Use of t-bet-positive ptreg cells as drug target

Abstract

Use of T-bet-positive pTreg cells as a drug target, relating to the field of immunology. Treg cells in the tumor microenvironment are mainly T-bet-positive pTreg cells. By targeting these cells, it is expected to significantly improve the efficacy of immunotherapy while reducing side effects on the immune system. Compared with conventional immunotherapy, precise targeting of pTreg cells enhances anti-tumor immune responses. In particular, by means of combination immunotherapy, anti-tumor immune responses are greatly boosted, immune homeostasis is ensured, and autoimmune problems caused by systemic depletion of Treg cells are avoided.

Description

Application of T-bet-positive pTreg cells as drug targets Technical Field

[0001] This invention relates to the field of immunology, and in particular to the application of T-bet-positive pTreg cells as drug targets. Background Technology

[0002] FOXP3-expressing regulatory T cells (Treg cells) play a crucial role in maintaining immune tolerance and homeostasis, primarily protecting the host from autoimmune damage by suppressing excessive immune responses (Ohkura, N., Y. Kitagawa, and S. Sakaguchi. 2013. Development and maintenance of regulatory T cells. Immunity 38: 414-423.). However, the suppressive effect of Treg cells in the tumor microenvironment allows tumor cells to evade host immune surveillance, promoting tumor development (Chen, X., Y. Du, X. Lin, et al. 2016. CD4). + CD25 + Regulatory T cells in tumor immunity. Int. Immunopharmacol 34: 244-249.). Existing studies have shown that the tumor microenvironment not only promotes the recruitment and proliferation of Treg cells ([1] Ohue, Y., and H. Nishikawa. 2019. Regulatory T(Treg) cells in cancer: Can Treg cells be a new therapeutic target? Cancer Sci 110: 2080-2089. [2] Tanaka, A., and S. Sakaguchi. 2019. Targeting Treg cells in cancer immunotherapy. Eur J Immunol 49: 1140-1146.), but also that a large number of Treg cells in the tumor significantly inhibit CD8. + The anti-tumor effect of T cells influences patient prognosis. Although Treg cells are a potential target for cancer immunotherapy, and several studies have attempted to enhance anti-tumor immune responses by targeting Treg cells (Ellis and Riley, 2020; Tay et al., 2023), existing immunotherapy strategies still have certain limitations.

[0003] Current immunotherapies, such as targeting the Treg cell surface marker CTLA-4, while effectively reducing Treg cell suppression, may also trigger severe autoimmune side effects. Furthermore, the Treg cell subsets in the tumor microenvironment are complex and diverse, and existing methods have not yet been able to effectively and selectively target the most immunosuppressive Treg cell subsets. Therefore, there is an urgent need for a new approach that can specifically target Treg cells in the tumor microenvironment without inducing side effects, thereby enhancing the anti-tumor immune response. Summary of the Invention

[0004] The purpose of this invention is to address the selectivity and side effects of targeted immunosuppressive cell therapy in existing tumor immunotherapy. While some studies have attempted to target Treg cells in the tumor microenvironment, the complexity of Treg cell subsets and their diversity in different microenvironments make it difficult for existing treatments to effectively target specific immunosuppressive subsets, thus affecting therapeutic efficacy. Furthermore, existing Treg cell-targeting therapies can also lead to severe autoimmune reactions. Therefore, this invention aims to address the issue of selective targeting of specific Treg cells within the tumor microenvironment. + pTreg cell subsets address the issues of poor selectivity and significant side effects in existing methods, and improve the efficacy of tumor immunotherapy.

[0005] The technical solution of this invention mainly involves, through in-depth analysis of the heterogeneity of regulatory T cells (Treg cells) in the tumor microenvironment, proposing a strategy to selectively target the major pTreg cell population in the tumor microenvironment to enhance anti-tumor immunity while avoiding the induction of autoimmune responses. The following are the technical solutions of this invention, including all the necessary technical features involved in achieving the objectives of the invention.

[0006] This invention underscores the importance of understanding Treg cell heterogeneity in the tumor microenvironment to selectively target the dominant Treg cell population within the tumor without triggering an autoimmune response. Through genome-wide transcriptomics and single-cell analysis in a mouse tumor model, we discovered T-bet... + pTreg cells are the most prevalent subset, highlighting their importance in tumor immunity. By elucidating T-bet... + This invention proposes a novel strategy to target pTreg cells to suppress anti-tumor immune responses, thereby enhancing anti-tumor immunity while reducing the risk of systemic autoimmune responses.

[0007] First, this invention characterized Treg cell subsets in various mouse tumor models. We used NRP1 as a marker to distinguish between thymus-derived tTreg cells and peripherally induced pTreg cells. Flow cytometry analysis revealed that most Treg cells in lymph nodes and spleen were NRP1-derived. + (i.e., tTreg cells), but in tumors, more than half of the Treg cells are NRP1. - (i.e., pTreg cells). This result was further verified through adoptive transfer experiments. The experiments showed that approximately 50% of the Treg cells in the tumor were pTreg cells, which originated from conventional CD4+ cells. + T cells. In contrast, Treg cells in lymph nodes and the spleen are mainly tTreg cells. Studies have shown that the tumor microenvironment may lead to immunosuppression by promoting the accumulation of pTreg cells.

[0008] This study then explores the role of pTreg cells in tumor immunity. We selectively removed pTreg cells using adoptive transfer and utilized Foxp3... DTR In mice, diphtheria toxin (DT) depleted pTreg cells without affecting tTreg cells, thereby preventing autoimmune responses. Results showed that tumor growth was inhibited after pTreg cell removal. Simultaneously, tumor-invasive CD8+ cells were reduced. + T cell numbers increased, exhibiting stronger proliferative capacity and cytolytic activity. Furthermore, the proportion of myeloid cells in the tumor microenvironment changed, with a decrease in TAMs and monocytes and an increase in neutrophils. Further treatment evaluation revealed that late-stage removal of pTreg cells in the E.G7 tumor model inhibited tumor growth and CD8+. + The proportion of T cells increased, and CD8 cells infiltrated by tumors. + The proportion of T cells in the total viable cells increased, particularly the proportion of IFN-γ, granzyme B, and CD107a cells. Overall, the removal of pTreg cells produced an effective therapeutic effect in tumor-bearing mice.

[0009] Next, this study analyzed whether intratumoral Treg cells were similar to any Th subset in terms of gene expression. Transcriptomic analysis of intratumoral Treg cells derived from Hepa1-6 and E.G7 mouse tumor models was performed using RNA sequencing. The results showed that intratumoral Treg cells exhibited upregulation of Th1 characteristic genes (such as Tbx21 and Ccr5), while Th2 and Th17 genes (such as Gata3, Rorc, and Ccr6) were downregulated. Furthermore, Nrp1 expression in intratumoral Treg cells was lower than that in lymph node Treg cells. Further flow cytometry analysis revealed that intratumoral Treg cells highly expressed T-bet but did not express RORγt, BCL6, or GATA3. + NRP1 - Treg cells account for 60% of Treg cells within the tumor. Overall, the study results indicate that T-bet... + NRP1 - Treg cells are the dominant population of intratumoral Treg cells in multiple cancer models.

[0010] RNA sequencing results showed that pTreg cells in tumors exhibited higher levels of Th1 gene upregulation compared to tTreg cells. Simultaneously, pTreg cells also showed upregulation of genes related to Treg cell inhibitory function, as well as genes related to activation, cell cycle regulation, and chemokine receptors. We also compared the gene expression characteristics of pTreg cells in tumors with those of intestinal Treg cells, finding that pTreg cells in tumors and intestinal Treg cells had similar gene expression profiles, especially in non-lymphoid tissue regions, such as the lamina propria containing a high proportion of pTreg cells. These results indicate that T-bet cells in tumors... + pTreg cells possess enhanced activation, proliferation, and inhibition functions, further emphasizing the role of T-bet. + The important role of pTreg cells in the tumor microenvironment.

[0011] Therefore, this invention provides, in one aspect, the use of T-bet-positive pTreg cells as drug targets in the development, screening, or preparation of drugs for the prevention and / or treatment of tumors. Preferably, the drug is capable of reducing the number or activity of T-bet-positive pTreg cells.

[0012] Specifically, the drug has at least one of the following effects:

[0013] (1) Inhibit the activity of T-bet-positive pTreg cells by targeting T-bet protein or related signaling pathways in T-bet-positive pTreg cells; (2) Enhance CD8 + The role of T cells in tumor killing activity.

[0014] Preferably, the tumor is a solid tumor; the solid tumor is lymphoma, hepatocellular carcinoma, melanoma, breast cancer, squamous cell carcinoma, colorectal cancer, non-small cell lung cancer, or head and neck cancer (nasopharyngeal carcinoma, oropharyngeal carcinoma, oral cancer, laryngeal cancer, pharyngeal cancer, tongue cancer, thyroid cancer, salivary gland cancer).

[0015] In another aspect, the present invention provides the use of molecules for reducing the number or activity of T-bet-positive pTreg cells in the preparation of drugs for the prevention and / or treatment of tumors.

[0016] Preferably, the molecule used to reduce the number or activity of T-bet-positive pTreg cells is a T-bet inhibitor and / or a CD39 inhibitor. The T-bet inhibitor is a sequence for knocking out or silencing the gene encoding T-bet (Tbx21 gene) or a compound for reducing T-bet expression; the CD39 inhibitor is a sequence for knocking out or silencing the gene encoding CD39 (Entpd1 gene) or a compound for reducing CD39 expression. When knocking out the Entpd1 gene, exons 2-7 of the Entpd1-201 (ENSMUST00000112231.8) transcript are used as the knockout region.

[0017] Preferably, when applied, the molecule used to reduce the number of T-bet-positive pTreg cells is used in combination with an immune checkpoint inhibitor; the immune checkpoint inhibitor is an anti-PD-1 antibody, an anti-CTLA-4 antibody, or a combination thereof.

[0018] By generating Foxp3 with Tbx21 specifically knocked out YFP-cre Tbx21 fl / fl Mice (KO group) were compared with wild-type mice (WT group). The results showed that tumor growth was significantly slowed in the KO group, and the number of Treg cells within the tumor was significantly reduced. Flow cytometry analysis showed that T-bet deletion reduced proliferative Treg cells within the tumor and CD8+. + T cell activation and proliferation are enhanced, and CD8+ in tumors are also increased. + The ratio of T cells to Treg cells was increased. Further adoptive transfer experiments validated the crucial role of T-bet in pTreg cell function. Furthermore, the combination of anti-PD-1 therapy and Treg cell-specific Tbx21 knockout significantly inhibited tumor growth and prolonged survival in mice. In summary, this study demonstrates that T-bet is essential for pTreg cell function in the tumor microenvironment, and its combination with anti-PD-1 therapy holds potential therapeutic efficacy.

[0019] Single-cell transcriptomics and flow cytometry revealed the role of CD39 in tumor T-bet.+ The study identified specific expression of CD39 in pTreg cells and determined that CD39-mediated immunosuppression is a unique mechanism of Th1-like Treg cells within tumors. The study found that Treg cells highly express CD39 in tumors and are associated with purine metabolism, suggesting its crucial role in immunosuppression. Co-expression of CD39 and CD73 was particularly prominent in tumor-bearing Treg cells, further indicating that T-bet... + pTreg cells possess unique immunosuppressive markers. Furthermore, CD39 expression was also found in other tissues containing a high proportion of pTreg cells, such as the lamina propria of the small and colonic intestines. These findings suggest that CD39 may act as a selective regulator of T-bets. + The potential therapeutic target of pTreg cells provides a new strategy for cancer immunotherapy.

