Congenital lymphocytes and biomarkers thereof for cell therapy

Abstract

Described herein is a method of isolating or enriching a population of cells for IL-10 producing type 2 innate lymphocytes, the method comprising: providing a population of cells comprising ILC2; cells expressing at least one of the following three markers: CD86, CD49d and retinoic acid related orphan alpha (RORA) are selected, and / or cells expressing GATA3 are rejected, including kits for implementing the methods. The use of isolated or enriched cell populations for cell therapy is also provided.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 469234, filed May 26, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to innate lymphocytes (intrinsic lymphocytes, ILCs), and more particularly to ILCs that produce IL-10, their use in cell therapy, and their biomarkers. Background Technology

[0004] Innate lymphocytes (ILCs) are a class of innate lymphocytes that play a central role in maintaining tissue and immune homeostasis (1, 2). Unlike T cells, ILCs lack antigen-specific receptors and instead respond rapidly to disturbances in the local microenvironment caused by cytokines, alarm molecules, neuropeptides, and hormones. The ILC family includes cytotoxic members (i.e., NK cells) and a “helper” subset (ILC1, ILC2, and ILC3) that coordinate immune responses and play important roles in tissue repair. Notably, ILC2 has been shown to recruit and activate regulatory T cells (Tregs) within tissues in inflammatory settings (3–5) and has been shown to protect against graft-versus-host disease (GVHD) (6) and ischemia-reperfusion injury (7) in mice, thus supporting its crucial role in promoting immune and tissue homeostasis.

[0005] Recent studies have identified a subset of ILC2 that produces IL-10 (called ILC2). 10 This subpopulation can suppress immune responses in studies of lung, neuroinflammation, and intestinal inflammation in mice, and it also expands in patients who respond to allergy immunotherapy (16-21). Furthermore, mouse ILC2... 10 It has also been shown to prevent islet rejection in a mouse islet transplantation model (56). Overall, these studies support the findings of ILC2. 10 It plays an important role in regulating harmful immune responses. Summary of the Invention

[0006] In some examples from this study, we explored the potential of human ILC2 for cell therapy in treating aGVHD and inflammatory / immune disorders. We developed a method for expanding human ILCs from peripheral blood and evaluated their therapeutic potential using a humanized mouse GVHD model. ILC2 expanded using our method expressed high levels of IL-10, which, along with other ILC2 expression patterns, significantly improved the therapeutic potential of human ILC2. 10 Phenotypic consistency allowed us to further identify amplified IL10. +The findings in this paper support the identification of ILC2 as a biomarker. 10 It is applied in cell-based therapies for GVHD, HSCT, transplantation, and inflammatory / immune conditions.

[0007] In one aspect, a method is provided for isolating or enriching a cell population for IL-10-producing type 2 innate lymphocytes (Group 2 innate lymphocytes, type 2 intrinsic lymphocytes, Group 2 intrinsic lymphocytes) (a method for isolating or enriching a cell population for IL-10-producing type 2 innate lymphocytes, a method for isolating or enriching a cell population for IL-10-producing type 2 innate lymphocytes), the method comprising: providing a cell population containing ILC2; selecting (selecting) cells expressing at least one of the following three markers: CD86, CD49d, and retinoic acid-associated orphan α (RORA), and / or deselecting (excluding) cells expressing GATA3.

[0008] In one respect, this paper provides a cell population enriched with IL-10-producing type 2 innate lymphocytes prepared by the method described herein.

[0009] In one aspect, an use of the cell population described herein is provided for use in the preparation of a medicine intended to improve, treat or prevent graft-versus-host disease or graft rejection, inflammatory conditions, autoimmune diseases, allergies, asthma, promote transplant graft function, and / or prevent or limit tissue damage in human subjects in need.

[0010] On one hand, a kit is provided for enriching cell populations for IL-10-producing type 2 innate lymphocytes (a kit for enriching cell populations for IL-10-producing type 2 innate lymphocytes, a kit for enriching cell populations of IL-10-producing type 2 innate lymphocytes), the kit comprising reagents for detecting cells expressing at least one of four markers CD86, CD49d, RORA and GATA3, and instructions for use.

[0011] In one aspect, a method is provided for generating or enriching a cell population for IL-10-producing type 2 innate lymphocytes (a method for generating or enriching a cell population for producing IL-10 type 2 innate lymphocytes, a method for generating or enriching a cell population for producing IL-10 type 2 innate lymphocytes), the method comprising: providing a cell population containing ILC2; and inducing overexpression of RORA in the cell population. Attached Figure Description

[0012] These and other features of the preferred embodiments of the present invention will become more apparent in the following detailed description with reference to the accompanying drawings, in which: Figure 1 Human ILC cells isolated from healthy blood and expanded in vitro maintain the expression of signature cytokines. To investigate the role of human ILC2 in GVHD, human ILCs were isolated from peripheral blood using flow cytometry and expanded in vitro using a subset of ILC-specific cytokines, as ILC2 is present in very low abundance in peripheral blood. (A) Representative gating strategies for ILC2 and ILC3. ILC2 cells were classified as live cells, lineage [missing information]. - CD94 - CD16 - NKG2D - CD127 + CRTh2 + CCR6 - Furthermore, ILC3 is classified as CD94. - CD16 - NKG2D - CD127 + CRTh2 - CD117 + CCR6 + Add lineage antibodies for removing non-ILCs, namely CD3 (OKT3), CD3 (UCHT1), CD4, CD8a, CD14, CD15, CD19, CD20, TCRαβ, and TCR. CD33, CD34, CD203c, FCεRI, CD79a, and CD138. (B) Cell expansion yield of ILC2 and ILC3 on days 20 and 3. On day 20 after stimulation with phorbol 12-myristate 13-acetate (PMA) and iomycin, CD56... dim Representative (C) and average (D) intracellular cytokines in NK cells (CD56-weakly expressing NK cells), ILC2, and ILC3. (E) Intracellular cytokines in ILC cells stained at 2x10⁻⁶. 5 After seeding (plating) at a concentration of 100 U / mL cells, and stimulation with 100 U / mL IL-2 for 16 hours, flow cytometry bead array analysis of secreted cytokines was performed.

[0013] Figure 2Cell therapy using human ILC2 to inhibit xenogeneic GVHD. To evaluate whether human ILC2 can limit GVHD, we employed an established xenogeneic GVHD model involving the transfer of PBMCs to NOD-scid-IL2Rg. null In NOD-scid-IL2Rg knockout mice (NSG). (A) Overview of the xenogeneic GVHD model to assess whether ILC2 can preventively limit GVHD. Briefly, NSG mice were irradiated and received PBS, human PBMCs (which induce multi-organ tissue lesions), or PBMCs with ILC2. Symptoms of xenogeneic GVHD in mice were monitored, including weight loss, survival, and a comprehensive xenogeneic GVHD score consisting of kyphosis, skin inflammation and hair loss, activity, pain, and percentage change in body weight. Blood was drawn every 7 days to confirm human immune cell transplantation and perform immunophenotyping. Mice were euthanized at the human or experimental endpoint, and tissues were collected for histological or flow cytometry analysis. (B) Representative transplanted human CD3 on day 14 after injection of human PBMCs. + CD45 + T cells (mostly human CD45) + (Cells). In blood 14 days after injection in three independent experiments, human CD3... + CD45 + (C), or CD4 + and CD8 + Mean T cell (D) transplantation (from 3 independent experiments, n=21 for NSG mice receiving human PBMCs and n=13 for NSG mice receiving human PBMCs plus allogeneic ILC2). E, F (E) representative xenogeneic GVHD score (n=3 / group in this experiment), and (F) representative weight loss from 3 independent experiments (n=3 / group in this experiment). G, H mean xenogeneic (G) GVHD score and (H) weight loss in mice on day 20 post-injection in 3 independent experiments (in 3 independent experiments, respectively, n=23 for PBS-treated NSG mice, n=19 for NSG mice receiving human PBMCs alone, and n=14 for NSG mice receiving human PBMCs plus allogeneic ILC2). (I) Survival rates of mice treated with ILC2 in three independent experiments (n=23, 19, and 14 for NSG mice receiving PBS, PBMC, and PBMC+ILC2, respectively). For any mouse that did not reach the humane endpoint, the experimental endpoint was day 40. (J) At the endpoint, CD4+ levels in the blood, bone marrow, and spleen of NSG mice. + and CD8 + T cells account for a significant portion of total human CD3+. +The mean proportion of T cells (n=11 and 13, from PBMC and PBMC+ILC2 in 3 independent experiments, respectively).

[0014] Figure 3 Cell therapy using ILC2 can inhibit CD4. + and CD8 + T cell proliferation, intestinal invasion, and CXCR3 expression. The effects of ILC2 transfer on T cell phenotype and proliferation in a xenogeneic GVHD model were assessed by flow cytometry. (A) Circulating human CD4+ in the blood at the endpoint. + Representative Ki-67 expression in T cells; (B) from 3 independent experiments, endpoint: human CD4 + Mean Ki-67 expression of T cells in blood, bone marrow, or spleen (from 3 independent experiments, n=11 mice per condition). (C) Circulating human CD8 + Representative Ki-67 expression on T cells. (D) From 3 independent experiments, endpoint was human CD8. + Mean Ki-67 expression of T cells in blood, bone marrow, or spleen (from 3 independent experiments, n=11 mice per condition). (E) Representative human CD3 immunohistochemistry of the colon of mice treated with PBMCs or PBMCs plus ILC2 (10x magnification). (F) Mean T cell infiltration in the colon of mice treated with PBS, PBMCs, or PBMCs + ILC2. Quantified using CD3 immunohistochemistry and the HALO algorithm, and normalized to the background in PBS mice.

[0015] Figure 4 Expanded human ILC3s can limit xenogeneic GVHD, but do not suppress in vivo T cell proliferation and intestinal invasion as observed with ILC2s. To investigate whether ILC3s can also limit GVHD, we evaluated the effects of cell therapy using ex vivo expanded human ILC3s in a xenogeneic GVHD model. G and H are present in circulating human CD4+. + (G) or CD8 + (H) Representative CXCR3 expression on T cells. At the experimental endpoint, human CD4+ expression was observed in blood, bone marrow, spleen, colon, and small intestine. + (I) or CD8 + (j) Mean CXCR3 expression on T cells (from 2 independent experiments, respectively, for PBMC condition, n=5, and for PBMC+ILC2 condition, n=5).

