Application of T-bet positive pTreg cell as drug target

Abstract

The invention discloses application of a T-bet positive pTreg cell as a drug target, and relates to the field of immunology. The invention discloses that Treg cells in a tumor microenvironment are mainly T-bet positive pTreg cells, and by targeting the cells, the immunotherapy effect is expected to be remarkably improved, and meanwhile, the side effects of an immune system are reduced. Compared with a traditional immunotherapy method, the immunotherapy method has the advantages that the anti-tumor immune response is greatly improved by precisely targeting the pTreg cells and enhancing the anti-tumor immune response, particularly through combined immunotherapy, the immune homeostasis is ensured, and the autoimmune problem caused by systematic removal of the Treg cells is avoided.

Description

Technical Field

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

[0002] FOXP3-expressing regulatory T cells (Treg cells) play a vital role in maintaining immune tolerance and homeostasis, mainly protecting the host from autoimmune damage by inhibiting excessive immune responses (Ohkura, N., Y. Kitagawa. and S. Sakaguchi. 201 3. Development and maintenance of regulatory T cells. Immunity 38: 414-423.). However, the inhibitory effect of Treg cells in the tumor microenvironment allows tumor cells to escape the host's immune surveillance and promotes 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 a large number of Treg cells in the tumor will significantly inhibit CD8 + The anti-tumor effect of T cells affects the patient's prognosis. Although Treg cells are potential targets for cancer immunotherapy, many 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 immunotherapy approaches, such as targeting the Treg cell surface marker CTLA-4, can effectively reduce the inhibitory effect of Treg cells, but may also cause serious autoimmune side effects. In addition, the Treg cell subpopulations 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 subpopulations. Therefore, there is an urgent need for a new method that can specifically target Treg cells in the tumor microenvironment without causing side effects and enhance anti-tumor immune responses. Summary of the invention

[0004] The purpose of the present invention is to solve the selectivity and side effects of immunosuppressive cell targeted therapy in existing tumor immunotherapy. In the prior art, although there are studies trying to target Treg cells in the tumor microenvironment, due to the complexity of Treg cell subpopulations and their diversity in different microenvironments, it is difficult for existing treatment methods to effectively target specific immunosuppressive subpopulations, thereby affecting the treatment effect. In addition, existing treatment methods targeting Treg cells may also cause severe autoimmune reactions. Therefore, the present invention aims to target specific T-bets in the tumor microenvironment. + The pTreg cell subset solves the problems of poor selectivity and severe side effects of existing methods and improves the efficacy of tumor immunotherapy.

[0005] The technical solution of the present invention mainly involves in-depth analysis of the heterogeneity of regulatory T cells (Treg cells) in the tumor microenvironment, and proposes a strategy of selectively targeting the main pTreg cell population in the tumor microenvironment to enhance anti-tumor immunity while avoiding inducing autoimmune reactions. The following is the technical solution of the present invention, including various necessary technical features involved in achieving the purpose of the invention.

[0006] This discovery highlights the importance of understanding the heterogeneity of Treg cells in the tumor microenvironment in order to selectively target the major Treg cell population within the tumor without triggering an autoimmune response. By performing whole-genome transcriptomics and single-cell analysis in mouse tumor models, we found that T-bet + pTreg cells are the most common subpopulation, highlighting their importance in tumor immunity. + How pTreg cells suppress anti-tumor immune responses. This invention proposes a new strategy to target these cells, thereby enhancing anti-tumor immunity while reducing the risk of systemic autoimmune responses.

[0007] First, the present invention characterized and analyzed Treg cell subsets in various mouse tumor models. We used NRP1 as a marker to distinguish thymus-derived tTreg cells from peripherally induced pTreg cells. Through flow cytometry, the study found that in lymph nodes and spleen, most Treg cells were NRP1. + However, in tumors, more than half of Treg cells are NRP1 - This result was further verified by adoptive transfer experiments. The experiments showed that about 50% of Treg cells in tumors are pTreg cells, which are derived from conventional CD4 + T cells. In contrast, Treg cells in lymph nodes and 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 explored the role of pTreg cells in tumor immunity. We selectively removed pTreg cells by adoptive transfer and used Foxp3 DTR The researchers used diphtheria toxin (DT) to remove pTreg cells without affecting tTreg cells, thereby avoiding autoimmune reactions. The results showed that tumor growth was inhibited after pTreg cells were removed. At the same time, tumor-infiltrating CD8 + The number of T cells increased and showed stronger proliferation and cytolytic activity. In addition, the proportion of myeloid cell populations in the tumor microenvironment changed, with a decrease in TAMs and monocytes and an increase in neutrophils. Further treatment evaluation found that after the removal of pTreg cells in the late stage of the E.G7 tumor model, tumor growth was inhibited, and CD8 + The proportion of T cells increased, and the tumor-infiltrating CD8 + The proportion of T cells in total live cells increased, especially the proportion of cells expressing IFN-γ, granzyme B, and CD107a. Overall, the removal of pTreg cells produced an effective therapeutic effect on tumor-bearing mice.

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

[0010] RNA sequencing results showed that pTreg cells in tumors showed higher levels of Th1 gene upregulation compared with tTreg cells. At the same time, pTreg cells also showed upregulation of genes related to the inhibitory function of Treg cells, as well as upregulation of 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 and found that pTreg cells in tumors had similar gene expression profiles to intestinal Treg cells, especially in non-lymphoid tissue areas, such as the lamina propria, which contained a higher proportion of pTreg cells. These results indicate that T-bet + pTreg cells have enhanced activation, proliferation, and suppression functions, further emphasizing the role of T-bet + The important role of pTreg cells in the tumor microenvironment.

[0011] Therefore, the present invention provides, on one hand, 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 can reduce 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 cancer, oropharyngeal cancer, oral cancer, laryngeal cancer, pharyngeal cancer, tongue cancer, thyroid cancer, salivary gland cancer).

[0015] In another aspect, the present invention provides the use of a molecule for reducing the number or activity of T-bet-positive pTreg cells in the preparation of a drug for preventing and / or treating tumors.

[0016] Preferably, the molecule for reducing 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 a gene encoding T-bet (Tbx21 gene) or a compound that reduces T-bet expression; the CD39 inhibitor is a sequence for knocking out or silencing a gene encoding CD39 (Entpd1 gene) or a compound that reduces CD39 expression. When knocking out the Entpd1 gene, exons 2-7 of the Entpd1-201 (ENSMUST00000112231.8) transcript are used as knockout regions.

[0017] Preferably, when used, 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 knockout cells specifically depleted of Tbx21 YFP-cre Tbx21 fl / fl The mice (KO group) were compared with wild-type mice (WT group). The results showed that the tumor growth in the KO group was significantly slowed down, and the number of Treg cells in the tumor was significantly reduced. Flow cytometry analysis showed that after T-bet loss, the proliferative Treg cells in the tumor decreased, and CD8 + T cell activation and proliferation were enhanced, and CD8 + The ratio of T cells to Treg cells increased. Further adoptive transfer experiments verified the key role of T-bet in the function of pTreg cells. In addition, the combination of anti-PD-1 therapy and Treg cell-specific knockout of Tbx21 significantly inhibited tumor growth and prolonged the survival of mice. Overall, the study shows that T-bet is essential for the function of pTreg cells in the tumor microenvironment, and its combination with anti-PD-1 therapy has potential therapeutic effects.