[0020] By analyzing RNA sequencing data from human breast cancer tissue, this study found that differentially expressed genes in tumor-bearing Treg cells were similar to those in mouse tumor models, particularly those involving nucleotide metabolism pathways. Furthermore, the study found that the ENTPD1 gene (encoding CD39) was universally upregulated in Treg cells across various cancer types. Single-cell RNA sequencing data from multiple human cancers further confirmed the specific co-expression of TBX21 (encoding T-bet) and ENTPD1 in Treg cells of various cancer types (such as nasopharyngeal carcinoma, breast cancer, melanoma, squamous cell carcinoma, hepatocellular carcinoma, and colorectal cancer). Flow cytometry analysis also verified the high co-expression of T-bet and CD39 in tumor-bearing Treg cells from liver cancer patients. These results indicate that the co-expression of T-bet and CD39 is an immunosuppressive mechanism spanning multiple human cancers, consistent with findings in mouse tumor models. This new discovery provides a basis for developing targeted T-bet expression mechanisms in tumor-bearing Treg cells. + Targeted therapy using pTreg cells provides a theoretical basis and holds promise for overcoming immunosuppression in cancer.

[0021] The study involved creating Foxp3. YFP-cre Entpd1 fl / fl In mice, specific knockout of the Entpd1 gene in Treg cells revealed no autoimmune pathological responses or changes in immune homeostasis after CD39 deficiency. In vitro experiments showed that Treg cells lacking CD39 expression exhibited resistance to CD8+. + The inhibitory capacity of T cells was significantly reduced. In vivo studies showed that knocking out Entpd1 slowed tumor growth and CD8... +T cell activity was enhanced. Furthermore, combining Treg cell-specific knockout of Entpd1 with anti-PD-1 therapy significantly enhanced tumor suppression and improved mouse survival. These results indicate that targeting CD39 in tumor-bearing Treg cells not only elicits an anti-tumor response but also produces a synergistic effect with anti-PD-1 therapy.

[0022] In another aspect, the present invention provides a drug for the prevention and / or treatment of tumors, said drug comprising an inhibitor capable of reducing the number or activity of T-bet-positive pTreg cells.

[0023] As a preferred embodiment, the inhibitor that can reduce the number or activity of T-bet-positive pTreg cells is a T-bet inhibitor and / or a CD39 inhibitor.

[0024] As a preferred embodiment, the drug further includes an immune checkpoint inhibitor, which is an anti-PD-1 antibody, an anti-CTLA-4 antibody, or a combination thereof.

[0025] T-bet + The combined use of pTreg cells and anti-PD-1 therapy has shown therapeutic potential. To further optimize this strategy, its combination with other immune checkpoint inhibitors could be explored. Immune checkpoint inhibitors can enhance tumor-specific CD8 uptake. + T-cell immune activity, while T-bet + pTreg cell-specific inhibition helps eliminate immunosuppression in the tumor microenvironment and is expected to significantly improve the efficacy of immunotherapy, especially in patients who do not respond well to anti-PD-1 therapy.

[0026] Specifically, the tumor is a solid tumor; the solid tumor is lymphoma, hepatocellular carcinoma, melanoma, breast cancer, squamous cell carcinoma, colorectal cancer, non-small cell lung cancer, or head and neck cancer (nasopharyngeal carcinoma, oropharyngeal carcinoma, oral cancer, laryngeal cancer, pharyngeal cancer, tongue cancer, thyroid cancer, salivary gland cancer).

[0027] This invention also provides the use of T-bet-positive pTreg cells as drug targets in the development, screening, or preparation of drugs for treating autoimmune diseases. The drugs can induce the generation of T-bet-positive pTreg cells or enhance the number or activity of T-bet-positive pTreg cells.

[0028] The autoimmune diseases mentioned are those associated with Treg dysfunction. Treg cells play a crucial role in maintaining immune tolerance and suppressing abnormal immune responses. When Treg cell function is impaired, the immune system may attack normal autologous tissues, leading to autoimmune diseases. Autoimmune diseases associated with Treg dysfunction include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, type 1 diabetes, autoimmune thyroid diseases (such as Hashimoto's thyroiditis and Graves' disease), inflammatory bowel diseases (including Crohn's disease and ulcerative colitis), psoriasis, autoimmune hepatitis, and allergic diseases. These diseases share the common characteristic of the immune system attacking itself, in which Treg cells play an important regulatory role; therefore, restoring or enhancing Treg function may help treat these diseases.

[0029] The core of this invention lies in selectively targeting pTreg cells in the tumor microenvironment to enhance anti-tumor immunity and avoid autoimmune reactions that may be triggered in traditional immunotherapies. This invention reveals that Treg cells in the tumor microenvironment are primarily pTreg cells. By targeting these cells, it is expected to significantly improve the efficacy of immunotherapy while reducing the side effects of the immune system. Compared to traditional immunotherapies, this invention enhances the anti-tumor immune response and ensures immune homeostasis by precisely targeting pTreg cells, avoiding the autoimmune problems caused by systemic removal of Treg cells. Furthermore, this invention provides a deeper understanding of the origin, developmental pathways, and immunosuppressive mechanisms of Treg cells within tumors, offering new theoretical support for the development of future immunotherapy strategies. This technology is of great significance in improving the safety and efficacy of immunotherapy.

[0030] This invention significantly improves the efficacy of anti-tumor immunotherapy and reduces side effects by precisely targeting pTreg cells, providing patients with higher survival rates and quality of life, thereby reducing treatment costs and alleviating the social burden of healthcare. Improvements in immunotherapy may reduce reliance on chemotherapy and radiotherapy, save medical resources, alleviate pressure on hospitals, and improve the efficiency of social healthcare.

[0031] Furthermore, improved treatment outcomes help patients recover sooner, return to work, and reduce economic losses caused by sick leave, thereby promoting social labor productivity and economic development. Overall, this invention not only advances the technology of tumor immunotherapy but also brings positive social and economic impacts, optimizes the allocation of medical resources, and improves social benefits. Attached Figure Description

[0032] Figure 1: NRP1 expression in Treg cells in lymph nodes (LN) and spleen (SPL) of tumor-free mice, or in lymph nodes, spleen, tumor draining lymph nodes (TDLN), and tumor-infiltrating lymphocytes (TILs) of E.G7 tumor-bearing mice (day 21) (n = 4-6 per group); where A is a flow cytometry plot; B is a statistical graph of expression.

[0033] Figure 2: NRP1 expression in Treg cells in tumor-free mice (LN and SPL) or in Hepa1-6 tumor-bearing mice (LN, SPL, TDLN, and TILs) on day 23 (n = 4-6 per group); where A is a flow cytometry plot and B is a statistical graph of expression.

[0034] Figure 3: NRP1 expression in Treg cells and cell adoptive transplantation experiment; where A shows the expression of NRP1 in Treg cells in tumor-free mice (LN) or in TILs of B16-OVA tumor-bearing mice (day 18) (n=11-12 per group); B shows the cell adoptive transplantation experiment procedure.

[0035] Figure 4: TCRbd in E.G7 tumor-bearing TCRbd - / - In recipient mice (day 24), tTreg (CD45.1) derived from LN and SPL... + ) cells and regular CD4 + T(CD45.1 - The proportion of cells (n=17 per group); where A is a flow cytometry graph; B is a statistical graph of expression.

[0036] Figure 5: TCRbd in E.G7 tumor-bearing TCRbd - / - In recipient mice, tTreg (CD45.1) derived from LN, SPL, and TILs... + ) cells and pTreg(CD45.1 - The proportion of cells (n=17-21 per group); where A is a flow cytometry graph; B is a statistical graph of expression.

[0037] Figure 6: TCRbd in E.G7 tumor-bearing TCRbd - / - In recipient mice, tTreg(CD45.1) + ) cells and pTreg(CD45.1 - NRP1 in cells + and NRP1 - Percentage of cells (n=12 per group); where A is a flow cytometry plot; B is a statistical graph of expression.

[0038] Figure 7: Adoptive cell transplantation experimental procedure (A) and E.G7 tumor-bearing TCRbd treated with diphtheria toxin (DT) or PBS. - / - Tumor growth curves (B) and tumor weight (C) of recipient mice.

[0039] Figure 8: TCRbd in E.G7 tumor-bearing TCRbd - / - In recipient mice, the absolute number of Treg cells in LNs(A), SPLs(B), and TILs(C) after treatment with DT or PBS.

[0040] Figure 9: TCRbd in E.G7 tumor-bearing TCRbd - / - In recipient mice, after treatment with DT or PBS, the total Treg cells in TILs (A and C), LN, and SPL (B and D) showed a significant increase in tTreg (CD45.1) levels. + ) cells and pTreg(CD45.1 - (Percentage of cells)

[0041] Figure 10: TCRbd in E.G7 tumor-bearing TCRbd - / - In recipient mice, after treatment with DT or PBS, the CD8 concentration per unit tumor weight (g) was... + Absolute T cell count and TILs; where A represents CD8 count per unit tumor weight (grams). + Absolute T cell count; B represents CD8+ in TILs. + The frequency of T cells in total living cells; C represents CD8 in TILs. + The ratio of T cells to Treg cells; DE represents CD44 in TILs. + Ki67 + CD8 + The frequency of T cells in total living cells.

[0042] Figure 11: TCRbd in E.G7 tumor-bearing TCRbd - / - In recipient mice, after treatment with DT or PBS, IFN-γ in TILs was reduced. + CD8 + T cells (AB) and CD107a + CD8 + The frequency of T cells (CD) in total living cells.

[0043] Figure 12: Volcano plots and T-bet expression in Treg cells; where the volcano plots show the upregulated (red dots) and downregulated (blue dots) genes in tumor-bearing Treg cells compared to gene expression in LN (n=4) from tumor-free mice in Hepa1-6 (n=4) (A) and E.G7 (n=2) (B) tumor models; T-bet expression in Treg cells in LN and SPL of tumor-free control wild-type (WT) C57BL / 6J mice, or in LN, SPL, TDLN, and TILs of Hepa1-6 tumor-bearing WT C57BL / 6J mice (C and D) (n=4-5 per group).

[0044] Figure 13: Expression of T-bet in LN and SPL of tumor-free wild-type (WT) C57BL / 6J mice, or in LN, SPL, TDLN and TILs of E.G7 tumor-bearing WT C57BL / 6J mice (n=4 per group); where A is a flow cytometry plot; B is a statistical graph of expression.

[0045] Figure 14: (A and B) Expression of T-bet in Treg cells in LN of tumor-free wild-type (WT) C57BL / 6J mice and TILs of B16-OVA tumor-bearing WT C57BL / 6J mice (n=3-4 per group); (C and D) Expression patterns of NRP1 and T-bet after co-staining in TILs of E.G7 tumor-bearing WT C57BL / 6J mice.

[0046] Figure 15: Flow cytometry plots (A) and quantitative data (B) showing RORγt expression in Treg cells from tumor-free mice WT C57BL / 6J and E.G7 tumor mice (including LN, SPL, TDLN and TILs) (n=3-4 per group).

