Modified t cell and use thereof

Abstract

Disclosed in the present invention is a T cell, wherein the expression of a given protein in the T cell is changed, and the given protein is suitable for regulating the expression of BCL6 in the T cell. The T cell is at least one of a CD8+T cell and a CD4+T cell, preferably a CD8+T cell specifically recognizing tumor cells. The given protein is suitable for regulating the expression of BCL6 by means of the IL-2-STAT5 pathway and / or TGF-beta-SMAD2 pathway. The expression of BCL6 in the T cell is up-regulated. The expression of the given protein in the T cell is changed. The given protein comprises at least one downstream protein on the IL-2-STAT5 pathway, and the expression of the given protein is down-regulated. The given protein comprises at least one selected from PRDM1 or a member of the same family thereof.

Description

A modified T cell and its application Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a modified T cell and an application thereof. Background Art

[0002] Immune checkpoint blockade therapy has shown good clinical promise in the treatment of solid tumors. However, due to severe exhaustion of T cells, only a small number of cancer patients can respond to this therapy. + T cells (Exhausted CD8 + T cells, T ex ) mainly includes two subpopulations, namely stem cell-like T cell subpopulation (T prog ) and terminally differentiated T cell subsets (T term ). prog The cells are in a relatively quiescent state and have a high level of proliferation potential. term The cells have high proliferation activity and strong tumor killing function, but they cannot survive continuously. prog T cells are the main T cell subset that responds to PD-1 / PD-L1 blockade therapy and can rapidly proliferate and differentiate into T term cells, exerting an effective anti-tumor effect. prog and T term Cells collaborate to mediate long-term and immediate antitumor effects, but how their differentiation is regulated is unclear.

[0003] Transcription factors play a core regulatory role in the exhaustion and differentiation of T cells. NR4A1 is a transcription factor associated with T cell activation and death. ex It is highly expressed in T cells and promotes T cell exhaustion and inhibits T cell function by inhibiting c-Jun function. ex In cells, continuous antigen stimulation and NFAT activation induce high levels of TOX, which regulates the exhaustion and maintenance of T cells at the transcriptional and epigenetic levels. + Transcription factors that are highly expressed in T cells and are also T prog Characteristic molecules of cells.

[0004] In addition to transcription factors, cytokines in CD8 + It also plays an important role in the exhaustion of T cells. Previous studies have reported that IL-27 and IL-2 are the induction factors of T cell depletion in tumor models. exIn addition, IL-27 can significantly increase TCF1 expression in chronic viral infection models. + T prog In tumor models, IL-27 can significantly enhance the anti-tumor ability of T cells. Type I interferon significantly enhances T cell survival by antagonizing TCF1. term IL-10 and IL-21 significantly enhance T cell development by activating the STAT3 signaling pathway. term However, how cytokines control T cell development and effector function in tumor models remains unclear. ex The differentiation of cell subpopulations is still unclear.

[0005] Therefore, it is urgent to study the specific mechanisms by which T cell differentiation is regulated by transcription factors and cytokines and then transform T cells to provide support and assistance for anti-tumor immunotherapy.

[0006] Summary of the Invention

[0007] The present invention aims to solve at least one of the technical problems in the related art to a certain extent. To this end, the present invention provides a modified T cell and its application, the modified T cell of the present invention can improve its stemness and maintain T prog The ability to or can not reduce T prog Under the premise of cell number, promote T term The development of the immune system can enhance its ability to kill tumors and provide support and assistance for anti-tumor immunotherapy.

[0008] The present invention is accomplished based on the following findings of the inventors:

[0009] BCL6 is a transcription factor of the BTB / POZ zinc finger protein family and also a follicular helper CD4 + T cells (Follicular helper CD4 + Previous studies have reported that BCL6 is a transcription factor for memory CD8 T cells (Tfh cells) and germinal center B cells in mouse models of acute viral infection and protein immunity. + In the model of chronic LCMV infection, BCL6 is highly expressed in TCF1 + CXCR5 + T prog cells, which are able to enter the B cell follicles in lymphoid tissue.

[0010] However, in the inventors' in-depth research, they unexpectedly found that in tumor models, BCL6 can be expressed in tumor-specific CD8 +High expression in T cells. Although tumor tissues generally do not have lymphoid structures, the aggregation areas of antigen-presenting cells contain many TCF1 + T prog Therefore, the inventors speculate that tumor-infiltrating T cells prog T cells may be involved in chronic infection models prog Cells have different mechanisms for their production and maintenance.

[0011] Based on this, the inventors further studied the relationship between BCL6 and T in tumor models. prog The inventors found that overexpression of BCL6 in T cells can damage the T cells themselves. In addition, the inventors unexpectedly found that upregulating BCL6 in T cells not only does not damage the T cells themselves, but also can improve the T prog In addition, the inventors unexpectedly discovered that simply increasing the number of copies of the BCL6 encoding gene in T cells to achieve BCL6 overexpression cannot achieve this technical effect. This effect can only be achieved by regulating the expression regulation mechanism of the BCL6 encoding gene.

[0012] Furthermore, the inventors found that downregulating PRDM1 expression could upregulate BCL6, and in tumor models, adoptive delivery of PRDM1-deficient CD8 + T cells combined with anti-PD-1 can significantly promote tumor infiltration of T cells. prog Cell proliferation produces more effective anti-tumor effects.