[0016] Figure 5 Increased circulating ILC2 and decreased CD4 in HSCT recipients +Th1 cell-associated HSCT recipients were protected from GVHD. Flow cytometry was used to assess the proportion and phenotype of ILCs in PBMCs from healthy donors and HSCT patients with or without GVHD. Total CD127 in A and B PBMCs. + Representative flow cytometry plots (A) and summary plots (B) for the ILC are used as CD45. + The proportion of lineage-negative viable cells. Lineage markers include CD3, CD4, CD8, CD14, CD19, CD20, CD33, CD34, CD123, CD138, CD303, FCεRI, and TCR. Representativeness (C) and mean (D) of PBMC in C and D CD117 - CRTh2 - CXCR3 + ILC1, CRTh2 + ILC2 and CRTh2 - CD117 + ILC3, as an active CD45 + The proportion of lineage-negative cells. (E) Representative CD4 counts in HSCT patients with or without aGVHD. + Th1 and CD8 + Tc1 cells. (F) CD4 in HSCT patients. + Th1 and CD8 + Tc1 cells, acting as CD4 cells, respectively + and CD8 + (G) The correlation between Th1 and Tc1 cells and ILC2 in all HSCT patients. (H) The correlation between Th1 and Tc1 cells and ILC3 in all HSCT patients.

[0017] Figure 6 IL-10-producing ILC2 expression of CD49d and CD86 was amplified in vitro. (A) UMAP representation of amplified ILC subsets by CITE-seq. ILC subsets were isolated by flow cytometry, amplified, and then stained with a CITE-seq antibody mixture. Each population was also labeled with a unique hashtag antibody to facilitate identification after sequencing. (B) Feature map showing protein levels of CD16, CD56, and CD117 on the amplified ILC subsets. PTGDR2 , GATA3 , IL17RB , GNLY , IL13 , IL5 and IL-10RNA level expression. (C) CD56 amplified after PMA / ionomycin stimulation. dim Representative intracellular IL-10 and bimodal protein expression in NK cells, ILC2, and ILC3. (D) Mean intracellular IL-10 expression (n=11–13) as assessed by flow cytometry, and IL-10 secreted by expanded subsets of ILCs (CD56) as assessed by flow cytometry bead array. dim NK cells, ILC2 and ILC3 (n=12, 19 and 20 respectively). (E)IL-10 + and IL-10 - Volcano plot of differentially expressed transcripts and antibody-derived tags (ADTs) among amplified human ILC2s. To validate IL-10... + ILC2-specific expression of surface markers, CD49dCD86, was analyzed by flow cytometry against other donors. FI, IL-10, was observed after PMA / ionomycin stimulation. + and IL-10 - Representative and mean expression of CD49d (F), CD86 (G), CD117 (H), and KLRG1 (I) in ILC2 (n=7). (J) Mean expression of IL-10 on ILC2 after PMA-ionomycin stimulation based on CD49d and CD86, as representative IL-10 expression, based on tSNE clustering of co-expression and mean plots (n=7).

[0018] Figure 7 ILC2 inhibits CD4 through a combination of IL-4 and IL-10. + and CD8 + T cell cytokine production. To determine whether the protective effect of cell therapy using ILC2 is due to ILC2's effect on allogeneic CD4... + and CD8 + We investigated the direct effects of T cells in vitro through co-culture. Expanded ILC2 cells were co-cultured with naive CD4 cells activated with anti-CD3 / anti-CD28 beads. + and CD8 + T cells were cultured together. Intracellular cytokine staining was assessed by flow cytometry after 4 days of culture. (A) Naïve CD4 cells cultured on day 4 with or without allogeneic ILC2. + Representative IFN-γ and TNF-α expression on T cells. (B) Compared with CD4 alone. +Compared to T cells, the mean decrease in IFN-γ production or co-expression of IFN-γ and TNF-α was expressed as a log2 fold change (n=17). (C) CD8+ co-cultured with allogeneic ILC2 and stimulated with anti-CD3 / CD28 beads for 4 days, as represented by flow cytometry. + Representative IFN-γ and TNF-α expression on T cells. (D) Compared with CD8 alone. + Compared to T cells, the mean decrease in IFN-g and co-expression of IFN-g and TNF-α was expressed as a log2 fold change (n=8). (E) Representative CD4 counts in the presence of specified blocking anti-IL-4 and / or anti-IL-10 antibodies, with or without ILC2 co-culture. + and CD8 + T cell IFN-γ expression. After culture with ILC2 cells supplemented with anti-IL-4 (n=10), anti-IL-10 (n=10), or anti-IL-4 and anti-IL-10 (n=7) blocking antibodies, CD4+ expression was increased. + (F) or CD8 + The effect of (G)T cells on the fold change of IFN-γ expression.

[0019] Figure 8 Cell therapy using ILC2 inhibits T-cell-mediated graft-versus-leukemia effects. (A) Circulating ILC2 from HSCT patients, as a proportion of lineage-negative PBMCs. Patients were grouped based on whether they experienced cancer recurrence. Follow-up at assessment ranged from 30 to 120 days. (B) Overview of a humanized GVL model in which leukemia cells (MV4-11 cells) were transplanted into NSG mice. On day 5 post-MV4-11 transfer, PBMCs were administered, and the transplanted T cells cleared the MV4-11 cells. (C) Representative MV4-11 cells in the bone marrow of NSG mice treated with PBS, PBMCs, or PBMCs with ILC2. (D) Mean MV4-11 transplantation from NSG mice treated with PBS, PBMCs, or PBMCs with ILC2 from two independent experiments (n=7 / group). (E) Mean xenogeneic GVHD score at day 19 from two independent experiments (n=7 / group). Day 19 corresponds to 14 days after injection of PBMC (with or without ILC2).

[0020] Figure 9 A general schematic diagram of the isolation and amplification of human ILC2. PBMCs were isolated using a Ficoll density gradient, and ILCs were enriched using lineage depletion combined with purification obtained via FACS sorting. ILC2 was classified as Lin. - CD45 + NKG2D, CD94 -NKp44 - CCR6 - CD127 + and CRTH2 + ILC2 cells were cultured in their activating cytokines for 20 days.

[0021] Figure 10 (A) A bidirectional lentiviral vector expressing the non-signaling nerve growth factor receptor (NGFR) or GFP marker gene and the ILC-associated transcription factor RORA has been cloned. The lentivirus was produced by transiently transfecting HEK 293T cells with a tetraplasmid for 48 hours. (B) A general protocol for transducing typical human ILC2.

[0022] Figure 11 RORA expression region isolate human ILC2. UMAP map of isolated ILC subset after transcriptome and epitope sequencing (CITEseq) cellular indexing, and corresponding RNA expression of GATA3 and RORA.

[0023] Figure 12 In vitro amplified IL-10-producing ILC2 cells expressed RORA and little or no GATA3, while IL-10-deficient ILC2 cells expressed GATA3. (A) UMAP plot of the amplified ILC subsets after CITE-seq. ILC subsets were isolated using FACS, amplified, and stained individually with a mixture of antibodies. Each cell population was stained with a unique tag antibody to allow for identification after sequencing. (B) UMAP plot showing the RNA levels of IL-10, RORA, and GATA3 on the amplified ILC subsets.

[0024] Figure 13 Human ILC2 cells expanded from healthy blood PBMCs maintained the expression of characteristic cytokines and produced the immunomodulatory cytokine IL-10 in vitro. A representative flowchart of intracellular cytokine staining in expanded human ILC2 cells is shown below. Two hours after stimulation, the protein inhibitors brefeldin A and monensin were added.

[0025] Figure 14 RORA expression is positively correlated with IL-10 production in ILC2. (A) Representative flowchart of transcription factor staining for amplified human ILC2. (B) IL-10 expression and amplified GATA3. + ILC2 and amplified RORA + Correlation of ILC2. In amplified GATA3 +and RORA + Flow cytometry expression of IL-10 in ILC2 cells after ICS is shown in the representative flowchart and correlation diagram. Secretory IL-10 and GATA3... + ILC2 and RORA + Correlation plot of ILC2. (C) ILC-related cytokines and GATA3 + ILC2 and RORA + Correlation plot of ILC2.

[0026] Figure 15 Human-typical ILC2 was transduced using NGFR-pCCL and GFP-pCCL.RORA at a multiplicity of infection (M2). Transduction efficiency was measured by NGFR or GFP expression 7 days post-transduction.

[0027] Figure 16 Overexpression of RORA induces the production of IL-10 and IL-22 in ILC2. (A) Log2 fold change in IL-10 production calculated using NGFR-pCCL control transduced ILC2, and (B) a summary plot of secreted IL-10 measured by GFP-pCCL.RORA transduced ILC2 after CBA, showing the Log2 fold change in secreted IL-10 and secreted IL-10 calculated using NGFR-pCCL control transduced ILC2.

[0028] Figure 17 Overexpression of RORA upregulates the surface expression of CD49d, a marker defining ILC2 that produces IL-10. A) Quantitative analysis of surface marker expression in GFP-pCCL.RORA ILC2 was performed by flow cytometry. ILC2 transduced with NGFR-pCCL control was tested in parallel to compare changes in surface marker expression (n=1), and B) flow cytometry expression of CD49d and CD86 in ILC2 transduced with GFP-pCCL.RORA, as well as flow cytometry expression of CD25, CD117, CD161, and KLRG1 in ILC2 transduced with GFP-pCCL.RORA, as shown in the summary figure.