[0019] Single-cell transcriptomics and flow cytometry revealed the role of CD39 in T-bet regulation in tumors+ The specific expression of CD39 in pTreg cells and the determination of CD39-mediated immunosuppression as a unique mechanism of Th1-like Treg cells in tumors. The study found that Treg cells highly expressed CD39 in tumors and were associated with purine metabolism, suggesting its key role in immunosuppression. The co-expression of CD39 and CD73 was particularly prominent in Treg cells in tumors, further indicating that T-bet + pTreg cells have unique immunosuppressive markers. In addition, CD39 expression has also been found in other tissues that contain a high proportion of pTreg cells, such as the lamina propria of the small intestine and colon. The results suggest that CD39 can serve as a selective regulator of T-bet + pTreg cells are potential therapeutic targets and provide a new strategy for cancer immunotherapy.

[0020] By analyzing RNA sequencing data from human breast cancer tissues, the study found that the differentially expressed genes of intratumoral Treg cells were similar to those in mouse tumor models, especially pathways related to nucleotide metabolism. In addition, the study also found that the ENTPD1 gene (encoding CD39) was commonly upregulated in intratumoral Treg cells of multiple cancer types. Through single-cell RNA sequencing data from a variety of human cancers, it was further confirmed that TBX21 (encoding T-bet) and ENTPD1 were specifically co-expressed in intratumoral Treg cells of multiple cancer types (such as nasopharyngeal carcinoma, breast cancer, melanoma, squamous cell carcinoma, hepatocellular carcinoma, and colorectal cancer). Flow cytometry analysis also verified that intratumoral Treg cells in liver cancer patient samples highly co-expressed T-bet and CD39. These results indicate that the co-expression of T-bet and CD39 is an immunosuppressive mechanism across a variety of human cancers, consistent with findings in mouse tumor models. This new discovery provides a basis for the development of drugs targeting intratumoral T-bet. + Targeted therapy of pTreg cells provides a theoretical basis for overcoming immunosuppression in cancer.

[0021] By creating Foxp3 YFP-cre Entpd1 fl / fl In vitro experiments showed that Treg cells lacking CD39 expression had no autoimmune pathological reactions or changes in immune homeostasis. + The inhibitory capacity of T cells was significantly reduced. In vivo studies showed that tumor growth was slowed after knocking out Entpd1, and CD8 +The activity of T cells was enhanced. In addition, the combination of Treg cell-specific knockout of Entpd1 and anti-PD-1 therapy significantly enhanced the tumor suppression effect and improved the survival rate of mice. The results showed that targeting CD39 in Treg cells within tumors can not only stimulate anti-tumor responses, but also produce synergistic effects with anti-PD-1 therapy.

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

[0023] As a preferred embodiment, the inhibitor capable of reducing 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 comprises an immune checkpoint inhibitor, which is an anti-PD-1 antibody, an anti-CTLA-4 antibody or a combination thereof.

[0025] T-bet + The combination of pTreg cells and anti-PD-1 therapy has shown some therapeutic potential. To further optimize this strategy, the combination with other immune checkpoint inhibitors can be explored. Immune checkpoint inhibitors can enhance tumor-specific CD8 + T cell immune activity, and T-bet + Specific inhibition of pTreg cells helps to eliminate immunosuppression in the tumor microenvironment and is expected to significantly enhance 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 cancer, oropharyngeal cancer, oral cancer, laryngeal cancer, pharyngeal cancer, tongue cancer, thyroid cancer, salivary gland cancer).

[0027] The present 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 drug 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 disease is an autoimmune disease associated with Treg dysfunction, and Treg cells play a key role in maintaining immune tolerance and inhibiting abnormal immune responses. When Treg cells are dysfunctional, the immune system may attack normal autologous tissues, leading to autoimmune diseases. The 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 disease (including Crohn's disease and ulcerative colitis), psoriasis, autoimmune hepatitis, allergic diseases. The common feature of these diseases is the self-attack of the immune system, in which Treg cells play an important regulatory role, so the recovery or enhancement of Treg function may help treat these diseases.

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

[0030] The present invention significantly improves the anti-tumor immune efficacy and reduces side effects by precisely targeting pTreg cells, providing patients with a higher survival rate and quality of life, thereby reducing treatment costs and alleviating the social medical burden. The improvement of immunotherapy may reduce dependence on chemotherapy and radiotherapy, save medical resources, relieve pressure on hospitals, and improve social medical efficiency.

[0031] In addition, the improvement of treatment effect helps patients recover early and return to work, reduces economic losses caused by sick leave, and promotes social labor productivity and economic development. Overall, the present invention not only promotes the technological progress of tumor immunotherapy, but also has a positive impact on society and the economy, optimizes the allocation of medical resources, and improves social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0035] Figure 4 :In E.G7 tumor-bearing TCRbd - / - In recipient mice (day 24), tTreg (CD45.1 + ) cells and conventional CD4 + T(CD45.1 - ) cells (n=17 in each group); A is the flow cytometry graph; B is the statistical graph of expression.

[0036] Figure 5 :In E.G7 tumor-bearing TCRbd- / - recipient mice, tTreg (CD45.1 + ) cells and pTreg (CD45.1-) cells (n=17-21 in each group); wherein, A is the flow cytometry graph; B is the expression statistics graph.

[0037] Figure 6 :In E.G7 tumor-bearing TCRbd- / - recipient mice, tTreg (CD45.1 + ) cells and pTreg (CD45.1-) cells NRP1 + and NRP1 - The percentage of cells (n=12 in each group); A is the flow cytometric graph; B is the statistical graph of expression.

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

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

[0040] Fig. 9 :In E.G7 tumor-bearing TCRbd - / - In recipient mice, tTreg (CD45.1 + ) cells and pTreg (CD45.1 - ) cells.

[0041] Fig.10 : CD8 per unit tumor weight (g) in E.G7 tumor-bearing TCRbd- / - recipient mice after DT or PBS treatment + The absolute number of T cells and the situation in TILs; A is the CD8 + Absolute number of T cells; B is CD8 + The frequency of T cells in the total living cells; C is the frequency of CD8 + The ratio of T cells to Treg cells; DE is the CD44 + Ki67 + CD8 + The frequency of T cells among total live cells.

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

[0043] Fig.12: Volcano plots and expression of T-bet in Treg cells; wherein the volcano plots show upregulated genes (red dots) and downregulated genes (blue dots) in Treg cells within tumors compared with gene expression in Treg cells in LNs (n=4) from tumor-free mice in Hepa1-6 (n=4) (A) and E.G7 (n=2) (B) tumor models; expression of T-bet in Treg cells in LNs and SPLs of tumor-free control wild-type (WT) C57BL / 6J mice, or in LNs, SPLs, TDLNs and TILs of Hepa1-6 tumor-bearing WT C57BL / 6J mice (C and D) (n=4-5 in each group).

[0044] Fig.13 : Expression of T-bet 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 E.G7 tumor-bearing WTC57BL / 6J mice (n=4 in each group); wherein A is a flow cytometric graph; B is a statistical graph of expression.

[0045] Fig.14 : (A and B) Expression of T-bet in Treg cells in LNs of tumor-free wild-type (WT) C57BL / 6J mice and in TILs of B16-OVA tumor-bearing WTC57BL / 6J mice (n=3-4 per group); (C and D) Expression pattern after co-staining of NRP1 and T-bet in TILs of E.G7 tumor-bearing WTC57BL / 6J mice.

[0046] Fig.15 : Flow cytometry images (A) and quantitative data (B) showing RORγt expression in Treg cells from tumor-free WT C57BL / 6J mice and E.G7 tumor-bearing mice (including LNs, SPLs, TDLNs, and TILs) (n=3-4 per group).

[0047] Fig.16 : Flow cytometry images (A) and quantitative data (B) showing BCL6 expression in Treg cells from tumor-free WT C57BL / 6J mice and E.G7 tumor-bearing mice (including LNs, SPLs, TDLNs, and TILs) (n=3-4 per group).