[0013] Therefore, in a first aspect of the present invention, the present invention provides a T cell. According to an embodiment of the present invention, the expression of a given protein in the T cell is changed, and the given protein is suitable for regulating the expression of BCL6 in the T cell. The T cell of the present invention can maintain T prog The T cells of the present invention can be used to treat and prevent cancer without reducing T cell stemness and maintaining long-term survival, thereby improving their ability to kill tumors. prog Under the premise of cell number, promote T term The T cells have a strong instantaneous killing ability against early tumors containing fewer tumor cells and can be used to treat early cancer.

[0014] In a second aspect of the present invention, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises a reagent and the T cells described in the first aspect of the present invention. The reagent is used to regulate the IL-2-STAT5 pathway and / or the TGF-beta-SMAD2 pathway to regulate the expression of BCL6 in T cells. As can be seen from the above, the T cells described in the first aspect can maintain T prog The state of T cells is enhanced, the stemness of T cells is enhanced and the long-term survival of T cells is maintained, which can improve their ability to kill tumors. Therefore, the use of a pharmaceutical composition containing the above reagents or T cells can achieve a long-lasting and effective anti-tumor immune response, which is helpful for the development of new anti-tumor immunotherapy to treat and prevent cancer. Alternatively, as can be seen from the above, the T cells described in the first aspect can be used without reducing T prog Under the premise of cell number, promote T term The T cells have a strong instantaneous killing ability against early-stage tumors containing fewer tumor cells. Therefore, a pharmaceutical composition containing the above-mentioned reagents or T cells can be used to treat early-stage cancer.

[0015] In a third aspect, the present invention provides use of the T cell described in the first aspect or the pharmaceutical composition described in the second aspect in preparing a medicament for treating or preventing cancer. The T cell or pharmaceutical composition of the present invention can effectively kill tumors, contributing to the development of novel anti-tumor immunotherapies for treating or preventing cancer.

[0016] In a fourth aspect, the present invention provides a drug combination. According to an embodiment of the present invention, the drug combination comprises an immune checkpoint inhibitor and the T cells described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention. According to an embodiment of the present invention, the combined administration of the two drugs has a high tumor-killing ability, can effectively kill tumor cells, inhibit tumor growth or reduce tumor volume, and can effectively prevent and treat tumors or cancer.

[0017] In its fifth aspect, the present invention provides a method for treating cancer. According to an embodiment of the present invention, the method comprises administering to a patient the T cells described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention. As previously noted, both the T cells described in the first aspect and the pharmaceutical composition described in the second aspect have high tumor-killing abilities, effectively killing tumor cells, inhibiting tumor growth, or reducing tumor volume. Thus, the method of the present invention is effective for treating and preventing cancer.

[0018] In a sixth aspect of the present invention, the present invention provides a method for upregulating the content of stem-like cells in immune cells. According to an embodiment of the present invention, the method comprises: administering to a patient the T cells described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect. As can be seen from the foregoing, the aforementioned T cells are in the stem-like T cell subpopulation (T prog ) stage, the proportion of stem cell-like cells in immune cells can be increased by adding these T cells. prog The cells are in a relatively dormant state and have a high level of proliferation potential. Therefore, the immune cells obtained by this method have a strong ability to kill tumor cells and can effectively treat and prevent cancer.

[0019] In a seventh aspect, the present invention provides a method for inhibiting T cells from term According to an embodiment of the present invention, the method comprises upregulating the expression of BCL6 in the T cells; the T cells comprise CD4 + T cells, CD8 + As previously known, upregulating BCL6 protein in T cells can maintain T cells in the stem cell-like state (T prog ) state, thereby inhibiting T cells from turning to T term Therefore, this method helps to maintain T cells in T prog state, producing more lasting and effective anti-tumor ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG1 shows the expression of BCL6 at the transcriptional and protein levels in Example 4 and the flow cytometry graph showing the expression of BCL6-RFP in CD8 + The expression of T. The vertical axis of the flow cytometer in Figure 1 indicates the amount of cells expressing BCL6-RFP.

[0021] Figure 2 is a graph showing the functional differences between BCL6 and BLIMP1 in Example 6 of the present invention. Figure 2A is a DNA binding heat map and Venn diagram centered on the binding sites of BCL6 and BLIMP1, Figure 2B is a Venn diagram of genes bound by BCL6 and BLIMP1, and Figure 2C is a diagram of BCL6. + and TIM-3 + CD8 + The expression of characteristic genes of T cells in the transcriptome of OT-I cells with PRDM1 overexpression and control group. Figure 2D shows the Gene Ontology (GO) analysis results of the top 20 significant biological signaling pathways enriched from genes induced by PRDM1 overexpression up-regulated and down-regulated.

[0022] Figure 3 is a graph showing the functional differences between BCL6 and BLIMP1 in Example 6 of the present invention. Figure 3A shows the correlation analysis of the expression levels of genes regulated by BCL6 and PRDM1 overexpression, Figure 3B shows the Venn diagram of genes directly regulated by BLIMP1, and Figure 3C shows the BCL6 regulated by BLIMP1. + and TIM-3 + CD8 + Venn diagram of T cell signature genes and gene regulatory network diagram centered on BCL6 and BLIMP1.