[0029] Figure 18RORA expression is associated with IL-10 production in ILC2. A) Representative flow cytometry images of expanded ILC2 cells after intracellular cytokine staining (ICS), showing three high IL-10 producing donors and three low IL-10 producing donors. Summary diagram of IL-10 production from 12 donors. H) Summary diagram of expanded low IL-10 producing ILC2 cells compared to high IL-10 producing ILC2 cells, showing the ILC2-producing cytokines IL-4, IL-9, and IL-13. B) GATA3 + and RORA + Characteristic map and violin plot of single-cell IL-10 expression at the protein level on ILC2. C) GATA3 + and RORA + A violin plot of single-cell cytokine expression levels of IL-5, IL-13, IL-4, AREG, IFNG, IL-22, and IL-17A on ILC2 cells.

[0030] Figure 19 Human ILC2 can prevent immune-mediated destruction of transplanted islets. NSG mice were injected with 150 mg / kg STZ and 1500 HLA-A2 cells were transplanted subcapsularly into the kidneys. + IEQ was used to monitor blood glucose levels. After 4 weeks, mice were infused with 1.5 x 10⁻⁶ mg / L. 6 1.5 x 10 HLA-A2-specific CD4 and CD8 CAR-T cells were used, with or without administration. 6 Induced rejection of transplanted islets in the case of allogeneic ILC2 cell therapy. A) Experimental overview. B) Representative fasting blood glucose levels over time from one independent experiment, and the percentage of mice with normal blood glucose in three independent experiments. Detailed Implementation

[0031] Numerous specific details are set forth in the following description to provide a full understanding of the invention. However, it will be understood that the invention may be practiced without these specific details.

[0032] The success of allogeneic hematopoietic stem cell transplantation (HSCT) as a therapy for hematologic malignancies remains limited by graft-versus-host disease (GVHD) complications. Type 2 and 3 innate lymphocytes (ILC2 and ILC3, respectively) are associated with defense against GVHD; however, the assessment of their cell therapy potential is limited by the scarcity of human ILCs in peripheral blood. In this study, we developed a method for isolating and expanding large quantities of circulating human ILC2s and evaluated their cell therapy potential in a xenogeneic acute GVHD model (xenoGVHD). Adoptive transfer of ILC2s reduced the severity of xenoGVHD and prolonged NOD when administered prophylactically or during GVHD flare-ups. scid IL2Rγ null (NSG) mice survived. Mechanistically, ILC2 therapy inhibited allogeneic human T cell proliferation and intestinal invasion, and reduced CD4 counts. + Th1 and CD8 + The proportion of Tc1 cells. In parallel patient studies, increased ILC2 and CD4 in HSCT recipients. + A decrease in Th1 cell proportion is associated with ILC2 and its ability to defend against aGVHD supports the possibility that ILC2 can antagonize the harmful T cell response in HSCT recipients. CITE-seq analysis showed high IL-10 expression in in vitro expanded ILC2, and the expanded IL-10-derived ILC2 expression of CD49d and CD86. In vitro studies support the role of ILC2-derived IL-10 and IL-4 in mediating allogeneic CD4+ expression. + Th1 and CD8 + Suppression of Tc1 cell cytokine production. Importantly, a high proportion of ILC2 was not associated with an increased cancer recurrence rate in HSCT recipients, and in humanized HSCT models, adoptive transfer of ILC2 did not eliminate graft-versus-leukemia effects. Overall, these findings support the use of human IL-10. + ILC2 shows potential for cell therapy in GVHD following allogeneic HSCT or other transplants, as well as in inflammatory / immune conditions.

[0033] In one aspect, a method is provided for isolating or enriching a cell population targeting type 2 innate lymphocytes that produce IL-10, the method comprising: providing a cell population containing ILC2; selecting cells expressing at least one of the following three markers: CD86, CD49d, and retinoic acid-associated orphan α (RORA); and / or knocking out cells expressing GATA3.

[0034] In some embodiments, cells expressing CD86 are selected. In some embodiments, cells expressing CD49d are selected. In some embodiments, cells expressing RORA are selected. In some embodiments, cells expressing both CD86 and CD49d are selected. In some embodiments, cells expressing all of CD86, CD49d, and RORA are selected.

[0035] In some embodiments, the method further includes knocking out cells expressing GATA3.

[0036] In some embodiments, the cell population is contained in a sample from the subject. Preferably, the sample is blood, bone marrow, tissue, or lymphatic organ.

[0037] In some embodiments, the method is used to isolate type 2 innate lymphocytes that produce IL-10.

[0038] In some implementations, the enriched cell populations are intended to improve, treat, or prevent graft-versus-host disease, transplant rejection, inflammatory conditions, autoimmune diseases, allergies, asthma, promote transplant graft function, and / or prevent or limit tissue damage in human subjects in need.

[0039] In some embodiments, the method further comprises administering a therapeutically effective amount of a cell population enriched with IL-10-producing type 2 innate lymphocytes to a subject in need to improve, treat, or prevent graft-versus-host disease, transplant rejection, inflammatory conditions, autoimmune diseases, allergies, asthma, promote transplant graft function, and / or prevent or limit tissue damage in the subject.

[0040] In some embodiments, the subject has received or will receive a cell transplantation procedure. Preferably, the cell transplantation is a stem cell transplantation, which is preferably a hematopoietic stem cell transplantation.

[0041] In some implementations, the subject has already received or will receive a bone marrow transplant.

[0042] In some implementations, the subject has received or will receive a solid organ transplant.

[0043] In some implementations, the subject has received or will receive a stem cell-derived tissue, cell, or organ transplant.

[0044] In one aspect, this article provides a cell population enriched with IL-10-producing type 2 innate lymphocytes prepared by the method described herein.

[0045] In one aspect, an use of the cell population described herein is provided for use in the preparation of a medicine intended to improve, treat or prevent graft-versus-host disease or graft rejection, inflammatory conditions, autoimmune diseases, allergies, asthma, promote transplanted graft function, and / or prevent or limit tissue damage in human subjects in need.

[0046] On one hand, a kit is provided for enriching cell populations for IL-10-producing type 2 innate lymphocytes, the kit comprising reagents for detecting cells expressing at least one of four biomarkers CD86, CD49d, RORA and GATA3, and instructions for use.

[0047] In some embodiments, the kit includes reagents for detecting cells expressing CD86. In some embodiments, the kit includes reagents for detecting cells expressing CD49d. In some embodiments, the kit includes reagents for detecting cells expressing both CD86 and CD49d. In some embodiments, the kit includes reagents for detecting cells expressing CD86, CD49d, and RORA. In some embodiments, the kit includes reagents for detecting cells expressing CD86, CD49d, RORA, and GATA3.

[0048] In one aspect, a method is provided for generating or enriching a cell population for type 2 innate lymphocytes that produce IL-10, the method comprising: providing a cell population containing ILC2; and inducing overexpression of RORA in said cell population.

[0049] As used in this article, Therapeutic effective dose Therapeutic effective dose refers to the amount of a pharmacological agent (pharmaceutical) that is effective in achieving the desired therapeutic outcome at the necessary dosage and within a specific timeframe. Therapeutic effective doses can vary depending on various factors, such as an individual's disease state, age, sex, weight, and the agent's ability to elicit the desired response in an individual. Therapeutic effective dose also refers to the amount of a pharmacological agent whose beneficial therapeutic effect outweighs any of its toxic or harmful effects.

[0050] The advantages of the present invention are further illustrated by the following embodiments. The embodiments and specific details described herein are for illustrative purposes only and should not be construed as limiting the scope of the claims of the present invention.

[0051] Example 1

[0052] Methods and Materials

[0053] Research Design

[0054] The aim of this study was to evaluate the therapeutic potential of in vitro expanded human ILC2 in a humanized mouse GVHD model and the mechanism by which ILC suppresses T cells. We validated our findings in the HSCT patient cohort using flow cytometry from PBMCs isolated from patients who had developed GVHD and those who had not.

[0055] To explore the cell therapy applications of ILC2, total ILCs were enriched from PBMCs of healthy donors using magnetic separation and then isolated using fluorescence-activated cell sorting. Flow cytometry was used to assess surface markers on ILCs and T cells, as well as intracellular cytokine expression. Secreted cytokines were analyzed using a flow cytometry bead array. In vivo effects of ILC2 on xenogeneic GVHD were studied in 6- to 10-week-old NSG mice, which were randomly assigned to receive PBS, PBMCs alone, or PBMCs with ILC2. Blinding was used to monitor weight loss and clinical symptoms in the mice. T cell infiltration into tissues was determined by flow cytometry and IHC, and evaluated in a blinded manner. For in vivo graft-versus-leukemia assays, mice were randomly assigned to receive MV4-11 cells or PBS. Five days later, mice receiving MV4-11 cells were randomly assigned to receive PBS, PBMCs, or PBMCs with ILC2. No power analysis was performed. The sample size for in vivo studies varied based on the availability of amplified ILCs, but at least three mice were used in each group in all independent experiments.

[0056] CD4 for in vitro experiments was isolated from PBMCs using magnetic separation. + and CD8 + T cells were co-cultured with expanded ILC2 cells. The levels of expanded ILC2, ILC3, and CD56 were assessed using single-cell CITE-seq. dim NK cells (NK cells with weak CD56 expression) and CD56 bright RNA and surface protein expression on NK cells (CD56-overexpressing NK cells). Pathways involved in the suppression of T-cell inflammatory cytokines were identified by adding neutralizing antibodies. All in vitro assays were performed in duplicate, and each experiment was run at least three times independently.

[0057] The sample size for patient studies was determined based on sample availability and was either freshly obtained or sourced from the Messner Allogeneic Transplantation Project biobank within the University Health Network. Human samples obtained from patients undergoing HSCT were approved by the University Health Network Research Ethics Committee in accordance with the Declaration of Helsinki (UHN REB 19-6351). Healthy peripheral blood was obtained from donors through the Canadian Blood Services Blood4Research program, with written informed consent provided by each donor (UHN REB 17-6229, CBS-approved study 2020-047). Animal research protocols were approved by the University Health Network Animal Care Committee (UHN AUP 6203) and all ethical guidelines were followed.