[0047] Figure 16: Flow cytometry plots (A) and quantitative data (B) showing BCL6 expression in Treg cells from tumor-free WT C57BL / 6J and E.G7 tumor mice (including LN, SPL, TDLN and TILs) (n=3-4 per group).

[0048] Figure 17: Representative flow cytometry plots (A) and quantitative data (B) showing GATA3 expression in Treg cells from tumor-free wild-type (WT) C57BL / 6J mice in LN and SPL, or from LN, SPL, TDLN, and TILs of E.G7 tumor-bearing C57BL / 6J mice (n = 3–4 per group).

[0049] Figure 18: (AC) Volcano plots show the expression of Th1 cell-related genes in (A) Hepa1-6 tumor pTreg cells (n=3) compared to Hepa1-6 tumor tTreg cells (n=5), and in (B) or (C) Hepa1-6 tumor pTreg cells (n=3) or Hepa1-6 tumor tTreg cells (n=5) compared to LN-derived Treg cells (n=4). Red dots indicate upregulated genes, and blue dots indicate downregulated genes.

[0050] Figure 19: (A) Gene heatmap shows the differential expression of genes related to Treg cell suppression mechanisms, activation, proliferation, and migration in tumor-bearing pTreg cells (n=3) and tumor-bearing tTreg cells (n=5) in the Hepa1-6 tumor model; (BD) Enrichment plots show the correlation between upregulated genes in (B) colonic repressive Treg cells, (C) colonic LT-like Treg cells, or (D) colonic NLT Treg cells and Hepa1-6 tumor-bearing pTreg cells (n=3) versus Hepa1-6 tumor-bearing tTreg cells (n=5), identified using GSEA calculations. Red indicates high expression levels, and blue indicates low expression levels. NES, normalized enrichment score.

[0051] Figure 20: (A) Gene heatmap showing the differential expression of colonic NLT Treg marker genes in intratumoral pTreg cells (n=3) versus intratumoral tTreg cells (n=5) in the Hepa1-6 tumor model. (B) Enrichment plot showing the correlation between upregulated genes in intestinal NLT Treg cells and Hepa1-6 intratumoral pTreg cells (n=3) versus Hepa1-6 intratumoral tTreg cells (n=5), identified using GSEA calculations. Red indicates high expression levels, blue indicates low expression levels. NES, normalized enrichment score. (C) Gene heatmap showing the differential expression of intestinal NLT Treg marker genes in Hepa1-6 intratumoral pTreg cells (n=3) versus intratumoral tTreg cells (n=5). (D) Enrichment plot showing the correlation between upregulated genes in intestinal NLT Treg cells and Hepa1-6 intratumoral pTreg cells (n=3) versus Hepa1-6 intratumoral tTreg cells (n=5), identified using GSEA calculations. Red indicates high expression levels, and blue indicates low expression levels. NES stands for Normalized Enrichment Score.

[0052] Figure 21: (A) B16-OVA tumor growth curves compared with Foxp3 YFP-cre Tbx21 fl1 / fl Mice (KO) and Foxp3 YFP-creMice (WT) (n = 5-6 per group). (B) E.G7 tumor growth curves, compared with Foxp3. YFP-cre Tbx21 fl / fl Mice (KO) and Foxp3 YFP-cre Mice (WT) (n = 12-16 per group).

[0053] Figure 22: Foxp3 in (AB)B16-OVA tumors YFP - cre Tbx21 fl / fl Mice (KO) and Foxp3 YFP-cre Treg cells from LN, SPL, TDLN, and TILs in mice (WT) account for a significant portion of total CD4+. + Frequency in T cells (n = 6-7 per group). (C) Foxp3 in B16-OVA tumors. YFP-cre Tbx21 fl / fl Mice (KO) and Foxp3 YFP-cre The absolute number of Treg cells in TILs in mice (WT) (n = 5-7 per group).

[0054] Figure 23: (A and C) Foxp3 tumors from B16-OVA-bearing tumors YFP-cre Tbx21 fl / / fl Mice (KO) and Foxp3 YFP-cre CD8 in mouse (WT) TILs + IFN-γ in T cells + TNF-α + Cell frequency (n = 6-7 per group). (B and D) from B16-OVA-bearing Foxp3 tumors. YFP-cre Tbx21 fl / fl Mice (KO) and Foxp3 YFP-cre CD8 in mouse (WT) TILs + TIM-3 in T cells + PD-1 + Cell frequency (n=6-7 per group).

[0055] Figure 24: (AB) Foxp3 tumors from B16-OVA-bearing tumors YFP-cre Tbx21 fl / fl Mice (KO) and Foxp3 YFP-cre CD8 in mouse (WT) TILs + TIM-3-Ly108 in T cells + Cell frequency (n=6-7 per group).

[0056] Figure 25: (AB) Foxp3 cells treated with anti-PD-1 antibody or vector control at 9, 12, and 15 days after B16-OVA tumor inoculation (as indicated by arrows). YFP-cre Tbx21 fl / fl Mice (KO) and Foxp3 YFP-cre Tumor growth curves (A) and survival rates (B) in mice (WT) (n = 6-8 per group).

[0057] Figure 26: (AB) Ifng from tumor-free tissue icre Rosa26 YFP CD4 produced by IFN-γ in mouse LN and SPL + T cells (YFP) + Percentage, and from E.G7 tumor-bearing Ifng icre Rosa26 YFP Percentages of LN, SPL, TDLN, and TIL in mice (n=11 per group).

[0058] Figure 27: (AC) From E.G7 tumor-bearing Ifng icre Rosa26 YFP CD4 in mouse TILs + YFP + Expression of T-bet and FOXP3 in cells (n=11 per group). (DE) from E.G7 tumor-bearing Ifng icre Rosa26 YFP Intratumoral CD4 in mice + YFP + Expression of NRP1 and FOXP3 in cells (n=11 per group).

[0059] Figure 28: (AB) From E.G7 tumor-bearing Ifng icre Rosa26 YFP In mouse TILs, CD4 + YFP + FOXP3 + T cell expression patterns after co-staining NRP1 and T-bet (n=10 per group). (CD) from E.G7 tumor-bearing Ifng icre Rosa26 YFP Intratumoral CD4 in mice + YFP + Expression of IFN-γ and FOXP3 in cells (n=11 per group).

[0060] Figure 29: (AC) OT-III fng from CD45.1 / CD45.2 (double positive) YFP CD45.1 in mice+ CD4 + T-bet and FOXP3 expression in donor cells in TILs of E.G7 tumor-bearing C57BL / 6J mice (n=9 per group).

[0061] Figure 30: (A) Transferred Cd4 Cre Tgfbr2 fl / fl (KO) or Tgfbr2 fl / fl (WT)OT-II cells bearing B16-OVA tumor TCRbd - / - Tumor growth curves in mice (n = 6–9 per group). (BC) from Cd4 Cre Tgfbr2 fl / fl (KO) or Tgfbr2 fl / fl (WT) group CD45.1 + CD4 + OT-II donor cells in B16-OVA tumor-bearing TCRbd - / - The frequency of TILs converting to pTreg cells in recipient mice (n = 6-9 per group).

[0062] Figure 31: (AB)B16-OVA tumor-bearing TCRbd - / - In recipient mouse TILs, from Cd4 Cre Tgfbr2 fl / fl (KO) or Tgfbr2 fl / fl (WT) group CD45.1 + CD4 + In OT-II donor cells, T-bet + FOXP3 - CD4 + T cell frequency (n = 6-9 per group). (CD)B16-OVA tumor-bearing TCRbd - / - In recipient mouse TILs, from Cd4 Cre Tgfbr2 fl / fl (KO) or Tgfbr2 fl / fl (WT) group CD45.1 + CD4 + IFN-γ in donor T cells + TNF-α + Cell frequency (n=6-7 per group).

[0063] Figure 32: (AC) scRNA-seq analysis of UMAP (AB) and the proportion of each cell population (C) of Tbx21-deficient Treg cells (Tbx21 KO), LN Treg cells (WT), and wild-type Treg cells (WT) in Hepa1-6 tumors.

[0064] Figure 33: Gene heatmap showing differentially upregulated gene expression in each identified cell population. Adjusted p-value < 0.05.

[0065] Figure 34: (A) Volcano plot showing upregulated genes (red dots) and downregulated genes (blue dots) in Th1-like Treg cells (C0) and Th2 / Th17-like Treg cells (C2) within the tumor. (B) Gene heatmap showing the expression of Tbx21, Gata3, Rorc, and genes involved in Treg cell suppression mechanisms in Th1-like Treg cells (C0) and Th2 / Th17-like Treg cells (C2) within the tumor. Adjusted p-value < 0.05.

[0066] Figure 35: (AC) Flow cytometry results showing the expression of TIGIT and CD39 in tumor-derived Tbx21-deficient Treg cells (KO) and tumor-derived wild-type Treg cells (WT). (B) Percentage. (C) MFI, data shown as fold change compared to the WT control group (n=7 per group). (D) RNA velocity showing the pseudo-temporal profile of WT Treg cells in Hepa1-6 tumors.

[0067] Figure 36: (A) Six gene clusters were identified by pathway enrichment analysis of RNA expression in intratumoral Treg cells in E.G7 (n=2) and Hepa1-6 (n=3) tumor models, and compared with those in extratumor LN (n=3). (BE) Nucleotide metabolic pathways enriched in intratumoral Treg cells in E.G7 (n=2) and Hepa1-6 (n=3) tumor models were compared with those in extratumor LN (n=3), and these pathways were identified in clusters 1 (B), 2 (C), 5 (D), and 6 (E), respectively.

[0068] Figure 37: (AB) Gene heatmap shows the expression of genes related to nucleotide metabolism in intratumoral Treg cells in Hepa1-6 (n=3) and E.G7 (n=2) tumor models, and compares them with Treg cells in extratumor LN (n=3).

[0069] Figure 38: (A) Gene heatmap showing the expression of genes related to nucleotide metabolism in tumor-bearing pTreg cells (n=3) and tumor-bearing tTreg cells (n=5) in the Hepa1-6 tumor model. (BC) Expression of T-bet and CD39 in TILs-derived Treg cells of Hepa1-6 tumor-bearing WT C57BL / 6J mice (n=6 per group).

[0070] Figure 39: Intratumoral NRP1 in (AB)E.G7 tumor-bearing wild-type mice + CD39 - and NRP1 - CD39 + Expression of T-bet in Treg cells (n=5). (CD)E.G7 tumor-bearing Foxp3 YFP-cre Tbx21 fl / fl Mice (labeled KO) and Foxp3 YFP-cre Intratumoral NRP1 in mice (labeled WT) + and NRP1 - CD39 expression in Treg cells (n=8-9).

[0071] Figure 40: (AB) Representative flow cytometry plots (A) and statistical plots (B) showing Treg cells in small intestinal IEL, colonic LPL and small intestinal LPL isolated from tumor-free C57BL / 6J mice, and TILs co-stained with CD39 and NRP1 isolated from E.G7 tumor-bearing C57BL / 6J mice (n = 6-7 per group).

[0072] Figure 41: (AB) Enrichment plots show the correlation of upregulated genes in human breast cancer tumor Treg cells compared to tumor-free LN Treg cells (A) (n=2) or Hepa1-6 tumor Treg cells (B) (n=4), identified using GSEA calculations. Red indicates high expression levels, and blue indicates low expression levels. (C) Nucleotide metabolic pathways enriched in tumor-free Treg cells, compared to PBMC Treg cells (n=6) derived from human breast cancer (n=4).