[0023] FIG4 shows the expression of BCL6 and BLIMP1 in CD8 tumors in Example 6 of the present invention. + Figure 4A shows the ChIP-seq and ATAC-seq images of the binding of BCL6 and BLIMP1 at specific gene loci, and Figure 4B shows the ChIP-seq images of the binding of BCL6 and BLIMP1 at specific gene loci.

[0024] FIG5 shows the expression of BCL6 and BLIMP1 in tumor CD8 in Example 6 of the present invention. + Figure 5A shows the results of antagonism during the differentiation of T cells. + 5B shows the expression of LY108 and TIM-3 in T cells, wherein the abscissa of the flow cytometer in FIG5B indicates the amount of cells expressing TIM-3, and the ordinate indicates the amount of cells expressing LY108.

[0025] Figure 6 shows the expression of BCL6 and BLIMP1 in tumor CD8 in Example 6 of the present invention. + Figure 6A shows the expression of BCL6-RFP, BLIMP1-EYFP, LY108 and TIM-3 in OT-I cells, wherein the horizontal axis in Figure 6A indicates the amount of cells expressing BLIMP1-EYFP, and the vertical axis indicates the amount of cells expressing BCL6-RFP, LY108 and TIM-3. Figure 6B is a UMAP diagram of the expression of specific genes, and Figure 6C is a UMAP diagram of the expression of specific genes in CD8 + Violin plots of expression levels in T cell subsets.

[0026] Figure 7 shows the expression of BLIMP1 in CD8 + Figure 7A shows the molecular mechanism of induction in T cells. Figure 7A shows the in vitro activation of CD8 +The expression of T cells in Figure 7A, where the horizontal axis indicates the amount of cells expressing BLIMP1-EYFP and the vertical axis indicates the amount of cells expressing TIM-3. Figure 7B shows the expression of CD25, TIM-3 and TCF1 in CD8 + The expression of T cells in Figure 7B, wherein the horizontal axis indicates the amount of cells expressing TIM-3 and the vertical axis indicates the amount of cells expressing CD25. Figure 7C shows the expression of CD8 T cells activated by LY108 and Granzyme B in vitro. + Expression in T cells, where the abscissa in Figure 7C indicates the expression levels of LY108 and Granzyme B, respectively.

[0027] FIG8 shows the effect of BLIMP1 on CD8 in tumors in Example 6 of the present invention. + Figure 8A shows the expression of BCL6 and TCF1 in OT-I cells. The horizontal axis in Figure 8A indicates the number of cells expressing CD8, and the vertical axis indicates the number of cells expressing TCF1 and BCL6. Figure 8B shows the expression of Granzyme B, IFN-γ, and TNF-α in OT-I cells. The horizontal axis in Figure 8B indicates the number of cells expressing TNF-α, and the vertical axis indicates the number of cells expressing IFN-γ and Granzyme B.

[0028] FIG. 9 shows the effect of BLIMP1 on CD8 in tumors in Example 6 of the present invention. + Figure 9A shows the expression of T-BET, TCF1 and BCL6 in OT-I cells, where the horizontal axis in Figure 9A indicates the amount of cells expressing T-BET, and the vertical axis indicates the amount of cells expressing TCF1 and BCL6. Figure 9B shows the expression of LY108 in tumor-infiltrating OT-I cells. + and TIM-3 + Figure 9C shows the dilution of CTV in OT-I cells in the spleen, where the abscissa in Figure 9C indicates the amount of cells expressing CTV.

[0029] FIG. 10 is a diagram of CD8 in Example 6 of the present invention. + Figure 10A shows the expression and function of BLIMP1 in the process of T cell immune response to endogenous tumor antigens. + The expression of TCF1 and Granzyme B in tumor-infiltrating CD8 T cells is shown in Figure 10A, where the horizontal axis indicates the amount of cells expressing BLIMP1-EYFP and the vertical axis indicates the amount of cells expressing CD44. +Expression in T cells, wherein the abscissa in FIG10B indicates the amount of cells expressing BLIMP1-EYFP, and the ordinate indicates the amount of cells expressing CD44.

[0030] Figure 11 is a graph showing the results of regulating the anti-tumor function of T cells by overexpression of BCL6 in Example 7 of the present invention. Figure 11A is a growth curve of E.G7 tumors, and Figure 11B is the expression of LY108 and TIM-3 in OT-I cells, wherein the abscissa in Figure 11B indicates the amount of cells expressing TIM-3, and the ordinate indicates the amount of cells expressing LY108. Figure 11C is the amount of OT-I cells, LY108 and TIM-3 per unit weight infiltrating the tumor. + and TIM-3 + The number of OT-I cells.

[0031] FIG12 shows that anti-PD-1 treatment in Example 7 of the present invention cannot significantly amplify T cells that overexpress BCL6. prog Figure 12A is a schematic diagram of adoptive delivery of OT-I cells, inoculation of E.G7 tumors, and anti-PD-1 treatment. Figures 12B and 12C are growth curves of E.G7 tumors.