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

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

[0050] Fig.19 : (A) Gene heatmap shows the differential expression of genes related to Treg cell suppression mechanism, activation, proliferation and migration in intratumoral pTreg cells (n=3) and intratumoral tTreg cells (n=5) in the Hepa1-6 tumor model; (B-D) Enrichment map shows the correlation of upregulated genes in (B) colon suppressive Treg cells, (C) colon LT-like Treg cells or (D) colon NLT Treg cells with Hepa1-6 intratumoral pTreg cells (n=3) compared with Hepa1-6 intratumoral tTreg cells (n=5), which were identified by GSEA calculation method. Red indicates high expression level and blue indicates low expression level. NES, normalized enrichment score.

[0051] Fig. 20 : (A) Gene heatmap shows the differential expression of colon NLT Treg marker genes in intratumoral pTreg cells (n=3) and intratumoral tTreg cells (n=5) in the Hepa1-6 tumor model. (B) Enrichment map shows the correlation of upregulated genes in intestinal NLTTreg cells with Hepa1-6 intratumoral pTreg cells (n=3) compared with Hepa1-6 intratumoral tTreg cells (n=5), which were identified by the GSEA calculation method. Red indicates high expression levels and blue indicates low expression levels. NES, normalized enrichment score. (C) Gene heatmap shows the differential expression of intestinal NLT Treg marker genes in Hepa1-6 intratumoral pTreg cells (n=3) and intratumoral tTreg cells (n=5). (D) Enrichment map shows the correlation of upregulated genes in intestinal LT Treg cells with Hepa1-6 intratumoral pTreg cells (n=3) compared with Hepa1-6 intratumoral tTreg cells (n=5), which were identified by the GSEA calculation method. Red indicates high expression levels, and blue indicates low expression levels. NES, normalized enrichment score.

[0052] Fig.21 :(A) B16-OVA tumor growth curve, comparison of Foxp3 YFP-cre Tbx21fl / fl Mouse (KO) and Foxp3 YFP -cre Mice (WT) (n=5-6 per group). (B) E.G7 tumor growth curves, compared with Foxp3 YFP-cre Tbx21 fl / fl Mouse (KO) and Foxp3 YFP-cre Mice (WT) (n=12-16 per group).

[0053] Fig. 22 :(AB) Foxp3 in B16-OVA tumors YFP-cre Tbx21 fl / fl Mouse (KO) and Foxp3 YFP-cre Treg cells from LN, SPL, TDLN and TILs in mice (WT) + The frequency of Foxp3 in T cells (n=6-7 per group). (C) The frequency of Foxp3 in T cells in B16-OVA tumors YFP-cre Tbx21 fl / fl Mouse (KO) and Foxp3 YFP-cre Absolute number of Treg cells in TILs in mice (WT) (n=5-7 per group).

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

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

[0056] Fig.25 : (AB) Foxp3 expression after anti-PD-1 antibody or vehicle control treatment (indicated by arrows) at 9, 12, and 15 days after B16-OVA tumor inoculation. YFP-cre Tbx21 fl / fl Mouse (KO) and Foxp3 YFP-cre Tumor growth curves (A) and survival rates (B) of mice (WT) (n=6-8 per group).

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

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

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

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

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

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

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

[0064] Fig.33 : Gene heatmap showing the expression of differentially upregulated genes in each identified cell population. Adjusted p-value < 0.05.

[0065] Fig.34 :(A) Volcano plot showing up-regulated genes (red dots) and down-regulated genes (blue dots) in intratumoral Th1-like Treg cells (C0) and Th2 / Th17-like Treg cells (C2). (B) Gene heat map showing the expression of Tbx21, Gata3, Rorc, and genes involved in the inhibitory mechanism of Treg cells in intratumoral Th1-like Treg cells (C0) and Th2 / Th17-like Treg cells (C2). Adjusted p value < 0.05.

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

[0067] Fig.36 : (A) Six gene clusters identified by pathway enrichment analysis of RNA expression in intratumoral Treg cells in E.G7 (n=2) and Hepal-6 (n=3) tumor models, compared with Treg cells in extratumoral LNs (n=3). (B-E) Nucleotide metabolism pathways enriched in intratumoral Treg cells in E.G7 (n=2) and Hepal-6 (n=3) tumor models, compared with Treg cells in extratumoral LNs (n=3), were identified in clusters 1 (B), 2 (C), 5 (D), and 6 (E), respectively.

[0068] Fig.37 : (AB) Gene heatmaps show 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 compared with Treg cells in extratumoral LNs (n=3).

[0069] Fig.38 :(A) Gene heatmap shows the expression of genes related to nucleotide metabolism in intratumoral pTreg cells (n=3) and intratumoral 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 in each group).

[0070] Fig.39 :(AB)NRP1 in tumors of 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 (marked as KO) and Foxp3 YFP-cre NRP1 in tumors of mice (labeled as WT) + and NRP1 - Expression of CD39 in Treg cells (n=8-9).

[0071] Fig.40 : (AB) Representative flow cytometry images (A) and statistical graphs (B) showing co-staining of CD39 and NRP1 in intestinal IELs, colonic LPLs, and Treg cells in intestinal LPLs isolated from tumor-free C57BL / 6J mice, and TILs isolated from E.G7 tumor-bearing C57BL / 6J mice (n = 6-7 per group).

[0072] Fig.41 : (AB) Enrichment plots showing the correlation of upregulated genes in human breast cancer intratumoral Treg cells with E.G7 intratumoral Treg cells (A) (n=2) or Hepa1-6 intratumoral Treg cells (B) (n=4), compared with tumor-free LN Treg cells (n=4), identified by GSEA computational approach. Red indicates high expression levels, blue indicates low expression levels. (C) Nucleotide metabolic pathways enriched in intratumoral Treg cells, compared with PBMCTreg cells (n=6), from human breast cancer (n=4).

[0073] Fig.42: (A) Gene heatmap shows the expression of genes involved in nucleotide metabolism in human breast cancer intratumoral Treg cells (n=4) and PBMC Treg cells (n=6) (determined by RNA sequencing). (BC) Gene heatmap shows the expression of genes involved in small molecule degradation (B) and small molecule degradation containing nucleotides (C) in human breast cancer intratumoral Treg cells (n=4) and PBMC Treg cells (n=6) (determined by RNA sequencing).

[0074] Fig.43 :(A) Venn diagram shows the overlap of nucleotide metabolism upregulated genes in intratumoral Treg cells in 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 intratumoral Treg cells of hepatocellular carcinoma (HCC).

[0075] Fig.44 : (AF) Heatmaps showing the expression of FOXP3, TBX21, ENTPD1, ​​NT5E, IL10, EBI3, IL124, TGFB1, IL17F, IL22, and IFNG in human tumor Treg cells 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 by 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] Fig.45 : (AC) Expression of T-BET (A and B) and CD39 (A and C) in Treg cells, respectively, from PBMC, para-tumor tissue or tumor infiltrating lymphocytes (TILs) of HCC patients (n=6-8 per group). (DE) Co-expression of T-BET and CD39 in Treg cells in TILs of HCC patients (n=6 per group). Data are presented as mean and standard error (SEM).

[0077] Fig.46 :(AB)CD39 in Foxp3 YFP-cre Entpdl fl / fl(KO) mice and Foxp3 YFP-cre CD4 + FOXP3 + Expression in Treg cells (n=6 per group). (C) Foxp3 YFP -cre Entpd1 fl / fl (KO) and Foxp3 YFP-cre (WT) Body weight of male and female mice (KO male, n=12; WT male, n=10; KO female, n=11; WT female, n=11). (DE) Body weight of 24-week-old Foxp3 YFP-cre Entpd1 fl / fl (KO) and Foxp3 YFP-cre Representative images of intestinal (D) and spleen (E) morphology of (WT) mice (n=4-6 per group).