[0073] Figure 42: (A) Gene heatmap showing gene expression related to nucleotide metabolism in human breast cancer Treg cells (n=4) and PBMC Treg cells (n=6) (as determined by RNA sequencing). (BC) Gene heatmap showing gene expression related to small molecule degradation processes (B) and nucleotide-containing small molecule degradation processes (C) in human breast cancer Treg cells (n=4) and PBMC Treg cells (n=6) (as determined by RNA sequencing).

[0074] Figure 43: (A) Venn plot shows the overlap of nucleotide metabolism upregulated genes in Treg cells within human breast cancer, hepatocellular carcinoma (HCC), colorectal cancer (CRC), and non-small cell lung cancer (NSCLC). (B) Volcano plot shows nucleotide metabolism upregulated genes (NUDT5, GLRX, GPX1, TYMP, ENTPD1) and Treg cell marker genes (IL2RA, CTLA4, FOXP3) in Treg cells within hepatocellular carcinoma (HCC) tumors.

[0075] Figure 44: (AF) Heatmap showing the expression of FOXP3, TBX21, ENTPD1, ​​NT5E, IL10, EBI3, IL12A, TGFB1, IL17F, IL22, and IFNG in Treg cells within human tumors, derived from human nasopharyngeal carcinoma (NPC) (A), breast cancer (BRCA) (B), melanoma (C), squamous cell carcinoma (SCC) (D), hepatocellular carcinoma (HCC) (E), and colorectal cancer (CRC) (F). These data were obtained through single-cell RNA sequencing (scRNA-seq) from the Pan-Cancer T-cell single-cell RNA sequencing data portal (http: / / cancer-pku.cn:3838 / PanC_T / ).

[0076] Figure 45: Expression of T-BET (A and B) and CD39 (A and C) in Treg cells from PBMCs, paratumor tissues, or tumor-infiltrating lymphocytes (TILs) of HCC patients (n = 6–8 per group). Co-expression of T-BET and CD39 in Treg cells of TILs from (DE) HCC patients (n = 6 per group). Data are presented as mean and standard error (SEM).

[0077] Figure 46: (AB)CD39 from Foxp3 YFP-cre Entpd1 fl / fl (KO) mice and Foxp3 YFP-cre CD4 in colonic LPL or E.G7 tumors in (WT) mice + FOXP3 + Expression in Treg cells (n=6 per group). (C) Foxp3 expression in 21-24 week old cells. YFP-cre Entpd1 fl / fl (KO) and Foxp3 YFP-cre (WT) Body weight of male and female mice (KO males, n=12; WT males, n=10; KO females, n=11; WT females, n=11). (DE) Showing the weights of mice from 24-week-old Foxp3 mice.YFP-cre Entpd1 fl / fl (KO) and Foxp3 YFP-cre Representative images of the morphology of the intestine (D) and spleen (E) of (WT) mice (n = 4–6 per group).

[0078] Figure 47: (A) Foxp3 YFP-cre Entpd1 fl / fl (KO) and Foxp3 YFP-cre Representative images of hematoxylin-eosin (H&E) staining of colonic and small intestinal tissue from mice (WT) (n=6 per group). At least five fields of view were taken for each slide using a 200x magnifying glass, and 40x or 100x magnifying glasses. Scale bar: 50 μm (200x magnification); 100 μm (40x and 100x magnification). (BC) Foxp3 YFP-cre Entpd1 fl / fl (KO) mice and Foxp3 YFP-cre (WT) Activated CD44 in LPL of the colon and small intestine of mice hi CD4 + Frequency of T cells (n=6 per group).

[0079] Figure 48: (AC)Foxp3 YFP-cre Entpd1 fl / fl (KO) mice and Foxp3 YFP-cre (WT) Effects of LN and SPL in mice (CD44) + CD62L - CD4 + T cells (A, B) and CD8 + Frequency of T cells (A, C) (n = 6-8 per group).

[0080] Figure 49: (A) E.G7 tumor-bearing Foxp3 YFP-cre Entpd1 fl / fl (KO) mice and Foxp3 YFP-cre (WT) Treg cell frequency in LN and TILs in mice (n = 6–8 per group). (B) E.G7 tumor-bearing Foxp3 YFP-cre Entpd1 fl / fl (KO) mice and Foxp3 YFP-cre NRP1 in TILs of (WT) mice - T-bet + pTreg cell frequency (n = 6-8 per group).

[0081] Figure 50: (AB) Foxp3 tumor-bearing tumors in Hepa1-6 YFP-cre Entpd1 fl / fl(KO) mice or Foxp3 YFP-cre (WT) Intratumoral NRP1 in mice - After Treg cell sorting, CD8 counts were measured using CTV staining. + T cell mitotic index (DI). (C) Using the formula [Inhibition (%) = 100 - (DI)] Treg / DI without Treg

[100] The determination of Hepa1-6 tumor-bearing Foxp3 YFP-cre Entpd1 fl / fl (KO) mice or Foxp3 YFP-cre (WT) Intratumoral NRP1 derived from mice - The percentage of Treg cells suppressed. (D)E.G7 tumor-bearing Foxp3 YFP-cre Entpd1 fl / fl (KO) mice and Foxp3 YFP-cre (WT) Tumor growth curves in mice (n = 5-6 per group). (E) B16-OVA-bearing Foxp3 tumors. YFP-cre Entpd1 fl / fl (KO) mice and Foxp3 YFP-cre (WT) Tumor growth curves in mice (n = 9-12 per group).

[0082] Figure 51: (A) E.G7 tumor-bearing Foxp3 YFP-cre Entpd1 fl / fl (KO) mice and Foxp3 YFP-cre (WT) Intratumoral CD8 in mice + IFN-γ in T cells + TNF-α + Cell frequency (n = 5-6 per group). (B)E.G7 tumor-bearing Foxp3 YFP-cre Entpd1 fl / fl (KO) mice and Foxp3 YFP-cre (WT) Intratumoral CD8 in mice + PD-1 in T cells + TIM-3 + Cell frequency (n = 5-7 per group). (CD)B16-OVA tumor-bearing Foxp3 YFP-cre Entpd1 fl / fl (KO) mice or Foxp3 YFP-cre (WT) Tumor growth curves (C) and survival rates (D) of mice treated with anti-PD-1 antibody or vector control at 9, 12 and 15 days after tumor inoculation (n = 11-12 per group). Detailed Implementation

[0083] Example 1: Enrichment of pTreg cells in the tumor microenvironment

[0084] Currently, the origin and functional mechanisms of Treg cells within tumors remain poorly understood. Therefore, we first analyzed Treg cell subsets in multiple mouse tumor models. Studies have shown that Neuropilin 1 (NRP1) is selectively expressed in thymus-derived native Treg cells (tTregs) but not in peripherally induced Treg cells (pTregs). Therefore, we used NRP1 as a marker for tTreg cells to perform phenotypic analysis of Treg cell subsets in the tumor microenvironment. We analyzed Treg cells isolated from lymph nodes (LNs), spleens (SPLs), tumor-draining lymph nodes (TDLNs), and tumor-infiltrating lymphocytes (TILs) using flow cytometry. These cells were derived from E.G7-OVA (Fig. 1, A and B), Hepa1-6 (Fig. 2, A and B), and B16-OVA (Fig. 3, A) mouse tumor models, respectively. LNs and SPLs from tumor-free mice served as controls. The results showed that nearly 80% of Treg cells from LN, SPL, and TDLN were NRP1. + This suggests that these cells are primarily tTreg cells. Conversely, over 50% of the Treg cells within the tumor are NRP1 cells. - The presence of Treg cells indicates the existence of a large number of newly induced pTreg cells in the tumor microenvironment of various tumor types.

[0085] We then validated this observation using the adoptive transfer method. We used CD45.2 Foxp3 GFP Mice (Jackson Laboratory, catalog number 006769) were mated with CD45.1 homologous mice (Jackson Laboratory, catalog number 002014) to obtain CD45.1 / CD45.2 (double-positive) Foxp3. GFP Mice. We obtained this from CD45.1 / CD45.2 double-positive Foxp3 mice. GFP tTreg cells were isolated from the spleen and LN of reporting mice, and tTreg cells were also isolated from CD45.2Foxp3. GFP Standard FOXP3 was isolated from report mice (Jackson Laboratory, catalog number 006769). - CD4 + T cells and CD8 + T cells. Subsequently, we added tTreg cells and standard CD4 cells. + T cells and CD8 + T cells were co-transferred to TCRbd at a ratio of 1:10:8. - / -In mice, physiological lymphoid organs mimicking those of normal mice were used. Then, E.G7 tumor cells were inoculated 3 days post-transfer (Figure 3, B). We used different CD45 markers carried by the donor cells to distinguish tTreg cells (CD45.1). + ) and pTreg cells (CD45.1) - ). tTreg cells versus regular CD4 + The ratio of T cells in LN and SPL was close to 1:10, compared to the ratio of tTreg cells and conventional CD4 cells used to sort from LN and SPL. + The initial 1:10 ratio of T cells was consistent (Figure 4, A and B). We found that at least 50% of the Treg cells within the tumor were derived from conventional CD4+. + pTreg cells (CD45.1) are T cells. - (Figure 5, A and B). Conversely, the majority of Treg cells in LN and SPL are tTreg cells (CD45.1). + (Figure 5, A and B). These results are consistent with observations from NRP1 staining. We further confirmed these findings by assessing NRP1 expression on donor cells. We found that most (over 70%) of CD45.1... + tTreg cells are NRP1 + And over 60% of CD45.1 - pTreg cells are NRP1 - (Figure 6, A and B). Therefore, the proportion of pTreg cells in the TME is comparable to, or even greater than, that of tTreg cells. This suggests that the tumor microenvironment may lead to immunosuppression by promoting the accumulation of pTreg cells.

[0086] Example 2: The important role of pTreg cells in promoting tumor growth

[0087] Removal of Treg cells can induce effective anti-tumor immunity, but it may also trigger an autoimmune response. To determine whether pTreg cells play a key role in tumor immunity, we used the aforementioned adaptive transfer method to selectively remove pTreg cells. We extracted pTreg cells from CD45.1 / CD45.2 double-positive Foxp3 cells. GFP tTreg cells were isolated from the spleen and lymph nodes of reporting mice, and derived from CD45.2Foxp3 cells. DTR Regular CD4 was isolated from mice + T cells and CD8 + T cells. Then, we added tTreg cells and regular CD4 cells. + T cells and CD8 +T cells were co-transferred to TCRbd at a ratio of 1:10:8. - / - Mice (Jackson Laboratory, catalog number 002122) were in vivo, followed by inoculation with E.G7 tumor cells 3 days after transfer. Foxp3 was used. DTR Mice as standard CD4 + To address the source of T cells, we selectively removed pTreg cells without affecting tTreg cells by injecting diphtheria toxin (DT), thus avoiding an autoimmune response. DT was administered every two days starting at day 12 until day 20. E.G7 tumor-bearing mice were sacrificed and analyzed at day 22 (Fig. 7, A). We found that tumor growth was significantly inhibited in DT-treated mice compared to control mice (Fig. 7, BC), and the number of Treg cells was significantly reduced in lymph nodes (LN), spleen (SPL), and tumors (Fig. 8, AC). Furthermore, pTreg cells (CD45.1)... - CD8+ cells were almost completely removed from LN, SPL, and tumors (Fig. 9, AD). By removing pTreg cells, CD8+ cells in tumor-infiltrating lymphocytes (TILs) were significantly reduced. + Both the absolute number and frequency of T cells increased (Figure 10, A and B), leading to an increase in CD8+ in TILs. + The ratio of T cells to total Treg cells is increased (Fig. 10, C). These CD8 cells... + T cells exhibit activation and proliferation (CD44) + Ki67 + This indicates that they are functional cells (Fig. 10, D and E). Regarding CD8... + Evaluation of interferon-γ (IFN-γ) and CD107a expression in T cells showed that they exhibited stronger cytolytic activity (CD107a) after pTreg cell removal. + IFN-γ + (Figure 11, AD). In summary, pTreg cells regulate anti-tumor immune cell types (such as CD8). + T cells play a crucial role in promoting tumor growth.