[0058] statistics

[0059] Statistical significance was determined using the Kruskal-Wallis test or the one-tailed Mann-Whitney test. The log-rank (Mantel-Cox) test was applied to the Kaplan-Meir curve. Spearman correlation analysis was used to calculate the correlation data. The number of repetitions was... n This is indicated and displayed in each legend. p<0.05; P<0.01; P<0.001; P<0.0001; ns The results were not significant. Unless otherwise stated, error bars represent standard deviation. Data analysis was performed using GraphPad Prism v9.

[0060] Human PBMC Separation

[0061] Fresh blood was collected in EDTA collection tubes (BD Biosciences). In some cases, frozen PBMC samples were obtained from the Messner Allogeneic Transplantation Project Biobank. PBMCs were isolated using Lymphoprep (StemCell Technologies) according to the manufacturer's instructions.

[0062] Flow cytometry

[0063] After blocking with human TruStain FcX (BioLegend) for 15 minutes, surface marker staining was performed for 30 minutes. Cells were washed in FACS buffer (PBS + 2% FCS) and fixed in 2% paraformaldehyde in PBS (ThermoFisherScientific). For intracellular staining, cells were fixed and permeabilized using FOXP3 / transcription factor staining set (eBioscience) and incubated with intracellular antibodies at room temperature (RT) for 30 minutes. Samples were collected at LSR Fretessa (BD Biosciences) and data were analyzed using FlowJo v10 software.

[0064] Human ILC sorting

[0065] PBMCs were stained with human TruStain FcX (BioLegend) and incubated with a mixture of FITC-conjugated lineage antibodies. Cells were washed in FACS buffer, resuspended in EasySep buffer (StemCell Technologies), and enriched using the EasySep FITC Positive Selection Kit II (StemCell Technologies) according to the manufacturer's instructions. The enriched cells were stained with the antibody mixture and sorted using FACSAria Fusion (BD Biosciences).

[0066] Human ILC amplification

[0067] Human ILC2 and ILC3 cells were cultured and sorted in complete X-Vivo 15 medium (Lonza) supplemented with 5% human AB serum (Sigma), 100 U / ml penicillin-streptomycin (Gibco), and 1x GlutaMAX (Gibco). CD56 cells were cultured in NK MACS medium (Miltenyi) supplemented with 5% human AB serum. bright and CD56 dim NK cells. ILC2 was amplified using 100 IU / mL IL-2 (SteriMax), 20 ng / mL IL-7, and 20 ng / mL IL-33 (BioLegend). ILC3 was amplified using 100 IU / mL IL-2, 20 ng / mL IL-1β, IL-7, and IL-23 (BioLegend). CD56 was amplified using 500 IU / mL IL-2 and 20 ng / mL IL-15 and IL-18 (Biolegend). bright and CD56 dim NK cells.

[0068] Cytokine and chemokine assay

[0069] For intracellular cytokine analysis, a 2x10⁻¹⁰ mixture of cell stimulation molecules (eBioscience) was used. 5 Cells were stimulated for 6 hours, and GolgiStop and GolgiPlug (BD Biosciences) were added 2 hours later. Intracellular staining was then performed using antibodies. For analysis of secreted factors, 1x102 cells were used. 5 Cells were seeded in complete X-Vivo medium and stimulated with 100 IU / mL IL-2. After 16 hours, the supernatant was collected and stored at -80°C. Analytes were measured using the 12-Triple LegendPlex Human Th Cytokine Panel (BioLegend) or the 13-Triple LegendPlex Human Pro-inflammatory Chemokine Panel 1 (BioLegend) according to the manufacturer's instructions.

[0070] Xenogeneic GVHD mouse model

[0071] 1x10 was injected into the tail vein. 7 1 x 10 freshly isolated PBMCs (injected simultaneously or 9 days later in PBS, or not) 7 The day before the amplified ILC2, NOD.Cg- in six- to ten-week-old infants was analyzed. Prkdc scid Il2rg tm1Wjl / SzJ (NSG) mice were irradiated with 150 cGy (23, 44). Control mice were injected with PBS. GVHD symptoms in mice were monitored daily, including weight loss, hair loss and skin inflammation, kyphosis, activity, and pain, on a scale of 0–3. At the endpoint, blood, bone marrow, and spleen were harvested as previously described (44, 45). For intestinal immune cells, the intestines were flushed, sectioned, and rinsed in 1×PBS. Tissue was cut into small segments, added to 2 mM EDTA in 1×PBS, and incubated at RT for 30 min. The tissue was filtered, and the remaining tissue was added to a calcium-containing Hank balanced salt solution (Wisent Bio Products) containing 1,650 NPA-U BP protease (VitaCyte), 2,500 CDA-U collagenase MA (VitaCyte), and 100 µg / mL DNase I (StemCell Technologies). The tissue was then excised and incubated at 37°C for another 30 min. The tissue was filtered through a 70µm filter and washed twice in RPMI (Gibco) + 5% FCS.

[0072] For graft-versus-leukemia experiments, 2x10 [units of something] were injected intravenously via the tail vein. 6 One day prior to MV4-11 cells / mouse, six- to ten-week-old NSG mice were irradiated with 250 cGy. Five days later, the mice were individually administered 5 x 10 cells / mouse. 6 One PBMC or with 5x10 6 Mice were administered amplified human ILC2 cells together with the PBMCs. Mice were sacrificed 14 days after PBMC injection, and bone marrow was collected.

[0073] Histology

[0074] Harvested spleen, lung, and colon tissues were fixed in 10% neutral buffered formalin for 3 days and then preserved in 70% ethanol. Paraffin embedding, tissue sectioning, H&E staining, and immunohistochemical staining were performed by the UHN Pathology Research Project Laboratory. Cells expressing IHC markers were quantified using the HALO image analysis platform, and reported as a percentage of all cells.

[0075] Co-cultivation

[0076] Using EasySep Human Infancy CD4 + T cells or EasySep human CD8 + T-cell isolation kit (StemCell Technologies) isolates immature CD4 cells from healthy donor blood. + Total CD8 + T cells. T cells were prepared at a rate of 5 x 10... 4 Each well was seeded and stimulated with the human CD3 / CD28 T activator DynaBeads (Gibco) at a 1:8 bead:T cell ratio. Expanded ILC2 cells were added to T cells at a 1:1 ratio. T cell biomarkers were assessed by flow cytometry after 72 hours. For ILC supernatant assays, expanded ILC2 cells were added at 5 x 10⁻⁶ cells per well. 4 Cells / mL were seeded in complete X-Vivo medium without cytokines. After 16 hours, the supernatant was harvested, centrifuged at 1500 rpm for 10 min, and stored at -80°C. On the day of T cell culture, the supernatant was thawed and added to the T cells at a 1:1 ratio. Fresh supernatant was added every 24 hours for 3 days. For inhibitor experiments, UltraLeaf anti-human IL-4, IL-9, IL-10, and IL-13 (BioLegend) was added at 10 µg / mL. ARL67156 (Tocris) and PBS12379 (Tocris) were added at 1 µg / mL to inhibit the activity of CD39 and CD73, respectively. Inhibitors were added on day 0, and then every 24 hours for 72 hours thereafter.

[0077] Single-cell CITE sequencing

[0078] 1x10 6 The amplified ILC cells were washed twice in 1x cell staining buffer (BioLegend) and incubated in TruStain FcX for 15 min. The cells were then incubated with TotalSeq-C antibody mixture (BioLegend) for 30 min. The cells were washed three times in cell staining buffer and resuspended in 1xPBS containing 0.04% BSA (Millipore Sigma). Samples for sequencing were prepared according to the manufacturer's instructions using the 10X Genomics Single Cell 5' v2 platform, capturing 12,000 cells per sample. Reverse transcription, cDNA amplification, and sequencing library construction were performed using 10X Genomics Single Cell 5' v2 reagents. The samples were sequenced to a depth of 40,000 reads. Read alignment and gene expression matrices were generated against the reference human genome (GRCh38 / hg38) using CellRanger v6.1.2. Individual cells were filtered to exclude those expressing >10% mitochondrial content, <1000 total transcripts, and <200 unique genes. Data were log-normalized, principal component analysis was performed, and cells were clustered based on the top 20 principal components using Seurat's FindNeighbors and FindClusters (46) with the Louvain community algorithm. Clusters were visualized using Uniform Manifold Approximation and Projection (UMAP) (47). Clusters were assigned to ILC subsets based on the expression of tagged antibodies, the lack of lineage markers, and the expression of subset-related markers. Figure 6 a). Cluster definition markers were identified using FindMarkers in Seurat. Differential gene expression was visualized using EnhancedVolcano (48).

[0079] Data availability

[0080] The CITEseq dataset will be made publicly available through NCBI GEO when it is released.

[0081] Research Approval

[0082] Patient research protocols were approved by the University Health Network Research Ethics Committee in accordance with the Declaration of Helsinki (UHN REB 19-6351), and all patients provided written informed consent. Healthy peripheral blood was obtained from donors through the Canadian Blood Services Blood4Research program, with each donor providing written informed consent (UHN REB 17-6229, CBS-approved study 2020-047). Animal research protocols were approved by the University Health Network Animal Care Committee (UHN AUP 6203) and all ethical guidelines were followed.

[0083] Results and Discussion

[0084] Human ILC2 and ILC3 can be amplified without altering the cytokine profile.