[0078] Fig.47 :(A)Foxp3 YFP-cre Entpd1 fl / fl (KO) and Foxp3 YFP-cre Representative images of hematoxylin-eosin (H&E) staining of colon and small intestine tissues of (WT) mice (n=6 per group). At least five fields of view were taken for each section, using a 200x magnification, as well as a 40x or 100x magnification. 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 Activated (CD44) in colon and small intestinal LPL of (WT) mice hi )CD4 + Frequency of T cells (n=6 per group).

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

[0080] Fig.49 :(A) E.G7 tumors bearing Foxp3 YEP-cre Entpd1fl / fl (KO) mice and Foxp3 YFP-cre (WT) Treg cell frequency in LN and TILs of 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] Fig.50 :(AB) In Hepa1-6 tumors bearing Foxp3 YFP-cre Entpd1 fl / fl (KO) mice or Foxp3 YFP-cre After NRP1-Treg cells were isolated from tumors of (WT) mice, CD8 + T cell division index (DI). (C) The formula [inhibition (%) = 100-(DI Treg / DI withoutTreg )100] measured Hepa1-6 tumor-bearing Foxp3 YFP-cre Entpd1 fl / fl (KO) mice or Foxp3 YFP-cre (WT) mouse-derived tumors with NRP1 - The inhibition percentage of Treg cells. (D) E. G7 tumors bearing Foxp3 YEP-cre Entpd1 fl / fl (KO) mice and Foxp3 YFP-cre (WT) Tumor growth curve of mice (n=5-6 per group). (E) B16-OVA tumor-bearing Foxp3 YFP-cre Entpd1 fl / fl (KO) mice and Foxp3 YFP-cre Tumor growth curves of (WT) mice (n=9-12 per group).

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

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

[0084] At present, the origin and functional mechanism of intratumoral Treg cells still lack in-depth research. 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 natural Treg cells (tTreg) but not in peripherally induced Treg cells (pTreg). Therefore, we used NRP1 as a marker of tTreg cells to phenotype Treg cell subsets in the tumor microenvironment. By flow cytometry, we analyzed Treg cells isolated from lymph nodes (LN), spleen (SPL), tumor-draining lymph nodes (TDLN), and tumor-infiltrating lymphocytes (TIL), which were derived from E.G7-OVA ( Figure 1 , A and B), Hepa1-6 ( Figure 2 , A and B) and B16-OVA ( Figure 3 , A) Mouse tumor model. The LN and SPL of tumor-free mice were used as the control group. The results showed that nearly 80% of Treg cells from LN, SPL and TDLN were NRP1 + , suggesting that these cells are mainly tTreg cells. In contrast, more than 50% of Treg cells within tumors are NRP1- Treg cells, indicating that a large number of newly induced pTreg cells exist in the tumor microenvironment of multiple tumor types.

[0085] We subsequently used adoptive transfer to confirm this observation. GFPMice (Jackson Laboratory, Cat. No. 006769) were mated with CD45.1 congenic mice (Jackson Laboratory, Cat. No. 002014) to obtain CD45.1 / CD45.2 (double positive) Foxp3 GFP We isolated CD45.1 / CD45.2 double positive Foxp3 GFP tTreg cells were isolated from spleen and LN of reporter mice and from CD45.2Foxp3 GFP Conventional FOXP3 was isolated from reporter mice (Jackson Laboratories, Cat. No. 006769). - CD4 + T cells and CD8 + T cells. Subsequently, we used tTreg cells, conventional CD4 + T cells and CD8 + T cells were co-transferred into TCRbd- / - mice at a ratio of 1:10:8 to simulate the physiological lymphoid organs of normal mice. Then, E.G7 tumor cells ( Figure 3 , B). We used different CD45 markers carried by donor cells to distinguish tTreg cells (CD45.1 + ) and pTreg cells (CD45.1-). tTreg cells and conventional CD4 + The ratio of T cells in LN and SPL was close to 1:10, which was consistent with the tTreg cells and conventional CD4 + The initial 1:10 ratio of T cells was consistent ( Figure 4 We found that at least 50% of Treg cells in tumors are derived from conventional CD4 + T cell pTreg cells (CD45.1-) Figure 5 , A and B). In contrast, the majority of Treg cells in LN and SPL are tTreg cells (CD45.1 + )( Figure 5 , A and B). These results are consistent with the observations of NRP1 staining. We further confirmed these findings by evaluating the expression of NRP1 on donor cells. We found that the majority (more than 70%) of CD45.1 + tTreg cells are NRP1 + , while more than 60% of CD45.1-pTreg cells were NRP1-( Figure 6 , A and B). Therefore, the proportion of pTreg cells in TME is comparable to that of tTreg cells, or even more. This shows 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 antitumor immunity, but it may also induce autoimmune reactions. To determine whether pTreg cells play a key role in tumor immunity, we used the above-mentioned adoptive transfer method to achieve selective removal of pTreg cells. We selected CD45.1 / CD45.2 double-positive Foxp3 GFP tTreg cells were isolated from spleen and lymph nodes of reporter mice and selected from CD45.2Foxp3 DTR Conventional CD4 + T cells and CD8 + Then, we used tTreg cells, conventional CD4 + T cells and CD8 + T cells were co-transferred into TCRbd- / - mice (Jackson Laboratories, Cat. No. 002122) at a ratio of 1:10:8, followed by inoculation of E.G7 tumor cells 3 days after transfer. DTR Mouse as conventional CD4 + As a source of T cells, we selectively removed pTreg cells by injecting diphtheria toxin (DT) without affecting tTreg cells, thereby avoiding autoimmune reactions. DT was injected every two days starting from day 12 until day 20, and E.G7 tumor-bearing mice were sacrificed on day 22 and analyzed ( Figure 7 , A). We found that tumor growth was significantly inhibited in DT-treated mice compared with control mice ( Figure 7 , BC), and the number of Treg cells was significantly reduced in lymph nodes (LN), spleen (SPL) and tumors ( Figure 8 , AC). In addition, pTreg cells (CD45.1-) were almost completely depleted in LN, SPL, and tumors ( Fig. 9 , AD). By removing pTreg cells, CD8 + The absolute number and frequency of T cells increased ( Fig.10 , A and B), leading to CD8 + The ratio of T cells to total Treg cells increased ( Fig.10 , C). These CD8 + T cells showed activation and proliferation (CD44 + Ki67 + ), indicating that they are functional cells ( Fig.10 , D and E). +Evaluation of interferon-γ (IFN-γ) and CD107a expression in T cells showed that they had stronger cytolytic activity (CD107a + IFN-γ + )( Fig.11 In summary, pTreg cells regulate anti-tumor immune cell types (such as CD8 + T cells), play a key 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 + Helper T cells (Th cells). Therefore, we analyzed whether intratumoral Treg cells were similar to any Th subset in gene expression. First, we performed transcriptome analysis of intratumoral Treg cells from Hepa1-6 and E.G7 mouse tumor models by RNA sequencing. Given that intratumoral Treg cells are composed of a large number of pTreg cells, while Treg cells from lymph nodes (LNs) are mainly composed of tTreg cells, we used Treg cells from tumor-free LNs as a control. Compared with Treg cells from tumor-free LNs, intratumoral Treg cells from both models had increased expression of Th1 cell characteristic genes (such as Tbx21 and Ccr5) ( Fig.12 , A and B). In contrast, intratumoral Treg cells barely expressed the Th2 cell lineage-determining transcription factor Gata3, and their expression of Th17 signature genes (such as Rorc and Ccr6) was also reduced ( Fig.12 , A and B). In addition, the results of NRP1 staining by flow cytometry analysis were consistent with those of flow cytometry analysis ( Figure 1 , A and B; Figure 2 , A and B), compared with LN-derived Treg cells, the expression level of NRP1 in tumor-derived Treg cells was lower ( Fig.12 , A and B).