[0088] Example 3: Treg cells in tumors highly express T-bet transcription factor

[0089] Treg cells contain multiple functional subsets, similar to CD4 cells. +Helper T cells (Th cells). Therefore, we analyzed whether intratumoral Treg cells were similar to any Th subset in terms of gene expression. First, we performed transcriptomic analysis on intratumoral Treg cells from Hepa1-6 and E.G7 mouse tumor models by RNA sequencing. Given that intratumoral Treg cells consisted largely of pTreg cells, while Treg cells from lymph nodes (LNs) were mainly composed of tTreg cells, we used Treg cells from tumor-free LNs as controls. Compared to Treg cells from tumor-free LNs, intratumoral Treg cells from both models showed increased expression of Th1 cell characteristic genes (such as Tbx21 and Ccr5) (Fig. 12, A and B). Conversely, intratumoral Treg cells expressed almost no Th2 cell lineage-determining transcription factor Gata3, and their expression of Th17 characteristic genes (such as Rorc and Ccr6) was also reduced (Fig. 12, A and B). Furthermore, consistent with the NRP1 staining results analyzed by flow cytometry (Fig. 1, A and B; Fig. 2, A and B), the expression level of NRP1 in tumor-derived Treg cells was lower than that in LN-derived Treg cells (Fig. 12, A and B).

[0090] Next, we further validated our findings using flow cytometry. In Hepa1-6 and E.G7 tumor models, we stained Treg cells from LN, SPL, TDLN, and tumors with T-bet, RORγt, BCL6, and GATA3, using Treg cells from tumor-free LN and SPL as controls. We also co-stained the tTreg cell marker NRP1 with these transcription factors. Consistent with our observations from RNA sequencing data, tumor-intratumor Treg cells highly expressed T-bet (Fig. 12, C and D; Fig. 13, A and B). Similar results were obtained in the B16-OVA tumor model (Fig. 14, A and B). We further explored the relationship between NRP1 and T-bet expression in tumor-intratumor Treg cells through co-staining. We found that tumor-intratumor Treg cells expressing T-bet originate from NRP1. - Intratumoral Treg cells (pTreg cells). Importantly, T-bet + NRP1 - Treg cells comprise approximately 60% of intratumoral Treg cells (Fig. 14, C and D). In contrast, intratumoral Treg cells express almost no RORγt (Fig. 15, A and B), BCL6 (Fig. 16, A and B), or GATA3 (Fig. 17, A and B). In summary, our results indicate that T-bet... + NRP1 - Treg cells are a major component of intratumoral Treg cells in multiple cancer models.

[0091] Example 4: Intratumoral T-bet + pTreg cells exhibit enhanced activation, proliferation, and inhibition functions.

[0092] In whole-genome transcriptome analysis of tumor-associated Treg cells, we found that Th1-related transcripts were upregulated in tumor-associated Treg cells (Figure 12, A and B). Next, by comparing the gene expression profiles of tumor-associated tTreg and pTreg cells, we further investigated whether these transcripts were upregulated by tumor-associated tTreg cells or pTreg cells. We obtained this information from CD45.1 Foxp3... YFP-cre tTreg cells were sorted from the spleen and lymph nodes (LN) of mice, and derived from CD45.2Foxp3. YFP-cre Sorting of conventional FOXP3 in mice - CD4 + T cells and CD8 + T cells. Subsequently, we added tTreg cells and standard CD4 cells. + T cells and CD8 + T cells co-transferred to TCRbd in a ratio of 1:10:8 - / - In mice, Hepa1-6 tumor cells were inoculated 3 days later. We generated RNA sequencing data from pTreg and tTreg cells within the tumor, and used Treg cells from tumor-free lymph nodes as a control.

[0093] Compared to tTreg cells within the tumor, pTreg cells within the tumor showed upregulation of Th1 cell-related transcripts (such as Tbx21, Ccr5, Cxcr3, Il12rb2, Il18r1, and Ifngr1) (Fig. 18, A). Compared to LN-derived Treg cells, pTreg cells within the tumor showed upregulation of all these transcripts, while tTreg cells within the tumor showed downregulation of all of them (except Ccr5) (Fig. 18, B and C). This result suggests that Th1-related T-bet cells within the tumor are highly sensitive to thrombosis. + Treg cells are mainly pTreg cells.

[0094] Next, we further compared the gene expression profiles of pTreg cells and tTreg cells within the tumor. Compared with tTreg cells, pTreg cells showed higher expression levels of genes related to the inhibitory function of Treg cells (such as Entpd1, Tigit, Pdcd1, Tgfb1, Il10, and Ebi3) (Figure 19, A). pTreg cells also showed upregulation of activation-related genes (such as Tnfrsf18, Icos, Cd28, Cd69, Klrg1, Prdm1, Tnfrsf4, and Tnfrsf9), cell cycle regulation (such as Pclaf, Rrm2, Smc2, Top2a, and Mcm4), and chemokine receptors that facilitate their migration (such as Ccr8, Ccr2, Ccr4, Ccr6, and Ccr9) (Figure 19, A).

[0095] Studies have shown that most Treg cells in the gut are pTreg cells. Therefore, we investigated whether pTreg cells in tumors share similar genetic characteristics with Treg cells in the gut. We compared differentially expressed genes (DEGs) of pTreg cells in tumors with characteristic genes of colonic Treg cells in publicly available databases (Miragaia, RJ, T. Gomes, A. Chomka, L. Jardine, et al. 2019. Single-Cell Transcriptomics of Regulatory T Cells Reveals Trajectories of Tissue Adaptation. Immunity 50: 493-504.e497.). GSEA analysis showed a positive correlation between DEGs of pTreg cells in tumors and DEGs of repressive Treg cells, lymphoid-tissue (LT)-like Treg cells, and non-lymphoid tissue (NLT) Treg cells derived from the colon (Figure 19, BD). However, the most significant positive correlation was found between DEGs in tumor-bearing pTreg cells and DEGs in colonic NLT Treg cells (Fig. 19, D). Common marker genes included Tnfrsf4 (OX40), Tnfrsf9 (4-1BB), Tnfrsf18 (GITR), Ccr8, Pdcd1, Batf, Hopx, Il2ra, and Klrg1 (Fig. 20, A).

[0096] We further compared the genetic characteristics of tumor-derived pTreg cells with those of wild-type mouse-derived intestinal lymphoid and non-lymphoid tissue Treg cells retrieved from another published single-cell RNA sequencing dataset (Gu, Y., R. Bartolomé-Casado, C. Xu, et al. 2024. Immune microniches shape intestinal Treg function. Nature 628: 854-862.). The LT region includes secondary lymphoid organs such as mesenteric lymph nodes (mLN), cecal patch (CP), and distal colon-organized lymphoid structures (OLS). The intestinal NLT region includes the lamina propria (LP) and small lymphoid aggregates (LA) located in the cecum and proximal colon. GSEA analysis showed a significant positive correlation between the DEGs of tumor-derived pTreg cells and those of Treg cells from intestinal NLT (LP and LA) cells (Figure 20, B). Shared marker genes included Lag3, Tigit, Tnfrsf4 (OX40), Ctla4, Hopx, and S100a4 (Fig. 20, C). Although there was a positive correlation between the DEGs of intratumoral pTreg cells and the DEGs of intestinal LT Treg cells (mLN, CP, OLS), the p-value was close to the critical value (Fig. 20, D). Overall, these results suggest that intratumoral pTreg cells and intestinal Treg cells, particularly those originating from non-lymphoid tissues (such as the lamina propria) and containing a high proportion of pTreg cells, share similar genetic characteristics.

[0097] In summary, our findings indicate that intratumoral T-bet... + pTreg cells exhibited enhanced activation, proliferation, and inhibition functions. This finding further emphasizes the role of T-bet. + The role of pTreg cells in the tumor microenvironment.

[0098] Example 5: The necessity of T-bet in the function of pTreg cells within tumors

[0099] Next, we investigated the function of T-bet in tumor-bearing pTreg cells. We constructed Foxp3... YFP-cre Tbx21 fl / flMice were used to specifically knock out the Tbx21 gene (gene number ENSMUSG00000001444) in Treg cells. Foxp3 YFP-cre Tbx21 fl / fl The mice were induced to develop Tbx21 fl / fl Mice (Jackson Laboratories, catalog number 022741) and Foxp3 YFP-cre Mice (Jackson Laboratory, catalog number 016959) were obtained through mating. As previously described (Di Giovangiulio, M., A. Rizzo, E. Franze, et al. 2019. Tbet Expression in Regulatory T Cells Is Required to Initiate Th1-Mediated Colitis. FrontImmunol 10: 2158.), these mice did not show signs of autoimmune pathology during their lifetime. We then established B16-OVA and E.G7 tumor models in the KO and WT groups, respectively. We observed that the growth of B16-OVA and E.G7 tumors in the KO group mice was significantly reduced compared to the WT group (Fig. 21, A and B). Tumor-bearing mice were euthanized between days 18 and 21, and LN, spleen (SPL), and tumor-infiltrating lymphocytes (TIL) were isolated for flow cytometry analysis. We obtained very similar results in the B16-OVA and E.G7 tumor models, showing that the total frequency and absolute number of Treg cells in the tumor decreased by at least 50% after Tbx21 gene knockout (gene number ENSMUSG00000001444). However, the total Treg cell frequency in the LN and spleen was not significantly affected (Figure 22, AC). This suggests that Treg cell proliferation in the tumor is reduced after T-bet gene knockout.

[0100] On the other hand, these mice had CD8 in their tumors. + Increased expression of IFN-γ and tumor necrosis factor-α (TNF-α) in T cells (Fig. 23, A and C). Consistently, intratumoral TIM-3... + PD-1 + Exhaustion CD8 + T cells were significantly reduced (Fig. 23, B and D), while stem-like CD8 cells were significantly reduced. + The frequency of T cells within the tumor is related to CD8. + Increased in T cells, manifested as CD8 + Ly108 expression was significantly increased in T cells (Fig. 24, A and B).

