors BATF3 and PU.1; h) a first construct or vector that, upon expression, encodes the transcription factor IRF8 and a second construct or vector that, upon expression, encodes the transcription factors BATF3 and SPIB; i) a first construct or vector that, upon expression, encodes the transcription factor BATF3, a second construct or vector that, upon expression, encodes the transcription factor IRF8, and a third construct or vector that, upon expression, encodes the transcription factor PU.1, and / or j) a first construct or vector that, when expressed, encodes the transcription factor BATF3; a second construct or vector that, when expressed, encodes the transcription factor IRF8; and a third construct or vector that, when expressed, encodes the transcription factor SPIB; Includes.

[0091] The TF may be as defined in the "Transcription Factor" section herein.

[0092] The promoter region may be as defined in the "Promoter" section herein.

[0093] The one or more constructs or vectors may be as defined in the "Compositions" section herein.

[0094] The cell may be of any cell type. In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is a human cell. In another embodiment, the cell is a mouse cell.

[0095] In one embodiment, the cell is selected from the group consisting of a stem cell, a differentiated cell, and a cancer cell.

[0096] In one embodiment, the stem cells are selected from the group consisting of pluripotent stem cells, endoderm-derived cells, mesoderm-derived cells, ectoderm-derived cells and multipotent stem cells, such as mesenchymal stem cells and hematopoietic stem cells.

[0097] In one embodiment, the differentiated cell is a cancer cell, e.g., a solid tumor cell, a hematopoietic tumor cell, a melanoma cell, a bladder cancer cell, a breast cancer cell, a lung cancer cell, a pleural cancer cell, a colon cancer cell, a rectal cancer cell, a colorectal cancer cell, a prostate cancer cell, a liver cancer cell, a pancreatic cancer cell, a cholangiocarcinoma cell, a gastric cancer cell, a testicular cancer cell, a brain cancer cell, an ovarian cancer cell, a lymphatic cancer cell, a lymphoma cell, a sarcoma cancer cell, a skin cell, a brain cancer cell, a bone cancer cell, an oral cancer cell, a head and neck cancer cell, or a soft tissue cancer cell, e.g., a glioblastoma cell, a rectal cancer cell, or a mesothelioma cell.

[0098] In one embodiment, the differentiated cell is any somatic cell.

[0099] In one embodiment, the somatic cells are selected from the group consisting of fibroblasts and hematopoietic cells, such as monocytes.

[0100] The cells disclosed herein can be further manipulated or modified to improve reprogramming efficiency according to the method disclosed herein in the "Method" section. Such modifications can include, for example, overexpression or silencing of genes encoding TFs that are related to successful reprogramming, respectively. It can also include overexpression or silencing of other genes that are related to the efficiency of reprogramming, for example, overexpression or silencing of genes that are differentially expressed upon expression of the TFs disclosed herein in the "Transcription Factor" section. Methods for overexpressing or silencing genes are well known in the art.

[0101] According to one embodiment, the cells are engineered to overexpress one or more genes encoding TFs associated with successful reprogramming, such as one or more genes encoding TFs associated with successful reprogramming listed in Table 1. In one embodiment, the cells are engineered to overexpress one or more genes encoding TFs associated with successful reprogramming, wherein the one or more genes encoding TFs associated with successful reprogramming are selected from the list in Table 1.

[0102] method Provided herein is a method for reprogramming or inducing a cell into a dendritic cell or an antigen presenting cell, the method comprising: a) Treating cells with a transcription factor, upon expression: i) BATF3, or a biologically active variant thereof that is at least 70% identical to SEQ ID NO: 10 (BATF3); ii) IRF8 or a biologically active variant thereof that is at least 70% identical to SEQ ID NO: 11 (IRF8); and iii) PU.1 or a biologically active variant thereof which is at least 70% identical to SEQ ID NO: 12 (PU.1); iv) IRF7 or a biologically active variant thereof that is at least 70% identical to SEQ ID NO: 21 (IRF7); v) BATF or a biologically active variant thereof that is at least 70% identical to SEQ ID NO: 19 (BATF); vi) SPIB or a biologically active variant thereof which is at least 70% identical to SEQ ID NO: 23 (SPIB); vii) SPIC or a biologically active variant thereof which is at least 70% identical to SEQ ID NO: 25 (SPIC), and / or viii) CEBPα or a biologically active variant thereof which is at least 70% identical to SEQ ID NO: 13 (CEBPα); transducing with one or more constructs or vectors encoding a transcription factor, wherein the one or more constructs or vectors comprise a promoter region capable of controlling transcription of the transcription factor, the promoter region comprising a spleen focus forming virus (SFFV) promoter, an MND (myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer binding site substitution) promoter, a CAG (CMV early enhancer / chicken beta actin) promoter, a cytomegalovirus (CMV) promoter, a ubiquitin C (UbC) promoter, an EF-1 alpha (EF-1α) promoter, an EF-1 alpha short (EF1S) promoter, an EF-1 alpha with intron (EF1i) promoter, a phosphoglycerate kinase (PGK) promoter, or a promoter that exhibits substantially the same effect; b) expressing a transcription factor; thereby obtaining a reprogrammed or derived cell; A method is provided that includes:

[0103] In one embodiment, the cell to be reprogrammed or derived is not a dendritic cell or an antigen-presenting cell.

[0104] In one embodiment, the reprogramming or induction occurs in vivo, such as in an animal or human.

[0105] In another embodiment, the reprogramming or induction is in vitro.

[0106] In another embodiment, the reprogramming or induction is ex vivo.

[0107] In one embodiment, the method further comprises culturing the transduced cells in cell culture medium. The step of culturing the transduced cells in cell culture medium can be performed before or after step b) of the method, i.e. before or after expressing the transcription factor. In one embodiment, the step of culturing the transduced cells in cell culture medium is performed before expressing the transcription factor, i.e. after step a) and before step b) in the method presented herein. In one embodiment, the transduced cells are cultured for at least 2 days, such as at least 5 days, such as at least 8 days, such as at least 10 days, such as at least 12 days.

[0108] For example, for the efficiency of reprogramming, it may be advantageous for the cell culture medium to contain one or more additional components.

[0109] In one embodiment, the method further comprises culturing the transduced cells in a medium comprising one or more cytokines. In one embodiment, the one or more cytokines are pro-inflammatory cytokines. In one embodiment, the one or more cytokines are hematopoietic cytokines. In one embodiment, the one or more cytokines are selected from the group consisting of IFNβ, IFNγ, TNFα, IFNα, IL-1β, IL-6, CD40I, Flt3I, GM-CSF, IFN-λ1, IFN-ω, IL-2, IL-4, IL-15, prostaglandin 2, SCF, and oncostatin M (OM). In a preferred embodiment, the one or more cytokines are selected from the group consisting of IFNβ, IFNγ, and TNFα.

[0110] The method may further comprise culturing the transduced cells in a cell culture medium containing a small molecule. The small molecule may be, for example, a small molecule that functions as an epigenetic modulator. The small molecule may also be, for example, a small molecule that targets epigenetic control of gene expression, such as an epigenetic modifier, such as a histone deacetylase inhibitor (HDACi), a DNA methyltransferase inhibitor, a histone methyltransferase (HMT) inhibitor or a histone demethylase inhibitor, or any small molecule that belongs to these categories, such as the small molecule that belongs to these categories disclosed herein.

[0111] In some embodiments, the method further comprises culturing the transduced cells in a cell culture medium containing one or more histone deacetylase inhibitor(s).

[0112] In one embodiment, the one or more histone deacetylase inhibitors is valproic acid.

[0113] In one embodiment, the cell comprises: a) one construct or vector that upon expression encodes the transcription factors BATF3, IRF8, and PU.1; b) one construct or vector that, upon expression, encodes the transcription factors BATF3, IRF8, and SPIB; c) a first construct or vector that, upon expression, encodes the transcription factors BATF3 and IRF8, and a second construct or vector that, upon expression, encodes the transcription factor PU.1; d) a first construct or vector that, upon expression, encodes the transcription factors BATF3 and IRF8, and a second construct or vector that, upon expression, encodes the transcription factor SPIB; e) a first construct or vector that, upon expression, encodes the transcription factor BATF3 and a second construct or vector that, upon expression, encodes the transcription factors IRF8 and PU.1; f) a first construct or vector that, upon expression, encodes the transcription factor BATF3 and a second construct or vector that, upon expression, encodes the transcription factors IRF8 and SPIB; g) a first construct or vector that, upon expression, encodes the transcription factor IRF8 and a second construct or vector that, upon expression, encodes the transcription factors BATF3 and PU.1; h) a first construct or vector that, upon expression, encodes the transcription factor IRF8 and a second construct or vector that, upon expression, encodes the transcription factors BATF3 and SPIB; i) a first construct or vector that, upon expression, encodes the transcription factor BATF3, a second construct or vector that, upon expression, encodes the transcription factor IRF8, and a third construct or vector that, upon expression, encodes the transcription factor PU.1, and / or j) a first construct or vector that, when expressed, encodes the transcription factor BATF3; a second construct or vector that, when expressed, encodes the transcription factor IRF8; and a third construct or vector that, when expressed, encodes the transcription factor SPIB; The gene is transduced with

[0114] The TF may be as defined in the "Transcription Factor" section herein.

[0115] The promoter region may be as defined in the "Promoter" section herein.

[0116] The one or more constructs or vectors may be as defined in the "Compositions" section herein.

[0117] The cell may be as defined in the "Cell" section herein.

[0118] The method may include additional steps to improve reprogramming efficiency.

[0119] In one embodiment, the method further comprises overexpressing in the transduced cells one or more genes encoding TFs associated with successful reprogramming, such as one or more genes encoding TFs associated with successful reprogramming listed in Table 1.

[0120] In one embodiment, the method further comprises overexpressing in the transduced cells one or more genes encoding TFs associated with successful reprogramming, wherein the one or more genes encoding TFs associated with successful reprogramming are selected from the list in Table 1.

[0121] In another embodiment, the method further comprises overexpressing in the transduced cells one or more genes encoding proteins associated with successful reprogramming, in one embodiment, the genes encoding proteins other than TFs.

[0122] Gene overexpression and silencing can be achieved using methods known in the art, for example, by expressing the gene from a vector or deleting a portion of the gene or the entire gene from the cell, respectively.

[0123] In one embodiment, the resulting reprogrammed cells are conventional dendritic cells of type 1 (DC1). cDC1 is a special subset of DCs that express, for example, human leukocyte antigen-DR isotype (HLA-DR) and the hematopoietic marker cluster of differentiation 45 (CD45). cDC1 also have a typical RNA expression profile and express the surface markers cluster of differentiation 141 (CD141), C-type lectin domain family 9 member A (CLEC9A), XC motif chemokine receptor 1 (XCR1), and cluster of differentiation 226 (CD226).

[0124] Thus, in one embodiment, the resulting reprogrammed cells are enriched for one or more surface markers selected from the list in Table 1.

[0125] In one embodiment, the resulting reprogrammed cells are CD45 positive. In one embodiment, the resulting reprogrammed cells are HLA-DR positive. In one embodiment, the resulting reprogrammed cells are CD141 positive. In one embodiment, the resulting reprogrammed cells are CLEC9A positive. In one embodiment, the resulting reprogrammed or derived cells are CD226 positive. In one embodiment, the resulting reprogrammed or derived cells are XCR1 positive. In one embodiment, the resulting reprogrammed or derived cells are CD45, HLA-DR, CD141, CLEC9A, XCR1 and / or CD226 positive.

[0126] The method for determining whether a cell or a plurality of cells are cDC1 cells is well known in the art.For example, the cell can be determined whether it expresses CD45, HLA-DR, CD226, CD141, XCR1 and / or CLEC9A by incubating the cell with fluorophore-conjugated antibodies specific to CD45, HLA-DR, CD226, CD141, XCR1 and / or CLEC9A, and then screen the cell using flow cytometry.For example, the cell can be determined whether it expresses the surface marker(s) selected from the list of table 1, and identified as successful reprogramming of the cell into cDC1, by incubating the cell with fluorophore-conjugated antibodies specific to one or more surface markers, and then screen the cell using flow cytometry.

[0127] Furthermore, the RNA profile of cells can be determined using single-cell RNA seq, and if the RNA profile is identical or similar to the RNA profile of natural cDC1 cells, it can be used to classify the cells as cDC1.Furthermore, the cells can be characterized with respect to their functional properties, such as their response to TLR stimulation and their ability to upregulate the surface expression of CD40, CD80 and other costimulatory molecules, their ability to secrete proinflammatory cytokines and chemokines, and their ability to activate antigen-specific T cells.

[0128] Thus, provided herein is a reprogrammed or derived cell obtained by the methods provided herein. In one embodiment, the cell is a dendritic cell or an antigen-presenting cell.

[0129] Transcription factors Transcription factors (TFs) are proteins that control the rate of transcription of genetic information from DNA to mRNA by binding to specific DNA sequences. The function of TFs is to control the expression of genes, i.e., turn them on and off. Groups of TFs work in concert to direct cell division, cell proliferation, and cell death throughout life, for cell migration and organization during embryonic development, and intermittently in response to extracellular signals such as hormones. There are up to 1600 TFs in the human genome. Transcription factors are members of the proteome as well as the regulome.

[0130] TFs act alone or in collaboration with other proteins in complexes by promoting (as activators) or blocking (as repressors) the recruitment of RNA polymerase to specific genes. The defining feature of TFs is that they contain at least one DNA-binding domain (DBD) that binds to specific sequences of DNA adjacent to the genes they regulate.

[0131] Provided herein are TFs that can be used to reprogram cells into dendritic cells or antigen-presenting cells, including BATF3, IRF8, PU.1, IRF7, BATF, SPIB, SPIC, and CEBPA.

[0132] BATF3 is a nuclear basic leucine zipper that belongs to the AP-1 / ATF superfamily of TFs. It mediates the expression of CD8 in the immune system. + It controls the differentiation of conventional dendritic cells in the thymus. It acts by forming heterodimers with JUN family proteins that recognize and bind specific DNA sequences to regulate the expression of target genes.

[0133] IRF8 is a TF that belongs to the interferon regulatory factor (IRF) family. It plays a role in the control of lineage commitment and maturation of myeloid cells. IRF8, as well as other TFs in the IRF family, bind to IFN-stimulated response elements and regulate the expression of genes stimulated by type I IFN.

[0134] PU.1 is a TF belonging to the erythroid transformation specific (ETS) domain family. It is a transcriptional activator that binds to the PU-box, a purine-rich DNA sequence that may function as a lymphocyte-specific enhancer. PU.1 may be specifically involved in the differentiation or activation of myeloid cells, such as macrophages and dendritic cells, as well as B cells.

[0135] IRF7 is a TF that belongs to the interferon regulatory factor (IRF) family. IRF7 has been shown to play a role in the transcriptional activation of virus-induced cellular genes, including type I interferon genes. IRF7 is constitutively expressed in lymphoid tissues and inducible in many other tissues throughout the body.

[0136] BATF is a nuclear basic leucine zipper that belongs to the AP-1 / ATF superfamily of TFs. BATF can interact with partner transcription factors, including IRF8 and IRF4, via its leucine zipper domain to mediate coordinate gene activation. Compensation between BATF factors has been previously demonstrated in the context of cDC1 development.

[0137] SPIB is a TF belonging to the erythroid transformation-specific (ETS) domain family. Like PU.1, SPIB is a sequence-specific transcriptional activator that binds to the PU-box, a purine-rich DNA sequence that may function as a lymphocyte-specific enhancer. It promotes the development of plasmacytoid dendritic cells (pDCs) and cDC precursors.

[0138] SPIC is a TF that belongs to the erythroid transformation-specific (ETS) domain family. Like PU.1 and SPIB, SPIC is a sequence-specific transcriptional activator that binds to the purine-rich DNA sequence PU-box. SPIC controls the development of red pulp macrophages, which are required for red blood cell recycling and iron homeostasis.

[0139] CΕΒΡα (CCAAT enhancer-binding protein alpha) is a TF that contains a basic leucine zipper (bZIP) domain and recognizes the CCAAT motif in the promoters of target genes. CΕΒΡα regulates growth arrest and differentiation of bone marrow progenitor cells, adipocytes, hepatocytes, and lung and placental cells.

[0140] Also disclosed herein are biologically active variants of BATF3, IRF8, PU.1, IRF7, BATF, SPIC, and SPIB. A biologically active variant is a variant of the TF that retains at least some of the activity of the parent TF. For example, a biologically active variant of SPIB, SPIC, BATF, BATF3, IRF8, or PU.1 can induce and / or inhibit the expression of the same genes as the parent BATF3, IRF8, or PU.1, respectively. The three biologically active variants of SPIB, SPIC, BATF, BATF3, IRF8, and PU.1 can reprogram or induce cells into dendritic cells or antigen-presenting cells according to the methods disclosed herein. However, the biologically active variants of each TF may be more or less efficient compared to the respective parent TF. For example, the efficiency of inducing and / or inhibiting the expression of genes and / or the efficiency of reprogramming or inducing cells into dendritic cells may be increased or decreased compared to the respective parent TF.

[0141] In one embodiment the biologically active variant of BATF3 is at least 60% identical to SEQ ID NO:10, such as at least 61%, for example at least 62%, such as at least 63%, for example at least 64%, such as at least 65%, for example at least 66%, such as at least 67%, for example at least 68%, such as at least 69%, for example at least 70%, such as at least 71%, for example at least 72%, such as at least 73%, for example at least 74%, such as at least 75%, for example at least 76%, such as at least 77%, for example at least 78%, such as at least 79%, for example at least 80%, such as at least 81%, for example at least 82%, such as at least 83%, for example at least 84%, for example at least 85%, for example at least 86%, for example at least 87%, for example at least 88%, for example at least 89%, 8%, such as at least 79%, for example at least 80%, such as at least 81%, for example at least 82%, such as at least 83%, for example at least 84%, such as at least 85%, for example at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99%, for example 100% identical.