[0090] Next, we further validated our findings by flow cytometry. We stained Treg cells from LN, SPL, TDLN, and tumors in Hepa1-6 and E.G7 tumor models for T-bet, RORγt, BCL6, and GATA3, and used 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, intratumoral 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 expression relationship between NRP1 and T-bet in intratumoral Treg cells by co-staining. We found that intratumoral Treg cells expressing T-bet were derived from NRP1 - Intratumoral Treg cells (pTreg cells). Importantly, T-bet + NRP1 - Treg cells account for approximately 60% of Treg cells in tumors ( Fig.14 , C and D). In contrast, intratumoral Treg cells barely expressed RORγt ( Fig.15 , A and B), BCL6 ( Fig.16 , A and B) or GATA3 ( Fig.17 , A and B). Taken together, our results indicate that T-bet+NRP1- Treg cells are the major component of intratumoral Treg cells in multiple cancer models.

[0091] Example 4: T-bet in tumors + pTreg cells show enhanced activation, proliferation and suppressive functions

[0092] In the whole-genome transcriptome analysis of intratumoral Treg cells, we found that Th1-related transcripts were upregulated in intratumoral Treg cells ( Fig.12 , A and B). Next, we further investigated whether these transcripts were upregulated by tTreg cells or pTreg cells within the tumor by comparing the gene expression profiles of tTreg cells and pTreg cells within the tumor. YFP-cre tTreg cells were isolated from the spleen and lymph nodes (LN) of mice and selected from CD45.2Foxp3 YFP -cre Isolation of conventional FOXP3-CD4 in mice + T cells and CD8 + T cells. Subsequently, we used tTreg cells, conventional CD4 + T cells and CD8 + T cells were co-transferred into TCRbd / - mice at a ratio of 1:10:8 and inoculated with Hepa1-6 tumor cells 3 days later. We generated RNA sequencing data from pTreg and tTreg cells within the tumors and included Treg cells from tumor-free lymph nodes as controls.

[0093] Compared with intratumoral tTreg cells, intratumoral pTreg cells showed upregulation of Th1 cell-related transcripts (such as Tbx21, Ccr5, Cxcr3, Il12rb2, Il18r1, Ifngr1) Fig.18 , A). Compared with LN-derived Treg cells, tumor-derived pTreg cells upregulated the expression of these transcripts, while tumor-derived tTreg cells downregulated these transcripts (except Ccr5) ( Fig.18 , B and C). This result indicates that T-bet + 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 associated with the suppressive function of Treg cells, such as Entpd1, Tigit, Pdcd1, Tgfb1, Il10, and Ebi3 ( Fig.19 pTreg cells also showed upregulation in activation-related genes (such as Tnfrsf18, Icos, Cd28, Cd69, Klrg1, Prdm1, Tnfrsf4, Tnfrsf9), cell cycle regulation (such as Pclaf, Rrm2, Smc2, Top2a, Mcm4), and chemokine receptors that contribute to their migration (such as Ccr8, Ccr2, Ccr4, Ccr6, Ccr9) ( Fig.19 , A).

[0095] Studies have shown that most Treg cells in the intestine are pTreg cells. Therefore, we studied whether pTreg cells in tumors have similar genetic characteristics to Treg cells in the intestine. We compared the differentially expressed genes (DEGs) of pTreg cells in tumors with the characteristic genes of colon Treg cells in public 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 that the DEGs of pTreg cells in tumors were positively correlated with the DEGs of colon-derived suppressive Treg cells, lymphoid tissue (LT)-like Treg cells, and non-lymphoid tissue (NLT) Treg cells ( Fig.19 , BD). However, the most significant positive correlation was found between the DEGs of intratumoral pTreg cells and those of colon NLT Treg cells ( Fig.19 , D). The shared marker genes include Tnfrsf4 (OX40), Tnfrsf9 (4-1BB), Tnfrsf18 (GITR), Ccr8, Pdcd1, Batf, Hopx, Il2ra and Klrg1 ( Fig. 20 , A).

[0096] We further compared the gene signatures of intratumoral pTreg cells with the characteristic genes of intestinal lymphoid tissue and non-lymphoid tissue Treg cells from wild-type mice retrieved from another public single-cell RNA sequencing data (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), caecal patches (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 that the DEGs of intratumoral pTreg cells were significantly positively correlated with the DEGs of Treg cells from intestinal NLT (LP and LA) ( Fig. 20 , B). The common marker genes include Lag3, Tigit, Tnfrsf4 (OX40), Ctla4, Hopx and S100a4 ( Fig. 20 , C). Although there was a positive correlation trend 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 share a similar genetic signature with intestinal Treg cells, particularly in regions derived from non-lymphoid tissues (such as the lamina propria) that contain a higher proportion of pTreg cells.

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

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

[0099] Next, we investigated the function of T-bet in pTreg cells within tumors. YFP- cre Tbx21 fl / flMice, used to specifically knock out the Tbx21 gene in Treg cells (gene number ENSMUSG00000001444). Foxp3 YFP-cre Tbx21 fl / fl Mice were induced by transfecting Tbx21 fl / fl Mouse (Jackson Laboratory, Cat. No. 022741) and Foxp3 YFP-cre The mice were obtained by mating with KO mice (Jackson Laboratory, Catalog No. 016959). As previously described (DiGiovangiulio, M., A. Rizzo, E. Franze, et al. 2019. Tbet Expression in Regulatory TCells Is Required to Initiate Th1-Mediated Colitis. Front Immunol 10: 2158.), these mice did not show signs of autoimmune pathology during their lives. Then, we established B16-OVA and E.G7 tumor models in the KO group and WT group, respectively. We observed that the growth of B16-OVA and E.G7 tumors in the KO group mice was significantly reduced compared with 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 cytometric analysis. We obtained very similar results in the B16-OVA and E.G7 tumor models, showing that the frequency and absolute number of total Treg cells in tumors were reduced by at least 50% after knockout of the Tbx21 gene (gene number ENSMUSG00000001444). However, the frequency of total Treg cells in LN and spleen was not significantly affected ( Fig. 22 , AC). This indicates that Treg cells in tumors proliferate less after T-bet gene knockout.

[0100] On the other hand, the tumor CD8 + 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 + CD8 depletion + A significant decrease in T cells ( Fig.23 , B and D), while stem-like CD8 + The frequency of T cells in tumors is CD8 + Increased in T cells, expressed as CD8 + The expression of Ly108 in T cells was significantly increased ( Fig.24 , A and B).

[0101] Next, we evaluated the therapeutic effect of Treg cell-specific knockout of Tbx21 combined with anti-PD-1 therapy. YFP-cre (WT) mice and Foxp3 YFP-cre Tbx21 fl / fl A B16-OVA tumor model was established in WT (KO) mice. Anti-PD-1 antibody was injected every three days starting from day 9. We evaluated tumor growth and survival of tumor-bearing mice. Anti-PD-1 treatment alone or Treg cell-specific deletion of Tbx21 gene significantly inhibited tumor growth in the B16-OVA tumor model. However, when Treg cell-specific knockout of Tbx21 gene was used in combination with anti-PD-1 treatment, the inhibitory effect on tumor growth was the most significant compared with WT mice ( Fig.25 , A). In addition, Treg cell-specific deletion of the Tbx21 gene significantly prolonged the survival time of B16-OVA tumor-bearing mice, with an effect comparable to that of anti-PD-1 therapy ( Fig.25 , B).