[0101] Next, we evaluated the therapeutic effect of Treg cell-specific knockout of the Tbx21 gene combined with anti-PD-1 therapy. We used Foxp3... YFP-cre (WT) mice and Foxp3 YFP-cre Tbx21 fl / fl A B16-OVA tumor model was established in KO mice. Anti-PD-1 antibodies were administered every three days starting from day 9. Tumor growth and survival in tumor-bearing mice were assessed. Both anti-PD-1 treatment alone and Treg cell-specific deletion of the Tbx21 gene significantly inhibited tumor growth in the B16-OVA tumor model. However, the combination of Treg cell-specific Tbx21 gene knockout and anti-PD-1 treatment showed the most significant inhibition of tumor growth compared to WT mice (Fig. 25, A). Furthermore, Treg cell-specific deletion of the Tbx21 gene significantly prolonged the survival time of B16-OVA tumor-bearing mice, with effects comparable to anti-PD-1 treatment (Fig. 25, B).

[0102] In summary, our findings indicate that T-bet is not only highly expressed in pTreg cells within tumors, but is also crucial for their function in the tumor microenvironment.

[0103] Example 6: Th1 cells transform into pTreg cells in tumors via TGF-β signaling.

[0104] Thus far, we have confirmed that a large number of Treg cells within tumors are peripherally induced and express T-bet. However, it remains unclear whether these Treg cells are directly generated from naïve CD4+ cells. + T cell induction, or transformation of Th1 cells from the tumor microenvironment, is crucial. Studies have shown that TGF-β can induce Th1 cells to express FOXP3 in vitro. However, whether Th1 cells transform into Treg cells, especially in the tumor environment, remains unclear. Therefore, we used IFN-γ fate mapping mice (Ifng) to investigate this phenomenon. icre Rosa26 YFP E.G7 and B16-OVA tumor models were established. icre Rosa26 YFP Mice are produced by ifng icre Mice (Beijing Biocytogen Gene Biotechnology Co., Ltd.) and Rosa26 YFPThe strain was obtained by mating mice (Srinivas, S., T. Watanabe, CSLin, et al. 2001. Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev Biol 1:4.). Ifng icre The mouse model was constructed by analyzing the structure of the Ifng gene and inserting IRES-iCre-PA between the Ifng coding frame and the 3'UTR, enabling normal expression of Ifng and iCre. Neo cassette was placed between IRES-iCre-PA and the 3'UTR.

[0105] Mice were sacrificed 16 to 18 days after tumor inoculation, and LN, SPL, TDLN, and tumor tissue were collected for flow cytometry analysis. Extratumor LN and SPL served as controls. CD4+ levels in LN, SPL, and TDLN were compared with those in other samples. + Compared to T cells, tumor-infiltrating CD4 + YFP in T cells + The frequency of cells was significantly increased (Fig. 26, A and B). Most CD4 cells... + YFP + Cells (approximately 80%) highly expressed T-bet (Fig. 27, A and B). Of these, approximately 20-30% expressed FOXP3 (Fig. 27, A and C). Therefore, FOXP3... - T-bet + (Single positive) and FOXP3 + T-bet + (Double-positive) cells in CD4 + YFP + The ratio in cells was 3:1 (Fig. 27, A). T-bet + FOXP3 + The percentage of double-positive cells was significantly higher than that of FOXP3. + Single positive cells (Fig. 27, E).

[0106] In CD4 + YFp + In the cell population, the vast majority (over 80%) of these Th1-like Treg cells are NRP1. - This indicates that they are pTreg cells (Fig. 27, D and E). Importantly, we found FOXP3. + YFp +Co-staining of NRP1 and T-bet in the cell population showed an expression pattern similar to that of the major subsets of Treg cells within the tumor (Fig. 14, C and D) (T-bet). + NRP1 - (Figure 28, A and B). Interestingly, approximately 90% of T-bets... + FOXP3 + Cells did not express IFN-γ (Fig. 28, C and D). These observations suggest that in the tumor microenvironment, IFN-γ-expressing Th1 cells can transform into pTreg cells and constitute NRP1 of intratumoral Treg cells. - T-bet + Major subgroups.

[0107] Our fate mapping experiments show that Th1 cells can transform into T-bet cells in tumors. + pTreg cells. Therefore, we further differentiated them in vitro from initial CD4 cells. + T cells differentiate into Th1 cells to further verify this process. We compared OT-II mice (Jackson Lab, catalog number 004194) with Ifng mice. YFP Mice (Jackson Laboratories, catalog number 017581) were mated to obtain OT-II Ifng. YFP Mice. Then, OT-II Ifng was administered. YFP Mice were crossed with CD45.1 homologous mice (Jackson Laboratory, catalog number 002014) to obtain CD45.1 / CD45.2 (double-positive) OT-II Ifng. YFP Mice. We obtained OT-II ifng from CD45.1 / CD45.2 (double positive) mice. YFP Initial CD4 sorting in mice + T cells were cultured, and CD4+ produced by IFN-γ was sorted out on day 4 of culture. + T cells were transferred to E.G7-inoculated mice. Seven days after tumor inoculation, the transfected mice were sacrificed on day 21 for flow cytometry analysis. Results showed that approximately 10% of the donor Th1 cells (CD45.1) were intact. + CD4 + T cells transform into T-bets in tumors. + FOXP3 + Treg cells (Fig. 29, A and B). As expected, over 80% of the donor Th1 cells expressed T-bet (Fig. 29, A and C). Therefore, allogeneic transplantation from in vitro differentiated Th1 cells further demonstrates the transformation of Th1 cells into pTreg cells in the TME.

[0108] Considering the role of TGF-β in regulating pTreg cell generation, we next tested whether the transformation of Th1 cells into pTreg cells requires the TGF-β signaling pathway. As previously described (Marie, JC, D. Liggitt, and A.Y. Rudensky. 2006. Cellular mechanisms of fatal early-onset autoimmunity in mice with the T cell-specific targeting of transforming growth factor beta receptor. Immunity 25: 441-454.), Cd4 Cre Tgfbr2 fl / fl Mice developed autoimmune pathology within 3 weeks of birth, with a mortality rate of 100%. Therefore, we constructed OT-IICd4. Cre Tgfbr2 fl / fl Mice. OT-IICd4 Cre Tgfbr2 fl / fl The mice were obtained by tyrosine oxo-II mice (Jackson Laboratories, catalog number 004194) and Tgfbr2 mice. fl / fl Mice (Chytil, A., MA Magnuson, CV Wright, et al. 2002. Conditional inactivation of the TGF-beta type II receptor using Cre:Lox.Genesis 32:73-75.) were mated and then subjected to Cd4 Cre The OT-II Cd4 was obtained by mating mice (TACONIC) (Nurieva, RI, A. Podd, Y. Chen, AM Alekseev, et al. 2012. STAT5 protein negatively regulates T follicular helper (Tfh) cell generation and function. J Biol Chem 287: 11234-11239.). Cre Tgfbr2 fl / fl Mice were then mated with CD45.1 homologous mice (Jackson Laboratory, catalog number 002014) to obtain CD45.1 / CD45.2 (double-positive) OT-IICd4. Cre Tgfbr2 fl / flMice. These mice exhibited only mild immunopathology and survived for several months or even longer. We obtained data from CD45.1 / CD45.2 (double-positive) OT-IICd4. Cre Tgfbr2 fl / fl (KO group) or CD45.1 / CD45.2 (double positive) OT-IITgfbr2 fl / fl Initial CD4 sorting in mice (WT group) + T cells. From CD45.2 Foxp3 GFP CD8 sorting in report mice + T cells and tTreg cells. Initially sorted CD4+ + T cells, tTreg cells, and CD8 + T cells co-transfer to TCRbd - / - In mice, B16-OVA tumor cells were inoculated into TCRbd 3 days later. - / - In recipient mice, tumor-bearing mice were sacrificed on day 16 for flow cytometry analysis. Results showed that tumor growth was significantly slowed in the KO group (Fig. 30, A). Consistent with our hypothesis, deletion of Tgfbr2 resulted in donor CD45.1... + Regular CD4 + The ability of T cells to transform into pTreg cells is suppressed (Fig. 30, B and C). Furthermore, the loss of Tgfbr2 leads to the reduction of Th1 cells (T-bet) to pTreg cells. + FOXP3 - Increased frequency of Th1 cells (Figure 31, A and B). + FOXP3 - The increase in frequency leads to CD4 + Increased IFN-γ expression levels were observed in T cells (Figure 31, C and D). In summary, our findings suggest that pTreg cells can transform from Th1 cells in the tumor microenvironment, a process dependent on the TGF-β signaling pathway.

[0109] Example 7: Th1-like Treg cells in tumors exhibit suppressive pro-tumor characteristics.

[0110] To further clarify T-bet + The developmental pathways of Treg cells in the tumor microenvironment and the role of T-bet in regulating this specific Treg cell population were investigated by Foxp3. YFP-cre and Foxp3 YFP-cre Tbx21 fl / fl Single-cell transcriptomic analysis was performed on Treg cells from mouse Hepa1-6 tumors. Foxp3 YFP-cre Tbx21fl / fl The mice were induced to develop Tbx21 fl / fl Mice (Jackson Laboratories, catalog number 022741) and Foxp3 YFP-cre Obtained by mating mice (Jackson Laboratory, catalog number 016959).

[0111] We are from Foxp3 YFP-cre and Foxp3 YFP-cre Tbx21 fl / fl Treg cells (CD4+) were isolated from Hepa1-6 tumors in mice. + YFP + ), and compared with Foxp3 YFP-cre Conventional CD4 in Hepa1-6 tumors in mice + T cells (CD4) + YFP - ) and Treg cells (CD4+) in tumor-free lymph nodes + YFP + Transcriptome data (only data from Treg cells are shown).

[0112] Based on these single-cell gene expression data, we identified five transcriptomically distinct cell populations (Fig. 32, A). Populations 0 (C0) and 4 (C4) were primarily enriched in Treg cells from wild-type (WT) tumors, while population 2 (C2) was primarily enriched in Tbx21-deficient tumor Treg cells. Population 1 (C1) was primarily enriched in WT lymph node-derived Treg cells, and population 3 (C3) was enriched in conventional CD4+ cells from WT tumors. + T cells (data not shown) showed similar proportions in WT tumor Treg cells and Tbx21-deficient tumor Treg cells (Fig. 32, B and C).

[0113] Population 0 exhibits characteristics of Th1 cells (Tbx21, Ccr5, Cxcr3, Il12rb2, Il18r1) and is the most dominant cell population, accounting for at least 70% of tumor-infiltrated Treg cells (Fig. 32, B and C). Population 0 is enriched with molecules associated with Treg cell inhibitory function, including Entpd1, Ctla4, Tigit, Tgfb1, Il10, and Ebi3, as well as activation markers such as Icos, Klrg1, Prdm1, Tnfrsf4 (OX40), and Tnfrsf9 (4-1BB) (Fig. 33, A). Overall, this population represents tumor-infiltrated Th1-like effector Treg cells with enhanced activation and inhibitory functions.

[0114] Population 1 is characterized by increased expression of Foxp3 and the tTreg cell marker Nrp1. This population also exhibits increased expression of genes supporting Treg cell differentiation and repressive function (such as Foxp1, Foxo1, Bach2, and Ikzf2). Furthermore, this population expresses transcripts from naive Treg cells, such as Sell, Slamf6, Tcf7, Lef1, and Ccr7 (Figure 33, A). Overall, this population belongs to the naive / resting Treg cell group. The majority of cells in Population 1 are likely tTreg cells.