[0142] In one embodiment the biologically active variant of IRF8 is at least 60% identical to SEQ ID NO:11, such as at least 61%, for example at least 62%, such as at least 63%, for example at least 64%, such as at least 65%, for example at least 66%, such as at least 67%, for example at least 68%, such as at least 69%, for example at least 70%, such as at least 71%, for example at least 72%, such as at least 73%, for example at least 74%, such as at least 75%, for example at least 76%, such as at least 77%, for example at least 78%, such as at least 79%, for example at least 80%, such as at least 81%, for example at least 82%, such as at least 83%, for example at least 84%, for example at least 85%, for example at least 86%, for example at least 87%, for example at least 88%, 8%, such as at least 79%, for example at least 80%, such as at least 81%, for example at least 82%, such as at least 83%, for example at least 84%, such as at least 85%, for example at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99%, for example 100% identical.

[0143] In one embodiment the biologically active variant of PU.1 is at least 60% identical to SEQ ID NO:12, such as at least 61%, for example at least 62%, such as at least 63%, for example at least 64%, such as at least 65%, for example at least 66%, such as at least 67%, for example at least 68%, such as at least 69%, for example at least 70%, such as at least 71%, for example at least 72%, such as at least 73%, for example at least 74%, such as at least 75%, for example at least 76%, such as at least 77%, for example at least 78%, for example at least 79%, for example at least 80%, for example at least 81%, for example at least 82%, for example at least 83%, for example at least 84%, for example at least 85%, for example at least 86%, for example at least 87%, for example at least 88%, for example at least 89%, 8%, such as at least 79%, for example at least 80%, such as at least 81%, for example at least 82%, such as at least 83%, for example at least 84%, such as at least 85%, for example at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99%, for example 100% identical.

[0144] In one embodiment the biologically active variant of IRF7 is at least 60% identical to SEQ ID NO:21 (IRF7), such as at least 61%, for example at least 62%, such as at least 63%, for example at least 64%, such as at least 65%, for example at least 66%, such as at least 67%, for example at least 68%, such as at least 69%, for example at least 70%, such as at least 71%, for example at least 72%, such as at least 73%, for example at least 74%, such as at least 75%, for example at least 76%, such as at least 77%, for example at least 78%, for example at least 79%, 78%, such as at least 79%, for example at least 80%, such as at least 81%, for example at least 82%, such as at least 83%, for example at least 84%, such as at least 85%, for example at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99%, for example 100% identical.

[0145] In one embodiment, the biologically active variant of BATF is at least 60% identical to SEQ ID NO:19 (BATF), such as at least 61%, for example at least 62%, such as at least 63%, for example at least 64%, such as at least 65%, for example at least 66%, such as at least 67%, for example at least 68%, such as at least 69%, for example at least 70%, such as at least 71%, for example at least 72%, such as at least 73%, for example at least 74%, such as at least 75%, for example at least 76%, such as at least 77%, for example at least 78%, such as at least 79%, for example at least 80%, such as at least 81%, for example at least 82%, such as at least 83%, for example at least 84%, for example at least 85%, for example at least 86%, 78%, such as at least 79%, for example at least 80%, such as at least 81%, for example at least 82%, such as at least 83%, for example at least 84%, such as at least 85%, for example at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99%, for example 100% identical.

[0146] In one embodiment the biologically active variant of SPIB is at least 60% identical to SEQ ID NO:23 (SPIB), such as at least 61%, for example at least 62%, such as at least 63%, for example at least 64%, such as at least 65%, for example at least 66%, such as at least 67%, for example at least 68%, such as at least 69%, for example at least 70%, such as at least 71%, for example at least 72%, such as at least 73%, for example at least 74%, such as at least 75%, for example at least 76%, such as at least 77%, for example at least 78%, for example at least 79%, 78%, such as at least 79%, for example at least 80%, such as at least 81%, for example at least 82%, such as at least 83%, for example at least 84%, such as at least 85%, for example at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99%, for example 100% identical.

[0147] In one embodiment the biologically active variant of SPIC is at least 60% identical to SEQ ID NO:25 (SPIC), such as at least 61%, for example at least 62%, such as at least 63%, for example at least 64%, such as at least 65%, for example at least 66%, such as at least 67%, for example at least 68%, such as at least 69%, for example at least 70%, such as at least 71%, for example at least 72%, such as at least 73%, for example at least 74%, such as at least 75%, for example at least 76%, such as at least 77%, for example at least 78%, for example at least 79%, 78%, such as at least 79%, for example at least 80%, such as at least 81%, for example at least 82%, such as at least 83%, for example at least 84%, such as at least 85%, for example at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99%, for example 100% identical.

[0148] In one embodiment the biologically active variant of CEBPA is at least 60% identical to SEQ ID NO: 13 (CEBPα), such as at least 61%, for example at least 62%, such as at least 63%, for example at least 64%, such as at least 65%, for example at least 66%, such as at least 67%, for example at least 68%, such as at least 69%, for example at least 70%, such as at least 71%, for example at least 72%, such as at least 73%, for example at least 74%, such as at least 75%, for example at least 76%, such as at least 77%, for example at least 78%, such as at least 79%, for example at least 80%, such as at least 81%, for example at least 82%, such as at least 83%, for example at least 84%, for example at least 85%, %, such as at least 78%, for example at least 79%, such as at least 80%, for example at least 81%, such as at least 82%, for example at least 83%, such as at least 84%, for example at least 85%, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, for example at least 98%, for example at least 99%, such as 100% identical.

[0149] In one embodiment BATF3 has at least 60% sequence identity to SEQ ID NO:14, such as at least 61%, for example at least 62%, such as at least 63%, for example at least 64%, such as at least 65%, for example at least 66%, such as at least 67%, for example at least 68%, such as at least 69%, for example at least 70%, such as at least 71%, for example at least 72%, such as at least 73%, for example at least 74%, such as at least 75%, for example at least 76%, such as at least 77%, for example at least 78%, such as at least 79% , such as at least 80%, for example at least 81%, such as at least 82%, for example at least 83%, such as at least 84%, for example at least 85%, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, for example at least 98%, such as at least 99%, for example 100% sequence identity.

[0150] In one embodiment IRF8 has at least 60% sequence identity to SEQ ID NO:15, such as at least 61%, for example at least 62%, such as at least 63%, for example at least 64%, such as at least 65%, for example at least 66%, such as at least 67%, for example at least 68%, such as at least 69%, for example at least 70%, such as at least 71%, for example at least 72%, such as at least 73%, for example at least 74%, such as at least 75%, for example at least 76%, such as at least 77%, for example at least 78%, such as at least 79%, For example encoded by a polynucleotide sequence having at least 80%, such as at least 81%, for example at least 82%, such as at least 83%, for example at least 84%, such as at least 85%, for example at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, for example at least 99%, such as 100% sequence identity.

[0151] In one embodiment PU.1 has at least 60% sequence identity to SEQ ID NO:16, such as at least 61%, for example at least 62%, such as at least 63%, for example at least 64%, such as at least 65%, for example at least 66%, such as at least 67%, for example at least 68%, such as at least 69%, for example at least 70%, such as at least 71%, for example at least 72%, such as at least 73%, for example at least 74%, such as at least 75%, for example at least 76%, such as at least 77%, for example at least 78%, such as at least 79%, For example encoded by a polynucleotide sequence having at least 80%, such as at least 81%, for example at least 82%, such as at least 83%, for example at least 84%, such as at least 85%, for example at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, for example at least 99%, such as 100% sequence identity.

[0152] In one embodiment IRF7 has at least 60% sequence identity to SEQ ID NO:20, such as at least 61%, for example at least 62%, such as at least 63%, for example at least 64%, such as at least 65%, for example at least 66%, such as at least 67%, for example at least 68%, such as at least 69%, for example at least 70%, such as at least 71%, for example at least 72%, such as at least 73%, for example at least 74%, such as at least 75%, for example at least 76%, such as at least 77%, for example at least 78%, such as at least 79%, For example encoded by a polynucleotide sequence having at least 80%, such as at least 81%, for example at least 82%, such as at least 83%, for example at least 84%, such as at least 85%, for example at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, for example at least 99%, such as 100% sequence identity.

[0153] In one embodiment BATF has at least 60% sequence identity to SEQ ID NO:18, such as at least 61%, for example at least 62%, such as at least 63%, for example at least 64%, such as at least 65%, for example at least 66%, such as at least 67%, for example at least 68%, such as at least 69%, for example at least 70%, such as at least 71%, for example at least 72%, such as at least 73%, for example at least 74%, such as at least 75%, for example at least 76%, such as at least 77%, for example at least 78%, such as at least 79%, For example encoded by a polynucleotide sequence having at least 80%, such as at least 81%, for example at least 82%, such as at least 83%, for example at least 84%, such as at least 85%, for example at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, for example at least 99%, such as 100% sequence identity.

[0154] In one embodiment the SPIB has at least 60% sequence identity to SEQ ID NO:22, such as at least 61%, for example at least 62%, such as at least 63%, for example at least 64%, such as at least 65%, for example at least 66%, such as at least 67%, for example at least 68%, such as at least 69%, for example at least 70%, such as at least 71%, for example at least 72%, such as at least 73%, for example at least 74%, such as at least 75%, for example at least 76%, such as at least 77%, for example at least 78%, such as at least 79%, For example encoded by a polynucleotide sequence having at least 80%, such as at least 81%, for example at least 82%, such as at least 83%, for example at least 84%, such as at least 85%, for example at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, for example at least 99%, such as 100% sequence identity.

[0155] In one embodiment the SPIC has at least 60% sequence identity to SEQ ID NO:24, such as at least 61%, for example at least 62%, such as at least 63%, for example at least 64%, such as at least 65%, for example at least 66%, such as at least 67%, for example at least 68%, such as at least 69%, for example at least 70%, such as at least 71%, for example at least 72%, such as at least 73%, for example at least 74%, such as at least 75%, for example at least 76%, such as at least 77%, for example at least 78%, such as at least 79%, For example encoded by a polynucleotide sequence having at least 80%, such as at least 81%, for example at least 82%, such as at least 83%, for example at least 84%, such as at least 85%, for example at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, for example at least 99%, such as 100% sequence identity.

[0156] In one embodiment CEBPα has at least 60% sequence identity to SEQ ID NO:17, such as at least 61%, for example at least 62%, such as at least 63%, for example at least 64%, such as at least 65%, for example at least 66%, such as at least 67%, for example at least 68%, such as at least 69%, for example at least 70%, such as at least 71%, for example at least 72%, such as at least 73%, for example at least 74%, such as at least 75%, for example at least 76%, such as at least 77%, for example at least 78%, such as at least 79% , such as at least 80%, for example at least 81%, such as at least 82%, for example at least 83%, such as at least 84%, for example at least 85%, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, for example at least 98%, such as at least 99%, for example 100% sequence identity.

[0157] promoter A promoter or promoter region is a sequence of DNA to which a protein binds and initiates transcription of a single RNA from the downstream DNA. This RNA may be an mRNA that codes for a protein, or may have a function such as transfer RNA (tRNA) or ribosomal RNA (rRNA). The promoter is located near the transcription start site of a gene and is upstream of the gene on the DNA.

[0158] In addition to the core promoter, the eukaryotic promoter region may further include other elements, such as a transcription start site (TSS), a binding site for RNA polymerase, a TF binding site, and other regulatory elements and / or structural elements. The eukaryotic gene promoter region is typically located upstream of the gene and may have regulatory elements several kilobases away from the TSS. Such regulatory elements may be, for example, enhancers.

[0159] The TF disclosed herein is controlled by a promoter region, including a core promoter.The inventors have surprisingly shown that the reprogramming of cells by the method disclosed herein can be significantly improved by expressing the TF disclosed herein under a specific promoter or promoter region.Such promoter regions include SFFV promoter, MND promoter, CAG promoter, CMV promoter, EF-1α promoter, EF1S promoter, EF1i promoter, PGK promoter, and other promoters that essentially show the same effect.

[0160] A promoter or promoter region that exhibits substantially the same effect is defined herein as a promoter or promoter region whose expression level of the gene(s) it controls exhibits the same expression level of the gene(s) as the promoter or promoter region disclosed herein. Therefore, whether a promoter region exhibits substantially the same effect as the promoter region disclosed herein can be measured by measuring the expression level of the gene(s) controlled by the promoter region and comparing it with the expression level of the same gene(s) controlled by the promoter region disclosed herein, and the expression level of the tested promoter region and the expression level of the promoter region disclosed herein are tested under the same conditions. Methods for measuring expression levels are well known in the art and can be performed using routine experimentation. For example, the expression level of a gene controlled by a particular promoter region can be measured by measuring the amount of messenger RNA (mRNA) produced by the expression of the gene. The amount of mRNA can be measured, for example, using reverse transcription polymerase chain reaction (RT-PCR) or transcriptomics. The expression level of a gene controlled by a particular promoter region can also be measured by measuring the amount of protein, i.e. the amount of gene product, produced by the expression of the gene using proteomics or Western blot.Herein, a promoter that shows substantially the same effect as the promoter region disclosed herein is defined as a promoter region that produces an expression level that is 50% higher or 50% lower than the expression level of the promoter region disclosed herein, for example 45%, for example 40%, for example 35%, for example 30%, for example 25%, for example 20%, for example 15%, for example 10%, for example 5% higher or lower than the expression level of the promoter disclosed herein.

[0161] The TFs disclosed herein can be controlled by any of the promoter regions disclosed. In one embodiment, the same promoter region controls the expression of at least one TF, for example, at least two TFs, for example, three TFs. In one embodiment, a first promoter region controls the expression of a first TF, a second promoter region controls the expression of a second TF, and a third promoter region controls the expression of a third TF. In one embodiment, a first promoter region controls the expression of a first and a second TF, and a second promoter region controls the expression of a third TF. The TFs can be as disclosed in the "transcription factors" section of this specification.

[0162] In one embodiment the SFFV promoter comprises or consists of a polynucleotide sequence which is at least 70% identical to SEQ ID NO:1, such as at least 75%, for example at least 80%, such as at least 81%, for example at least 82%, such as at least 83%, for example at least 84%, such as at least 85%, for example at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, for example at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, for example at least 99%, such as 100% identical to SEQ ID NO:1.

[0163] In one embodiment the MND promoter comprises or consists of a polynucleotide sequence which is at least 70% identical to SEQ ID NO:2, such as at least 75%, for example at least 80%, such as at least 81%, for example at least 82%, such as at least 83%, for example at least 84%, such as at least 85%, for example at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, for example at least 97%, such as at least 98%, for example at least 99%, such as 100% to SEQ ID NO:2.

[0164] In one embodiment, the CAG promoter comprises or consists of a polynucleotide sequence which is at least 70% identical to SEQ ID NO:3, such as at least 75%, for example at least 80%, such as at least 81%, for example at least 82%, such as at least 83%, for example at least 84%, such as at least 85%, for example at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, for example at least 97%, such as at least 98%, for example at least 99%, such as 100% to SEQ ID NO:3.

[0165] In one embodiment the CMV promoter comprises or consists of a polynucleotide sequence which is at least 70% identical to SEQ ID NO:4, such as at least 75%, for example at least 80%, such as at least 81%, for example at least 82%, such as at least 83%, for example at least 84%, such as at least 85%, for example at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99%, for example 100% to SEQ ID NO:4.

[0166] In one embodiment the UbC promoter comprises or consists of a polynucleotide sequence which is at least 70% identical to SEQ ID NO:5, such as at least 75%, for example at least 80%, such as at least 81%, for example at least 82%, such as at least 83%, for example at least 84%, such as at least 85%, for example at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99%, for example 100% to SEQ ID NO:5.

[0167] In one embodiment, the EF-1α promoter comprises or consists of a polynucleotide sequence which is at least 70% identical to SEQ ID NO:6, such as at least 75%, for example at least 80%, such as at least 81%, for example at least 82%, such as at least 83%, for example at least 84%, such as at least 85%, for example at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99%, for example 100% to SEQ ID NO:6.

[0168] In one embodiment, the EF1S promoter comprises or consists of a polynucleotide sequence which is at least 70% identical to SEQ ID NO:7, such as at least 75%, for example at least 80%, such as at least 81%, for example at least 82%, such as at least 83%, for example at least 84%, such as at least 85%, for example at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, such as at least 99%, for example 100% to SEQ ID NO:7.

[0169] In one embodiment, the EF1i promoter comprises or consists of a polynucleotide sequence which is at least 70% identical to SEQ ID NO:8, such as at least 75%, for example at least 80%, such as at least 81%, for example at least 82%, such as at least 83%, for example at least 84%, such as at least 85%, for example at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99%, for example 100% to SEQ ID NO:8.

[0170] In one embodiment the PGK promoter comprises or consists of a polynucleotide sequence which is at least 70% identical to SEQ ID NO:9, such as at least 75%, such as at least 80%, for example at least 81%, such as at least 82%, for example at least 83%, such as at least 84%, for example at least 85%, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, for example at least 99%, such as 100% to SEQ ID NO:9.

[0171] Treatment method The compositions and methods disclosed herein can be used for the treatment and / or prevention of diseases or disorders, such as, for example, tumors and cancers or infectious diseases.

[0172] In one embodiment, the compositions and methods disclosed herein are used to treat tumors and / or cancer or infectious diseases.

[0173] In one embodiment, the tumor and / or cancer is a benign tumor, a malignant tumor, an early stage cancer, a basal cell carcinoma, a cervical dysplasia, a sarcoma, a germ cell tumor, a retinoblastoma, a glioblastoma, a lymphoma, a Hodgkin's lymphoma, a non-Hodgkin's lymphoma, a blood cancer, a prostate cancer, an ovarian cancer, a cervical cancer, an esophageal cancer, a uterine cancer, a vaginal cancer, a breast cancer, a head and neck cancer, a gastric cancer, an oral cancer, a nasopharyngeal cancer, a tracheal cancer, a laryngeal cancer, a bronchial cancer, a bronchial cancer, a pulmonary cancer, a pulmonary fibrosis ... The cancer is selected from the group consisting of cancer, bronchiolar cancer, lung cancer, pleural cancer, bladder urothelial cancer, hollow organ cancer, esophageal cancer, stomach cancer, bile duct cancer, intestinal cancer, colon cancer, colorectal cancer, rectal cancer, bladder cancer, ureter cancer, kidney cancer, liver cancer, gallbladder cancer, spleen cancer, brain cancer, lymphatic system cancer, bone cancer, pancreatic cancer, leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia, acute myelogenous leukemia, skin cancer, melanoma or myeloma. In a preferred embodiment, the cancer is selected from the group consisting of melanoma, head and neck cancer, bladder urothelial cancer, pancreatic cancer, and glioblastoma.