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

[0103] Example 6: Th1 cells are transformed into pTreg cells in tumors through TGF-β signaling

[0104] Thus far, we have confirmed that a large number of tumor-resident Treg cells are peripherally induced and express T-bet. However, it is unclear whether these Treg cells are directly derived from naive CD4 + Whether T cells are induced or transformed by Th1 cells in the tumor microenvironment, studies have shown that in vitro, TGF-β can induce Th1 cells to express FOXP3. However, whether there is a phenomenon of Th1 cells transforming into Treg cells, especially in the tumor environment, remains unclear. To this end, we used IFN-γ fate mapping mice (Ifng icre Rosa26 YFP ) established E.G7 and B16-OVA tumor models. icre Rosa26 YFP Mice were induced by icre Mouse (Beijing Biocytogen Biotech Co., Ltd.) and Rosa26 YFPThe mice (Srinivas, S., T. Watanabe, C.S Lin, et al. 2001. Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev Biol 1: 4.) were obtained by mating. icre The mouse was constructed by inserting IRES-iCre-PA between the Ifng coding frame and the 3'UTR after analyzing the structure of the Ifng gene, so that Ifng is expressed normally and iCre is expressed at the same time. The Neo cassette is 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 tissues were collected for flow cytometry analysis. LN and SPL outside the tumor served as controls. + Compared with tumor-infiltrating CD4 + YFP in T cells + The frequency of cells increased significantly ( Fig.26 , A and B). Most CD4 + YFP + Cells (about 80%) highly express T-bet ( Fig. 27 , A and B). Among them, about 20-30% of cells express 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 the cells is 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 + The vast majority (more than 80%) of these Th1-like Treg cells were NRP1 - , indicating that they are pTreg cells ( Fig. 27 Importantly, we found that FOXP3 + YFP +Co-staining of NRP1 and T-bet in the cell population showed that it was related to the main subpopulation of intratumoral Treg cells ( Fig.14 , C and D) similar expression patterns (T-bet+NRP1-)( Fig.28 , A and B). Interestingly, about 90% of T-bets + FOXP3 + Cells do not express IFN-γ ( Fig.28 , C and D). These observations suggest that in the tumor microenvironment, IFN-γ-expressing Th1 cells can be transformed into pTreg cells and constitute the NRP1-T-bet of intratumoral Treg cells. + Main subgroups.

[0107] Our fate mapping experiments show that Th1 cells can transform into T-bet in tumors + Therefore, we further differentiated pTreg cells from naive CD4 + To further verify the process of T cell differentiation into Th1 cells, we crossed OT-II mice (Jackson Laboratory, Cat. No. 004194) with Ifng YFP OT-II Ifng mice (Jackson Laboratory, Cat. No. 017581) were mated to obtain YFP Then, OT-II Ifng YFP Mice were crossed with CD45.1 congenic mice (Jackson Laboratory, Cat. No. 002014) to obtain CD45.1 / CD45.2 (double positive) OT-II Ifng YFP We isolated OT-II Ifng mice from CD45.1 / CD45.2 (double positive) YFP Isolation of naive CD4 + T cells were cultured and CD4 T cells that produced IFN-γ were sorted on the 4th day of culture. + T cells and transferred them into E.G7-inoculated mice. Seven days after tumor inoculation, the transferred mice were sacrificed on day 21 and flow cytometry analysis was performed. The results showed that about 10% of the donor Th1 cells (CD45.1 + CD4 + T cells) transform into T-bet in tumors + FOXP3 + Treg cells Fig.29 , A and B). As expected, more than 80% of donor Th1 cells expressed T-bet ( Fig.29 , A and C). Therefore, allogeneic transplantation of in vitro differentiated Th1 cells further demonstrated the conversion of Th1 cells into pTreg cells in the TME.

[0108] Considering the role of TGF-β in regulating the generation of pTreg cells, we next tested whether the TGF-β signaling pathway is required for the conversion of Th1 cells into pTreg cells. 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 develop autoimmune pathology within 3 weeks after birth, with a 100% mortality rate. Cre Tgfbr2 fl / fl Mouse. OT-II Cd4 Cre Tgfbr2 fl / fl Mice were generated by crossing OT-II mice (Jackson Laboratories, Cat. No. 004194) with Tgfbr2 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.) and then mated with Cd4 Cre OT-II Cd4 Cre Tgfbr2 fl / fl The mice were then mated with CD45.1 congenic mice (Jackson Laboratory, Cat. No. 002014) to obtain CD45.1 / CD45.2 (double positive) OT-II Cd4 Cre Tgfbr2 fl / flThese mice showed only mild immunopathology and could survive for several months or even longer. We isolated CD45.1 / CD45.2 (double positive) OT-II Cd4 Cre Tgfbr2 fl / fl (KO group) or CD45.1 / CD45.2 (double positive) OT-II Tgfbr2 fl / fl Initial CD4 + T cells. From CD45.2Foxp3 GFP Isolation of CD8 in reporter mice + T cells and tTreg cells. + T cells, tTreg cells and CD8 + T cells were co-transferred into TCRbd / - mice. Three days later, B16-OVA tumor cells were inoculated into TCRbd - / - In recipient mice. On day 16, tumor-bearing mice were sacrificed for flow cytometric analysis. The results showed that the tumor growth of mice in the KO group was significantly slowed down ( Fig.30 , A). Consistent with our hypothesis, loss of Tgfbr2 resulted in donor CD45.1 + Conventional CD4 + The ability of T cells to transform into pTreg cells is inhibited ( Fig.30 , B and C). In addition, the loss of Tgfbr2 leads to the proliferation of Th1 cells (T-bet + FOXP3 - )The increase in frequency ( Fig.31 , A and B). Th1 cells (T-bet + FOXP3 - ) frequency leads to an increase in CD4 + The expression level of IFN-γ in T cells increased ( Fig.31 , C and D). Taken together, our results suggest that pTreg cells can transform from Th1 cells in the tumor microenvironment and this process is dependent on the TGF-β signaling pathway.

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

[0110] To further explain T-bet + The developmental pathway of Treg cells in the tumor microenvironment and the role of T-bet in regulating this specific Treg cell population were investigated. YEP-ce and Foxp3 YEP-cre Tbx21 fl / fl Single-cell transcriptomic analysis of Treg cells in mouse Hepa1-6 tumors.YFP-cre Tbx21 fl / fl Mice were induced by transfecting Tbx21 fl / fl Mouse (Jackson Laboratory, Cat. No. 022741) and Foxp3 YFP-cre The mice were obtained by mating with the Jackson Laboratory, Cat. No. 016959.

[0111] We start from Foxp3 YFP-cre and Foxp3 YFP-cre Tbx21 fl / fl Treg cells (CD4 + YFP + ), and compared with the YFP-cre Conventional CD4 in Hepa1-6 tumors of mice + T cells (CD4 + YFP - ) and Treg cells in tumor-free lymph nodes (CD4 + YFP + )’s transcriptome data (only Treg cell data are shown).

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

[0113] Population 0 exhibits the characteristics of Th1 cells (Tbx21, Ccr5, Cxcr3, H12rb2, Il18r1) and is the most dominant cell population, accounting for at least 70% of Treg cells in tumors ( Fig.32 , B and C). Group 0 is enriched in molecules associated with the suppressive function of Treg cells, 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). Collectively, this population represents tumor-infiltrating Th1-like effector Treg cells with enhanced activation and suppressive functions.

[0114] Population 1 is characterized by increased expression of Foxp3, as well as increased expression of the tTreg cell marker Nrp1. This population also shows increased expression of genes that support Treg cell differentiation and suppressive function (such as Foxp1, Foxo1, Bach2, Ikzf2). In addition, this population also expresses transcripts of naive Treg cells, such as Sell, Slamf6, Tcf7, Lef1, and Ccr7 ( Fig.33 , A). Taken together, this population belongs to naive / resting Treg cells. Most of the cells in population 1 are likely tTreg cells.