[0115] Population 2 was the most enriched population of Tbx21-deficient Treg cells, exhibiting characteristics of Th2 / Th17 cells. Population 2 upregulated Th2 cell-specific transcription factor (Gata3) and Th2 cell cytokine transcripts (Il4, Il13, Il5). This population also upregulated Th17 cell lineage-determining transcription factor (Rorc), Th17 cell cytokine transcripts (ll17a), and the transcription factor Irf4, which promotes the differentiation of Th2 and Th17 cells (Figure 33, A).

[0116] Population 3 was defined as the Th1 cell population. Population 3 shared characteristic genes of population 0, including Tbx21, Ccr5, Il12rb2, Il18r1, and Entpd1 (Fig. 33, A). Population 3 also highly expressed Th1 effector cell cytokine transcripts (Ifng, Tnf1) and granzyme family members (Gzmd, Gzme, Gzmk), accompanied by cytotoxicity-related molecules (Prf1, Nkg7). This population also upregulated marker genes associated with activated effector cells, such as Icos, Prdm1, and Cx3cr1 (Fig. 33, A). In summary, population 3 represents the most differentiated effector Th1 cells.

[0117] Population 4 was the most proliferative, exhibiting an increase in the proliferation marker gene Mki67. This population was enriched with a range of genes involved in DNA replication, transcription, and cell cycle regulation, including Exo1, Rrm1, Rrm2, the microchromosome maintenance gene family (Mcm3, Mcm5, Mcm10), Pclaf, Smc2, and Top2a. Population 4 shared the expression of Treg cell activation markers Icos, Tnfrsf4 (OX40), and Tnfrsf9 (4-1BB) with Population 0 (Figure 33, A). Therefore, this population was defined as activated proliferative Treg cells.

[0118] Next, we further investigated the role of T-bet in tumor Treg cells. Since the proportion of Th1-like Treg cells (C0) was significantly reduced in Tbx21-deficient Treg cells compared to WT tumor Treg cells (KO vs WT, 0.28 vs 0.71), but the proportion of Th2 / Th17-like Treg cells (C2) was significantly increased (KO vs WT, 0.45 vs 0.02), we further compared the DEGs between Th1-like Treg cells (C0) and Th2 / Th17-like Treg cells (C2) (Figure 34, A). Compared to Th2 / Th17-like Treg cells (C2), Th1-like Treg cells (C0) upregulated Th1-related molecules (Tbx21, Ccr5, Cxcr3), proliferation (Mki67), and activation marker genes (Icos), as well as genes associated with Treg cell suppression mechanisms (Entpd1, Nt5e, Tgfb1, Ebi3, Ctla4, Lag3, Tigit, Gzmb). Simultaneously, Th2 / Th17-like Treg cells upregulated Th2 cell (Gata3, Il4, Il5, Il13) and Th17 cell (Rorc, Il17a) marker genes (Figure 34, A). These results indicate that T-bet+ Treg cells suppress the Th2 / Th17 cell response in the TME. These results also suggest that in Tbx21-deficient Treg cells, the transcriptomic program undergoes dynamic changes, leading to a transformation of the Treg cell lineage from Th1-like Treg cells to Th2 / Th17-like Treg cells.

[0119] Compared to Th1-like Treg cells (population C0), Th2 / Th17-like Treg cells (population C2) exhibited less inhibitory properties. The reduced proportion of C0 cells and the increased proportion of C2 cells may have weakened inhibitory function in the tumor microenvironment of Tbx21 conditionally knocked-out mice compared to wild-type mice (WT). Further flow cytometry analysis showed a significant decrease in the frequency and absolute number of Treg cells within the tumors of Tbx21 conditionally knocked-out mice compared to WT mice (Figure 22, AC). Therefore, it can be inferred that the proportion of inhibitory and pro-tumor Treg cell populations is generally reduced in Tbx21 conditionally knocked-out mice.

[0120] Furthermore, compared to Th1-like Treg cells (population C0), Th2 / Th17-like Treg cells (population C2) showed downregulated expression of transcripts related to Treg cell repression mechanisms (Fig. 34, A). We further analyzed the co-expression of these transcripts with Tbx21 (Fig. 34, B). Entpd1 and Tigit showed higher fold changes between C0 and C2 (Fig. 34, A), and both were co-expressed with Tbx21. Conversely, Entpd1 and Tigit were not co-expressed with Gata3 or Rorc (Fig. 34, B). Flow cytometry results also confirmed that the expression of both CD39 and TIGIT was reduced in Tbx21-deficient Treg cells. However, compared to TIGIT expression, CD39 expression showed a more significant loss in Tbx21-deficient Treg cells, both in percentage and mean fluorescence intensity (MFI) (Fig. 35, AC). These findings suggest that CD39-mediated metabolic disruption may be a specific pathway by which Th1-like Treg cells exercise inhibitory mechanisms in the TME.

[0121] In summary, T-bet plays an important role in the activation and proliferation of Treg cells in tumors, the inhibitory function of Treg cells, especially the maintenance of CD39 function mediated by nucleotide metabolism, and the inhibition of Th2 / Th17 cell responses.

[0122] To further elucidate intratumoral T-bet + To investigate the development of Treg cells, we used pseudotime (RNA velocity) analysis to examine the temporal dynamics of Treg cells in the tumor tumor microenvironment (TME) of WT mice (Figure 35, D). We found that the Th1-like Treg cell population (C0) was the dominant terminally differentiated cell population. C0 primarily originated from population C3, which upregulated Th1 cell characteristic genes, indicating a transition from C3 to C0. Most proliferating cells from population C4 eventually differentiated into Th1-like Treg cells (C0). These findings further strengthen the evidence that tumor-derived Th1-like Treg cells originate from peripheral Treg cells (pTreg cells) derived from Th1 cells in the tumor microenvironment.

[0123] Example 8: Intratumoral T-bet + pTreg cells highly express CD39

[0124] This study aims to elucidate the role of CD39-mediated inhibition in tumor-bearing Treg cells and further explore the specific expression of CD39 in the tumor microenvironment. Through single-cell transcriptomics analysis and flow cytometry of Treg cells from Tbx21-deficient mice, we found that CD39-mediated metabolic interference may be a unique inhibitory mechanism of Th1-like Treg cells. Therefore, we further investigated the role of CD39-mediated inhibition in tumor-bearing Treg cells. Literature indicates that CD39 is an exogenous nucleotidase capable of hydrolyzing the phosphate bonds in ATP, converting ATP to AMP. CD39-mediated phosphate bond hydrolysis is crucial for nucleotide metabolism, especially purine and pyrimidine metabolism. Purine metabolism is essential for adenosine production, which inhibits CD8 by binding to the A2A receptor (A2AR). + The function of T cells.

[0125] First, we performed genome-wide transcriptomic analysis of Treg cells and RNA sequencing data from mouse E.G7 and Hepa1-6 tumor models to analyze differentially expressed genes (DEGs) and pathways, resulting in six distinct gene clusters (Fig. 36, AE). Clusters 1 and 2 contained genes upregulated in Treg cells within E.G7 and Hepa1-6 tumors (Fig. 36, AC). Clusters 5 and 6 contained genes upregulated in Treg cells within E.G7 and Hepa1-6 tumors, respectively (Fig. 36, A, D, and E). Pathway analysis of the upregulated genes in clusters 1, 2, 5, and 6 revealed enrichment of pathways related to nucleotide metabolism (Fig. 36, BE). Consistent with these findings, we further revealed that Treg cells within E.G7 and Hepa1-6 tumors largely upregulated genes related to purine metabolism. These genes include those encoding exogenous adenosine triphosphate diphosphate hydrolases (Entpd1, Entpd7), purine nucleotide phosphorylases (Pnp), the Nudix hydrolase family (Nudt1, Nudt4, Nudt9, Nudt21), the adenosine kinase family (Ak1, Ak2, Ak3, Ak4, Ak5, Ak6, Ak8), the NME / NM23 nucleotide diphosphate kinase family (Nme1, Nme4), and hypoxanthine-guanine phosphoryltransferase (Hprt) (Fig. 37, A and B). Furthermore, these gene clusters were found to be enriched in response pathways to hypoxia and oxidative stress (Fig. 36, BE). In addition, a comparison of gene expression profiles in pTreg and tTreg cells within the Hepa1-6 tumor model revealed that pTreg cells in the tumor upregulated genes involved in nucleotide degradation, including the Entpd1 gene encoding CD39 (Fig. 38, A).

[0126] Next, we explored the expression relationships of CD39, T-bet, and NRP1 in tumor-bearing Treg cells. Consistent with our single-cell transcriptomics analysis (Fig. 37, A), flow cytometry results showed that in the Hepa1-6 tumor model, tumor-bearing Treg cells exhibited co-expression of T-bet and CD39 (Fig. 38, B and C). We further analyzed the expression of T-bet and NRP1 in Foxp3 cells. YFP-cre Tbx21 fl / fl Mice (designated as KO group) and control Foxp3 YFP-cre Molecular phenotypic analysis was performed on intratumoral Treg cells in mice (designated as the WT group). Surprisingly, we found that T-bet cells showed CD39 activity within tumors of WT mice. + NRP1 - CD39 expression was significantly higher in pTreg cells (Fig. 39, A and B). Furthermore, in WT mice, CD39 expression was significantly higher than NRP1 expression within tumors. - Selective expression of NRP1 in Treg cells. By comparing with Treg cell-specific T-bet KO mice, we found that NRP1 was present in tumors. - CD39 expression levels were significantly higher in pTreg cells, a phenomenon not observed in T-bet KO mice (Fig. 39, C and D). Therefore, T-bet and CD39 expression are highly correlated. T-bet expression is consistently associated with NRP1 expression. - The presence of pTreg cells within the tumor was associated (Fig. 14, C and D), and CD39 was also selectively expressed in NRP1 within the tumor. - pTreg cells (Fig. 39, A, C, and D). These findings further enhanced intratumoral NRP1. - T-bet + Evidence suggests that pTreg cells exert inhibitory functions through CD39 expression.

[0127] To determine whether CD39 expression in Treg cells is tumor-specific, we assessed and compared CD39 expression in Treg cells from different tissue sources. These tissues included inguinal lymph nodes (iLN), thymus, Peyer's spots (PP), spleen, mesenteric lymph nodes (mLN), intraepithelial lymphocytes (IEL) of the colon, intraepithelial lymphocytes (IEL) of the small intestine, intraepithelial lymphocytes (LPL) of the colon, and intraepithelial lymphocytes (LPL) of the small intestine, all derived from tumor-free mice. We stained for CD39 expression using tumor-derived Treg cells as a positive control. Surprisingly, we found that, similar to tumor-derived Treg cells, CD39 was also highly expressed in Treg cells derived from tissues containing abundant pTreg cells, including intraepithelial IEL, intraepithelial LPL, and intraepithelial LPL of the small intestine. We further validated these observations by co-staining CD39 with NRP1. Consistently, we found that CD39 was selectively expressed in NRP1. - These observations were consistent in tumors, small intestinal IEL, colonic LPL, and small intestinal LPL (Fig. 40, A and B). In summary, our study suggests that T-bet... + pTreg cells exhibit high levels of CD39 expression, and CD39 is a key mediator of the adenosine signaling pathway in purine metabolism. CD39 may serve as a therapeutic target, selectively targeting intratumoral T-bets. + pTreg cells, without triggering a systemic autoimmune response.