[0032] FIG13 shows that anti-PD-1 treatment in Example 7 of the present invention cannot significantly amplify T cells that overexpress BCL6. prog Figure 13A shows the expression of TCF1 and TIM-3 in OT-I cells, where the horizontal axis in Figure 13A indicates the amount of cells expressing TIM-3 and the vertical axis indicates the amount of cells expressing TCF1. Figure 13B shows the expression of OT-I cells, TCF1 and TIM-3 in OT-I cells infiltrating E.G7 tumors. + and TIM-3 + Figure 13C shows the number of OT-I cells and Granzyme B + The ratio of cells and Granzyme B per unit weight of E.G7 tumor infiltration + 13C , wherein the abscissa indicates the amount of cells expressing TIM-3, and the ordinate indicates the amount of cells expressing Granzyme B.

[0033] FIG. 14 shows the expression of PRDM1 in tumor-specific CD8 T cells in Example 7 of the present invention. + Figure 14A shows the results of the combination of T cell deletion and anti-PD-1. Figure 14A is a schematic diagram of adoptive delivery of OT-I cells, E.G7 tumor inoculation and anti-PD-1 treatment, Figure 14B is the growth curve of E.G7 tumor, Figure 14C is the expression of TCF1 and TIM-3 in OT-I cells, and the expression of OT-I cells and TCF1 in E.G7 tumor infiltration per unit.+ and TIM-3 + 14C , the abscissa indicates the amount of cells expressing TIM-3, and the ordinate indicates the amount of cells expressing TCF1.

[0034] FIG. 15 shows the expression of PRDM1 in tumor-specific CD8 T cells in Example 7 of the present invention. + Figure 15A shows the results of the combination of T cell deletion and anti-PD-1. + The proportion of OT-I cells and Granzyme B per unit tumor infiltration + 15A shows the number of OT-I cells, wherein the abscissa indicates the amount of cells expressing TIM-3, and the ordinate indicates the amount of cells expressing Granzyme B. FIG15B shows the expression of IFN-γ and TNF-α in OT-I cells, wherein the abscissa in FIG15B indicates the amount of cells expressing TNF-α, and the ordinate indicates the amount of cells expressing IFN-γ. DETAILED DESCRIPTION

[0035] The following embodiments of the present invention are described in detail. The embodiments described below are exemplary and are only used to explain the present invention, and are not to be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature in this field or the product specifications are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be obtained commercially.

[0036] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0037] In this document, the terms “contain”, “include” or “include” are open expressions, that is, they include the contents specified in the present invention but do not exclude other contents.

[0038] As used herein, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of combining the active ingredient (T cell) with a carrier that constitutes one or more accessory ingredients. Generally, the composition is prepared by uniformly and thoroughly combining the active compound with a liquid carrier, a finely divided solid carrier, or both.

[0039] As used herein, the term "pharmaceutically acceptable" refers to substances that are suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic response), ie, substances with a reasonable benefit / risk ratio.

[0040] As used herein, the term "pharmaceutically acceptable amount" or "pharmaceutically acceptable dose" refers to a dose suitable for use in humans and / or mammals without excessive adverse side effects (e.g., toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio. For example, it may be an "effective amount" or "effective dose," where "effective amount" or "effective dose" refers to an amount that can produce a function or activity in humans and / or animals and is acceptable to humans and / or animals.

[0041] As used herein, the term "pharmaceutically acceptable excipient" may include any solvent, diluent, or other liquid excipient, etc., suitable for the particular target dosage form. Except to the extent that any conventional excipient is incompatible with the compound of the present invention, such as any adverse biological effect produced or interaction with any other component of the pharmaceutically acceptable composition in a deleterious manner, their use is also contemplated by the present invention.

[0042] As used herein, the term "administer" refers to the introduction of a predetermined amount of a substance into a patient via a suitable means. The T cells or pharmaceutical compositions of the present invention can be administered via any common route, as long as they can reach the desired tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, and the like, but the present invention is not limited to these exemplified modes of administration. Preferably, the T cells or pharmaceutical compositions of the present invention are administered via intravenous injection.

[0043] As used herein, the term "treatment" refers to obtaining a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the progression of the disease; or (c) alleviating the disease, such as alleviating the symptoms associated with the disease. "Treatment" as used herein covers any medication that administers T cells or pharmaceutical compositions to an individual to treat, cure, alleviate, improve, reduce or inhibit the individual's disease, including but not limited to administering a drug containing the T cells described herein to an individual in need.

[0044] The present invention provides a T cell, a pharmaceutical composition, a use of a T cell or a pharmaceutical composition in preparing a drug, a drug combination, a method for treating cancer, a method for increasing the content of stem cell-like cells in immune cells, a method for inhibiting T cell-mediated T cell proliferation, and a method for inhibiting T cell-mediated T cell proliferation.term Methods of cell transformation.

[0045] T cells

[0046] In a first aspect of the present invention, a T cell is provided. According to an embodiment of the present invention, the expression of a given protein in the T cell is altered, and the given protein is suitable for regulating the expression of BCL6 in the T cell. The T cell of the present invention can maintain T prog The T cells of the present invention can be used to treat and prevent cancer without reducing T cell stemness and maintaining long-term survival, thereby improving their ability to kill tumors. prog Under the premise of cell number, promote T term The T cells have a strong instantaneous killing ability against early tumors containing fewer tumor cells and can be used to treat early cancer.