[0174] Thus, the cells produced by the methods described herein may be used to treat or prevent cancers including breast cancer, prostate cancer, lymphoma, skin cancer, pancreatic cancer, colon cancer, melanoma, malignant melanoma, esophageal cancer, ovarian cancer, brain cancer, primary brain cancer, head and neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer, head and neck cancer, breast cancer, ovarian cancer, lung cancer, small cell lung cancer, Wilms' tumor, cervical cancer, testicular cancer, bladder cancer, pancreatic cancer, gastric cancer, colon cancer, prostate cancer, genitourinary cancer, thyroid cancer, esophageal cancer, myeloma, multiple myeloma, adrenal cancer, renal cell carcinoma, endometrial cancer, adrenal cortical carcinoma, malignant pancreatic insulinoma, malignant carcinoid cancer, trophoblastoma, pleural effusion cancer ... The cells can be used to prepare cells for treating or alleviating several cancers and tumors, including, but not limited to, carcinoma, mycosis fungoides, malignant hypercalcemia, cervical hyperplasia, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, chronic granulocytic leukemia, acute granulocytic leukemia, hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma, polycythemia vera, essential thrombocytosis, Hodgkin's disease, non-Hodgkin's lymphoma, soft tissue sarcoma, osteogenic sarcoma, primary macroglobulinemia, and retinoblastoma.

[0175] Provided herein are the compositions, cells, pharmaceutical compositions, and / or reprogrammed or derived cells provided herein for use in medicine.

[0176] Also provided herein are the compositions, cells, pharmaceutical compositions, and / or reprogrammed or derived cells provided herein for use in the treatment of cancer or an infectious disease.

[0177] Further provided herein are methods of treating cancer or an infectious disease, comprising administering to an individual in need thereof a composition, cell, pharmaceutical composition, and / or a reprogrammed or derived cell as provided herein.

[0178] Also provided herein is the use of a composition, cell, pharmaceutical composition, and / or a reprogrammed or induced cell as provided herein for the manufacture of a medicament for treating cancer or an infectious disease. EXAMPLES

[0179] Example 1. General methods and materials cell culture Human embryonic kidney HEK293T cells, human embryonic fibroblasts (HEF) (passages 3–8) and human dermal fibroblasts (HDF) (passages 3–8) were maintained in growth medium Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v / v) heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine and antibiotics (10 U / ml penicillin 10 μg / ml streptomycin) (DMEM complete). Mouse embryonic fibroblasts (MEF) (passages 3–5) were isolated from E13.5 embryos of Clec9a-tdTomato reporter mice as previously described (Rosa et al. 2020) and cultured in DMEM complete in 0.1% gelatin-coated dishes. Monocyte-derived dendritic cells (moDCs) were cultured in RPMI 1640 (RPMI complete) supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, and antibiotics. cDC1s isolated from peripheral blood were maintained in RPMI complete supplemented with 50 μM 2-mercaptoethanol, 1 mM sodium pyruvate, and antibiotics. Mesenchymal stromal cells (MSCs, passages 3–5) were cultured in minimum essential medium (MEM) supplemented with 10% pooled human platelet lysate, 2 U / ml heparin (STEMCELL Technologies), 2 mM L-glutamine, and antibiotics. All tissue culture reagents were obtained from Thermo Fisher Scientific unless otherwise stated.

[0180] mouse C57BL / 6J and OT-I mice were obtained from Janvier and Taconic, respectively. Cre / Cre Rosa tdTomato / tdTomato(Clec9A-tdTomato) animals were a kind gift of Caetano Reis e Sousa, Francis Crick Institute, London, United Kingdom (Rosa et al., 2018) and were re-derived by Janvier before transporting across the country to the animal house at Lund University. All animals were housed under a controlled temperature (23 ± 2 °C) under a fixed 12-h light-dark cycle with free access to food and water. Animal care and experimental procedures were carried out in accordance with Swedish guidelines and regulations and after approval of the local committee.

[0181] Lentivirus production HEK293T cells were co-transfected with a mixture of transfer plasmids, packaging, and envelope constructs encoding VSV-G with polyethylenimine (PEI) as previously described (Rosa et al., 2020). Viral supernatants were harvested after 36, 48, and 72 hours, filtered (0.45 μm, low protein binding), concentrated 40-fold with a Lenti-X concentrator, and stored at -80°C.

[0182] Viral transduction and reprogramming HEFs, HDFs, and Clec9a-tdTomato MEFs were seeded at a density of 40,000 cells per well and MSCs at a density of 50,000 cells per well on 6-well plates coated with 0.1% gelatin. The next day, cells were incubated overnight with either a 1:1 ratio of TetO-PIB to M2rtTA, SFFV-PIB-GFP, or lentiviral particles of SFFV-GFP in medium supplemented with 8 μg / ml polybrene. Cells were transduced overnight on two consecutive days, with medium changes in between. After the second transduction, medium was replaced with normal growth medium (day 0). When using TetO-PIB, medium was supplemented with Dox (1 μg / ml). Medium was changed every 2–3 days over the culture period. Where mentioned, medium was supplemented with LPS (100 ng / ml, Enzo), poly I:C (25 μg / ml, InvivoGen) and R848 (3 μg / ml, InvivoGen) overnight. Cytokines were added on day 2 and maintained throughout the culture period. For reprogramming in xeno-free conditions, MSCs were cultured in X-Vivo 15 (Lonza) after transduction and medium was changed every 2-3 days throughout the culture period.

[0183] Molecular cloning For the doxycycline (Dox)-inducible overexpression system, the coding regions of human PU.1, IRF8, and BATF3 (PIB) were cloned into the pFUW-TetO plasmid in this order, separated by a 2A self-cleaving peptide. The first two coding sequences lacked a stop codon. The coding regions of PU.1, IRF8, BATF3, ID2, TXNIP, ZFP36PLEK, SUB1, JUNB, CREM, KLF4, MXD1, LITAF, IRF7, FOS, NMI, TFEC, SP110, IRF5, STAT2, BATF, ZNF267, IRF1, RELB, and BATF2 were individually cloned into the pFUW-TetO plasmid. A lentiviral vector (pFUW-UbC-M2rtTA) containing the reverse tetracycline transactivator M2rtTA under the control of the constitutively active human ubiquitin C promoter was used for co-transduction (Rosa et al. 2018). For constitutive overexpression, the human PIB polycistronic cassette was subcloned into lentiviral vectors with constitutive promoters pFUW-UbC, pRRL.PPT-SFFV, pRRL.PPT-PGK, pRRL.PPT-EF1S, pHAGE2-EF1, and pWPXL-EF1i (Addgene plasmid no. 12257) (Sommer et al. 2009, Dahl et al. 2015, Schambach et al. 2006).

[0184] Generation of human MSC, moDC, and cDC1 cultures Human bone marrow (BM) cells were collected at the Department of Hematology, Lund University (Sweden) from consenting healthy donors by aspiration from the iliac crest. The use of human samples was approved by the Institutional Review Board of Lund University in accordance with the Declaration of Helsinki. Mononuclear cells derived from BM aspirate samples were isolated by density gradient centrifugation (LSM 1077 lymphocytes, PAA) after preincubation with RosetteSep Human Mesenchymal Stem Cell Enrichment Cocktail (STEMCELL Technologies) for lineage depletion by magnetic activated cell sorting (MACS) (CD3, CD14, CD19, CD38, CD66b, glycophorin A) as previously described (Li et al. 2014). MSC purification was followed by FACS sorting (additional information follows below). To generate moDCs, fresh leukocyte concentrates were diluted 1:1 in phosphate-buffered saline (PBS) and peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Lymphoprep (STEMCELL Technologies). CD14 were isolated by positive selection using MACS with CD14 microbeads (Miltenyi Biotec) according to the manufacturer's protocol. + Monocytes were expanded from PBMCs. CD14 + + Monocytes were cultured for 7 days in X-VIVO 15 medium (Lonza) supplemented with 5% FBS. Cells were cultured at 1 × 10 6Cells were seeded at a density of 1000 cells / ml, and 8 ml of cell suspension was added to a T75 flask. The medium was supplemented with IL-4 (350 ng / ml) and GM-CSF (850 ng / ml) on day 0, and the medium was changed every 2–3 days. On day 6, IL-6 (15 ng / ml), PGE2 (10 μg / ml), TNF-α (10 ng / ml), and IL1β (5 ng / ml) were added to the medium for 24 h to generate mature moDCs. Mature moDCs were dissociated using TrypLE Express (Gibco) and used for functional characterization. To isolate cDC1s for functional assays, DCs were enriched from PBMCs by MACS using a Pan-DC enrichment kit (Miltenyi Biotec) and subsequently further purified with anti-CLEC9A antibody conjugated to biotin and anti-biotin microbeads (Miltenyi Biotec).

[0185] Flow cytometry analysis For analysis of surface marker expression, human and mouse cells were dissociated and incubated with antibodies diluted in 5% FBS in PBS for 30 min at 4°C in the presence of mouse or rat serum (1 / 100, GeneTex) to block non-specific binding for human and mouse cells, respectively. Cells were washed, resuspended in 5% FBS in PBS, and analyzed on a BD FACSCanto II or BD LSRFortessa flow cytometer (BD Biosciences) unless otherwise stated. DAPI was used for dead cell exclusion. Flow cytometry data were analyzed using FlowJo software (FLOWJO, LLC, version 10.6.1). All flow cytometry analyses were performed with a live single cell gate.

[0186] Fluorescence-activated cell sorting (FACS) DCs were enriched from PBMCs by negative selection with MACS using the Pan-DC Enrichment Kit (Miltenyi Biotec) according to the manufacturer's protocol. + CD11C + CD141 +cDC1, HLA-DR + CD11C + CD141 - CD1C + cDC2 and HLA-DR + CD11C - CD123 + pDCs were purified using a FACSAria III (BD Biosciences) and used for single-cell RNA-seq profiling. + , CD45 + HLA-DR - , CD45 + HLA-DR + , and CD45 + HLA-DR + CD226 + To purify hiDCs, cells were dissociated using TrypLE Express, resuspended in PBS with 5% FBS, incubated with anti-CD45, anti-HLA-DR, and anti-CD226 antibodies in the presence of mouse serum for 30 min at 4°C, and purified on a FACSAria III. For isolation of human primary MSCs, lineage-depleted BM mononuclear cells were incubated in blocking buffer [Ca2 / Mg2-free PBS, 3.3 mg / ml human normal immunoglobulin (Octapharma), 1% FBS] followed by antibody staining. CD45 - CD271 + MSCs were purified using a FACSAria III (BD Biosciences) and used for reprogramming experiments. Dead cells were excluded by staining with 7-amino-actinomycin (7-AAD) or 4',6-diamidino-2-phenylindole (DAPI).

[0187] Single-cell RNA sequencing HEF, hiDCs on days 3, 6, and 9 (CD45 + HLA-DR - and CD45 + HLA-DR +), cDC1, cDC2 and pDC from peripheral blood (from three individual donors) were FACS sorted for scRNA-seq. Purified cells were placed in 10x Chromium (10x Genomics) according to the manufacturer's protocol. scRNA-seq indexed libraries were prepared using Chromium Single Cell 3'v2 and v3 Reagent Kits (10x Genomics) according to the manufacturer's protocol. Day 9 hiDCs reprogrammed with and without cytokines from HEF and HDF, as well as CD45 + HLA-DR + CD226 + hiDCs were similarly profiled. Library quantification and quality assessment were determined by Agilent Bioanalyzer using the High Sensitivity DNA Analysis Kit (Agilent). Indexed libraries were pooled at equimolar concentrations and sequenced on an Illumina NextSeq 500. The resulting read rate was approximately 130,000 reads per single cell. Details regarding scRNA-seq data analysis can be found in the Supplementary Material.

[0188] Analysis of RNA sequencing data In total, the transcriptomes of 51,903 single cells were profiled with approximately 130,000 reads per cell (R1 reads: technical, length: 26–28 bp; R2 reads: biological, length: 90–98 bp). Single-cell RNA-seq paired-end sequencing reads were processed using 10x Genomics software Cell Ranger v2.2.0 (https: / / support.10xgenomics.com / single-cell-gene-expression / software). First, cellranger mkfastq was used to convert binary base call files into FASTQ files to simultaneously decode multiplexed samples. Next, cellranger counts were applied to the FASTQ files and alignment to the human (hg38) genome assembly was performed using STAR v2.5.3a. The output files from each run were then combined and one single matrix was generated using cellranger aggr. The sparse expression matrix generated by the cellranger analysis pipeline was used as input to the Scater library (http: / / bioconductor.org / packages / release / bioc / html / scater), and cells and genes that met quality control thresholds were included according to the following criteria: 1) the total number of detected unique molecular identifiers (UMIs) per sample was greater than 3 lower median absolute deviations (MAD), 2) the number of genes detected in each single cell was greater than 3 lower MAD, and 3) the count rate within mitochondrial genes <7.5%. The resulting expression matrix was filtered by the Scater analysis pipeline and used as input to the Seurat library v4 (https: / / satijalab.org / seurat). To account for technical variations, batch integration was performed. First, each batch was normalized individually using "LogNormalize" with a scale factor of 10,000, and 9,000 variable features were identified. Then, batch integration was performed by finding corresponding anchors between batches using 30 dimensions. After that, 50 principal components were calculated and their importance was tested by JackStraw.The first 30 principal components were selected for subsequent tSNE visualization. For differential expression analysis between cell types, the Seurat v4 FindAllMarkers function was used with LR test with specification of latent variables (sequencing run and donor) to reduce batch effects and define the following parameters: logfc.threshold=0.5, min.pct=0.5, and BH-adjusted p<0.05. Additionally, scaling was performed with specification of latent variables in the vars.to.regess parameter, and the resulting genes were visualized using the Seurat v4 DoHeatmap function. For additional samples, all data were normalized together using "LogNormalize" with a scale factor of 10,000. The first 30 principal components were selected for subsequent tSNE and UMAP visualization. For differential expression analysis comparing imputed hiDCs with controls, the Seurat v4 FindMarkers function was used with a Wilcox test, defining the following parameters: logfc.threshold=0.25, min.pct=0.25, BH-adjusted p<0.05. The resulting intersection points were visualized using the Vennerable R library (https: / / github.com / js229 / Vennerable).

[0189] Example 2. PU.1, IRF8, and BATF3 induce a general cDC1 gene expression program in human fibroblasts. To characterize DC reprogramming of human fibroblasts at the transcriptional level, we cultured non-transduced HEF (d0), day 3 (CD45 + , d3), day 6 (CD45 + d6), and day 9 (CD45 + HLA-DR + , d9 DR + , CD45 + HLA-DR - , d9 DR - ) human iDCs (hiDCs), as well as peripheral blood cDC1s, cDC2s, and pDCs, were profiled by single-cell RNA-seq using the 10X Chromium system (Figure 1A).

[0190] method vector To induce DC fate in HEFs, a polycistronic construct encoding PIBs (PU.1, IRF8, and BATF3) separated by 2A sequences was cloned into a doxycycline (Dox)-inducible lentiviral vector (tetO-PIB) and transduced into cells ( Rosa et al., 2018 ) ( Figure 1A ).

[0191] Single-cell RNA sequencing Transduced and untransduced HEF cells were FACS sorted for scRNA-seq. Purified cells were placed in 10x Chromium (10x Genomics) according to the manufacturer's protocol. scRNA-seq libraries were prepared using the Chromium Single Cell 3'v2 Reagent Kit (10x Genomics) according to the manufacturer's protocol. Indexed sequencing libraries were constructed using reagents from the Chromium Single Cell 3'v2 Reagent Kit. Library quantification and quality assessment were determined by Agilent Bioanalyzer using a high sensitivity DNA analysis kit. Indexed libraries were pooled in equimolar amounts and sequenced on an Illumina NextSeq 500 using paired-end 26 x 98 bp sequencing mode. The resulting read rate was approximately 100,000 reads per single cell.

[0192] Scanning Electron Microscope (SEM) PIB factor-transduced HEFs were sorted on day 8 (CD45 + HLA-DR +), seeded on 0.1% gelatin-coated coverslips and analyzed alongside HEFs transduced with M2rtTA on day 9. Samples were washed in 0.1 M Sorensen's phosphate buffer and fixed in 0.1 M Sorensen's phosphate buffer pH 7.4, 1.5% formaldehyde, and 2% glutaraldehyde for 30 min at room temperature. After fixation, samples were washed in 0.1 M Sorensen's buffer. Samples were then dehydrated in a graded series of ethanol (50%, 70%, 80%, 90%, and 100% twice), critical point dried, and mounted on 12.5 mm aluminum stubs. Samples were then sputtered with 10 nm Au / Pd (80 / 20) in a Quorum Q150T ES turbo-pumped sputter coater and examined in a Jeol JSM-7800F FEG-SEM.

[0193] DC subset classification The scPred library (Alquicira-Hernandez et al., 2019) and publicly available DC single-cell expression data (Villani et al., 2017) were used for subset assignment. To train the classifier using the scPred method (implemented as an R library), default parameters of getFeatureSpace, trainModel were used as defined in the tool vignette. To predict the assignment of DCs isolated from PBMCs to publicly available DC subsets, the scPredict function was used with default parameters. For classification of hiDCs, the scPredict function was used separately for each donor with a threshold = 0.99, and then the number of cells assigned to the respective subset was combined.