[0115] Population 2 is the most enriched population in Tbx21-deficient Treg cells, and this population has the characteristics of Th2 / Th17 cells. Population 2 upregulated Th2 cell-specific transcription factors (Gata3) and Th2 cell cytokine transcripts (Il4, Il13, Il5). This population also upregulated Th17 cell lineage-determining transcription factors (Rorc), Th17 cell cytokine transcripts (Il17a), and transcription factors Irf4 ( Fig.33 , A).

[0116] Population 3 was defined as the Th1 cell population. Population 3 shared the characteristic genes of population 0, including Tbx21, Ccr5, Il12rb2, Il18r1, and Entpd1 ( Fig.33 , A). Group 3 also highly expressed Th1 effector cell cytokine transcripts (Ifng, Tnf1) and granzyme family members (Gzmd, Gzme, Gzmk), along with cytotoxicity-related molecules (Prf1, Nkg7). This group 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 population, showing an increase in the proliferation marker gene Mki67. This population was enriched for a series of genes involved in DNA replication, transcription, and cell cycle regulation, including Exo1, Rrm1, Rrm2, the minichromosome 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 ( Fig.33 , A). Therefore, this population was defined as activated proliferative Treg cells.

[0118] Next, we further investigated the role of T-bet in intratumoral Treg cells. Since the proportion of Th1-like Treg cells (C0) in Tbx21-deficient Treg cells was significantly reduced compared with that in 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). Fig.34 , A). Compared with 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 related to Treg cell inhibitory mechanisms (Entpd1, Nt5e, Tgfb1, Ebi3, Ctla4, Lag3, Tigit, Gzmb). At the same time, Th2 / Th17-like Treg cells upregulated Th2 cell (Gata3, Il4, Il5, I113) and Th17 cell (Rorc, Il17a) marker genes ( Fig.34 , A). These results indicate that T-bet+ Treg cells suppress the response of Th2 / Th17 cells in the TME. These results also suggest that in Tbx21-deficient Treg cells, the transcriptome program undergoes dynamic changes, resulting in the lineage conversion of Treg cells from Th1-like Treg cells to Th2 / Th17-like Treg cells.

[0119] Compared with Th1-like Treg cells (population C0), Th2 / Th17-like Treg cells (population C2) exhibited less suppressive properties. The reduction in the proportion of C0 cells and the increase in the proportion of C2 cells may lead to the weakened suppressive function in the tumor microenvironment of Tbx21 conditional KO mice compared with wild-type mice (WT). Further flow cytometric analysis showed that the frequency and absolute number of Treg cells in tumors of Tbx21 conditional KO mice were significantly decreased compared with WT mice ( Fig. 22 , AC). Therefore, it can be speculated that the proportion of suppressive tumor-promoting Treg cell population is generally reduced in Tbx21 conditional KO mice.

[0120] In addition, compared with Th1-like Treg cells (population C0), Th2 / Th17-like Treg cells (population C2) showed downregulation of transcripts related to Treg cell suppression mechanisms ( Fig.34 , A), we further analyzed the co-expression of these transcripts with Tbx21 ( Fig.34, B). Among them, Entpd1 and Tigit had higher fold changes between C0 and C2 ( Fig.34 , A), and both are co-expressed with Tbx21. In contrast, Entpd1 and Tigit are not co-expressed with Gata3 or Rorc ( Fig.34 , B). Flow cytometry results also confirmed that the expression of CD39 and TIGIT was reduced in Tbx21-deficient Treg cells. However, compared with the expression of TIGIT, the expression of CD39 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 interference may be a specific pathway for Th1-like Treg cells to exercise suppressive mechanisms in the TME.

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

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

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

[0124] This example aims to clarify the role of CD39-mediated inhibitory mechanisms in intratumoral Treg cells and further explore the specific expression of CD39 in the tumor microenvironment. Through single-cell transcriptomics analysis and flow cytometry detection of intratumoral Treg cells in Tbx21-deficient mice, we found that CD39-mediated metabolic interference may be a unique inhibitory mechanism for Th1-like Treg cells. Therefore, we further studied the role of CD39-mediated inhibitory mechanisms in intratumoral Treg cells. Literature has shown that CD39 is an exogenous nucleotidase that can hydrolyze phosphate bonds in ATP and convert ATP into AMP. CD39-mediated phosphate bond hydrolysis is essential for nucleotide metabolism, especially purine metabolism and pyrimidine metabolism. Purine metabolism is essential for the production of adenosine, which inhibits CD8 by binding to the A2A receptor (A2AR). + Functions of T cells.

[0125] First, we performed genome-wide transcriptomic analysis of Treg cells and performed differentially expressed genes (DEGs) and pathway analysis on RNA sequencing data of mouse E.G7 and Hepa1-6 tumor models, and obtained six different gene clusters ( Fig.36 Clusters 1 and 2 contain genes that are upregulated in E.G7 and Hepa1-6 intratumoral Treg cells ( Fig.36 , AC). Clusters 5 and 6 contain genes that are upregulated in E.G7 and Hepa1-6 intratumoral Treg cells, respectively ( Fig.36 , A, D, and E). Pathway analysis of the up-regulated 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 in E.G7 and Hepa1-6 tumors mostly upregulated genes related to purine metabolism. These genes included genes encoding exogenous ATP diphosphohydrolases (Entpd1, Entpd7), purine nucleotide phosphorylases (Pnp), Nudix hydrolase family (Nudt1, Nudt4, Nudt9, Nudt21), adenosine kinase family (Ak1, Ak2, Ak3, Ak4, Ak5, Ak6, Ak8), NME / NM23 nucleotide diphosphokinase family (Nme1, Nme4) and hypoxanthine-guanine phosphorylase (Hprt) ( Fig.37 , A and B). In addition, these gene clusters were found to be enriched in pathways that respond to hypoxia and oxidative stress ( Fig.36In addition, we compared the gene expression profiles of intratumoral pTreg and tTreg cells in the Hepa1-6 tumor model and found that intratumoral pTreg cells upregulated genes involved in nucleotide degradation, including the Entpd1 gene encoding CD39 ( Fig.38 , A).

[0126] Next, we explored the expression relationship between CD39, T-bet, and NRP1 in intratumoral Treg cells. Fig.37 , A), flow cytometry results showed that in the Hepa1-6 tumor model, intratumoral Treg cells showed co-expression of T-bet and CD39 ( Fig.38 , B and C). We further investigated the expression of YEP- cre Tbx21 fl / fl Mice (designated as KO group) and control Foxp3 YFP-cre We molecularly phenotyped intratumoral Treg cells in WT mice (designated as WT group). Surprisingly, we found that T-bet was expressed in CD39 + The expression in NRP1-pTreg cells was significantly higher ( Fig.39 , A and B). In addition, in WT mice, CD39 was selectively expressed in NRP1-Treg cells within the tumor. By comparing with Treg cell-specific T-bet KO mice, we found that the expression level of CD39 in NRP1-pTreg cells within the tumor was significantly higher, while this phenomenon did not occur in T-bet KO mice ( Fig.39 , C and D). Therefore, the expression of T-bet and CD39 is highly correlated. The expression of T-bet is always associated with the presence of pTreg cells in NRP1-tumors ( Fig.14 , C and D), and CD39 is also selectively expressed in NRP1-pTreg cells in tumors ( Fig.39 , A, C, and D). These findings further reinforce the role of NRP1-T-bet in tumors. + Evidence that pTreg cells exert suppressive function through CD39 expression.