[0047] According to an embodiment of the present invention, the T cell may further include at least one of the following technical features:

[0048] In some optional embodiments of the present invention, the T cells are modified T cells.

[0049] In some optional embodiments of the present invention, the T cells are derived from T cells isolated from a healthy individual or patient.

[0050] Exemplarily, the patient may be a tumor patient or a patient with other diseases. In a preferred embodiment of the present invention, the patient is a tumor patient.

[0051] According to an embodiment of the present invention, the T cells are CD8 + T cells and CD4 + At least one of the T cells, preferably CD8 T cells, which specifically recognizes tumor cells + T cells.

[0052] In this article, the term “CD8 + T cells are CD44 positive (CD44 + ) of CD8 + T cells.

[0053] For example, in a specific embodiment of the present invention, flow cytometry is used to detect CD8 + T cells, CD44 + CD8 + T cells (CD44 positive CD8 + CD8 T cells that specifically recognize tumor cells + T cells.

[0054] According to an embodiment of the present invention, the given protein is suitable for regulating the expression of BCL6 via the IL-2-STAT5 pathway and / or the TGF-beta-SMAD2 pathway.

[0055] According to an embodiment of the present invention, the expression of BCL6 in the T cells is upregulated. Thus, the T cells can maintain T prog state, enhancing their T cell stemness and maintaining long-term T cell survival can improve their ability to kill tumors, thereby contributing to the development of new anti-tumor immunotherapies for the treatment and prevention of cancer.

[0056] Herein, the term "BCL6 expression is upregulated" means that the expression level of BCL6 protein in the transformed T cells is increased compared with that in the untransformed T cells.

[0057] Exemplarily, the T cells before modification are T cells isolated from tumor-infiltrated individuals; "BCL6 expression is upregulated" means that the expression level of BCL6 protein in the T cells of the present invention is increased compared to the isolated T cells.

[0058] According to an embodiment of the present invention, the expression of the given protein in the T cells is altered so as to inhibit the IL-2-STAT5 pathway and / or activate the TGF-beta-SMAD2 pathway.

[0059] According to an embodiment of the present invention, the given protein includes at least one downstream protein on the IL-2-STAT5 pathway, and the given protein is downregulated.

[0060] According to an embodiment of the present invention, the given protein includes at least one selected from the following: PRDM1 or a member of its homologous family.

[0061] Herein, the term "the expression of the given protein is downregulated" means that the expression level of the given protein in the transformed T cells is reduced compared to that in the untransformed T cells.

[0062] Exemplarily, the T cells before transformation are T cells isolated from tumor-infiltrated individuals; "the expression of the given protein is downregulated" means that the expression level of the given protein in the T cells of the present invention is reduced compared to the isolated T cells.

[0063] According to an embodiment of the present invention, the downregulation of the expression of the given protein is achieved by at least one of gene silencing, gene editing, small molecule inhibitors, and antibody drugs.

[0064] In some optional embodiments of the present invention, the small molecule inhibitor includes at least one of the small molecule inhibitors of STAT5a / b, STAT3, STAT1, and STAT4.

[0065] In some optional embodiments of the present invention, the antibody drug includes at least one of IL-2, IL-2Rα, IL-2β and IL-2Rγ blocking antibodies.

[0066] According to an embodiment of the present invention, the gene silencing or gene editing includes at least one of CRISPR-Cas9 virus and non-virus, shRNA, and siRNA.

[0067] According to an embodiment of the present invention, the PRDM1 gene in the T cells is knocked down.

[0068] According to an embodiment of the present invention, the PRDM1 gene in the T cells is knocked out.

[0069] According to an embodiment of the present invention, the expression of BCL6 in the T cells is downregulated. prog Under the premise of cell number, promote T term The T cells have a strong instantaneous killing ability against early tumors containing fewer tumor cells and can be used to treat early cancer.

[0070] Herein, the term "downregulated expression of BCL6" means that the expression level of BCL6 protein in the transformed T cells is reduced compared with that in the untransformed T cells.

[0071] Exemplarily, the T cells before transformation are T cells isolated from tumor-infiltrated individuals; "BCL6 expression is downregulated" means that the expression level of BCL6 protein in the T cells of the present invention is reduced compared to the isolated T cells.

[0072] According to an embodiment of the present invention, the expression of the given protein in the T cells is altered so as to activate the IL-2-STAT5 pathway and / or inhibit the TGF-beta-SMAD2 pathway.

[0073] According to an embodiment of the present invention, the given protein includes at least one downstream protein on the IL-2-STAT5 pathway, and the expression of the given protein is upregulated.

[0074] Herein, the term "the expression of the given protein is upregulated" means that the expression level of the given protein in the transformed T cells is increased compared to that in the untransformed T cells.

[0075] Exemplarily, the T cells before transformation are T cells isolated from tumor-infiltrated individuals; "the expression of the given protein is upregulated" means that the expression level of the given protein in the T cells of the present invention is increased compared to the isolated T cells.

[0076] According to an embodiment of the present invention, the given protein includes at least one selected from the following: PRDM1 or a member of its homologous family.

[0077] According to an embodiment of the present invention, the up-regulation of the expression of the given protein is achieved through at least one of gene transfection, promoter enhancer, and auxiliary expression factor.