[0085] To examine the potential of ILC2 and ILC3 cell therapies, we first needed to develop a method for isolating and expanding ILC2 from peripheral blood in a manner that preserved the expression of characteristic cytokines, as they were present at very low abundance. We isolated ILC2 from normal donor peripheral blood mononuclear cells (PBMCs) using flow cytometry sorting, employing a combination of ILC markers and exclusion lineage-positive cells. Figure 1 A). CD56 dim NK cells were sorted into live lineages - CD45 + CD56 + CD16 + ILC2 was sorted into a live lineage. - CD45 + CD94 - CD16 - NKG2D - CD127 + CRTh2 + CCR6 - Furthermore, ILC3 was sorted into a living lineage. - CD45 + CD94 - CD16 - NKG2D - CD127 + CRTh2 - CD117 + CCR6 +NK cells were expanded in NK MACS medium supplemented with IL-2, IL-15, and IL-18, followed by ILC2 expansion in X-Vivo medium supplemented with IL-2, IL-7, and IL-33, and ILC3 expansion in X-Vivo medium supplemented with IL-1β, IL-2, IL-7, and IL-23. Using this protocol, ILC2 and ILC3 showed robust expansion, resulting in an average of 2.3 x 10⁻⁶ cells per 10⁻⁶ cells after 34 days of culture. 4 ± 7.2x10 3 and 9.8x10 3 ± 3.6x10 3 Double amplification ( Figure 1 B). After amplification, upon stimulation with PMA-ionomycin, ILC2 strongly co-expressed IL-4, IL-13, IL-9, and GM-CSF, while the expression of IFN-γ, IL-17A, or IL-22 was low or absent. Figure 1 C, D). In contrast, amplified CD56 dim NK cells co-express IFN-γ and TNF-α, and ILC3 cells express IL-22 and GM-CSF, while IL-17A expression is low. Figure 1 (C, D) After stimulating a subset of ILCs with IL-2, the production of secreted cytokines was quantified using flow cytometry bead array (CBA). ILC2 cells secreted large amounts of IL-4, IL-9, IL-5, and IL-13, while NK cells and ILC3 cells did not produce significant amounts of these cytokines, but instead produced typical cytokines including IFN-γ and TNF-α or IL-22, respectively. Figure 1 E). The expanded ILC2 also produced the chemokines CCL2, CCL11, CCL20, and CXCL10. Furthermore, the expanded ILC2 maintained cytokine expression when cultured in ILC3 or NK cell medium, and the ILC3 maintained the ILC3-related cytokine profile when cultured in ILC2 or NK cell-related cytokine medium, supporting that both ILC populations exhibited stable phenotypes.

[0086] Adoptive transfer of allogeneic human ILC2 can suppress xenogeneic GVHD

[0087] Because previous mouse studies support the potential application of ILC2 transfer for GVHD (15, 22), we first evaluated the potential of human ILC2 cell therapy using a xenogeneic GVHD model (xenoGVHD). NOD-scid-IL2Rg was used in 6–8 week old females. nullMice were irradiated and injected with human PBMCs after HSCT to induce aGVHD-like histopathology (23). XenoGVHD signs in NSG mice were monitored using weight loss and a composite xenoGVHD score (23). Figure 2 A). Human CD45 was observed in the blood 14 days later. + CD3 + T-cell transplantation showed no significant difference in T-cell ratio between NSG mice given human PBMCs alone and mice treated with ILC2, indicating that ILC2 does not inhibit T-cell transplantation. Figure 2 BD).

[0088] The signs of xenoGVHD in NSG mice were monitored, including weight loss and a composite xenoGVHD score, which measured hair loss, skin inflammation, kyphosis, mobility, and pain. In three independent experiments, NSG mice treated with ILC2 significantly delayed the onset of GVHD and alleviated GVHD symptoms, including weight loss. Figure 2 EH). These effects were also observed in parallel experiments in male NSG mice, although the kinetics differed. This single infusion of ILC2 not only reduced the severity of xenoGVHD but also significantly improved survival in the model. Notably, ILC2-treated mice reached the endpoint only due to weight loss, but other GVHD symptoms remained mild. Figure 2 Otherwise, the endpoint would not be reached. To assess whether ILC2 could also treat GVHD during flare-ups, allogeneic ILC2 was adopted after T-cell transplantation and onset of xenoGVHD symptoms, resulting in defense against xenoGVHD similar to that achieved with prophylactic ILC2 administration on day 0. Overall, cell therapy using human ILC2 limited pathology and enhanced overall survival in this GVHD model.

[0089] Allogeneic ILC2 suppressed T cell responses in the xenoGVHD model.

[0090] Next, we attempted to determine how ILC2 defends against GVHD. Similar to previous reports, among all the organs analyzed, transplanted human CD45... + T cells account for >96% of the cells ( Figure 2 (BD)(23). Although there was no difference in transplantation on day 14, we noted that at the endpoint, NSG mice receiving ILC2 had significantly higher levels of CD4 in their blood, bone marrow, and spleen. + T cells increased moderately, and CD8 + T cells decrease accordingly ( Figure 2(J), indicating that ILC2 therapy altered T cell responses. We observed that in NSG mice treated with ILC2, CD4+ in blood, bone marrow, and spleen was increased. + and CD8 + T cells showed decreased Ki-67 expression, indicating reduced T cell proliferation. Figure 3 AD). This was not accompanied by changes in T cell expression of checkpoint molecules, including PD-1, CTLA-4, and CD25.

[0091] Previous studies have shown that in GVHD mouse models, CXCR3+ T cells drive migration to target organs, including the intestine (24, 25). CXCR3 also tags CD4. + Th1 and CD8 + Tc1 cells (26–28) induce intestinal damage in a GVHD model and are associated with disease in patients (2, 4). Therefore, we investigated whether ILC2 affects T cell phenotype and intestinal migration in a xenoGVHD model. Immunohistochemical staining of CD3 tissues showed a significant reduction in T cell infiltration in the colon of ILC2-treated mice, while no changes in T cell infiltration were observed in the lungs, spleen, or liver. Figure 3 E, F). In each of the compartments examined, CD4 in mice receiving ILC2 cell therapy + and CD8 + Decreased CXCR3 expression in T cells ( Figure 3 AD), while CD4 + The markers CRTh2 and CCR6 in Th2 and Th17 cells, respectively, did not increase (27-29). Therefore, adoptive transfer of ILC2 cells does not promote differentiation into other subsets of T cells, but it does suppress CD4. + Th1 and CD8 + Tc1 cells ( Figure 3 AD)(27-29).

[0092] HSCT recipients without GVHD have an elevated proportion of ILC2.

[0093] Previous studies have reported that, compared with HSCT patients who developed GVHD, HSCT patients who did not develop GVHD had lower levels of CD56 in their peripheral blood. bright Increased numbers of NK cells, ILC2, and ILC3 (12). This is due to ILC2-mediated CD4+ in the xenoGVHD model. + Th1 and CD8 +Regarding Tc1 cell suppression, we were interested in the relationship between ILC and T cell subsets in HSCT recipients with different clinical outcomes. We analyzed the proportions of ILC and T cell subsets in the blood of HSCT patients at the time of aGVHD diagnosis and compared the proportions of these populations in patients without aGVHD with those in healthy donors. All HSCT recipients were receiving prophylactic post-transplant cyclophosphamide therapy, and two patients received additional immunosuppression at the time of sample collection. Consistent with previous literature, patients without aGVHD had a reduced proportion of CD56... dim NK cells and an increased proportion of CD56 bright NK cells (10, 11). Similarly, patients who developed aGVHD had a reduced proportion of helper ILC (Lin - CD127 + () Figure 5 (A, B) Compared with healthy controls, the proportions of ILC1, ILC2, and ILC3 were reduced ( Figure 5 (C, D). However, HSCT patients without aGVHD had a significantly higher proportion of ILC2, but the difference in ILC1 or ILC3 compared to patients with aGVHD was not significant in our sample size. Figure 5 C, D).

[0094] Next, we examined the correlation between ILC and T cell subsets in HSCT recipients. Similar to what we observed with ILC2 therapy in the xenoGVHD model, HSCT recipients with aGVHD had increased CD4 counts. + Th1 and CD8 + Tc1 cells, as representatives of total CD4 + and CD8 + The proportion of T cells ( Figure 5 E, F), Th17 cells showed no significant difference (26-28). In all HSCT patients, elevated ILC2 cells were associated with a low proportion of Th1 cells (as CD3 cells). + The percentage of T cells was strongly correlated with ( Figure 5 G). However, the proportion of ILC3 was not significantly correlated with changes in Th1 or Tc1 cells in HSCT patients (G). Figure 5 H). Combined with our findings that ILC2 therapy can inhibit xenoGVHD and suppress allogeneic CD4, + Th1 and CD8 + Tc1 cells, the proportion of normal human ILC2 after HSCT can defend against aGVHD and are associated with a reduced pathogenic T cell response.

[0095] Amplified ILC2 exhibits ILC210 Phenotype

[0096] To confirm the potential mechanism by which ILC2 suppresses allogeneic T cells, we performed single-cell transcriptome and epitope sequencing-based cell indexing (CITE-seq) on amplified ILCs. Amplified ILCs were stained with a TotalSeq-C antibody mixture and labeled with tag antibodies for easy identification after sequencing. Figure 6 A). All examined ILCs expressed pedigree-defining markers, distinguishing them from other family members. CD56 bright and CD56 dim NK cell expression GNLY And CD56, and CD56 dim NK cells express CD16. ILC3 cells strongly express CD117, and ILC2 cells also express CD16. PTGDR2, GATA3, IL17RB, IL13 and IL5 ( Figure 6 B).

[0097] We evaluated the relationship with regulatory CD4. + FOXP3 + Expression of Treg and T regulatory 1 (Tr1) cell-related molecules. Low expression or background expression of checkpoint molecules such as PD-1, PD-L1, CTLA-4, or LAG3 were observed at both RNA and protein levels. FOXP3 Gene This is also true (30, 31). Furthermore, we did not detect... IL12A and EBI3 These constitute the regulatory cytokine IL-35. Although TFGB1 TGF-β1 is universally transcribed in ILC subsets, but no active TGF-β1 protein level is expressed in expanded NK cells, ILC2, or ILC3. Despite the absence of these regulatory molecules, both expanded ILC2 and ILC3 express TGF-β1. ENTPD1 This refers to the CD39 gene. ILC2 and ILC3 are also weakly expressed. NT5E (CD73), but only ILC2 and CD56 are available. bright NK cells exhibited expression of the surface CD73 protein. Flow cytometry analysis confirmed the expression of these enzymes on expanded ILCs, with ILC2 co-expressing high levels of CD39 and CD73, which are known to inhibit T cell functional activity (32, 33).