[0194] result To induce DC fate in human embryonic fibroblasts (HEFs), we used a polycistronic construct encoding PU.1, IRF8, and BATF3 (PIB) separated by a 2A sequence (Rosa et al. 2018) cloned into a doxycycline (Dox)-inducible lentiviral vector (TetO-PIB) (Figure 1A). After transduction of HEFs with PIB, CD45 + Cell populations and CD45 with DC-like morphology from days 6 to 9 + HLA-DR + The emergence of a small population of cells was observed (Figure 1B-D), which were named human inducible DCs (hiDCs). To reveal transcriptional changes, scRNA-seq was performed using the 10X Chromium system. Peripheral blood cDC1, cDC2, pDC, non-transduced HEF (d0), day 3 (CD45 + , d3), day 6 (CD45 + , d6), and hiDCs (CD45 + HLA-DR - , d9 DR - , CD45 + HLA-DR + , d9 DR + 45,870 cells from three donors were profiled, including hiDC d3, d4, d5, d6, and d7. t-Distributed Stochastic Neighbor Embedding (t-SNE) visualization of the dataset highlighting four clusters: HEF, cDC1, cDC2, and pDC (Figure 1E). hiDC d9 mapped with cDC1 and DR, whereas hiDC d3 and d6 did not map to any clusters in particular. + DR - These data indicate that human cDC1 reprogramming requires a 9-day time frame and is more similar to CD45 + HLA-DR -The single-cell data obtained were then integrated with publicly available DC datasets (Villani et al. 2017) using scPred (Alquicira-Hernandez et al. 2019). As expected, we observed that 53.8% of purified cDC1s were assigned to the cDC1 subset, 66.9% and 29.8% of cDC2s were assigned to the DC2 and DC3 subsets, respectively, and 66.5% of pDCs were assigned to the DC6 subset (Figure S3). HEFs were independent, but not significant, as 1.3% of d3, 5.3% of d6, and DR of d9 were significantly more abundant than DR of d1. - 14.4% of hiDC and DR of d9 + 36.7% of hiDCs were specifically assigned to the cDC1 subset, suggesting that PU.1, IRF8, and BATF3 progressively impose a cDC1 signature with a degree of heterogeneity that does not cross subset boundaries (Figure 1F-G). cDC1-assigned cells express higher levels of the cDC1-specific genes CADM1 and WDFY4 compared to their non-assigned counterparts (Villani et al. 2017) (Figure 1H). We then extracted the most variable genes across the dataset and grouped them into five clusters (Figure 1I). Cluster 1 contains genes that are highly expressed in HEFs and silenced during reprogramming. Cluster 2 highlights early transcriptional changes during reprogramming, and cluster 3 contains cDC1-specific genes C1orf54, ANPEP, TACSTD2, and SLAMF8, which are highly expressed in d9 hiDCs and cDC1s (Heidkamp et al. 2016, See et al. 2017, Villani et al. 2017) (Figure 1J, Table 2). Cluster 4 and cluster 5 contain genes highly expressed in cDC2s and pDCs, respectively. Interestingly, d9 hiDCs expressed high levels of antigen processing and cross-presentation genes, including PSMB9, TAP1, and HLA-C (Figure 1K-M), suggesting that the reprogrammed cells acquired cross-presentation capability.

[0195] [Table 2]

[0196] conclusion Taken together, these data demonstrate that PIB factors impose a cDC1 signature on human fibroblasts.

[0197] Example 3. Single cell analysis highlights pathways associated with successful and unsuccessful cDC1 reprogramming It was hypothesized that single-cell RNA-seq could be used to analyze the trajectory of human DC reprogramming and uncover pathways or factors that correlate with successful reprogramming, thus enabling optimization of cDC1 reprogramming in human cells.

[0198] method Pseudo-time reconstruction The Monocle3 library (Cao et al. 2019) was used to order cells by reprogramming pseudotime. Monocle3 was run on tSNE with the following parameters: use_partition=FALSE. This assumes that all cells in the dataset are derived from a common transcriptional ancestor. The root of the trajectory was automatically selected. To mitigate batch effects in identifying genes fluctuating along the trajectory, batch correction was performed using the regressBatches function from the batchelor R library (http: / / bioconductor.org / packages / release / bioc / html / batchelor.html). Genes varying across the trajectory were then identified using the graph_test function, grouped into 21 distinct modules using the find_gene_modules function, and clustered using the Ward.D2 method in the pheatmap R library (https: / / cran.r-project.org / web / packages / pheatmap / index.html). Genes were defined as successful and unsuccessful reprogramming groups according to the clustering results. These gene lists were then additionally filtered by calculating the means of each gene in each cell population and using the following criteria: successful reprogramming - hiDC d9 DR + (Attribution)>hiDC d9 DR + (unattributed) and hiDC d9 DR - (Attribution)>hiDC d9 DR + (unattributed) and hiDC d9 DR + (Attribution)>HEF and hiDC d9 DR -(Attribution) > HEF. All comparisons are reversed for failed reprogramming. To reconstruct reprogramming dynamics scVelo v0.2.4 (Bergen et al. 2020) was used. For these analyses, sparse expression matrices generated by the cellranger analysis pipeline were converted to spliced ​​and unspliced ​​matrices using Velocyto v0.17.17 (http: / / velocyto.org). scVelo was run with default settings. Additionally, scVelo was also used to recover latency, and the top 1000 genes that change along latency were selected and displayed on a heatmap. In an additional sample (Figure 3), the Monocle3 library was used to order cells over a pseudo-time course during HEF to hiDC reprogramming using the same approach.

[0199] Transcription factor (TF) co-regulation network analysis To construct the TF network, genes associated with the success or failure of cDC1 reprogramming were submitted for analysis using ChEA3 (https: / / maayanlab.cloud / chea3 / ) and integrated by average rank across the entire library. The TF network was visualized as a network plot with points representing human TFs based on their co-expression similarity.

[0200] result We used Monocle3 to reconstruct cDC1 reprogramming trajectories (Cao et al. 2019). HEFs and cDC1s were placed at the beginning and end of pseudotime, respectively (Figure 2A-2B). d9 hiDCs were placed at the end of the trajectory with cDC1s, whereas d3 and d6 hiDCs were located in the middle, highlighting the stepwise transition of single-cell transcriptomes during cDC1 reprogramming. Importantly, we placed the attributed d9 hiDCs later in pseudotime compared to the non-attributed d9 hiDCs, suggesting that the successful cDC1 reprogramming pathway was captured by the trajectory reconstruction. We also observed a "dead-end" trajectory with non-attributed hiDCs mapping close to HEFs, suggesting that these cells do not enter a successful reprogramming pathway. To infer the expression dynamics and directionality of individual cells during reprogramming, we applied scVelo analysis (Bergen et al. 2020) to the reprogramming trajectories. hiDC velocities are primarily consistent with the cDC1 reprogramming trajectory projected by Monocle3 (Figure 2C). Nevertheless, we observed hiDC mapping closer to the dead-end trajectory display velocities pointing to HEF, consistent with previous reports that described failed reprogramming pathways characterized by the expression of genes related to the original cell state (Biddy et al. 2018, Zhou et al. 2019, Treutlein et al. 2016). Reconstruction of gene expression dynamics with latency showed downregulation of cell cycle genes during cDC1 reprogramming (Figure 2D-E), suggesting that hiDCs exit the cycle. We also observed enrichment in cytokines and IFN-type I and II (IFN-γ) signaling pathways later in the latency period. The downregulation of cell cycle and upregulation of IFN gene signatures is consistent with our previous findings in mouse systems (Rosa et al. 2018), suggesting a species-conserved role for IFN signaling in cDC1 reprogramming.To map stage-specific genetic changes along reprogramming, we clustered differentially expressed genes along pseudotime in 21 modules (Figure 2F). Non-assigned hiDCs fail to downregulate several gene modules enriched in HEFs, including modules 1, 2, 4, and 7 (failed reprogramming). Assigned hiDCs and cDC1s are enriched in genes expressed in modules 3, 9, 13, 15, and 17 (successful reprogramming). Extraction of genes encoding surface molecules and transcriptional regulators from these modules highlighted fibroblast genes enriched in failed reprogramming (CD248 and PRRX1) and DC genes, including cDC1 markers CD226 (Heidkamp et al. 2016) and IRF7 (Honda et al. 2005), that are upregulated in successfully reprogrammed cells (Figure 2G, Table 1). Transcription factor enrichment analysis for failed reprogramming genes identified previously described direct reprogramming impediments (Tomaru et al. 2014), including TWIST1, TWIST2, PRRX1, PRRX2, and OSR1 (Figure 2H). Interestingly, the same analysis for successful reprogramming genes reinforced the importance of PU.1, IRF8, and BATF in establishing a successful cDC1 reprogramming gene signature.

[0201] conclusion These data demonstrate that forced expression of transcription factors PU.1, IRF8, and BATF3 can improve reprogramming efficiency and highlight the role of cytokine and IFN signaling in cDC1 reprogramming.

[0202] Example 4. Single cell analysis highlights surface markers associated with successful reprogramming, allowing the identification and future isolation of successfully reprogrammed hiDCs. We hypothesized that surface markers enriched in successful cDC1 reprogramming (Table 1) would allow for the identification and future isolation of successfully reprogrammed hiDCs. As a proof of concept, we selected one of these surface markers, CD226, and identified CD226 + Reprogrammed hiDCs were purified and their cDC1 identity was confirmed by CD226 - Compared with reprogrammed hiDCs.

[0203] method First, we investigated the effect of partially reprogrammed CD45 + HLA-DR - , and reprogrammed CD45 + HLA-DR + Surface expression of CD226 on hiDCs was assessed. Then, CD45 + HLA-DR + CD226 + Purify hiDCs and confirm their cDC1 identity by CD45 expression using the scPred system. + HLA-DR + CD226 - The identity of hiDC was compared. For experimental details, see the previous example.

[0204] result First, CD45 + HLA-DR + hiDCs express CD45 + HLA-DR - We observed that hiDCs expressed higher levels of CD226 than cDCs (Figure 2I). To verify the usefulness of CD226 for identifying cDC1-like cells, we next used CD45 + HLA-DR + CD226 + hiDCs were purified and profiled by scRNA-seq. Interestingly, CD226 + The cells showed an increase in cDC1 affiliation (from 19.5% to 40.9%) (Figure 2J). + hiDCs express CD226 in phagocytosis of dead cells -We observed that CD226 functioned better compared to hiDCs, suggesting that CD226 marks functional hiDCs (Figure 2K).

[0205] conclusion These data suggest that surface markers associated with successful reprogramming, including CD226, allow the isolation of more functional hiDCs with a refined cDC1 identity.

[0206] Example 5. Single cell analysis identifies transcription factors associated with successful cDC1 reprogramming that can cooperate with PU.1, IRF8, and BATF3 to increase the efficiency of cDC1 reprogramming We hypothesized that transcription factors enriched in successful cDC1 reprogramming (Table 1) could cooperate with PU.1, IRF8, and BATF3 to enhance the reprogramming efficiency of cDC1. As proof of concept, 22 transcription factors were selected (ID2, TXNIP, ZFP36, PLEK, SUB1, JUNB, CREM, KLF4, MXD1, LITAF, IRF7, FOS, NMI, TFEC, SP110, IRF5, STAT2, BATF, ZNF267, IRF1, RELB, and BATF2). These were predicted to be associated with successful cDC1 reprogramming or regulate successful cDC1 reprogramming gene signature (see transcription factor (TF) co-regulation network analysis described in Example 3), and their ability to increase cDC1 reprogramming efficiency when co-expressed with PU.1, IRF8, and BATF3 was evaluated.

[0207] method The coding regions of ID2, TXNIP, ZFP36, PLEK, SUB1, JUNB, CREM, KLF4, MXD1, LITAF, IRF7, FOS, NMI, TFEC, SP110, IRF5, STAT2, BATF, ZNF267, IRF1, RELB, and BATF2 were individually cloned into pFUW-TetO plasmid. Lentiviral particles encoding individual transcription factors, i.e., PU.1, IRF8, and BATF3 (pFUW-tetO-PIB), or the reverse tetracycline transactivator M2rtTA (pFUW-UbC-M2rtTA), under the control of the constitutively active human ubiquitin C promoter, were used for co-transduction (Rosa et al. 2018). Reprogramming efficiency was assessed by flow cytometry in HEFs 9 days after transcription factor overexpression.

[0208] result To evaluate whether additional regulators enhance reprogramming, we recruited individual transcription factors associated with successful cDC1 reprogramming to PU.1, IRF8, and BATF3, and observed that IRF7 and BATF increased cDC1 reprogramming efficiency (Figure 2L). IRF7 is a transcription regulator of downstream inflammatory signaling (Honda et al. 2005). BATF is highly homologous to BATF3 and has been shown to compensate for BATF3 during cDC1 development (Tussiwand et al. 2012).

[0209] conclusion These data suggest that transcription factors associated with successful reprogramming, including IRF7 and BATF, may increase the reprogramming efficiency of cDC1.

[0210] Example 6. Increasing cDC1 reprogramming efficiency using exogenous cytokines Motivated by the enrichment of cytokine signaling in reprogrammed hiDCs and its role in human DC specificity, we hypothesized that cytokines may synergize with PU.1, IRF8, and BATF3 in DC reprogramming.

[0211] method Two days after PIB induction in HEFs, 17 human hematopoietic cell cytokines, including inflammatory mediators (Table 3), were individually added to the culture medium and reprogramming efficiency was measured on day 9. Changes in reprogramming efficiency were analyzed using both single cytokines and combinations of two or three cytokines.

[0212] [Table 3]

[0213] result IFN-γ inhibits CD45 + HLA-DR + IFN-γ had the greatest impact promoting a 20-fold increase in cell generation (7.9% ± 2.2% vs. 0.4% ± 0.2% without cytokines) (Figure 3A). Other inflammatory cytokines including IL-1β (3-fold), IL-6 (2.5-fold), Oncostatin M (4-fold), TNF-α (3-fold), and IFN-β (4-fold) also increased reprogramming efficiency. FLT3L, IL-4, and GM-CSF, used for in vitro differentiation of DCs from progenitor cells (Balan et al., 2018) or monocytes (Chapuis et al., 1997), did not affect reprogramming efficiency. Combining IFN-γ with IFN-β or TNF-α further increased reprogramming efficiency by 2.5-fold and 2-fold, respectively. Combining the three cytokines IFN-γ, IFN-β, and TNF-α yielded 28.1% ± 3.4% hiDCs, a 70-fold increase compared to cytokine-free conditions (Figure 3B).

[0214] conclusion These data strongly suggest that the efficiency of cDC1 reprogramming increases with the supply of inflammatory cytokines.

[0215] Example 7. Using a stronger constitutive promoter to enhance the reprogramming efficiency of cDC1 Given that cDC1 reprogramming trajectory reconstruction correlated PU.1, IRF8, and BATF3 expression with successful establishment of the cDC1 fate, we investigated whether forced expression of reprogramming factors using stronger constitutive promoters would increase reprogramming efficiency.

[0216] method The PIB polycistronic cassette, followed by IRES-GFP, was cloned into a constitutive vector utilizing multiple promoters in a lentiviral backbone to evaluate DC reprogramming efficiency (Rosa et al., 2018) in MEFs carrying the Clec9a-tdTomato reporter. The following vector backbones and promoters were used: pFUW-UbC, pRRL.PPT-SFFV, pRRL.PPT-PGK, pRRL.PPT-EF1S, pHAGE2-EF1, and pWPXL-EF1i.

[0217] result PIB overexpression driven by the SFFV promoter was shown to be highly efficient (46.6% ± 16.7% tdTomato + MHC-II + In HEFs, the development of 21.3 ± 6.1% hiDCs was observed in the SFFV system (Figure 4B). Furthermore, constitutive overexpression of PIB in the majority of fibroblasts induced surface expression of CD45, suggesting that a larger population of cells initiates the DC reprogramming process compared to the Dox-induced system (Figure 4B).

[0218] conclusion These data indicate that lentiviral vectors carrying the SFFV promoter can be used to improve reprogramming in human cells.

[0219] Example 8. Enhancement of cDC1 reprogramming efficiency using a combination of cytokines and stronger constitutive promoters Given that IFN-γ, IFN-β, and TNF-α signaling and SFFV-mediated constitutive overexpression of PU.1, IRF8, and BATF3 increased cDC1 reprogramming efficiency, we investigated whether inflammatory cytokine signaling acts synergistically with constitutive overexpression of reprogramming factors to achieve higher reprogramming efficiency.

[0220] method The effect on reprogramming efficiency after combination of cytokine treatment and SFFV-driven PIB induction was evaluated. The scPred system was used for integration with "natural" DCs. See previous examples for experimental details.

[0221] result Combining SFFV-driven induction with treatment with IFN-γ, IFN-β, and TNF-α resulted in 76.9 ± 11.9% hiDCs, a 190-fold improvement in cDC1 reprogramming efficiency compared to the original protocol (Figure 4B). Furthermore, an increase in the absolute number of generated hiDCs was observed with the improved protocol (Figure 4C). To characterize the cDC1 identity of hiDCs generated by the improved reprogramming protocol, we profiled hiDCs obtained using inducible (tetO-PIB) and constitutive (SFFV-PIB) systems with and without cytokines and used scPred for integration with peripheral blood DCs (Figure 4E). Interestingly, 61.4% and 53.2% of hiDCs generated with SFFV with and without cytokines, respectively, were assigned to the cDC1 lineage. In contrast, only 33.4% and 22.0% were generated in the inducible system (tetO-PIB). These data suggest that cytokine signaling and forced expression of PU.1, IRF8, and BATF3 act synergistically for successful establishment of cDC1-like cell fates. IL-10 did not affect reprogramming efficiency, and TGF-β reduced it two-fold (Figure 5). IL-10 and TGF-β signaling did not affect CD40 expression in hiDCs.

[0222] conclusion These data suggest that 1) the improved protocol increases both cDC1 reprogramming efficiency and cDC1 identity, and 2) cytokines and forced expression of PU.1, IRF8, and BATF3 act synergistically for successful reprogramming.

[0223] Example 9. Functional reprogramming of human cDC1-like cells cDC1s regulate adaptive immunity through multiple mechanisms, including cytokine secretion and antigen presentation to T cells. Motivated by the induction of a cDC1-like gene expression profile in human fibroblasts following PU.1, IRF8, and BATF3 overexpression, we investigated whether hiDCs could function as naturally occurring cDC1s.