[0127] To determine whether CD39 expression in Treg cells is tumor-specific, we evaluated and compared CD39 expression in Treg cells derived from different tissues. These tissues included inguinal lymph nodes (iLN), thymus, Peyer's patches (PP), spleen, mesenteric lymph nodes (mLN), colonic intraepithelial lymphocytes (IEL), small intestinal IEL, colonic lamina propria lymphocytes (LPL), and small intestinal LPL, all of which were derived from tumor-free mice. We stained intratumoral Treg cells for CD39 expression 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 a large number of pTreg cells, including small intestinal IEL, colonic LPL, and small intestinal LPL. We further validated these observations by co-staining CD39 with NRP1. Consistently, we found that CD39 was selectively expressed in NRP1- Treg cells. These observations were consistent in tumors, small intestinal IEL, colonic LPL, and small intestinal LPL ( Fig.40 , A and B). In summary, our research shows that T-bet + pTreg cells have high levels of CD39 expression, which is a key mediator of adenosine signaling in purine metabolism. CD39 may serve as a therapeutic target to selectively target intratumoral T-bet + pTreg cells without triggering a systemic autoimmune response.

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

[0129] This example aims to further verify whether nucleotide metabolism is a key mechanism for the role of intratumoral Treg cells in tumor immunosuppression. First, we used publicly available RNA sequencing data of human breast cancer Treg cells for pathway analysis. Gene set enrichment analysis (GSEA) showed that the differentially expressed genes (DEGs) of intratumoral Treg cells in E.G7 and Hepa1-6 mouse tumor models were positively correlated with the differentially expressed genes of intratumoral Treg cells in human breast cancer tumors ( Fig.41 , A and B). As expected, pathways related to nucleotide metabolism in intratumoral Treg cells were enriched in these tumor models ( Fig.41 , C).

[0130] We further validated that in human breast cancer, intratumoral Treg cells upregulated most genes related to nucleotide metabolism, including the ENTPD1 gene ( Fig.42, A). The ENTPD1 gene is upregulated during the degradation of small molecules and purine-containing nucleotides, both of which are important pathways of nucleotide metabolism ( Fig.42 , B and C). Some genes involved in nucleotide metabolism, such as TYMP, NUDT5, PGM2, HPRT1, GLRX, GPX1, and ENTPD1, ​​were also upregulated in intratumoral Treg cells in 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 intratumoral Treg cells in different types of human cancers ( Fig.43 , A). Volcano plot analysis revealed that ENTPD1 gene expression was significantly upregulated in Treg cells in hepatocellular carcinoma (HCC) tumors ( Fig.43 , B).

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

[0132] In summary, the research results of this example indicate that nucleotide metabolism, especially the expression of the ENTPD1 gene and the co-expression of T-bet, play an important role in intratumoral Treg cells. This phenomenon has been verified in different types of human cancers and is consistent with our observations in mouse tumor models.

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

[0134] In this example, the purpose is to further clarify the function of CD39 in Treg cells in tumors. We constructed Foxp3 YFP -cre Entpd1 fl / fl Mouse. Foxp3YFP-cre Entpd1 fl / fl Mice were induced by expressing Entpd1 fl / fl Mouse (Jicui Yaokang, Catalog No. T011367) and Foxp3 YFP-cre The Entpd1 gene has 11 transcripts. According to the structure of the Entpd1 gene, exons 2-7 of the Entpd1-201 (ENSMUST00000112231.8) transcript were selected as the knockout region. Knockout of this region resulted in the destruction of protein function. fl / fl The mice were constructed by modifying the Entpd1 gene using CRISPR / Cas9 technology. The brief process is as follows: sgRNA is transcribed in vitro and the donor vector is constructed. Cas9, sgRNA and Donor are microinjected into C57BL / 6J mouse fertilized eggs. The fertilized eggs are transplanted to obtain positive F0 mice, which are confirmed by PCR and sequencing, and the positive F0 mice are mated with C57BL / 6J mice to obtain a stable F1 generation mouse model. These flox mice are knocked out after mating with mice expressing Cre recombinase, resulting in the loss of target gene function in specific genes or tissues.

[0135] This is the first time that this transgenic mouse has been described. Flow cytometry results confirmed that knockout of the Entpd1 gene (gene number ENSMUSG00000048120) was validated by the loss of CD39 expression in Treg cells (i.e., a 92%-96% reduction), a finding that was observed in Treg cells from mice with colon LPL and E.G7 tumors and Foxp3 YFP-cre The results obtained from the comparison of mice ( Fig.46 , A and B). These mice did not show any autoimmune pathology. Foxp3 YFP - cre Entpd1 fl / fl Body weight and Foxp3 expression in KO mice YFP-cre (hereinafter referred to as WT) mice, both in male and female mice (21-24 weeks old) ( Fig.46 , C). In addition, the intestine and spleen (SPL) of Entpd1 KO mice showed no morphological changes ( Fig.46 , D and E). Histological analysis of the colon and small intestine of Entpd1KO mice revealed no signs of inflammation ( Fig.47 , A). FOXP3 in colonic LPL and small intestinal LPL - or FOXP3 + CD4 + In T cells, there was a significant difference between Entpd1 KO and WT mice in activated cells (CD44hi ) had no significant difference in frequency ( Fig.47 , B and C). Consistently, Entpd1 KO mice showed significant differences in effector (CD44 + CD62L-)CD4 + and CD8 + There was no significant difference in the frequency of T cells ( Fig.48 , AC). In addition, 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, after deletion of Entpd1, T-bet of Treg cells in tumors + NRP1 - The frequency of pTreg cells was not affected ( Fig.49 , B). These observations suggest that CD39 expression in Treg cells is not essential for maintaining immune homeostasis.

[0136] Next, we first performed in vitro studies to explore the function of CD39 in intratumoral Treg cells. - We isolated Foxp3 from Hepa1-6 tumor-bearing cells. YFP-cre (WT) mice and Foxp3 YFP-cre Entpd1 fl / fl Intratumoral CD4 + YFP + NRP1 - Treg cells. The sorted Treg cells were compared with the initial CD8 + T cells were co-cultured in 96-well culture plates for 72 hours. As a negative control, CD8 + CD8 T cells co-cultured with intratumoral Treg cells of the WT group + The cell division index (DI) of T cells was significantly higher than that of 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) Compared with WT NRP1-Treg cells in tumors, Entpd1-deficient NRP1 - Treg cells in vitro + The suppressive capacity of T cells was significantly reduced.

[0137] To further investigate the role of CD39 in intratumoral Treg cells in the tumor microenvironment, we expressed CD39 in Foxp3 YFP-cre Entpd1 fl / fl E.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 a reduction in tumor growth ( Fig.50 , D and E). After knocking out Entpd1, CD8 + T cell function was enhanced, as shown by increased expression of TNF-α and IFN-γ ( Fig.51 , A), while the expression of PD-1 and TIM-3 decreased ( Fig.51 , B).

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

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

Claims

1. 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 can reduce the number or activity of T-bet-positive pTreg cells.

2. The use 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) Enhance CD8 + The role of T cells in tumor-killing activity.

3. The use according to claim 1, characterized in that: 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.

4. Use of a molecule for reducing the number or activity of T-bet-positive pTreg cells in the preparation of a drug for preventing and / or treating tumors.

5. The use according to claim 4, characterized in that: 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.

6. The use according to claim 4, characterized in that: When applied, molecules used to 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 preventing and / or treating tumors, characterized in that: The drug includes an inhibitor capable of reducing the number or activity of T-bet-positive pTreg cells.

8. The drug for preventing and / or treating tumors according to claim 7, characterized in that: The inhibitor capable of reducing the number or activity of T-bet-positive pTreg cells is a T-bet inhibitor and / or a CD39 inhibitor.

9. The drug for preventing and / or treating tumors according to claim 7, characterized in that: The drug also includes an immune checkpoint inhibitor, which is an anti-PD-1 antibody, an anti-CTLA-4 antibody or a combination thereof.

10. 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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