[0078] In some optional embodiments of the present invention, the promoter enhancer includes at least one of a CMV promoter enhancer, an EF1α promoter enhancer, a CD2 promoter enhancer, and an IL-2 promoter enhancer.

[0079] In some optional embodiments of the present invention, the auxiliary expression factor includes at least one of T cell activating factor, NF-κB, CD28 costimulatory factor, and IL-2 cytokine.

[0080] According to an embodiment of the present invention, the gene transfection is achieved by at least one of liposome, calcium phosphate, and PEI.

[0081] According to an embodiment of the present invention, the PRDM1 gene in the T cells is upregulated.

[0082] According to an embodiment of the present invention, the T cell is at least one of a CAR-T cell, a TIL cell and a TCR-T cell.

[0083] According to an embodiment of the present invention, the expression of immune checkpoint-related proteins in the T cells is downregulated.

[0084] As used herein, the term "downregulated expression of immune checkpoint-related proteins" refers to a decrease in the expression level of immune checkpoint-related proteins. In particular, the expression of immune checkpoint-related proteins in engineered T cells is increased compared to unengineered T cells.

[0085] It should be noted that "downregulation of the expression of immune checkpoint-related proteins" in the present invention can be achieved by conventional methods in the art, including but not limited to gene silencing or gene editing systems (CRISPR-Cas9 viruses and non-viruses, shRNA, siRNA), small molecule inhibitors and antibody drugs (immune checkpoint-related protein antibodies).

[0086] According to an embodiment of the present invention, the immune checkpoint-related protein includes at least one of PD-1, CTLA-4, LAG-3, TIM-3, TIGIT and VISTA.

[0087] Pharmaceutical composition

[0088] In a second aspect of the present invention, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition includes an agent for regulating the IL-2-STAT5 pathway and / or the TGF-beta-SMAD2 pathway to regulate the expression of BCL6 in T cells; or the T cells described in the first aspect of the present invention. As mentioned above, the T cells described in the first aspect can maintain T prog The state of T cells is enhanced, the stemness of T cells is enhanced and the long-term survival of T cells is maintained, which can improve their ability to kill tumors. Therefore, the use of a pharmaceutical composition containing the above-mentioned T cells can achieve a long-lasting and effective anti-tumor immune response, which is helpful for the development of new anti-tumor immunotherapy to treat and prevent cancer. Alternatively, as can be seen from the above, the T cells described in the first aspect can be used without reducing T prog Under the premise of cell number, promote T term The T cells have a strong instantaneous killing ability against early-stage tumors containing fewer tumor cells. Therefore, a pharmaceutical composition containing the above-mentioned reagents or T cells can be used to treat early-stage cancer.

[0089] According to an embodiment of the present invention, the agent is used to inhibit the IL-2-STAT5 pathway.

[0090] According to an embodiment of the present invention, the agent inhibits the IL-2-STAT5 pathway by downregulating the expression of a given protein, wherein the given protein comprises at least one selected from the following: PRDM1 or a member of its homologous family.

[0091] According to an embodiment of the present invention, the reagent includes at least one of small molecule inhibitors, antibody drugs, CRISPR-Cas9 viruses and non-viruses, TALEN, ZFN, shRNA, and siRNA.

[0092] According to an embodiment of the present invention, the reagent is used to activate the TGF-beta-SMAD2 pathway, and the reagent includes a TGF-beta-SMAD2 pathway activator and a TGF-beta-SMAD3 pathway activator.

[0093] According to an embodiment of the present invention, the agent is used to activate the IL-2-STAT5 pathway.

[0094] According to an embodiment of the present invention, the agent activates the IL-2-STAT5 pathway by upregulating the expression of a given protein, wherein the given protein comprises at least one selected from the following: PRDM1 or a member of its homologous family.

[0095] According to an embodiment of the present invention, the reagent includes at least one of liposomes, calcium phosphate, and PEI.

[0096] According to an embodiment of the present invention, the reagent is used to inhibit the TGF-beta-SMAD2 pathway, and the reagent includes a TGF-beta-SMAD2 pathway inhibitor and a TGF-beta-SMAD3 pathway inhibitor.

[0097] According to an embodiment of the present invention, the pharmaceutical composition further comprises: an immune checkpoint blocker.

[0098] In some optional embodiments of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0099] In some optional embodiments of the present invention, the pharmaceutical composition is a combination drug or a drug kit.

[0100] It should be noted that the immune checkpoint blockers in the present invention should be understood in a broad sense, including but not limited to pharmaceutical reagents, treatment methods, etc. As long as they can block the immune checkpoints, they are all within the scope of protection of the present invention.

[0101] According to an embodiment of the present invention, the immune checkpoint blocker includes at least one of PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, LAG-3 antibody, TIM-3 antibody, TIGIT antibody, and VISTA antibody.

[0102] In some optional embodiments of the present invention, the immune checkpoint blocker can be further achieved by the following methods, including but not limited to tyrosine kinase inhibitors, co-stimulatory molecule monoclonal antibodies, epigenetic therapy drugs, chemotherapeutic drugs, radiotherapy preparations, vaccines, PDCD1 knockout therapy, PD1, PDL1 antibodies, or viral vector-based gene knockout methods.