[0224] method To investigate whether these hiDCs share the same functions as naturally occurring DCs, these hiDCs were challenged with Toll-like receptor 4 (TLR4) [lipopolysaccharide (LPS)], TLR3 [polyinosinic-polycytidylic acid (poly I:C)], or TLR7 / 8 [resiquimod (R848)] or a combination of all TLRs. Surface expression of the costimulatory molecules CD40 and CD80 (required for T cell activation) was analyzed by flow cytometry and used as a marker for T cell activation.

[0225] To access the phagocytosis of dead cells, HEK293T cells were exposed to ultraviolet (UV) irradiation (50 J / m2) to induce cell death and labeled with CellVue Claret Far Red Fluorescent Cell Linker Kit (Sigma). hiDCs (day 9), HEFs, and cDC1s were incubated with far-red labeled dead cells for 2 h, washed with PBS 5% FBS, and analyzed on a BD LSRFortessa X-20. Live CD45 + HLA-DR + hiDCs, CD45 + HLA-DR + CD226 - hiDCs, CD45 + HLA-DR + CD226 + hiDCs, CD141 + CLEC9A + Dead cell uptake was quantified in peripheral blood cDC1s or in control populations using far-red. For time-lapse fluorescence microscopy imaging of dead cell phagocytosis, far-red labeled dead cells were transfected with CD45 mAbs derived from FACS-sorted HEFs immediately prior to the start of image acquisition on a Zeiss Celldiscoverer 7. + HLA-DR + were added to the hiDC cultures, and microscopic images were taken every 10 min for 16 h.

[0226] To access inflammatory cytokine secretion, FACS-sorted CD45 cells generated in the presence or absence of cytokines + HLA-DR + Day 9 hiDCs, HEFs cultured in the presence or absence of cytokines, moDCs, and FACS-sorted CD141 + CLEC9A + XCR1 + Human cytokine levels were quantified in 25 μl of cDC1 culture supernatant using a cytometric bead array kit (LEGENDplex Human Anti-Virus Response Panel, BioLegend) according to the manufacturer's instructions. In particular, LPS, poly I:C or R848, or the three combined stimuli were added overnight before analysis. Acquisition was performed on a FACSCanto and data were analyzed using LEGENDplex software (BioLegend).

[0227] To access cross-presenting capacity, HEFs, moDCs, and magnetic-activated cell sorting (MACS) enriched Clec9a + cDC1 and hiDC were reprogrammed on day 8 and stimulated with LPS (3 ng / ml), poly I:C (25 μg / ml), and R848 (3 ng / ml). After overnight stimulation, cells were washed in PBS containing 2% FBS and pulsed with 2 μl / ml CMV protein (Miltenyi Biotec). After 3 hours, cells were washed and pulsed with MACS-enriched CD8 mAbs isolated from CMV-seropositive donors. + T cells. CMV positivity was confirmed by flow cytometry using CMV dextramer (Immudex). 1 × 10 5 CD8 + T cells and 5 × 10 4DCs were co-cultured in 200 μl of X-VIVO 15 in 96-well plates. After 24 h, T cell activation was measured by quantifying IFN-γ levels in the supernatants using ELISA (BD). Absorbance was read at 490 nm on a GloMax Discover Microplate Reader (Promega).

[0228] result We observed that both hiDCs and cDC1s upregulated costimulatory molecules after TLR3 or combined stimulation (Figure 6A). Moreover, hiDCs responded to TLR4 induction to a greater extent than cDC1s. Thus, CD34 +cDC1s differentiated from hematopoietic progenitors responded to LPS, pointing out a general feature of in vitro generated cDC1-like cells (Balan et al. 2018). To assess phagocytic capacity, a short incubation with labeled dead cells was performed. We observed hiDCs (47.5 ± 12.0%), hiDCs generated in the presence of IFN-γ, IFN-β, and TNF-α (19.4 ± 7.7%), as well as cDC1s (10.9 ± 3.5%), incorporated into dead cell material (Figure 6B-6D), a key feature of cross-presenting DCs. DC maturation and phagocytosis are often inversely correlated (Broz et al. 2014). Accordingly, hiDCs generated in the presence of cytokines expressed higher levels of costimulatory molecules and showed a reduced ability to incorporate dead cells (Figure 6A-C). To confirm that hiDCs were also providing a third signal required for T cell activation, cytokine secretion was assessed (Figure 6F). First, it was observed that hiDCs and cDC1s responded to TLR3 challenge by secreting the human cDC1-specific cytokine IFN-λ1 (Hubert et al. 2020). This is in contrast to moDCs, which did not respond to TLR3 agonists (Lauterbach et al. 2010). Furthermore, hiDCs also responded to TLR4 and 3 by secreting IL12p70, CXCL10, and TNF-α. Furthermore, it was observed that IFN-γ, IFN-β, and TNF-α increased the magnitude of cytokine secretion. We then demonstrated that hiDCs upregulate CD8 + We investigated whether CMV proteins could cross-present antigens to T cells. HEFs, moDCs, cDC1s, and hiDCs were pulsed with CMV proteins and then expressed as CMV antigens. + CD8 isolated from donors + As expected, cDC1s, in contrast to moDCs or HEFs, secreted IFN-γ, whereas CD8+ / -10 ... + We observed that hiDCs efficiently cross-presented CMV antigens to T cells. Surprisingly, we found that hiDCs generated with or without cytokines expressed CD8 +We observed that reprogrammed hiDCs established the ability to cross-present antigens to T cells. Collectively, these data support that reprogrammed hiDCs are functional cross-presenting DCs.

[0229] conclusion Taken together, these data support the membership of hiDCs to the cDC1 subset and their acquired capacity to respond to inflammatory stimuli, phagocytosis of dead cells, secretion of cytokines, and expression of antigen-specific CD8 + It supports cross-presentation of antigens that allows for T cell activation.

[0230] Example 10. Efficient reprogramming of human adult somatic cells Generation of cDC1 from accessible human cell types could be an additional source of DCs for cancer immunotherapy. Therefore, we attempted to reprogram human primary dermal fibroblasts (HDFs) and mesenchymal stromal cells (MSCs) into cDC1-like cells by improving the DC reprogramming protocol.

[0231] method HDFs from three healthy donors were obtained and evaluated for cDC1 reprogramming efficiency. Single-cell transcriptomes were generated for HDF-derived hiDCs, and scPred analysis was used for DC subset assignment. Purified MSCs from three healthy donors were transduced with SFFV-PIB lentiviral particles and cultured in chemically defined serum-free X-VIVO 15 medium (Figure 8A). Cells were evaluated for cDC1 reprogramming efficiency. Please refer to the previous example for experimental details.

[0232] result The efficiency of hiDC generation ranged from 20 to 35% across donors using SFFV-PIB (Figures 7A-7B). When combined with IFN-γ, IFN-β, and TNF-α, the reprogramming efficiency increased approximately two-fold (Figures 7A-B), also resulting in an increase in CD40 and CD80 (Figure 7C). By scPred analysis, 60.6% and 59.3% of hiDCs derived from HDFs with and without cytokines, respectively, were assigned to the cDC1 subset (Figure 7D). The identification of cDC1 was further confirmed by the expression of cDC1-specific genes C1orf54 and HLA-DPA1 and antigen processing and presentation genes CD74, HLA-C, B2M, PSMB9, NAAA, and TAP1 (Figures 7E-F). 60-75% of MSCs from the three donors were hiDCs co-expressing CD40 and CD80 (CD45 + HLA-DR + ) (FIGS. 8B-8D). IFN-γ, IFN-β, and TNF-α did not further improve the generation of hiDC1 from MSC cultures.

[0233] conclusion These data suggest that PU.1, IRF8, and BATF3 induce cDC1 fate in human adult cells and MSCs, highlighting the consistency of reprogramming methods across multiple cell types and donors. The lack of effect of adding cytokines to MSCs suggests that inflammatory cytokine signaling may promote cDC1 reprogramming in a cell type-specific manner.

[0234] Example 11. Induction of anti-tumor immunity in vivo Given the interest of assessing whether iDCs generated by direct cell reprogramming function in vivo, we utilized a mouse system to generate mouse iDCs (derived from Clec9a-tdTomato reporter MEFs, tdTomato + We investigated whether these cells induce antitumor immunity in a syngeneic mouse model of cancer.

[0235] method To access antigen cross-presentation, CD8 + T cells were isolated from naive mouse CD8 + T cells were enriched using a T cell isolation kit (Miltenyi). + T cells were labeled with 5 μM Cell Trace Violet CTV (Thermo Fisher) for 20 min at room temperature, washed, and counted. FACS-sorted tdTomato+ (generated with SFFV-PIB) and cDC1-like BM-DCs at the indicated time points were incubated with OVA protein (10 μg / ml) in the presence of poly I:C (1 μg / ml) for 10 h at 37°C. After extensive washing, 20,000 DCs were incubated with 100,000 CTV-labeled OT-I CD8 + T cells were incubated with poly I:C (1 μg / ml) in 96-well round-bottom tissue culture plates. After 3 days of co-culture, T cells were harvested, stained, and analyzed on a BD LSR Fortessa. T cell proliferation (dilution of CTV staining) was assessed by the expression of live single TCR + CD8 + Determined by gating on T cells.

[0236] Mouse IFN-α and Cxcl10 levels were assayed using the LEGENDplex Mouse Antiviral Response Panel (BioLegend) on day 9 purified tdTomato. + The assay was performed in 50 μl of cell culture supernatant. LPS (100 ng / ml) or poly I:C (1 μg / ml) was added overnight. Acquisition was performed on a FACSCanto and data were analyzed using LEGENDplex (BioLegend) software.

[0237] For in vivo experiments, B16-OVA (0.5 × 10 6 ) Tumor cells were injected subcutaneously into the left flank of 6- to 10-week-old C57Bl / 6 female mice. On day 9, FACS-sorted tdTomato tumor cells generated with SFFV-PIB were + The cells were mixed with B16-OVA cells prior to tumor implantation. +cells, MEFs transduced with SFFV-GFP control or CD103 + BM-DCs were injected intratumorally into established tumors on day 8 after tumor establishment. The day before, cells were stimulated overnight with LPS (100 ng / ml) and poly I:C (1 μg / ml). On the day of injection, cells were stimulated with OVA. 257-264 The cells were pulsed with peptide (5 μg / ml) for 30 min at 37°C. After washing twice with PBS, 80,000 cells were resuspended in 60 μl of PBS and injected intratumorally for each tumor-bearing mouse on day 8 after implantation of B16-OVA tumors. Tumor size was measured with a caliper [volume = 0.5 × length × width × height] every 1-2 days for the indicated time periods. For evaluation of T cell infiltration and activation, 1.5 × 10 cells were injected after injection of iDCs into tumor-bearing animals. 6 CTV labeling of OT-I CD8 + T cells were injected intravenously. After 4 days, animals were sacrificed and tumors and tumor-draining lymph nodes were collected and mechanically and chemically digested with collagenase D (1 mg / ml) and DNAse I (10 mg / ml). Dead cells were excluded using Percoll. For intracellular cytokine analysis, cells were restimulated in complete RPMI medium in the presence of phorbol 12-myristate 13-acetate (100 ng / ml) and ionomycin (1 μg / ml) for 4 h at 37°C and 5% CO2. GolgiPlug solution (1 μl / ml) was added to the medium for the last 2.5 h. Cells were stained with the fixable live cell dye FITC for 30 min at 4°C. Intracellular staining for IFN-γ and granzyme B was performed using the Intracellular Fixation & Permeabilization Buffer Set. Data were acquired using a Gallios and BD LSRFortessa.

[0238] result iDCs were already capable of antigen cross-presentation on days 4 and 6 of reprogramming (Figures 9A-9B). +iDCs secreted Cxcl10 and IFNα, previously described as essential for cDC1-mediated tumor rejection (Figure 9C) (Diamond et al., 2011). Furthermore, coinjection with iDCs was observed to reduce tumor growth during tumor formation (Figure 9D). Notably, a single intratumoral injection of 80,000 iDCs into established tumors was sufficient to slow tumor growth (Figure 9E). Non-reprogrammed MEFs and CD103 + Intratumoral injection of BM-DCs was not very efficient in controlling tumor growth. Moreover, injection of iDCs suppressed the expression of antigen-specific CD8 + It increased T cell infiltration and promoted an increased cytotoxic profile of T cells in tumor-draining lymph nodes in both models (Figure 9F).

[0239] conclusion These data support the hypothesis that iDCs induce antitumor immune responses. Collectively, these data suggest that iDCs express antigen-specific CD8 + It has been suggested that it controls tumor growth by activating T cells and promoting their infiltration into tumors.

[0240] Example 12. Efficient DC reprogramming requires the combined action of PU.1, IRF8, and BATF3 To shed light on the molecular mechanisms underlying DC reprogramming via PU.1, IRF8, and BATF3, we transduced HDFs with DOX-inducible lentiviral particles encoding the three reprogramming factors simultaneously or individually, and performed ChIP-seq for PU.1, IRF8, and BATF3 48 h after TF transduction (Figure S1A).

[0241] method ChIP sequencing TFs were delivered with a polycistronic lentiviral vector (pFUW-tetO-PIB) carrying pFUW-M2rtTA or individual vectors (pFUW-tetO-PU.1, pFUW-tetO-IRF8, or pFUW-tetO-BATF3). ChIP was performed 48 hours after addition of Dox.

[0242] Chromatin of cultured cells was fixed by adding 1 / 10 volume of freshly prepared formaldehyde solution [11% formaldehyde (Sigma), 0.1 M NaCl, 1 mM EDTA and 50 mM HEPES] to each cell suspension in complete DMEM. The tubes were left at room temperature for 15 min with stirring. Fixation was stopped by adding 1 / 20 volume of 2 mM glycine solution (Sigma). After 5 min incubation, cells were centrifuged at 800 g for 10 min at 4 °C. Cell pellets were resuspended in 10 ml of chilled PBS-Igepal, 100 μl of PMSF was added to each tube and centrifuged at 800 g for 10 min at 4 °C. Cell pellets were snap frozen on dry ice and stored at -80 °C. Chromatin was prepared by Active Motif (Carlsbad, CA), ChIP was performed, libraries were generated and libraries were sequenced. Briefly, chromatin was isolated by addition of lysis buffer and disruption with a Dounce homogenizer. Lysates were sonicated to shear DNA to an average length of 300–500 bp (Active Motif's EpiShear probe sonicator). Genomic DNA (input) was prepared by treating an aliquot of chromatin with RNase, proteinase K, and heat to decrosslink, followed by washing using solid-phase reversible immobilization (SPRI) beads (Beckman Coulter) and subsequent quantification by Clariostar (BMG Labtech). Extrapolation to the original chromatin volume allowed determination of total chromatin yield. 30 μg of chromatin was precleared with protein A / G agarose beads (Invitrogen). Immunoprecipitation was performed with 4 μg of antibodies against human PU.1, IRF8, and BATF3 (rabbit anti-human PU.1, rabbit anti-human IRF8, or sheep anti-human BATF3). Complexes were washed and eluted from the beads with SDS buffer and subjected to RNase and proteinase K treatment. Crosslinks were reversed by incubation at 65° C. overnight, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation.To confirm ChIP enrichment, quantitative PCR (QPCR) reactions were performed in triplicate for specific genomic regions using SYBR Green Supermix (Bio-Rad). The resulting signals were normalized for primer efficiency by performing QPCR for each primer pair using input DNA. Illumina sequencing libraries were prepared from ChIP and Input DNA by standard sequential enzymatic steps of end-polishing, dA addition, and adapter ligation. The steps were performed on an automated system (Apollo 342, Wafergen Biosystems / Takara). After the final PCR amplification step, the resulting DNA libraries were quantified and sequenced (75 nt reads, single-ended) on an Illumina NextSeq500.

[0243] ChIP-sequencing analysis and data visualization ChIP-seq analysis was performed on raw FASTQ files. FASTQ files were mapped to the human hg38 genome, which allows for 2 base pair mismatches, using the Bowtie 2 program. The mapped output files were processed through MACS v2.1.0 analysis software to determine peaks. Peak annotation was performed using the ChIPseeker R library. For genome tracks, bigwig files were created from bam files using deeptools (https: / / deeptools.readthedocs.io / en / develop / ) and explored using the UCSC genome browser. For chromatin state fold enrichment analysis, enrichment scores for genomic features such as PU.1, IRF8, and BATF3 ChIP-seq peaks and histone marks were calculated using ChromHMM Overlap Enrichment (http: / / compbio.mit.edu / ChromHMM / ). ChromHMM segmentations containing 18 different chromatin states were downloaded from the Roadmap website (http: / / www.roadmapepigenomics.org / tools) and used for the analysis. Enrichment scores were calculated as the ratio between observed and expected overlap for each feature and chromatin state based on their size and the size of the human genome. For de novo motif discovery, the findMotifsGenome.pl procedure from HOMER was used with PU.1, IRF8, and BATF3 individually. HOMER was run using default parameters and input sequences containing + / - 100bp from the center of the top 2500 peaks. Co-bound regions by PU.1, IRF8, and BATF3 were found using the circuit findOverlapsOfPeaks function in the ChIPpeakAnno R library (http: / / www.biomedcentral.com / 1471-2105 / 11 / 237). Co-bound regions were used for de novo motif discovery using HOMER.To assess the similarity of the two sets based on intersection points, we calculated the Jaccard statistic using MACRO-APE (https: / / opera.autosome.ru / macroape / compare).To generate heatmaps and profile plots, we used deeptools in fiducial mode, where each feature (e.g., TF peaks or histone marks) was aligned to the apex of PU.1, IRF8, or BATF3, and the adjacent upstream and downstream regions were tiled within ±4 kb.