[0128] Example 9: Treg cells in cancer patients significantly co-express T-bet and CD39.

[0129] This embodiment aims to further verify whether nucleotide metabolism is a key mechanism by which intratumoral Treg cells play a role in tumor immunosuppression. First, we performed pathway analysis using publicly available RNA sequencing data of human breast cancer Treg cells. Gene set enrichment analysis (GSEA) showed a positive correlation between differentially expressed genes (DEGs) in intratumoral Treg cells of E.G7 and Hepa1-6 mouse tumor models and differentially expressed genes in intratumoral Treg cells of human breast cancer (Figure 41, A and B). As expected, pathways related to nucleotide metabolism in intratumoral Treg cells were enriched in these tumor models (Figure 41, C).

[0130] Further validation revealed that in human breast cancer, tumor-associated Treg cells upregulated most genes related to nucleotide metabolism, including the ENTPD1 gene (Fig. 42, A). The ENTPD1 gene was upregulated in both small molecule degradation and purine-containing nucleotide degradation, both important pathways of nucleotide metabolism (Fig. 42, B and C). Several genes involved in nucleotide metabolism, such as TYMP, NUDT5, PGM2, HPRT1, GLRX, GPX1, and ENTPD1, ​​were also upregulated in tumor-associated Treg cells of other human cancer types (Fig. 43, A). These cancer types include hepatocellular carcinoma (HCC), colorectal cancer (CRC), and non-small cell lung cancer (NSCLC). Among these genes, ENTPD1 is a key upregulated gene in tumor-associated Treg cells across different types of human cancer (Fig. 43, A). Volcano plot analysis revealed a significant upregulation of the ENTPD1 gene in tumor-associated Treg cells of hepatocellular carcinoma (HCC) (Fig. 43, B).

[0131] Using publicly available single-cell RNA sequencing data from human cancers, we further investigated whether intratumoral Treg cells in human cancers co-express T-bet (encoded by the TBX21 gene) and CD39 (encoded by the ENTPD1 gene). TBX21 gene expression was found in intratumoral Treg cells from various human cancers, and co-expression of TBX21 and ENTPD1 was confirmed in intratumoral Treg cells from multiple human cancers, including nasopharyngeal carcinoma (NPC), breast cancer (BRCA), melanoma, squamous cell carcinoma (SCC), hepatocellular carcinoma (HCC), and colorectal cancer (CRC) (Figure 44, AF). This observation is consistent with our flow cytometry analysis, which found significant co-expression of T-bet and CD39 in intratumoral Treg cells from hepatocellular carcinoma (HCC) patients (Figure 45, AE).

[0132] In summary, the results of this study indicate that nucleotide metabolism, particularly the expression of the ENTPD1 gene and its co-expression with T-bet, plays an important role in Treg cells within tumors. This phenomenon has been validated in different types of human cancers and is consistent with our observations in a mouse tumor model.

[0133] Example 10: Targeting CD39 to induce anti-tumor response and enhance the effect of anti-PD-1 therapy

[0134] This embodiment aims to further elucidate the function of CD39 in tumor-bearing Treg cells. We constructed Foxp3... YFP-cre Entpd1 fl / fl Mice. Foxp3 YFP-cre Entpd1fl / fl Mice are induced to develop Entpd1 fl / fl Mice (Jicui Yaokang, catalog number T011367) and Foxp3 YFP-cre This mouse (Jackson Laboratory, catalog number 016959) was bred. The Entpdl gene has 11 transcripts. Based on the structure of the Entpdl gene, exons 2-7 of the Entpd1-201 (ENSMUST00000112231.8) transcript were selected as the knockout region. Knockout of this region leads to protein dysfunction. Entpd1 fl / fl The mice were constructed by modifying the Entpd1 gene using CRISPR / Cas9 technology. The process can be summarized as follows: sgRNA was transcribed in vitro, and a donor vector was constructed. Cas9, sgRNA, and Donor were microinjected into C57BL / 6J mouse zygotes. The zygotes were transplanted to obtain positive F0 mice, which were confirmed by PCR and sequencing. Stable F1 generation mice were obtained by mating these positive F0 mice with C57BL / 6J mice. These flux mice were then knocked out after mating with mice expressing Cre recombinase, resulting in the loss of function of the target gene in a specific gene or tissue.

[0135] This is the first description of information related to this transgenic mouse. Flow cytometry results confirmed that the knockout of the Entpd1 gene (gene number ENSMUSG00000048120) was validated by the absence of CD39 expression (i.e., a reduction of 92%-96%) in Treg cells. This result was observed in mouse Treg cells derived from colonic LPL and E.G7 tumors compared to Foxp3. YFP-cre The results were obtained from comparisons in mice (Figure 46, A and B). These mice did not exhibit any autoimmune pathological phenomena. Foxp3 YFP-cre Entpd1 fl / fl (hereinafter referred to as KO) mouse body weight and Foxp3 YFP-cre The results were comparable in both male and female WT mice (21–24 weeks old) (Fig. 46, C). Furthermore, no morphological changes were observed in the intestines and spleen (SPL) of Entpd1 KO mice (Fig. 46, D and E). Histological analysis of the colon and small intestine of Entpd1 KO mice revealed no signs of inflammation (Fig. 47, A). FOXP3 derived from colonic LPL and small intestinal LPL was also observed. - Or FOXP3 + CD4 + In T cells, Entpd1 KO and WT mice showed differences in activated cells (CD44). hi No significant differences were observed in the frequency of ) (Fig. 47, B and C). Consistently, Entpd1 KO and WT mice showed similar effector activity (CD44) in lymph nodes (LNs) and spleen (SPL).+ CD62L - CD4 + and CD8 + No significant difference was observed in the frequency of T cells (Fig. 48, AC). Furthermore, in the E.G7 tumor model, there was no significant difference in the frequency of Treg cells in tumors and lymph nodes between Entpd1 KO and WT mice (Fig. 49, A). In the tumor microenvironment, deletion of Entpd1 resulted in a decrease in the T-bet frequency of Treg cells within the tumor. + NRP1 - The frequency of pTreg cells was not affected (Fig. 49, B). These observations suggest that CD39 expression in Treg cells is not necessary for maintaining immune homeostasis.

[0136] Next, we first conducted in vitro studies to investigate the function of CD39 in tumor-bearing Treg cells. Given that CD39 is mainly involved in NRP1... - pTreg cells express this gene in a subset of Hepa1-6 tumor-bearing Foxp3 cells. YFP-cre (WT) mice and Foxp3 YFP-cre Entpd1 fl / fl Intratumor CD4 was isolated from (KO) mice. + YFP + NRP1 - Treg cells. The sorted Treg cells were then compared with the initial CD8... + T cells were co-cultured in 96-well plates for 72 hours. As a negative control, CD8 cells not co-cultured with Treg cells were also included. + T cells were also treated concurrently. CD8 cells were co-cultured with intratumoral Treg cells from the WT group. + The T cell division index (DI) was significantly higher in the KO group than in the KO group (Fig. 50, A and B). Therefore, the inhibitory effect of intratumoral Treg cells in the KO group was significantly lower than that in the WT group (Fig. 50, C). This was in contrast to the intratumoral NRP1 levels in the WT group. - Compared to Treg cells, NRP1 cells lacking Entpd1 - Treg cells against CD8 in vitro + The inhibitory capacity of T cells decreased significantly.

[0137] To further investigate the role of CD39 in the tumor microenvironment of intratumoral Treg cells, we conducted a study on Foxp3... YFP-cre Entpd1 fl / flE.G7 and B16-OVA tumor models were established in mice. Tumor-bearing mice were euthanized on days 18–21 and analyzed by flow cytometry. In the E.G7 and B16-OVA tumor models, we observed reduced tumor growth (Fig. 50, D and E). After Entpd1 knockout, CD8+ within the tumor was significantly reduced. + T cell function was enhanced, as evidenced by increased expression of TNF-α and IFN-γ (Fig. 51, A), while the expression of PD-1 and TIM-3 was reduced (Fig. 51, B).

[0138] Next, we evaluated the therapeutic effect of combining Treg cell-specific deletion of Entpd1 with anti-PD-1 therapy. We used Foxp3... YFP-cre (WT) mice and Foxp3 YFP-cre Entpd1 fl / fl A B16-OVA tumor model was established in KO mice. Anti-PD-1 antibodies were administered every three days starting on day 9. We evaluated tumor growth and survival in B16-OVA-bearing WT and KO mice under anti-PD-1 treatment. Both anti-PD-1 treatment alone and Treg cell-specific Entpd1 knockout significantly reduced tumor growth in the B16-OVA tumor model. However, the reduction in tumor growth was most significant in combination with anti-PD-1 treatment compared to WT mice when Treg cell-specific Entpd1 knockout was used in combination (Fig. 51, C). Furthermore, the combination of anti-PD-1 treatment and Treg cell-specific Entpd1 knockout significantly enhanced the survival rate of B16-OVA-bearing mice (Fig. 51, D).

[0139] Through this embodiment, we demonstrated the function of CD39 in tumor Treg cells and showed that targeting CD39 can induce an anti-tumor response and significantly enhance the anti-tumor effect when used in combination with anti-PD-1 therapy.

Claims

1. The use of T-bet-positive pTreg cells as drug targets in the development, screening, or preparation of drugs for the prevention and / or treatment of tumors, wherein the drugs are capable of reducing the number or activity of T-bet-positive pTreg cells.

2. The application according to claim 1, characterized in that, The drug has at least one of the following effects: (1) Inhibit the activity of T-bet-positive pTreg cells by targeting T-bet protein or related signaling pathways in T-bet-positive pTreg cells; (2) Enhanced CD8 + The role of T cells in tumor killing activity.

3. The application according to claim 1, characterized in that, The tumor is a solid tumor; the solid tumor may be lymphoma, hepatocellular carcinoma, melanoma, breast cancer, squamous cell carcinoma, colorectal cancer, non-small cell lung cancer, or head and neck cancer.

4. The use of molecules that reduce the number or activity of T-bet-positive pTreg cells in the preparation of drugs for the prevention and / or treatment of tumors.

5. The application according to claim 4, characterized in that, The molecules used to reduce the number or activity of T-bet-positive pTreg cells are T-bet inhibitors and / or CD39 inhibitors.

6. The application according to claim 4, characterized in that, When applied, molecules that reduce the number of T-bet-positive pTreg cells are used in combination with immune checkpoint inhibitors; The immune checkpoint inhibitor is an anti-PD-1 antibody, an anti-CTLA-4 antibody, or a combination thereof.

7. A drug for the prevention and / or treatment of tumors, characterized in that, The drug includes inhibitors that can reduce the number or activity of T-bet-positive pTreg cells.

8. The medicament for preventing and / or treating tumors according to claim 7, characterized in that, The inhibitors that can reduce the number or activity of T-bet-positive pTreg cells are T-bet inhibitors and / or CD39 inhibitors.

9. The drug for preventing and / or treating tumors according to claim 7, characterized in that, The drug also includes immune checkpoint inhibitors, which are anti-PD-1 antibodies, anti-CTLA-4 antibodies, or combinations thereof.

10. The use of T-bet-positive pTreg cells as drug targets in the development, screening or preparation of drugs for the treatment of autoimmune diseases, wherein the drugs can induce the generation of T-bet-positive pTreg cells or enhance the number or activity of T-bet-positive pTreg cells.

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