[0103] In some optional embodiments of the present invention, the immune checkpoint blocker further includes CAR-T, CAR-NK, tumor vaccine, oncolytic virus vaccine, TLR7 / 8 agonist, anti-CD47 or IL-2 receptor agonist.

[0104] According to an embodiment of the present invention, the immune checkpoint blocker is formulated to be suitable for simultaneous administration with the T cells or separate administration.

[0105] Use of T cells or pharmaceutical compositions in the preparation of drugs

[0106] In a third aspect, the present invention provides use of the T cell described in the first aspect or the pharmaceutical composition described in the second aspect in preparing a medicament for treating or preventing cancer. The T cell or pharmaceutical composition of the present invention can effectively kill tumors, contributing to the development of novel anti-tumor immunotherapies for treating or preventing cancer.

[0107] According to an embodiment of the present invention, the cancer includes at least one selected from the following: gastric cancer, lung cancer, pancreatic cancer, liver cancer, breast cancer, cervical cancer, skin cancer, prostate cancer, melanoma, thyroid cancer, uterine fibroids, lymphoma, esophageal cancer, intestinal cancer, bone marrow cancer, nasal cancer, bone cancer, head and neck cancer, oral cancer, kidney cancer, and Kaposi's sarcoma.

[0108] Drug combination

[0109] In a fourth aspect, the present invention provides a drug combination. According to an embodiment of the present invention, the drug combination comprises an immune checkpoint inhibitor and the T cells described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention. According to an embodiment of the present invention, the combined administration of the two drugs has a high tumor-killing ability, can effectively kill tumor cells, inhibit tumor growth or reduce tumor volume, and can effectively prevent and treat tumors or cancer.

[0110] Methods for treating cancer

[0111] In its fifth aspect, the present invention provides a method for treating cancer. According to an embodiment of the present invention, the method comprises administering to a patient the T cells described in the first aspect of the present invention and the pharmaceutical composition described in the second aspect of the present invention. As previously noted, both the T cells described in the first aspect and the pharmaceutical composition described in the second aspect have high tumor-killing abilities, effectively killing tumor cells, inhibiting tumor growth, or reducing tumor volume. Thus, the method of the present invention is effective for treating and preventing cancer.

[0112] According to an embodiment of the present invention, it further includes administering at least one of PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, LAG-3 antibody, TIM-3 antibody, TIGIT antibody, and VISTA antibody to the patient simultaneously or sequentially.

[0113] The effective amount of the active ingredients of the present invention (T cells, and at least one of PD-1 antibodies, PD-L1 antibodies, CTLA-4 antibodies, LAG-3 antibodies, TIM-3 antibodies, TIGIT antibodies, and VISTA antibodies) may vary depending on the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the active ingredients, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, etc. For example, depending on the urgency of the treatment, several divided doses may be administered daily, or the dose may be reduced proportionally.

[0114] Methods for upregulating the content of stem-like cells in immune cells

[0115] In a sixth aspect of the present invention, the present invention provides a method for upregulating the content of stem-like cells in immune cells. According to an embodiment of the present invention, the method comprises: administering to a patient the T cells described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect; the expression of BCL6 in the T cells is upregulated. As can be seen from the above, the aforementioned T cells are in the stem-like T cell subset (T prog ) stage, the proportion of stem cell-like cells in immune cells can be increased by adding these T cells. prog The cells are in a relatively quiescent state and have a high level of proliferation potential. Therefore, according to an embodiment of the present invention, the immune cells obtained by this method have a strong tumor cell-killing ability and can effectively treat and prevent cancer or tumors.

[0116] Inhibit T cells to T term Cell transformation methods

[0117] In a seventh aspect, the present invention provides a method for inhibiting T cells from term According to an embodiment of the present invention, the method comprises upregulating the expression of BCL6 in the T cells; the T cells comprise CD4 + T cells, CD8 + As previously known, upregulating BCL6 protein in T cells can maintain T cells in the stem cell-like state (T prog ) state, thereby inhibiting T cells from turning to T term Therefore, according to an embodiment of the present invention, the method helps to maintain T cells T prog state, producing more lasting and effective anti-tumor ability.

[0118] According to an embodiment of the present invention, the upregulation of BCL6 expression in the T cells is achieved by downregulating the expression of a given protein in the T cells, and the given protein is suitable for regulating the expression of BCL6 through the IL-2-STAT5 pathway; or the upregulation of BCL6 expression in the T cells is achieved by contacting the T cells with a reagent, and the reagent is used to activate the TGF-beta-SMAD2 pathway.

[0119] According to an embodiment of the present invention, the reagents include a TGF-beta-SMAD2 pathway activator and a TGF-beta-SMAD3 pathway activator.

[0120] According to an embodiment of the present invention, the given protein includes at least one selected from the following: PRDM1 or a member of its homologous family.

[0121] According to an embodiment of the present invention, the downregulation of the expression level of a given protein in the T cells is achieved by at least one of gene silencing or gene editing, small molecule inhibitors and antibody drugs.

[0122] According to an embodiment of the present invention, the gene silencing or gene editing includes at least one of CRISPR-Cas9 virus and non-virus, shRNA, and siRNA.

[0123] According to an embodiment of the present invention, the upregulation of BCL6 expression in the T cells is achieved by knocking out the PRDM1 gene in the T cells.