[0244] Co-immunoprecipitation (Co-IP) Whole cell extracts were prepared from HEK293T cells transfected with SFFV-PIB at three cell densities (1, 2, and 5 million cells) in IP lysis buffer (Thermo Fisher) supplemented with protease inhibitors [1x Halt protease inhibitor cocktail (Thermo Fisher), 1 mM PMSF, 5 mM NaF]. ChIP-grade protein A / G magnetic beads were incubated with 5 μg of the respective antibody (rabbit anti-human PU.1, rabbit anti-human IRF8, or sheep anti-human BATF3) for 2 h. Cell lysates were pretreated with non-antibody treated ChIP-grade protein A / G beads for 1 h and then incubated with antibody treated beads for 1 h. Supernatants were removed and beads were washed three times with Tris-buffered saline containing 0.1% Tween 20 detergent (TBST). Input controls were performed using 10% (2 million cell density) of the non-immunoprecipitated sample. As a control, lysates (2 million cell density) were immunoprecipitated with 5 μg of rabbit IgG isotype (Invitrogen). Samples were eluted by boiling in Laemmli sample buffer and processed for Western blotting. For immunoblotting, membranes were blocked with TBST buffer containing 3% milk, incubated overnight with primary antibodies, washed five times with PBS containing 0.1% Tween 20 detergent (PBST), blocked for 45 min with TBST buffer containing 3% milk, incubated with HRP-conjugated secondary antibodies for 1 h, washed four times with PBST, and then detected by ECL (Thermo Scientific) in a Chemidoc (Bio-Rad).

[0245] result PU.1 dominant chromatin targeting ability in cDC1 reprogramming To shed light on the molecular mechanisms underlying DC reprogramming via PU.1, IRF8, and BATF3, we expressed the three reprogramming factors in combination or individually in HDFs and performed chromatin immunoprecipitation sequencing (ChIP-seq) at the early stage of reprogramming (48 h, FIG. 10A). First, when the factors were coexpressed, PU.1 showed the highest chromatin binding (75,593 peaks), followed by IRF8 (18,962 peaks) and BATF3 (11,505 peaks) (FIG. 10B). Interestingly, we observed that more than 40% of the PU.1 binding peaks were similar between individual and combined expression, suggesting that PU.1 has an independent targeting ability that is enhanced when IRF8 and BATF3 are available. In sharp contrast, IRF8 and BATF3 peaks were scarce when these transcription factors were expressed individually (less than 3% of peaks compared to combined expression), suggesting that IRF8 and BATF3 must bind cooperatively with PU.1 to bind chromatin and induce cDC1 fate. De novo motif prediction analysis of PU.1 peaks showed strong enrichment for PU.1 motifs when expressed individually or in combination (Figure 10C). IRF8 and BATF3 showed enrichment for IRF and AP-1 motifs, respectively, when expressed individually, whereas PU.1 motifs were highly enriched for these transcription factors when expressed in combination. These data are in line with recent findings describing PU.1 as a non-classical pioneer transcription factor that can redistribute partner transcription factors in human myeloid and lymphoid cells (Minderjahn et al. 2020), highlighting transcription factor cooperative dynamics during the early stages of cDC1 reprogramming.

[0246] Cooperative binding of PU.1, IRF8, and BATF3 at promoters and enhancers in open chromatin Next, we investigated the overlap between PU.1, IRF8, and BATF3 chromatin targets. When the three reprogramming factors were expressed together, 5,383 genomic positions were shared between them, representing 28% and 47% of the total peaks of IRF8 and BATF3, respectively (Figure 11A). De novo motif prediction for the PIB overlap peaks showed enrichment for PU.1-IRF and BATF motifs (Figure 11B), which also showed some overlap and similarity (Jaccard similarity index = 0.02) (Figure 11C). These data suggest that PU.1, IRF8, and BATF3 physically interact. To test this hypothesis, we performed co-immunoprecipitation (co-IP) and confirmed the interaction between the three factors (Figure 11D). We then plotted the differentially expressed genes between HDFs and hiDC d9 that were bound by at least one of the reprogramming factors and observed that they included both downregulated fibroblast genes and upregulated cDC1-associated genes, including SLAMF8 and TACSTD2 (Figure 11E). To examine whether PU.1, IRF8, and BATF3 binding occurs at open or closed chromatin regions, we utilized publicly available ChIP-seq datasets for histone marks in HDFs and used ChromHMM chromatin partitioning (Ernst and Kellis 2012) for visualization. We observed that PIB co-binding peaks were mainly enriched at promoter and enhancer regions (Figure 11F). We also observed a small percentage (12%) of peaks associated with bivalent chromatin marked with either H3K4me1, H3K4me3, and H3K27me3, or H3K4me1 and H3K27me3.

[0247] conclusion Taken together, our data support a model in which PU.1 binds to active promoters and enhancers located primarily at open chromatin sites, recruits IRF8 and BATF3, and silences native fibroblast genes, gradually imposing the cDC1 transcriptional program ( Figure S11G ).

[0248] Example 13. Efficient DC reprogramming of mouse and human cancer cells Considering that ectopic expression of PU.1, IRF8, and BATF3 induces a cDC1-like fate in mouse and human fibroblasts, we hypothesized that forced expression of the same combination of TFs in cancer cells would convert them into antigen-presenting cDC1s, overcoming one of the major problems of tumor immunity: the loss of antigen presentation machinery.

[0249] method Cancer cell reprogramming Cancer cell lines were seeded at a density of 60,000 cells / mL in 6-well plates and incubated overnight with SFFV-PIB-GFP lentiviral supernatant supplemented with polybrene (8 μg / mL). Over the culture period, medium was changed every 2 days. Cells were seeded at a dilution of 1:6 on 10 cm plates whenever cells reached 80-90% confluence. Flow cytometry was used to examine the DC reprogramming efficiency of mouse and human cancer cells.

[0250] T cell priming and antigen cross-presentation assays CD8 derived from spleens of OT-I mice + T cells were isolated from naive mouse CD8 + T cells were enriched using a T cell isolation kit (Miltenyi). + T cells were labeled with CTV according to the manufacturer's protocol. MACS-sorted reprogrammed cells, non-reprogrammed cancer cells, eGFP-transduced cancer cells, and CD103 + BM-DCs were incubated with OVA peptide (SIINFEKL, T cell priming assay) or protein (cross-presentation assay) at 37°C. OVA-expressing cells were not incubated with exogenous OVA. Where indicated, cells were incubated overnight in the presence of poly(I:C) or IFN-γ. 5 × 10 3 1 x 10 antigen presenting cells were plated in 96-well round-bottom untreated tissue culture plates. 5 CTV labeled OT-I CD8+ After 3 days of co-culture, T cells were harvested and stained for viability (fixable live cell recognition dye eFluor-520, eBioscience), CD8α, TCR-β, and CD44, and analyzed by flow cytometry. T cell proliferation (CTV dilution) and activation (CD44 expression) were analyzed by staining live single TCR-β. + T cells and CD8 + The threshold for data plotting was fixed at 1000 events within the live cell gating.

[0251] T cell killing assay CD8 derived from spleens of OT-I mice + T cells were cultured using mouse CD8 + T cells were enriched using a T cell isolation kit (Miltenyi). Anti-CD3 and anti-CD28 were added to 6-well untreated plates at 2 × 10 -3 mg mL -1 After coating with 1x10 cells / mL for 2 h at 37 °C and washing three times, 6 T cells were seeded in complete growth medium (RPMI) supplemented with mouse IL-2 (Peprotech, 100 U mL-1) and mouse IL-12p70 (Peprotech, 2.5 x 10-3 mg mL-1). After 24 h activation, T cells were seeded at 1 x 10 in fresh complete RPMI supplemented with mouse IL-2. 6 The reprogrammed mOrange T cells were then re-seeded at 200 ng / mL onto fresh untreated plates for 48 h to allow expansion of the T cells. +B16-OVA cells or cells treated with IFN-γ were seeded at equal numbers with non-fluorescent B16-OVA (mOrange-) and co-cultured with T cells 24 h later. Expanded T cells were added at T cell to target cell ratios of 0:1, 1:1, 5:1, and 10:1. Assay specificity was assessed using B16 cells that do not express OVA. For flow cytometry analysis, cells were resuspended and stained for viability (DAPI) and anti-CD3 at the indicated time points after co-culture with T cells.

[0252] Tumor induction and injection B16-OVA tumors were established by subcutaneous injection of 2–5 × 105 tumor cells into the right flank of 6- to 10-week-old C57BL / 6 female mice. Reprogrammed tumor-APCs were generated by transducing B16 with SFFV-PIB. Five days after transduction and on days 7, 10, and 13 after tumor establishment, tumor-APCs were purified by MACS with anti-MHC-II antibody and 2 × 10 5 ~3×10 5 Cells were resuspended in 100 μL PBS and injected intratumorally. PBS or control lentivirus-transduced cells were injected into tumors as controls. Tumor cells or tumor-APCs were stimulated with poly(I:C) and challenged with OVA 24 h prior to injection. Mice were followed for survival and tumor size was measured with a caliper [volume=π / 6×L×W×H] every 2 days after tumor formation until the end point. Mice were sacrificed when tumors exceeded 1500 mm3 in volume.

[0253] result First, we used SFFV-PIB-IRES-GFP lentiviral supernatant to overexpress PIB in 3LL and B16, murine lung adenocarcinoma and melanoma cells, respectively. Both murine cancer cell lines originated from a C57BL / 6 background and are widely used in syngeneic mouse models for tumor immunity. We observed the emergence of MHC-II and CD45 double positive populations 9 days after transduction (Figure 12A). Recent CRISPR screening approaches have highlighted the importance of IFN-γ signaling in antitumor immunity and cytotoxic T lymphocyte (CTL) sensitivity release. Interestingly, gene signatures of the IFN and STING pathways were upregulated in both B16 and LLC cells transduced with PIB (Figure 12B), suggesting an immunogenic profile obtained during reprogramming. To further investigate whether tumor-APCs express immunogenicity and present endogenously expressed antigens, we utilized the B16 cell line expressing ovalbumin (OVA) (B16-OVA) and magnetic activated cell sorting (MACS) enriched CD45 + MHC-II + OVA-expressing tumor APCs were cultured using naïve OT-I CD8 + Priming was assessed by co-culturing with T cells. Control eGFP-transfected B16-OVA and LLC-OVA cells showed low OVA antigen-presenting ability after stimulation with IFN-γ or P(I:C), whereas tumor-APCs were able to express OVA antigen-presenting naive OT-I CD8 T cells independently of IFN-γ or P(I:C) treatment. + The tumor-APCs were remarkably efficient in priming T cells (Figure 12C). Next, to assess whether tumor-APCs were susceptible to CTL killing, we utilized B16-OVA cells expressing the fluorescent protein mOrange. We generated tumor-APCs or administered IFN-γ (target, mOrange + )-treated B16-OVA cells were mixed with untreated B16-OVA cells (non-target, mOrange-) and co-cultured for 3 days to induce activation of OT-I CD8 + First, tumor-APCs increased the proportion of CD8 T cells compared with untreated B16-OVA cells. +We observed that tumor-APCs were more susceptible to killing by T cells (Figure 12D). Moreover, tumor-APCs were killed more efficiently by T cells (42.42±6.2%) than IFN-γ-stimulated B16-OVA cells (12.31±7.1%), especially at a low (1:1) ratio. Interestingly, we also observed that non-target populations were killed in tumor-APC cocultures at higher T cell to target cell ratios and at later time points (72 hours). Such bystander killing effects may reflect sustained activation of T cells by reprogrammed cells, which can increase the clearance of non-target cancer cells. Next, we evaluated the cross-presentation of tumor-APCs after pulsing with OVA protein. Surprisingly, tumor-APCs were more CD8 + We observed that OVA-loaded tumor-APCs were able to cross-present antigens to T cells, which was further enhanced by TLR3 stimulation (63.5 ± 8.5 vs. 27.5 ± 20.9%) (Figure 12E). We then investigated whether tumor-APCs loaded with OVA induced tumor growth control in vivo after intratumoral injection in established B16-OVA tumors (Figure 12F). Notably, injection of tumor-APCs led to reduced tumor growth and significantly improved survival when compared to mice injected with PBS or control virus (Figure 12G-H).

[0254] Next, to assess whether cDC1 fate could be directly induced in human cancer cells, expression of PU.1, IRF8, and BATF3 was performed in a panel of 28 human cancer cell lines. After 9 days, transduced EGFP cells co-expressing CD45 and HLA-DR, reflecting hematopoietic restriction and antigen-presenting capacity. + Reprogramming efficiency as a percentage of cells was assessed (Figure 13A-B). All cell lines transduced with hPIB-IRES-EGFP showed reprogrammed CD45 + HLA-DR +We observed the emergence of CD45+ / -100 cells in the EGFP-transduced control but not in the EGFP-transduced control, suggesting that cDC1 reprogramming is broadly applicable to human cancer cells. Furthermore, these data revealed that the reprogramming efficiency of cDC1 ranged from 0.2±0.1% to 94.5±7.6% across cancer cell lines, independent of transduction level and proliferation rate. Interestingly, despite the low reprogramming efficiency in lung and breast cancer-derived cancer cell lines, we detected a large population of cells that had acquired either CD45 or HLA-DR expression, which may represent partially reprogrammed cells that had acquired dendritic cell characteristics (Figures 13A-B). Human cancer cell-derived CD45+ / -100 cells were significantly more potent than the control cells, suggesting that the reprogramming efficiency of cDC1 was broadly applicable to human cancer cells. + HLA-DR + The cells expressed cDC1 surface markers, including CLEC9A (59.1 ± 3.6%), CD226 (67.5 ± 1.8%), and CD11c (54.4 ± 3.6%) (Figure 13C). Given that productive activation of naive T cells requires the expression of costimulatory molecules, surface expression of CD40, CD80, and CD86 was assessed. Human cancer cell-derived CD45 + HLA-DR + The cells expressed these costimulatory molecules in a gradually increasing manner starting from day 4 to day 9. Importantly, tumor-APCs responded to TLR3 / 4 triggers (LPS and poly I:C) by increasing surface expression of costimulatory molecules, particularly CD40 (88.2±3.8% vs. 31.3±1.8%) (Figure 13D,E).

[0255] An important consideration for converting tumor-APCs into therapy is whether reprogramming can be induced in human primary cancer cells (Figure 13F,G). To validate cDC1 reprogramming of primary cancer cells, 17 samples were taken from seven different tumors obtained from patients with melanoma, lung, tonsil, tongue, pancreatic, breast and bladder cancer as well as lung cancer-associated fibroblasts (CAFs) derived from PDX. Upon transduction with hPIB, all primary cancer cells showed major phenotypic changes, reflecting reprogramming as they initiated expression of CD45 and HLA-DR. Reprogramming efficiency ranged from 0.6% ± 0.4 to 47.0% ± 2.0. Samples from the same tumor type showed similar phenotypic profiles and relatively small variability between patients. Interestingly, when compared to a panel of cell lines, primary cells were less resistant to reprogramming as shown by lung cancer cell lines (0.5% ± 0.1) and primary cells (47.0% ± 2.0). These data suggest that epigenetic barriers limiting reprogramming are not reinforced in primary cells, paving the way for broad applicability of cDC1 reprogramming to human tumors (Figure S13F, G).

[0256] conclusion These data support the hypothesis that PU.1, IRF8, and BATF3 can reprogram mouse and human cancer cells into cDC1-like cells that can present tumor antigens and induce antitumor immune responses. We also verified that reprogramming into cDC1 is conserved across species and tissues and can be achieved from primary cancer cells from patients.

[0257] Example 14. Epigenetic modifiers enhance the reprogramming efficiency of cDC1. Given that ectopic expression of PU.1, IRF8, and BATF3 induces a cDC1-like fate in unrelated cell types by imposing transcriptional and epigenetic changes, we hypothesized that the efficiency of cDC1 reprogramming could be enhanced by epigenetic modifiers, namely histone deacetylase inhibition.

[0258] method ATAC-seq library preparation To investigate epigenetic changes induced by reprogramming, 5,000–10,000 cells were isolated from a given population by FACS and processed to prepare sequencing libraries. Quality was assessed using a high-sensitivity DNA chip (Agilent Technologies), and libraries were sequenced on a NextSeq 500 (Illumina) using the NextSeq 500 / 550 High Output Kit (150 cycles).

[0259] ATAC-seq data analysis A total of 1,384,592,926 ATAC-seq reads were obtained, yielding a median sample coverage of approximately 46.7 million reads. NGmerge61 was used with adapter removal mode set to remove Illumina universal adapters. Reads were mapped to the GRCh38 reference genome using HISAT2 v2.0.462 with the following parameters: --very-sensitive -k 20. Peak calling was performed for each sample individually by Genrich (v0.6.1, available at https: / / github.com / jsh58 / Genrich, parameters: -m 30 -j -y -r -e chrM). A combined peak list for all samples was obtained by using the PEPATACr R library. Finally, the read counts of the combined peak list were calculated with bedtools multicov56. The obtained read counts were processed with the R package DESeq252 and normalized using the RLE method. PCA was performed using the plotPCA function from the DESeq2 package. ChIPseeker R library64 was used for peak annotation. To map common chromatin changes, a modified procedure of ATAC-seq data described for the tumor-APC gene expression signature was used. Briefly, for each peak associated with an individual gene from the tumor-APC signature, the average difference between day 9 and day 0 was calculated, which was normalized to the difference between cDC1 and day 0 for each individual phenotype / reprogramming time point. We then took and plotted the median normalized peak value for each phenotype / time point of reprogramming separately. For genomic tracks, bigwig files were created from bam files by deeptools. Genomic tracks were explored using the WashU epigenomic browser. For motif discovery, the findMotifsGenome.pl procedure from HOMER67 was used with default parameters for differential ATAC-seq peaks.Functional enrichment analysis for the differential ATAC-seq peaks was performed by Great software68 using the GO biological process ontology.

[0260] Assessment of the effect of epigenetic modifiers on reprogramming efficiency As a control, cancer cell lines were transduced with PIB-IRES-EGFP lentiviral particles or EGFP and cultured in the presence or absence of VPA from days 1 to 4 of reprogramming, and CD45 + , and MHC-II or HLA-DR surface expression, live EGFP on day 9 of reprogramming + Reprogramming efficiency was quantified by flow cytometry on the cells, and the reprogrammed cells were then analyzed as previously described.