[0098] Interestingly, one of the first genes identified as being uniquely expressed by ILC2 is IL-10 ( Figure 6 B), we verified this using flow cytometry and flow cytometry bead arrays ( Figure 6(C, D) In ​​all donors, amplified ILC2 supernatant and consistently high levels of IL-10 secretion were observed, and a high proportion of IL-10 was noted in most of the tested donors. + cell( Figure 6 (C, D). Therefore, the ILC2 amplified using our method is always IL-10. + ILC2. It is worth noting that several recent studies have found IL-10... + ILC2 possesses immunomodulatory properties, including the ability to suppress autologous and allogeneic T cells (17, 21, 34).

[0099] Although IL-10 levels were consistently high, we noted differences in IL-10 levels between ILC2 isolates from different donors. + The cell proportions were non-uniform. We investigated IL-10 that could be identified in our expanded ILC2 cultures. + Can ILC2 markers better identify IL-10? + ILC2. By IL10 + ILC2 and IL10 - ILC2 comparisons were performed using differential gene expression analysis to reveal possible distinguishing genes. IL-10 + and IL10 - The markers of ILC2. In IL10 + ILC2 noted CD86 and ITGA4 Increased expression of (CD49d) Figure 6 E). Flow cytometry confirmed the difference relative to IL-10. - ILC2, IL-10 + ILC2 cells showed high expression of CD49d and CD86. Figure 6 F, G). We also examined CD117 expression, as CD117 expression differed among isolated ILC2 cells during the isolation process, as well as KLRG1, which had previously been reported to be expressed by ILC2 cells that produce IL-10 (21). IL-10 + and IL-10 - ILC2 expression of these markers was very low, although we noted a small but significant fold increase in CD117 expression. Figure 6 H, I). Expanded ILC2 cells were clustered based on CD49d and / or CD86 expression, revealing the co-expression of CD49d and CD86 relative to CD86. - CD49d - ILC2 had the highest IL-10 expression ( Figure 6 J). Therefore, CD49d and CD86 expression labeled IL-10 amplified in vitro. + ILC2, which can be used to add IL-10 to an amplification protocol. + Purity of ILC2.

[0100] To evaluate the role of these biomarkers in identifying ILC2 in patient-based studies 10 To explore the potential utility, we examined publicly available scRNA-seq datasets of ILCs from patients with hay fever who were treated with either sublingual hay fever allergen-specific immunotherapy (GP-SLIT) or placebo (PL-SLIT) (21). Here, the authors report that patients treated with GP-SLIT had improved outcomes and increased ILC2 at 12 and 24 months of follow-up compared to those treated with placebo. 10 Proportion. Furthermore, the authors report that KLRG1 expression defines ILC2. 10 Furthermore, at both time points following GP-SLIT, pathways related to IL-10 signaling and regulation were upregulated. We reanalyzed this scRNA-seq dataset to assess whether the increased IL-10 expression in ILC2 was associated with increased CD49d and CD86. After clustering ILC2 from patients treated with GP-SLIT, as reported, the expression of both IL10 and IL4 was increased (…). Figure 6 K). Furthermore, patients treated with GP-SLIT showed increased expression of IL2RA, IL7R, and IL1RL1, which are beneficial for our ILC2. 10 Amplified cytokine receptors ( Figure 6 K). It is worth noting that, in addition to the reported increase KLRG1 In addition to expression, ILC2 levels were elevated in patients treated with GP-SLIT. ITGA4 (CD49d) and CD86 Express( Figure 6 K). This elevated expression of CD49d and CD86 is associated with ILC2. 10 This supports the use of these biomarkers to distinguish ILC2 in human studies. 10 And the potential for wide-ranging applications of inflammatory ILC2.

[0101] Amplified ILC2 inhibits CD4 through IL-4 and IL-10. + and CD8 + T cells

[0102] To determine whether ILC2 or ILC3 can directly regulate allogeneic T cells, a bulk of ILC2 or ILC3 was used in combination with allogeneic naive CD4 cells. + or CD8 + T cells were co-cultured in vitro. Culture with ILC2 induced IFN-γ. + and IFN-γ + / TNF-α + T cells were significantly reduced ( Figure 7 A, B). When allogeneic ILC2 and CD8 + When T cells were cultured together, a decrease in IFN-γ was also observed. Figure 7 (C, D). To confirm this in vivo finding that ILC2 reduces Th1 and Tc1 cells without increasing the proportion of Th2 or Th17 cells, we also examined cytokines expressed by other T cell subsets. (In cells containing CD4...) + or CD8 + No increase in IL-17A, IL-22, IL-4, IL-9, or IL-13 was observed after adding ILC2 to T cell cultures. Therefore, the decreased IFN-γ does not appear to be due to altered T cell differentiation, but rather to ILC2-mediated CD4+ reduction. + Th1 and CD8 + This is due to the suppression of Tc1 cells. Compared to ILC2, it is more pronounced compared to CD4. + and CD8 + ILC3 co-cultured with T cells did not affect cytokine expression, further validating that ILC3 is regulated through a different mechanism than ILC2. These data, combined with xenogeneic GVHD data, support the conclusion that while ILC3 may have a protective role in GVHD, its function differs from that of ILC2.

[0103] Next, we explored whether various molecules associated with ILC2 or identified by CITE-seq control the regulation of ILC2-mediated T cell interactions. We noted that the inhibition of T cells by allogeneic T cells activated in the presence of the supernatant of ILC2 stimulated by IL-2 was comparable to that by directly adding ILC2 to T cell cultures, suggesting that one or more secreted factors produced by ILC2 are the basis for its T cell-suppressing ability.

[0104] Next, ILC2 cells were co-cultured with allogeneic T cells (35-37°C) with or without neutralizing antibodies or inhibitors targeting the target molecules (molecules of interest) (IL-10, CD39, CD73) and ILC2 cytokines (IL-4, IL-9, IL-13). In vitro, the addition of IL-4 neutralizing antibodies strongly reduced ILC2-mediated CD4+.+ T cells inhibit IFN-γ, but blocking IL-9, IL-13, or CD39 / CD73 has no effect. Figure 7 E, F). Neutralizing IL-10 also reduces CD4. + T cell-IFN-γ inhibition, but to a lesser extent than IL-4 ( Figure 7 E, F). Conversely, the combination of anti-IL-4 and anti-IL-10 eliminated ILC2-mediated CD8. + T cells inhibit IFN-γ, but this was not observed when using anti-IL-4 or anti-IL-10 alone. Figure 7 E, G). Therefore, IL-4 and IL-10 are ILC2 inhibitors of allogeneic CD4. + and CD8 + At least one mechanism of T cells, in which IL-4 and IL-10 affect CD8 + and CD4 + The contributions of T cells to regulation differ.

[0105] Allogeneic human ILC2 does not inhibit T cell-mediated graft-versus-leukemia effects.

[0106] Graft-mediated leukemia (GVL) effects are crucial for the effectiveness of HSCT. Given our observation of direct regulation of T-cell responses by ILC2 in a xenograft GVHD model and the increased proportion of ILC2 in patients who defended against GVHD development, a key question is whether ILC2 adversely affects T-cell-mediated GVL effects. In our clinical cohort, we assessed circulating ILC2 proportions and how they correlated with relapse status. No significant difference in ILC2 proportions was observed in the blood of patients who continued to experience relapse of their malignancies using our sample size, indicating that an increase in ILC2 proportions did not suppress GVL effects. Figure 8 A).

[0107] Next, we evaluated whether ILC2 cell therapy negatively impacted T cell-mediated GVL effects in a humanized mouse model. In short, MV4-11 cells (an acute myeloid leukemia cell line) were injected into mice 5 days prior to the transfer of individual PBMCs or PBMCs with expanded allogeneic ILC2. Figure 8 B). Subsequently, the ability of T cells to kill MV4-11 cells was assessed at 14 days. As expected, injection of PBMCs resulted in a reduction of MV4-11 cells in the bone marrow compared to mice receiving MV4-11 cells alone. Figure 8 C, D). The reduction in MV4-11 cells in the bone marrow of mice treated with PBMCs and allogeneic ILC2 was comparable to that observed in mice receiving PBMCs alone. Figure 8 C, D). Importantly, in the same mice, ILC2 treatment reduced xenoGVHD symptoms, similar to those observed in mice without MV4-11 metastasis. Figure 8 (E), which clearly demonstrates that the pathogenic T cell response underlying GVHD is defended without impairing the T cell-mediated GVL effect. Overall, our findings support that while ILC2 can defend against GVHD, it does not limit the antileukemic effects of allogeneic T cells, thus representing a strong candidate for controlling ILC2 in cell-based therapies for HSCT.

[0108] In addition to GVHD, ILC2 also has cell therapy applications for transplantation and autoimmune diseases, because adoptive cell therapy of ILC2 can suppress immune-mediated rejection in humanized transplantation models. Figure 19 In this model, non-diabetic 8-14 week old NOD.Cg- Prkdc scid Il2rg tm1Wjl / SzJ (NSG) mice were intraperitoneally (IP) injected with streptozotocin (STZ) at a concentration of 150 mg / kg dissolved in 0.1 mol / L citrate buffer (pH 4.5) to induce diabetes and destroy endogenous mouse β-islet cells. One to two days later, human HLA-A+2 cadaveric islets were transplanted subcapsularly into the left kidney of mice with confirmed elevated blood glucose levels (>11 mmol / L). Each mouse received 1500 IEQ HLA-A2... + Islets were selected and transplantation was confirmed within 28 days. After 28 days, mice with successful transplantation (determined by sustained blood glucose (BG) < 11 mmol / L) received an intravenous (IV) injection of 1.5 x 10⁻⁶ islets. 6 Individual antibody-HLA-A2 CAR T cells (equal CD4 and CD8 ratio) were used, with or without 1.5 x 10⁻⁶ cells. 6 In the case of amplified ILC2, rejection of transplanted islets was induced. The ability of ILC2 to prevent transplanted islet rejection was assessed by 4-hour fasting blood glucose monitoring and an intraperitoneal glucose tolerance test (IPGTT), in which mice were subsequently challenged with 200 mg / kg glucose. Mice were considered to have diabetes if their fasting glucose reading reached >17 mmol / L. All glucose readings were measured on an Accu-Chek Guide blood glucose meter (Roche). Figure 19 As shown in B, mice receiving cell therapy using human ILC2 were protected from T-cell-mediated destruction of transplanted islets, supporting the application of human ILC2 in suppressing harmful immune responses in transplantation and in autoimmunity (type 1 diabetes in this example).