[0261] result To map the dynamics of reprogramming of human cancer cells at the transcriptional and epigenetic levels, we investigated the reprogramming process over time by using CD45 + HLA-DR + ) and partially reprogrammed (CD45-HLA-DR +) T98G cells were profiled using assays for mRNA sequencing and transposase accessible chromatin (ATAC) sequencing (Figure 14A). PCA separated all reprogramming stages (days 3, 5, 7, and 9) from parental cells, with days 7 and 9 mapping to the most native cDC1s, indicating a gradual acquisition of the cDC1 transcriptional program (Figure 14B). Consistent with this, partially reprogrammed cells were delayed in the time course, supporting the notion that these cells were on their way to successful reprogramming. Interestingly, reprogramming of human embryonic fibroblasts (HEF) followed a similar reprogramming trajectory (Figure 14B), indicating that reprogramming dynamics are conserved across malignant and non-cancerous primary cells. PCA of differentially open chromatin regions demonstrated that epigenetic remodeling occurred rapidly, with large changes (62% variation) between days 0 and 3, followed by fine tuning at subsequent time points (days 3, 5, 7, and 9), moving cells closer to the open chromatin pattern of cDC1 (Figure 14B). To confirm these observations, we utilized the tumor-APC gene signature and the mapped changes over time. Indeed, the signature was gradually imposed at the transcriptional level and rapidly established at the chromatin level (Figure 14C). These data indicate that hPIB-mediated reprogramming induces a stepwise rewiring of the cDC1 transcriptional program followed by rapid epigenetic remodeling.

[0262] To test whether the reprogramming efficiency of cDC1 was limited by epigenetic barriers, B16 and LLC cells were treated with valproic acid (VPA) and the reprogramming efficiency was assessed on day 9. VPA treatment reduced the reprogramming efficiency by approximately three-fold in LLC (45.9±25.5% vs. 15.8±4.79%) and approximately five-fold in B16 cells (29.9±19.3% vs. 5.9±4.8%), and CD45 + MHC-II +The generation of tumor-APCs was promoted (Figure 15A-B). We also confirmed that transduced cells in the presence of VPA upregulated MHC-I (Figure 15C), indicating that a larger population of cancer cells became immunogenic. Functionally, tumor-APCs generated in the presence of VPA were OT-I CD8 + Naive CD8 T cells were primed after incubation with exogenous antigens, which presented endogenous antigens to T cells (Fig. S15D) and served as targets for T cell-mediated cytotoxicity (Fig. S15E). + T cells (Figure 15F). Next, we investigated the effect of VPA treatment on cDC1 reprogramming of human cancer cells. We observed that VPA treatment increased the reprogramming efficiency in all lines tested (Figure 15G).

[0263] conclusion The data indicate that promoting chromatin accessibility during cDC1 reprogramming can enhance cancer cell reprogramming.

[0264] Example 14. SPIB and SPIC compensate for the role of PU.1 in cDC1 reprogramming. The human genome encodes nearly 2000 different transcription factors organized in multiple families and subfamilies. Transcription factors that share significant homology are usually included in the same family / subfamily of transcription factors. Under certain conditions, transcription factors can compensate for the absence of a particular transcription factor from the same family or subfamily. In this regard, we hypothesized that homologs from PU.1, IRF8, and BATF3 may compensate for their role in cDC1 reprogramming. As a proof of concept, the ability of SPIB and SPIC, two PU.1 homologs, to replace the role of PU.1 in cDC1 reprogramming was tested.

[0265] method The coding regions of SPIB and SPIC were cloned individually into the pFUW-TetO plasmid. Lentiviral particles encoding individual transcription factors or the reverse tetracycline transactivator M2rtTA (pFUW-UbC-M2rtTA) under the control of the constitutively active human ubiquitin C promoter were used for co-transduction (Rosa et al. 2018). Reprogramming efficiency was assessed by flow cytometry in Clec9a-tdTomato mouse embryonic fibroblasts (MEFs) 9 days after transcription factor overexpression.

[0266] result We observed that both SPIB and SPIC could replace PU.1 in the context of cDC1 reprogramming (Figure 16A). Importantly, SPIB and SPIC alone were unable to activate DC-specific reporters in transduced MEFs. Interestingly, SPIB-induced reporter activation was to a greater extent than PU.1 or SPIC, and was significantly higher than tdTomato. + They represented approximately 8.14±1.16% of the cells, whereas PU.1 and SPIC represented only 2.87±0.18% and 1.46±0.73%, respectively.

[0267] Next, tdTomato + We analyzed the expression of CD45 and MHC-II in the cells. Surprisingly, SPIB was significantly more abundant than PU.1 (17.15 ± 2.04%), which co-expressed CD45 and MHC-II, compared to tdTomato (17.15 ± 2.04%). + There was a two-fold increase in the expression of IFN-γ in cells (33.63±3.76%) (FIG. 16B).

[0268] conclusion These data suggest that SPIB and SPIC can compensate for the role of PU.1 in cDC1 reprogramming.

[0269] Example 15. Adenovirus- and adeno-associated virus-mediated delivery of PU.1, IRF8, and BATF3 enables cDC1 reprogramming of healthy and cancer cells. Cell reprogramming strategies based on overexpression of cell type-specific transcription factors have traditionally relied on the use of retroviral or lentiviral vectors. Nevertheless, the integrative nature of these viral vectors raises safety concerns for clinical use. The use of non-integrative viral systems is a good alternative to circumvent these safety concerns and deliver transcription factors to target cells for therapeutic use. Here, we hypothesize that the delivery of PU.1, IRF8, and BATF3 mediated by non-integrative adenovirus and adeno-associated virus (AAV) allows the reprogramming of cDC1 in non-related cell types.

[0270] method Cell Reprogramming Mouse embryonic fibroblasts isolated from Clec9a-tdTomato reporter mice, B2905 mouse melanoma cell line, IGR-39 melanoma and T98G glioblastoma human cell lines, and 2778 human primary melanoma cells were seeded at a density of 12,500 cells / well in 12-well plates and incubated overnight with lentivirus (Lenti), adenovirus (Ad5 or Ad5 / F35), or AAV (AAV-DJ or AAV2-QuadYF), encoding PIB-GFP or GFP alone, using a multiplicity of infection of 50,000 RNA copies / cell, 5,000 infectious units / cell, and 250,000 genome copies / cell, respectively. The medium was supplemented with polybrene (8 μg / mL) when cells were incubated with lentivirus. The medium was changed every 2 days throughout the culture period. cDC1 reprogramming efficiency was assessed according to the surface expression of CD45 and MHC-II or HLA-DR, live, GFP + Cells were quantified by flow cytometry on day 9 of reprogramming.

[0271] result We observed that adenovirus and AAV encoding PIB-GFP could induce activation of Clec9a-tdTomato reporter (Figure 17A) and surface expression of CD45 and MHC-II in mouse embryonic fibroblasts (Figure 17B). As expected, no tdTomato expression was observed in cells transduced with a viral vector encoding GFP. We next investigated whether adenovirus and AAV vector encoding PIB-GFP could reprogram mouse and human cancer cells. We observed surface expression of CD45 and MHC-II in B2905 mouse melanoma cancer cells (Figure 17C), and CD45 and HLA-DR in human cancer cell lines (IGR-39 and T98G) and human primary melanoma cells 2778 (Figure 17D).

[0272] conclusion These data suggest that adenovirus- and AAV-mediated delivery of PU.1, IRF8, and BATF3 enables cDC1 reprogramming in healthy and cancer, mouse, and human cell types.

[0273] [Table 4]

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[0275] item 1. Transcription factors when expressed: i) BATF3 or a biologically active variant thereof which is at least 70% identical to SEQ ID NO: 10 (BATF3), such as at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95%, such as at least 96%, for example at least 97%, such as at least 98%, for example at least 99%, such as 100% identical to SEQ ID NO: 10 (BATF3); ii) IRF8 or a biologically active variant thereof which is at least 70% identical to SEQ ID NO: 11 (IRF8), such as at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95%, such as at least 96%, for example at least 97%, such as at least 98%, for example at least 99%, such as 100% identical to SEQ ID NO: 11 (IRF8), and iii) PU.1 or a biologically active variant thereof which is at least 70% identical to SEQ ID NO: 12 (PU.1), such as at least 75%, for example at least 80%, such as at least 85%, for example at least 90%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99%, for example 100% identical to SEQ ID NO: 12 (PU.1); 1. A composition comprising one or more constructs or vectors encoding a transcription factor, the one or more constructs or vectors comprising a promoter region capable of controlling the transcription of the transcription factor, the promoter region comprising a spleen focus forming virus (SFFV) promoter, an MND (myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer binding site substitution) promoter, a CAG (CMV early enhancer / chicken beta actin) promoter, a cytomegalovirus (CMV) promoter, a ubiquitin C (UbC) promoter, an EF-1 alpha (EF-1α) promoter, an EF-1 alpha short (EF1S) promoter, an EF-1 alpha with intron (EF1i) promoter, a phosphoglycerate kinase (PGK) promoter, or a promoter that exhibits substantially the same effect.

[0276] 2. Upon expression: a) IRF7 or a biologically active variant thereof which is at least 70% identical to SEQ ID NO: 21 (IRF7), such as at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95%, such as at least 96%, for example at least 97%, such as at least 98%, for example at least 99%, such as 100% identical to SEQ ID NO: 21 (IRF7); b) BATF or a biologically active variant thereof which is at least 70% identical to SEQ ID NO: 19 (BATF), such as at least 75%, for example at least 80%, such as at least 85%, for example at least 90%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99%, for example 100% identical to SEQ ID NO: 19 (BATF); c) SPIB or a biologically active variant thereof which is at least 70% identical to SEQ ID NO: 23 (SPIB), such as at least 75%, for example at least 80%, such as at least 85%, for example at least 90%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99%, for example 100% identical to SEQ ID NO: 23 (SPIB); d) SPIC or a biologically active variant thereof which is at least 70% identical to SEQ ID NO: 25 (SPIC), such as at least 75%, for example at least 80%, such as at least 85%, for example at least 90%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99%, for example 100% identical to SEQ ID NO: 25 (SPIC); The composition of claim 1, further comprising one or more constructs or vectors encoding one or more transcription factors selected from the group consisting of: ...

[0277] 3. a) said SFFV promoter comprises or consists of a polynucleotide sequence which is at least 80% identical to SEQ ID NO:1, such as at least 85%, for example at least 90%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, for example at least 99%, for example 100% identical to SEQ ID NO:1, b) said MND promoter comprises or consists of a polynucleotide sequence which is at least 80% identical to SEQ ID NO:2, such as at least 85%, for example at least 90%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99%, for example 100% identical to SEQ ID NO:2; c) the CAG promoter comprises or consists of a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 3, such as at least 85%, for example at least 90%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99%, for example 100% identical to SEQ ID NO: 3; d) said CMV promoter comprises or consists of a polynucleotide sequence which is at least 80% identical to SEQ ID NO:4, such as at least 85%, for example at least 90%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, for example at least 99%, such as 100% identical to SEQ ID NO:4; e) the UbC promoter comprises or consists of a polynucleotide sequence which is at least 80% identical to SEQ ID NO:5, such as at least 85%, for example at least 90%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, for example at least 99%, for example 100% identical to SEQ ID NO:5; f) the EF-1α promoter comprises or consists of a polynucleotide sequence which is at least 80% identical to SEQ ID NO:6, such as at least 85%, for example at least 90%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, for example at least 99%, for example 100% identical to SEQ ID NO:6, g) the EF1S promoter comprises or consists of a polynucleotide sequence which is at least 80% identical to SEQ ID NO:7, such as at least 85%, for example at least 90%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99%, for example 100% identical to SEQ ID NO:7; h) the EF1i promoter comprises or consists of a polynucleotide sequence which is at least 80% identical to SEQ ID NO:8, such as at least 85%, for example at least 90%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, for example at least 99%, for example 100% identical to SEQ ID NO:8, i) The composition according to any one of items 1 or 2, wherein said PGK promoter comprises or consists of a polynucleotide sequence which is at least 80% identical to SEQ ID NO:9, such as at least 85%, such as at least 90%, for example at least 95%, such as at least 96%, such as at least 97%, for example at least 98%, such as at least 99%, for example 100% identical to SEQ ID NO:9.

[0278] 4. The composition comprises: a) one construct or vector that upon expression encodes the transcription factors BATF3, IRF8, and PU.1; b) one construct or vector that, upon expression, encodes the transcription factors BATF3, IRF8, and SPIB; c) a first construct or vector that, upon expression, encodes the transcription factors BATF3 and IRF8, and a second construct or vector that, upon expression, encodes the transcription factor PU.1; d) a first construct or vector that, upon expression, encodes the transcription factors BATF3 and IRF8, and a second construct or vector that, upon expression, encodes the transcription factor SPIB; e) a first construct or vector that, upon expression, encodes the transcription factor BATF3 and a second construct or vector that, upon expression, encodes the transcription factors IRF8 and PU.1; f) a first construct or vector that, upon expression, encodes the transcription factor BATF3 and a second construct or vector that, upon expression, encodes the transcription factors IRF8 and SPIB; g) a first construct or vector that, upon expression, encodes the transcription factor IRF8 and a second construct or vector that, upon expression, encodes the transcription factors BATF3 and PU.1; h) a first construct or vector that, upon expression, encodes the transcription factor IRF8 and a second construct or vector that, upon expression, encodes the transcription factors BATF3 and SPIB; i) a first construct or vector that, when expressed, encodes the transcription factor BATF3; a second construct or vector that, when expressed, encodes the transcription factor IRF8; and a third construct or vector that, when expressed, encodes the transcription factor PU.1; and / or j) a first construct or vector that, when expressed, encodes the transcription factor BATF3; a second construct or vector that, when expressed, encodes the transcription factor IRF8; and a third construct or vector that, when expressed, encodes the transcription factor SPIB; 2. The composition of any one of the preceding claims, comprising:

[0279] 5. The composition comprising: a) one construct or vector that upon expression encodes the transcription factors BATF3, IRF8, PU.1, and IRF7; b) a first construct or vector that, upon expression, encodes the transcription factors BATF3 and IRF8, and a second construct or vector that, upon expression, encodes the transcription factors PU.1 and IRF7; c) a first construct or vector that, upon expression, encodes the transcription factors BATF3 and PU.1, and a second construct or vector that, upon expression, encodes the transcription factors IRF8 and IRF7; d) a first construct or vector that, upon expression, encodes the transcription factors PU.1 and IRF8, and a second construct or vector that, upon expression, encodes the transcription factors BATF3 and IRF7; e) a first construct or vector that, upon expression, encodes the transcription factors BATF3, IRF8, and PU.1, and a second construct or vector that, upon expression, encodes the transcription factor IRF7; f) a first construct or vector that, upon expression, encodes the transcription factor BATF3 and a second construct or vector that, upon expression, encodes the transcription factors IRF8, PU.1, and IRF7; g) a first construct or vector that, upon expression, encodes the transcription factor IRF8 and a second construct or vector that, upon expression, encodes the transcription factors BATF3, PU.1, and IRF7; h) a first construct or vector that, upon expression, encodes the transcription factor PU.1 and a second construct or vector that, upon expression, encodes the transcription factors BATF3, IRF8, and IRF7; and / or i) a first construct or vector that, when expressed, encodes the transcription factor BATF3, a second construct or vector that, when expressed, encodes the transcription factor IRF8, a third construct or vector that, when expressed, encodes the transcription factor PU.1, and a fourth construct or vector that, when expressed, encodes the transcription factor IRF7; 2. The composition of any one of the preceding claims, comprising:

[0280] 6. The composition comprising: a) one construct or vector that, upon expression, encodes the transcription factors BATF3, IRF8, PU.1, and BATF; b) a first construct or vector that, upon expression, encodes the transcription factors BATF3 and IRF8, and a second construct or vector that, upon expression, encodes the transcription factors PU.1 and BATF; c) a first construct or vector that, upon expression, encodes the transcription factors BATF3 and PU.1, and a second construct or vector that, upon expression, encodes the transcription factors IRF8 and BATF; d) a first construct or vector that, upon expression, encodes the transcription factors PU.1 and IRF8, and a second construct or vector that, upon expression, encodes the transcription factors BATF3 and BATF; e) a first construct or vector that upon expression encodes the transcription factors BATF3, IRF8, and PU.1, and a second construct or vector that upon expression encodes the transcription factor BATF; f) a first construct or vector that, upon expression, encodes the transcription factor BATF3 and a second construct or vector that, upon expression, encodes the transcription factors IRF8, PU.1, and BATF; g) a first construct or vector that, upon expression, encodes the transcription factor IRF8 and a second construct or vector that, upon expression, encodes the transcription factors BATF3, PU.1, and BATF; h) a first construct or vector that, upon expression, encodes the transcription factor PU.1 and a second construct or vector that, upon expression, encodes the transcription factors BATF3, IRF8, and BATF; and / or i) a first construct or vector that, when expressed, encodes the transcription factor BATF3, a second construct or vector that, when expressed, encodes the transcription factor IRF8, a third construct or vector that, when expressed, encodes the transcription factor PU.1, and a fourth construct or vector that, when expressed, encodes the transcription factor BATF; 2. The composition of any one of the preceding claims, comprising:

[0281] 7. The composition of any one of the preceding items, wherein BATF3 is encoded by a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO:14, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99%, for example 100% sequence identity to SEQ ID NO:14.

[0282] 8. The composition of any one of the preceding items, wherein IRF8 is encoded by a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO:15, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99%, for example 100% sequence identity to SEQ ID NO:15.

[0283] 9. The composition of any one of the preceding items, wherein PU.1 is encoded by a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO:16, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99%, for example 100% sequence identity to SEQ ID NO:16.

[0284] 10. The composition of any one of the preceding items, wherein BATF is encoded by a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO:18, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99%, for example 100% sequence identity to SEQ ID NO:18.

[0285] 11. The composition of any one of the preceding items, wherein IRF7 is encoded by a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO:20, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99%, for example 100% sequence identity to SEQ ID NO:20.