[0109] discuss

[0110] This study demonstrates the cell therapy potential of human IL-10-generated ILC2 to regulate harmful T cell responses, particularly those driving GVHD pathology. In a humanized mouse model, ILC2 therapy suppressed the development of GVHD without impairing GVL effects crucial for HSCT success. These findings are relevant to patient-based studies where high ILC2 proportions were associated with defense against GVHD without increasing the risk of cancer recurrence. CD4+ in xenoGVHD models + and CD8 + T cells exhibited reduced proliferation, tissue migration, intestinal infiltration, and a decreased proportion of CD4+ in multiple tissue sites treated with ILC2 therapy. + Th1 and CD8 + Tc1 cells. ILC2 cells expanded using our method expressed high levels of IL-10, similar to ILC2 cells. 10 The identity was consistent and highly stable. CD86 and CD49d labeled the in vitro amplified IL-10. + ILC2, which inhibits CD4 through a combination of IL-10 and IL-4. + and CD8 + IFN-γ expression on T cells. In summary, our findings support the presence of IFN-γ in human ILC2 cells. 10 It limits harmful allogeneic T-cell responses and may have applications in adoptive cell therapies designed to limit harmful immune responses.

[0111] Various prophylactic treatments have been explored for the treatment and prevention of aGVHD, including post-transplant cyclophosphamide, calcineurin inhibitors (such as tacrolimus or cyclosporine), and other immunosuppressants (such as mycophenolate mofetil, anti-thymocyte globulin, and sirolimus). While these treatments have significantly improved aGVHD outcomes, the development of severe aGVHD remains a clinical challenge, and treatment options are very limited for patients who do not respond to these therapies. In a recent multicenter review encompassing various treatment modalities, more than half of HSCT patients required hospitalization within 100 days post-transplant, primarily due to the development of aGVHD, with severe GVHD (grade III-IV) occurring in 41.9% of patients (42, 43). Patients diagnosed with severe aGVHD were primarily treated with increased steroid doses (51.3%); however, 52.8% of these patients died during follow-up (42, 43). Therefore, new therapies remain urgently needed to improve the success rate of HSCT.

[0112] Previous studies have reported that ILCs are particularly sensitive to pre-transplant conditioning therapy and are depleted from peripheral blood and tissues (15, 38, 39). Unlike NK cells, helper ILC remodeling is slow, further leading to a reduction in helper-like ILCs after HSCT. Our results complement these previous studies, demonstrating the interaction between ILC2 and CD4. + A negative correlation was found between Th1 cells (associated with GVHD pathology) and human ILC2 administration, either prophylactically or during GVHD flare-ups, was demonstrated to suppress xenogeneic GVHD. Analysis of the effects of ILC2 on T cells revealed a novel protective mechanism of ILC2: direct inhibition of allogeneic CD4 via a combination of IL-4 and IL-10. + Th1 and CD8 + The ability of Tc1 cells. This differs from previous studies on mouse ILC2, in one major GVHD MHC-mismatch BMT model where ILC2 recruits immunosuppressive MDSCs to the gut for protection via IL-13, and when mouse ILC2 loses IL-13, T cells are not suppressed due to the lack of MDSC recruitment (15, 22). Using this xenogeneic GVHD model, the vast majority of transplanted human CD45 cells... + The cells in question are T cells, with limited contributions from myeloid cells, NK cells, and B cells. Therefore, while we were able to assess the effects of ILC2 on T cells, there may be other protective mechanisms that cannot be captured using this xenogeneic mouse model.

[0113] CD127 + CRTh2 + ILC2s are heterogeneous, and their cytokine production is influenced by activated cytokines (16, 17, 19–21, 34). While massively expanded ILC2s successfully limited the development of xenoGVHD, this could be enhanced by isolating only ILC2s expressing IL-4 and IL-10. Considering ILC2s... 10 In one of the few studies on unique markers, Golebski et al. reported KLRG1 + ILC2 produced IL-10 and other characteristic ILC2 cytokines (21). Here, the amplified human IL-10... + CITE-seq analysis of ILC2 revealed limited KLRG1 expression, but conversely, high levels of CD49d and CD86. Flow cytometry confirmed that ILC2 cells co-expressing CD49d and CD86 exhibited the highest IL-10 expression, supporting the use of these biomarkers for isolating or enhancing ILC2 cells in amplification protocols. 10 Purity.

[0114] Tregs can significantly reduce the severity of aGVHD in preclinical mouse models, and clinical trials have noted signs (evidence) that Tregs can limit allogeneic immune responses. However, the use of polyclonal Tregs has not achieved significant response rates, and most patients still develop GVHD or require systemic immunosuppression. （refs） Furthermore, Treg therapy is limited by several issues, including hindering the GVL effect, difficulties in the stability and purity of Tregs during amplification, and limited efficacy when using polyclonal Tregs (40). In addition, Tregs are typically derived from transplant donors or recipients, requiring time-sensitive isolation and amplification (41). Here, we demonstrate that amplified human ILC2, which is allogeneic to PBMCs, suppresses xenoGVHD severity while preserving the GVL effect, supporting the use of third-party donors as a “ready-to-use” ILC2 source for cell therapy. Furthermore, mouse studies have demonstrated that ILC2-derived bimodal proteins can defend against tissue damage during GVHD, representing an additional repair benefit compared to Tregs (15, 22). This is due to the amplified human IL-10... + ILC2 also expresses high levels of bimodal proteins, and its dual immune regulation and repair functions may represent the potential of ILC2. 10 Additional advantages for use in GVHD and other indications.

[0115] ILC2 exhibits significant heterogeneity and can exert pro-inflammatory or tissue-protective functions based on cytokines and microenvironmental factors. 10 It has been shown to suppress immune responses in a variety of contexts, including inflammation in the lungs, gut, and nervous system. In humans, individuals with hay fever have ILC2s with a reduced ability to produce IL-10 upon stimulation, and patients who respond to allergy immunotherapy have an increased proportion of IL-10. + ILC2 (21). In the context of transplantation, Huang et al. demonstrated that mouse ILC2 10 Survival of islet allogeneic transplants can be prolonged by inhibiting T cell attack on transplanted islets (34). Although increasing reports demonstrate that ILC2 10 ILC2 plays a crucial role in regulating immunity or, alternatively, promoting pro-inflammatory responses, but we have lacked the ability to accurately identify ILC2 from routine or inflammatory ILC2. 10 The markers. In attempting to identify ILC2 10 In one of the few studies on unique markers, Golebski et al. reported KLRG1 +ILC2 produces IL-10 and other characteristic ILC2 cytokines (21). However, KLRG1 is also associated with inflammatory ILC2 and may instead label activated or memory-like ILC2 populations rather than specifically labeling ILC2. 10 (57-60). For activated and amplified human ILC2 10 CITE-seq analysis showed limited KLRG1 expression, but high levels of CD49d and CD86 expression. Flow cytometry confirmed that ILC2 cells co-expressing CD49d and CD86 had the highest IL-10 expression. To assess the broader utility of these biomarkers, we evaluated their correlation with ILC2 cells observed by Golebski et al. in patients with hay fever who responded to allergen immunotherapy. 10 Related. In addition to previously reported KLRG1 In addition, after allergy immunotherapy ITGA4 (CD49d) and CD86 It also increases. However, ILC2 amplified in vitro from peripheral blood... 10 The absence of KLRG1 expression may indicate that KLRG1 is downregulated in vitro, or that KLRG1 is expressed by ILC2 in different tissues or contexts. 10 Differential expression. However, the combination of CD49d and CD86 was able to express IL-10 in both studies. + ILC2 is distinguishable from regular ILC2, demonstrating its effectiveness in tracking ILC2 in human studies. 10 The effectiveness of the response.

[0116] ILC2 10 It has been shown to limit inflammation in various contexts. Lung ILC2s in mice with limited inflammation after IL-2 administration showed significant IL-10 induction, and in intestinal mice, ILC2s were the major producers of IL-10 during homeostasis and disease (17). Individuals with pollen allergies had ILC2s with reduced capacity to produce IL-10 upon stimulation, while patients responding to allergy immunotherapy had an increased proportion of IL-10. + ILC2 (21). In the context of transplantation, Huang et al. demonstrated that mouse IL-10-generating ILC2 can prolong the survival of islet allogeneic transplants by limiting allogeneic T cell attack on transplanted islets (34). These increasing reports demonstrate the efficacy of ILC2. 10 It plays an important role in regulating immunity, and combined with our research, it provides insights into the role of ILC2-based immune systems in other contexts (including autoimmunity and transplantation). 10 Cell therapy provides a strong justification.

[0117] Therefore, we also demonstrated that human ILC2 can prevent T cell-mediated attack in a mouse transplantation model. Figure 19 Islet transplantation is a potentially curative therapy for individuals with type 1 diabetes (T1D) because it restores insulin-producing beta cells and has the ability to normalize glycemic control. However, individuals receiving islet transplantation require a lifelong, harmful immunosuppressive regimen to suppress recurrent autoimmune and allogeneic immune responses. While other tolerability-enhancing adoptive cell therapies are being explored, ILC2 represents a promising approach due to its ability to suppress T cells in an allogeneic context. Here, we utilize human ILC2 to prevent transplanted islet rejection, supporting its potential application in both transplantation and autoimmunity.

[0118] Example 2

[0119] The applicant further explored the ILC2 subsets that produce IL-10 and their ability to limit T-cell responses in islet transplantation, helminth expulsion, and allergic rhinitis. [The text abruptly shifts to a seemingly unrelated topic:] IL-10 + The characteristics that distinguish ILC2 from those that do not produce IL-10 are largely unknown, and the transcription factors that induce IL-10 production in ILC2 have not yet been identified. To explore IL-10... + Based on the biological characteristics of ILC2 and its application in tolerable cell therapy, we developed a method for isolating and expanding human IL-10. + The ILC2 method was used to identify IL-10. + Biomarkers for selective ILC2 expression. Using CITEseq, we evaluated biomarkers for selective ILC2 expression in human ILC2 cells, particularly IL-10. + ILC2 transcription factor expression. Isolated ILC2 cells derived from blood selectively express RAR-associated orphan receptor A (RAR-associated orphan receptor A). RORA ),but GATA-3 It is expressed by all human ILCs at the transcriptional level ( Figure 11 IL-10 expression was detected after ILC2 was activated using the aforementioned cytokines for amplifying human ILC2. When assessing IL-10 expression in activated human ILC2, IL-10 expression was found to be significantly higher than previously thought. RORA But not express GATA-3 IL-10 expression was selectively detected in ILC2. Figure 12 BC).

[0120] Materials and Methods

[0121] Figure 9A general schematic diagram of the isolation and amplification of human ILC2 is shown. PBMCs were isolated using a Ficoll density gradient, and ILCs were enriched using a combination of lineage depletion and purification by FACS sorting. ILC2 was sorted as Lin - CD45 + NKG2D, CD94 - NKp44 - CCR6 - CD127 + and CRTH2 + ILC2 cells were cultured in their activating cytokines for 20 days.

[0122] refer to Figure 10 A. These studies used bidirectional lentiviral vectors that expressed non-signaling nerve growth factor receptor (NGFR) or GFP marker genes and ILC-associated transcription factor RORA. The lentiviruses were generated by transiently transfecting HEK293T cells with a tetraplasmid for 48 hours.

[0123] Figure 10 B shows a general protocol for transducing typical human ILC2.

[0124] Results and Discussion

[0125] refer to Figure 11 This demonstrates that RORA expression can distinguish in vitro human ILC2. A UMAP plot of the in vitro ILC subset following transcriptome and epitope sequencing cell indexing (CITEseq), and... GATA3 and RORA The corresponding RNA expression.

[0126] In vitro amplification and activation of ILC2 expression that produces IL-10 RORA And rarely or never express GATA-3 The lack of IL-10 leads to the expression of ILC2 and GATA3 ( Figure 12 BC). Figure 12 A shows a UMAP plot of the ILC subsets amplified after CITE-seq. ILC subsets were isolated using FACS, amplified, and stained individually with a mixture of antibodies. Each cell population was stained with a unique tag antibody to allow for identification after sequencing. Figure 12 B shows the UMAP diagram, illustrating the expanded ILC subset. IL-10 RNA level expression. Figure 12 C shows that cells expressing IL-10 selectively express... RORA .

[0127] Figure 18The study demonstrated the variable proportion of IL-10-expressing ILC2 in ILC2 preparations from different donors. Representative flow cytometry plots of amplified ILC2 after intracellular cytokine staining (ICS) show a summary plot of IL-10 expression in ILC2 cell preparations from three high-IL-10-producing donors, three low-IL-10-producing donors, and 12 independent PBMC donors. Figure 18 A). CITEseq analysis of ILC2 cells expressing IL-10 demonstrated that IL-10-expressing cells express transcription factors. RORA ( Figure 18 B), but not expressed GATA3 However, in expressing RORA ILC2 contrastive expression GATA3 In ILC2, no differences in the expression of other ILC2-related molecules (including IL-4, IL-5, IL-13, and AREG) or ILC1 or ILC3-related cytokines (respectively) were observed. IFN-g and IL22 / IL-17A The upward adjustment of ).

[0128] Human ILC2, expanded from healthy blood PBMCs in vitro, maintains the expression of characteristic cytokines and produces the immunomodulatory cytokine IL-10. Figure 13 B shows a representative flowchart of intracellular cytokine staining in amplified human ILC2 cells after 6 hours of stimulation with PMA / ionomycin. Two hours after stimulation, the protein inhibitors brefedipine A and monensin were added.

[0129] RORA expression is positively correlated with IL-10 production in ILC2 cells. Figure 14 A shows a representative flowchart of transcription factor staining of amplified human ILC2, demonstrating that human ILC2 differentially expresses GATA-3 and RORA at the protein level after cytokine stimulation. Figure 14 B shows human GATA3 + and RORA + A representative flowchart of intracellular cytokine staining in ILC2 cells. Figure 14 B demonstrates that IL-10 expression is positively correlated with RORA- expression, but negatively correlated with GATA-3 expression. RORA- expression is also negatively correlated with IL-13 expression.

[0130] To determine whether RORA expression promotes IL-10 production in human ILC2, human ILC2 was transduced using NGFR-pCCL and GFP-pCCL-RORA lentiviral vectors at infection multiplicity 2. Transduction efficiency was measured by assessing NGFR or GFP expression at 7 days post-transduction. Figure 15 C).

[0131] Overexpression of RORA can induce the production of IL-10 in ILC2 cells. This can be measured by flow cytometry. Figure 16 B) and flow cytometry bead arrays ( Figure 16 C), cytokine expression in GFP-pCCL.RORA ILC2 was quantified by intracellular cytokine production. NGFR-pCCL control-transduced ILC2 was tested in parallel to compare changes in cytokine production. RORA transduction upregulated IL-10 production in human ILC2 (C). Figure 16 B, C).

[0132] In addition to upregulating IL-10 expression, RORA overexpression also upregulates CD49d (a marker of ILC2 that produces IL-10). Figure 17 Surface expression of GFP-pCCL.RORA ILC2, including CD161, CD117, CD25, and KLRG1, was measured. Surface marker expression of GFP-pCCL.RORA ILC2 was quantified by flow cytometry. NGFR-pCCL control-transduced ILC2 was tested in parallel to compare changes in surface marker expression.

[0133] In summary, we demonstrated the in vitro activation of human ILC2 expression that produces IL-10. RORA ILC2 expression lacking IL-10 GATA3 At the protein level, RORA expression is positively correlated with IL-10 production. Using a lentiviral gene transfer system, we demonstrated the forced expression of RORA in human ILC2. RORA Expression promotes IL-10 production in ILC2, which supports the forced expression of RORA to support IL-10-based ILC2 production. Therefore, RORA-overexpression represents a method for inducing or engineering IL-10-based ILC2 production, which can be applied to cell therapies for GVHD, autoimmune diseases, transplantation, and inflammation.

[0134] While preferred embodiments of the invention have been described herein, those skilled in the art will understand that variations may be made without departing from the spirit of the invention or the scope of the appended claims. All documents disclosed herein, including those in the following list of references, are incorporated herein by reference.

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Claims

1. A method for isolating or enriching a cell population targeting type 2 innate lymphocytes that produce IL-10, the method comprising: Provide a cell population containing ILC2; Select cells that express at least one of the following three biomarkers: CD86, CD49d, and retinoic acid-associated orphan α (RORA), and / or remove cells that express GATA3.

2. The method according to claim 1, wherein, Select cells that express CD86.

3. The method according to claim 1, wherein, Select cells that express CD49d.

4. The method according to claim 1, wherein, Select cells that express RORA.

5. The method according to claim 1, wherein, Select cells that express both CD86 and CD49d.

6. The method according to claim 1, wherein, Select cells that express all of CD86, CD49d, and RORA.

7. The method according to any one of claims 1-6, further comprising knocking out cells expressing GATA3.

8. The method according to any one of claims 1-7, wherein, The cell population was contained in a sample from the subject.

9. The method according to claim 8, wherein, The samples are blood, bone marrow, tissues, or lymphatic organs.

10. The method according to any one of claims 1-9, for isolating type 2 innate lymphocytes that produce IL-10.

11. The method according to any one of claims 1-10, wherein, The enriched cell populations are intended to improve, treat, or prevent graft-versus-host disease, transplant rejection, inflammatory conditions, autoimmune diseases, allergies, asthma, promote transplant graft function, and / or prevent or limit tissue damage in human subjects in need.

12. The method according to any one of claims 1-11, further comprising administering to a subject in need a therapeutically effective amount of a cell population enriched with IL-10-producing type 2 innate lymphocytes for the purpose of improving, treating or preventing graft-versus-host disease, transplant rejection, inflammatory conditions, autoimmune diseases, allergies, asthma, promoting transplant graft function, and / or preventing or limiting tissue damage.

13. The method according to claim 12, wherein, The subjects have received or will receive cell transplantation.

14. The method according to claim 13, wherein, The cell transplantation is a stem cell transplantation, preferably a hematopoietic stem cell transplantation.

15. The method according to claim 12, wherein, The subjects have received or will receive a bone marrow transplant.

16. The method according to claim 12, wherein, The subjects have received or will receive solid organ transplants.

17. The method according to claim 12, wherein, The subjects have received or will receive stem cell-derived tissue, cell, or organ transplants.

18. A cell population enriched with IL-10-producing type 2 innate lymphocytes prepared by the method of any one of claims 1-11.

19. Use of the cell population of claim 18 for use in the preparation of a medicament intended to improve, treat or prevent graft-versus-host disease or graft rejection, inflammatory conditions, autoimmune diseases, allergies, asthma, promote graft function and / or prevent or limit tissue damage in human subjects in need.

20. A kit for enriching cell populations for IL-10-producing type 2 innate lymphocytes, the kit comprising reagents for detecting cells expressing at least one of four biomarkers CD86, CD49d, RORA and GATA3, and instructions for use.

21. The kit of claim 20, comprising reagents for detecting cells expressing CD86.

22. The kit of claim 20, comprising reagents for detecting cells expressing CD49d.

23. The kit of claim 20, comprising reagents for detecting cells expressing both CD86 and CD49d.

24. The kit of claim 20, comprising reagents for detecting cells expressing CD86, CD49d and RORA.

25. The kit of claim 20, comprising reagents for detecting cells expressing CD86, CD49d, RORA, and GATA3.

26. A method for generating or enriching a cell population targeting type 2 innate lymphocytes that produce IL-10, the method comprising: Provide a cell population containing ILC2; Overexpression of RORA was induced in the cell population.

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