[0286] 12. The composition of any one of the preceding items, wherein the SPIB is encoded by a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO:22, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99%, for example 100% sequence identity to SEQ ID NO:22.

[0287] 13. The composition of any one of the preceding items, wherein the SPIC is encoded by a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO:24, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99%, for example 100% sequence identity to SEQ ID NO:24.

[0288] 14. The composition of any one of the preceding items, wherein the one or more constructs or vectors, upon expression, further encode a transcription factor CCAAT / enhancer binding protein alpha (CEBPα) or a biologically active variant thereof which is at least 70% identical to SEQ ID NO: 13 (CEBPα), such as at least 75%, for example at least 80%, such as at least 85%, for example at least 90%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99%, for example 100% identical to SEQ ID NO: 13.

[0289] 15. The composition according to any one of the preceding items, wherein CEBPα is encoded by a polynucleotide sequence having at least 90% sequence identity to SEQ ID NO:17, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99%, for example 100% sequence identity to SEQ ID NO:17.

[0290] 16. The composition of any one of the preceding items, wherein the one or more constructs or vectors further comprise a self-cleaving peptide operably linked to at least two of the at least three coding regions, thus forming a single open reading frame.

[0291] 17. The composition described in item 16, wherein the self-cleaving peptide is a 2A peptide.

[0292] 18. The composition described in item 17, wherein the 2A peptide is selected from the group consisting of Equine rhinitis A virus (E2A), foot and mouth disease virus (F2A), porcine teschovirus-1 (P2A) and Thosea signa virus (T2A) peptides.

[0293] 19. The composition of any one of the preceding items, wherein the one or more constructs or vectors is a viral vector.

[0294] 20. The composition of any one of the preceding items, wherein the one or more constructs or vectors is a viral vector selected from the group consisting of a lentiviral vector, a retroviral vector, an adenoviral vector, a herpes virus vector, a poxvirus vector, an adeno-associated virus vector, a paramyxoviridae vector, a rhabdovirus vector, an alphavirus vector, and a flavivirus vector.

[0295] 21. The composition described in item 20, wherein the viral vector is a lentiviral vector.

[0296] 22. The composition of item 20, wherein the adenoviral vector is selected from the group consisting of a wild-type Ad vector, a hybrid Ad vector, and a mutant Ad vector.

[0297] 23. The composition of item 22, wherein the wild-type Ad vector is Ad5 and the hybrid Ad vector is Ad5 / F35.

[0298] 24. The composition described in item 20, wherein the adeno-associated virus vector is selected from the group consisting of a wild-type AAV vector, a hybrid AAV vector, and a mutant AAV vector.

[0299] 25. The composition described in item 24, wherein the hybrid AAV vector is AAV-DJ and the mutant AAV vector is AAV2-QuadYF.

[0300] 26. The composition of any one of the preceding items, wherein the one or more constructs or vectors is a plasmid.

[0301] 27. The composition of any one of the preceding items, wherein the backbone of the one or more constructs or vectors is selected from the group consisting of FUW, pRRL-cPPT, pRLL, pCCL, pCLL, pHAGE2, pWPXL, pLKO, pHIV, pLL, pCDH, and pLenti.

[0302] 28. The composition of any one of the preceding items, wherein the one or more constructs or vectors further comprise a post-transcriptional regulatory element (PRE) sequence.

[0303] 29. The composition described in item 28, wherein the PRE sequence is a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

[0304] 30. The composition of any one of the preceding items, wherein the one or more constructs or vectors further comprise a central polypurine tract (cPPT).

[0305] 31. The composition of any one of the preceding items, wherein the one or more constructs or vectors further comprise 5' and 3' terminal repeats.

[0306] 32. The composition described in item 31, wherein at least one of the 5' and 3' terminal repeats is a self-inactivating (SIN) design with a partial deletion of U3 of the lentiviral long terminal repeat or 3' long terminal repeat.

[0307] 33. The composition of any one of the preceding items, wherein the one or more constructs or vectors further comprise a nucleocapsid protein packaging target site.

[0308] 34. The composition described in item 33, wherein the protein packaging target site comprises an HIV-1 psi sequence.

[0309] 35. The composition of any one of the preceding items, wherein the one or more constructs or vectors further comprise a REV protein response element (RRE).

[0310] 36. The composition of any one of the preceding items, further comprising one or more cytokines selected from the group consisting of IFNβ, IFNγ, TNFα, IFNα, IL-1β, IL-6, CD40I, Flt3I, GM-CSF, IFN-λ1, IFN-ω, IL-2, IL-4, IL-15, prostaglandin 2, SCF, and oncostatin M (OM).

[0311] 37. The composition of any one of the preceding items, further comprising one or more epigenetic modifiers, such as a histone deacetylase inhibitor.

[0312] 38. The composition described in item 37, wherein the one or more histone deacetylase inhibitors is valproic acid.

[0313] 39. A composition described in any one of the preceding items, wherein the composition is a pharmaceutical composition.

[0314] 40. A cell comprising one or more of the constructs or vectors described in any one of the preceding items.

[0315] 41. The cell according to item 40, wherein the cell is a mammalian cell, such as a human cell or a mouse cell.

[0316] 42. The cell is selected from the group consisting of a stem cell, a differentiated cell, and a cancer cell; a) the stem cells are selected from the group consisting of pluripotent stem cells and multipotent stem cells, e.g. mesenchymal stem cells or hematopoietic stem cells; b) the differentiated cell is any somatic cell, such as a fibroblast, or a hematopoietic cell, such as a monocyte; The cell according to any one of items 40 to 41.

[0317] 43. The cell according to any one of items 40 to 42, wherein the cell is a human dendritic cell, such as a reprogrammed human type 1 conventional dendritic cell, or an antigen-presenting cell.

[0318] 44. The cell of any one of items 40 to 43, wherein the cell further expresses one or more surface markers selected from the surface markers of Table 1.

[0319] 45. The cell of any one of items 40 to 44, wherein the cell is positive for one or more surface markers listed in Table 1.

[0320] 46. ​​The cell according to any one of items 40 to 45, wherein the cell is CD226 positive.

[0321] 47. A method for reprogramming or inducing a cell into a dendritic cell or an antigen-presenting cell, said method comprising: c) transducing the cell with a composition comprising the construct or vector according to any one of items 1 to 32; d) expressing a transcription factor; thereby obtaining a reprogrammed or derived cell; The method comprising:

[0322] 48. The method of claim 47, wherein the reprogramming or induction is in vivo, in vitro, or ex vivo.

[0323] 49. The method according to any one of items 47 to 48, wherein the method further comprises a step of culturing the transduced cells in a cell culture medium, the step being carried out before or after expression of the transcription factor.

[0324] 50. The method according to any one of items 47 to 49, further comprising culturing the transduced cells in a cell culture medium containing one or more cytokines selected from the group consisting of IFNβ, IFNγ, TNFα, IFNα, IL-1β, IL-6, CD40I, Flt3I, GM-CSF, IFN-λ1, IFN-ω, IL-2, IL-4, IL-15, prostaglandin 2, SCF, and oncostatin M (OM).

[0325] 51. The method according to any one of items 47 to 50, further comprising culturing the transduced cells in a cell culture medium containing one or more epigenetic modifiers, such as a histone deacetylase inhibitor.

[0326] 52. The method of claim 51, wherein the histone deacetylase inhibitor is valproic acid.

[0327] 53. The method according to any one of items 47 to 52, wherein the cell is a mammalian cell, such as a human cell or a mouse cell.

[0328] 54. The cell is selected from the group consisting of a stem cell, a differentiated cell, and a cancer cell; e) the stem cells are selected from the group consisting of pluripotent stem cells and multipotent stem cells, such as mesenchymal stem cells or hematopoietic stem cells; f) the differentiated cell is any somatic cell, such as a fibroblast, or a hematopoietic cell, such as a monocyte; 54. The method according to any one of items 47 to 53.

[0329] 55. The method according to any one of items 47 to 54, wherein the transduced cells are cultured for at least 2 days, such as at least 5 days, for example at least 8 days, such as at least 10 days, for example at least 12 days.

[0330] 56. The method according to any one of items 47 to 55, wherein the resulting reprogrammed or induced cells are type 1 conventional dendritic cells.

[0331] 57. The method of any one of items 47 to 56, wherein the resulting reprogrammed or induced cells are cluster differentiation 45 (CD45) positive.

[0332] 58. The method according to any one of items 47 to 57, wherein the resulting reprogrammed or induced cells are XC motif chemokine receptor 1 (XCR1) positive.

[0333] 59. Any one of items 47 to 58, wherein the resulting reprogrammed or induced cells are cluster differentiation 226 (CD226) positive.

[0334] 60. The method of any one of items 47 to 59, wherein the resulting reprogrammed or induced cells are human leukocyte antigen-DR isotype (HLA-DR) positive.

[0335] 61. A reprogrammed or derived cell obtained according to the method defined in any one of items 47 to 60.

[0336] 62. The reprogrammed or derived cell of item 61, wherein the cell is a dendritic cell, such as a type 1 conventional dendritic cell, or an antigen-presenting cell.

[0337] 63. The reprogrammed or induced cell of any one of paragraphs 61-62, wherein the resulting reprogrammed or induced cell is positive for one or more surface markers listed in Table 1.

[0338] 64. The reprogrammed or induced cell of any one of paragraphs 61 to 63, wherein the resulting reprogrammed or induced cell is CD45, HLA-DR, CD141, CLEC9A, XCR1, and / or CD226 positive.

[0339] 65. A composition according to any one of items 1 to 39, a cell according to any one of items 40 to 46, and / or a reprogrammed or derived cell according to any one of items 61 to 64 for use in medicine.

[0340] 66. The composition of any one of items 1 to 39, the cell of any one of items 40 to 46, and / or the reprogrammed or derived cell of any one of items 61 to 64 for use in the treatment of cancer or an infectious disease.

[0341] 67. The cancer is selected from the group consisting of basal cell carcinoma, cervical dysplasia, sarcoma, germ cell tumor, retinoblastoma, glioblastoma, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, blood cancer, prostate cancer, ovarian cancer, cervical cancer, esophageal cancer, uterine cancer, vaginal cancer, breast cancer, head and neck cancer, gastric cancer, oral cancer, nasopharyngeal cancer, tracheal cancer, laryngeal cancer, bronchial cancer, bronchiolar cancer, lung cancer, pleural cancer, bladder urothelial cancer, hollow organ cancer, esophageal cancer, gastric cancer, bile duct cancer, intestinal cancer, colon cancer, colorectal cancer, rectal cancer, bladder cancer, ureter cancer, renal cancer, 67. The composition, cell and / or reprogrammed cell according to item 66, wherein the cancer is selected from the group consisting of: liver cancer, gallbladder cancer, spleen cancer, brain cancer, lymphatic system cancer, bone cancer, pancreatic cancer, leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia, acute myelogenous leukemia, skin cancer, melanoma or myeloma, preferably wherein the cancer is selected from the group consisting of melanoma, head and neck cancer, breast cancer, colorectal cancer, liver cancer, lymphoma, bladder urothelial carcinoma, pancreatic cancer and glioblastoma.

[0342] 68. A method for treating cancer or an infectious disease, the method comprising administering to an individual in need thereof the composition of any one of items 1 to 38, the cell of any one of items 40 to 46, the pharmaceutical composition of item 39, and / or the reprogrammed or induced cell of any one of items 61 to 64.

[0343] 69. Use of the composition according to any one of items 1 to 38, the cell according to any one of items 40 to 46, the pharmaceutical composition according to item 39, and / or the reprogrammed or induced cell according to any one of items 61 to 64 for the manufacture of a medicament for treating cancer or an infectious disease.

Claims

1. A composition comprising one or more constructs or vectors encoding transcription factors that, upon expression: a) BATF3, or a biologically active variant thereof that is at least 90% identical to SEQ ID NO: 10 (BATF3); b) IRF8, or a biologically active variant thereof that is at least 90% identical to SEQ ID NO: 11 (IRF8); and c) PU.1, or a biologically active variant thereof that is at least 90% identical to SEQ ID NO: 12 (PU.1), wherein the one or more constructs or vectors comprise a promoter region capable of controlling the transcription of the transcription factor, and the promoter region comprises a spleen focus-forming virus (SFFV) promoter.

2. Upon expression, further comprising one or more constructs or vectors encoding one or more transcription factors selected from: a) IRF7, or a biologically active variant thereof that is at least 90% identical to SEQ ID NO: 21 (IRF7); b) BATF, or a biologically active variant thereof that is at least 90% identical to SEQ ID NO: 19 (BATF); c) SPIB, or a biologically active variant thereof that is at least 90% identical to SEQ ID NO: 23 (SPIB); d) SPIC, or a biologically active variant thereof that is at least 90% identical to SEQ ID NO: 25 (SPIC), wherein the one or more constructs or vectors comprise a promoter region capable of controlling the transcription of the transcription factor, and the promoter region comprises a spleen focus-forming virus (SFFV) promoter. The composition according to claim 1.

3. The composition according to any one of claims 1 or 2, wherein the SFFV promoter comprises or consists of a polynucleotide sequence that is at least 90% identical to SEQ ID NO:

1.

4. The composition is a) one construct or vector encoding transcription factors BATF3, IRF8, and PU.1 upon expression; b) one construct or vector encoding transcription factors BATF3, IRF8, and SPIB upon expression; c) a first construct or vector encoding transcription factors BATF3 and IRF8 upon expression, and a second construct or vector encoding transcription factor PU.1 upon expression; d) a first construct or vector encoding the transcription factors BATF3 and IRF8 upon expression, and a second construct or vector encoding the transcription factor SPIB upon expression, e) a first construct or vector encoding the transcription factor BATF3 upon expression, and a second construct or vector encoding the transcription factors IRF8 and PU.1 upon expression, f) a first construct or vector encoding the transcription factor BATF3 upon expression, and a second construct or vector encoding the transcription factors IRF8 and SPIB upon expression, g) a first construct or vector encoding the transcription factor IRF8 upon expression, and a second construct or vector encoding the transcription factors BATF3 and PU.1 upon expression, h) a first construct or vector encoding the transcription factor IRF8 upon expression, and a second construct or vector encoding the transcription factors BATF3 and SPIB upon expression, i) a first construct or vector encoding the transcription factor BATF3 upon expression, a second construct or vector encoding the transcription factor IRF8 upon expression, and a third construct or vector encoding the transcription factor PU.1 upon expression, and / or j) a first construct or vector encoding the transcription factor BATF3 upon expression, a second construct or vector encoding the transcription factor IRF8 upon expression, and a third construct or vector encoding the transcription factor SPIB upon expression, The composition according to any one of claims 1 or 2, comprising the same.

5. The composition according to any one of claims 1 or 2, wherein the one or more constructs or vectors are viral vectors selected from the group consisting of adenoviral vectors, lentiviral vectors, retroviral vectors, herpesviral vectors, poxviral vectors, adeno-associated viral vectors, paramyxovirus family vectors, rhabdoviral vectors, alphavirus vectors, and flavivirus vectors.

6. A cell comprising one or more constructs or vectors according to any one of claims 1 or 2.

7. The cell according to claim 6, wherein the cell is a mammalian cell.

8. The cell according to claim 7, wherein the mammalian cell is a human cell or a mouse cell.

9. The cell is selected from the group consisting of stem cells, differentiated cells, and cancer cells, a) the stem cell is selected from the group consisting of pluripotent stem cells and multipotent stem cells, b) the differentiated cell is any somatic cell, the cell according to claim 6.

10. The cell according to claim 9, wherein the pluripotent stem cell or the multipotent stem cell is a mesenchymal stem cell or a hematopoietic stem cell.

11. The cell according to claim 9, wherein the any somatic cell is a fibroblast or a hematopoietic cell.

12. A method of reprogramming or inducing a cell into a dendritic cell or an antigen-presenting cell, the method comprising the following: a) transducing the cell with a composition comprising the construct or vector according to any one of claims 1 to 2; b) expressing a transcription factor, thereby obtaining a reprogrammed or induced cell, the method comprising the above steps.

13. The reprogramming or induction according to claim 12 is in vivo, in vitro, or ex vivo.

14. The method according to claim 12 further comprises culturing the transduced cell in a cell culture medium, and the step is performed before or after the expression of the transcription factor.

15. The method according to claim 12 further comprises culturing the transduced cell in a cell culture medium containing one or more epigenetic modifiers.

16. The method according to claim 15, wherein the one or more epigenetic modifiers are histone deacetylase inhibitors.

17. The method according to claim 16, wherein the histone deacetylase inhibitor is valproic acid.

18. A reprogrammed or induced cell obtained according to the method defined in claim 12.

19. A pharmaceutical composition for use in medicine, comprising one or more constructs or vectors according to any one of claims 1 to 2.

20. A pharmaceutical composition for use in the treatment of cancer or an infectious disease, comprising one or more constructs or vectors according to any one of claims 1 to 2.

21. The pharmaceutical composition for use according to claim 20, wherein the cancer is selected from the group consisting of basal cell carcinoma, cervical dysplasia, sarcoma, germ cell tumor, retinoblastoma, glioblastoma, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, blood cancer, prostate cancer, ovarian cancer, cervical cancer, esophageal cancer, uterine cancer, vaginal cancer, breast cancer, head and neck cancer, gastric cancer, oral cancer, nasopharyngeal cancer, tracheal cancer, laryngeal cancer, bronchial cancer, bronchioloalveolar carcinoma, lung cancer, pleural cancer, urothelial cancer, luminal organ cancer, esophageal cancer, gastric cancer, cholangiocarcinoma, intestinal cancer, colon cancer, colorectal cancer, rectal cancer, bladder cancer, ureteral cancer, kidney cancer, liver cancer, gallbladder cancer, spleen cancer, brain cancer, lymphatic cancer, bone cancer, pancreatic cancer, leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia, acute myelogenous leukemia, skin cancer, melanoma, and multiple myeloma.

22. The pharmaceutical composition for use according to claim 21, wherein the cancer is selected from the group consisting of melanoma, head and neck cancer, breast cancer, colorectal cancer, liver cancer, lymphoma, bladder urothelial cancer, pancreatic cancer, and glioblastoma.

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