[0124] In some optional embodiments of the present invention, the knockout of the PRDM1 gene in the T cells is achieved by at least one of CRISPR-Cas9, shRNA and siRNA.

[0125] For example, in a specific embodiment of the present invention, CRISPR-Cas9 technology is used to knock out PRDM1 in OT-I cells, and the T cells after PRDM1 knockout are combined with anti-PD-1 to detect the killing ability of E.G7 tumors in CD45.1 receptor mice.

[0126] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0127] Example 1: BCL6 is selectively overexpressed in tumor stem cell-like CD8 + T cells

[0128] Previous studies have reported that BCL6 is selectively expressed in stem cell-like CD8 + Therefore, this example needs to verify the expression of BCL6 in tumor models. The specific steps are as follows:

[0129] 1. First, dimensionality reduction and cluster analysis were performed on single-cell RNA-seq data of head and neck cancer and melanoma patients (GEO No.: GSE103322 and GSE120575). The dimensionality reduction and cluster analysis software was Seurat (version 3.2.2), Find Neighbors and Find Clusters function and Feature Plot function. Second, CD8 was identified by the expression of CD3E, CD8B, CD4 and FOXP3 molecules. + Two major cell populations were obtained, one of which significantly overexpressed TCF1, a stem-like cell-infiltrating cell in the mouse B16-GP33 tumor. + The characteristic genes of P14 cells, including TCF7, CCR7 and IL7R, were identified as T cells. prog cell subsets; another cell subset significantly overexpressed terminally differentiated TCF1 infiltrating mouse B16-GP33 tumors - P14 cells were identified as T cells because of their characteristic genes including GZMB, PRF1, HAVCR2, PRDM1 (encoding BLIMP1 protein), PDCD1 and TOX. term Cell subsets. term Cell subsets and T prog Analysis of BCL6 expression levels in cell subsets showed that BCL6 expression was significantly enriched in T cells of head and neck cancer and melanoma patients. prog In cells.

[0130] 2. Further verify the expression level of BCL6 in mouse tumor models. The specific steps are as follows:

[0131] C57BL / 6 mice were divided into two groups, with 8 mice in each group. E.G7 tumor cells and B16-OVA tumor cells were subcutaneously inoculated in the two groups of mice, respectively. On the 21st day after inoculation, the E.G7 and B16-OVA tumors, tumor-draining lymph nodes and spleens of the mice were removed. Tumor-infiltrating lymphocytes were obtained by density gradient centrifugation using 30% and 70% Percoll (GE) solutions, and single cell suspensions were obtained by grinding tumor-draining lymph nodes and spleens to obtain isolated CD8 + Then, flow cytometry was used to detect naive T cells (CD44- ) peripheral CD8 + T cells, activated (CD44 + ) peripheral CD8 + T cells and tumor-infiltrating PD-1 - CD8 + T cells and PD-1 + CD8 + The expression of BCL6 in T cells. The results showed that on the 21st day after vaccination, the PD-1 + CD8 + There was significant expression of BCL6 in T cells, whereas PD-1 in tumor-infiltrating - CD8 + CD8 T cells in the naive type (T cells that have matured and left the thymus but have not yet encountered antigens) in the spleen and lymph nodes + T cells and activated CD8 + T cells showed almost no significant expression of BCL6.

[0132] 3. Further analysis of BCL6 in tumor antigen-specific CD8 + The expression level in T cells is as follows:

[0133] Will specifically recognize OVA 257-264 Naive OT-I cells were adoptively transferred into C57BL / 6 recipient mice (8 mice), and then E.G7 tumor cells were inoculated one day later. Spleens and lymph nodes were then removed from CD45.1 OT-I mice and then treated with Dynabeads. TM FlowComp TM Mouse CD8 Kit (Invitrogen) was used to separate and purify CD8 + T cells. Then flow cytometry was used to detect CD25 - CD44lowCD62Lhigh naive OT-I cells were labeled and then sorted using a FACSAria (BD) flow cytometer to obtain isolated naive OT-I cells. The following experiments and tests were performed using the method described in step 2 of this example. The results showed that BCL6 was continuously highly expressed in E.G7 tumor-infiltrating OT-I cells from day 11 to day 20. In addition, BCL6 was selectively expressed in T prog cells, but not in T term In cells.

[0134] 4. To further detect BCL6 + CD8 +The inventors isolated the infiltrating BCL6-RFP (BCL6 tandem red fluorescent protein) T cell transcriptome characteristics from E.G7 tumors of BCL6-RFP reporter mice. + TIM-3 - cells (BCL6 + CD8 + T) and BCL6-RFP - TIM-3 + cells (TIM-3 for T term Cell marker molecules), Magzol reagent (Magen) was used to + Total RNA was extracted from T cells. Transcriptome library construction and high-throughput sequencing were sent to BGI Genomics. The cDNA library was sequenced by Illumina Hiseq4000 from BGI Genomics. The sequenced data were analyzed using Trim Galorev0.4.4, bowtie2, and feature counts. Differentially expressed genes were screened using DESeq2 with an FDR adjusted p-value of 0.05. The results showed that BCL6 + CD8 + The transcriptome of T cells highly expresses TCF1 in B16-GP33 tumor-infiltrating + The characteristic genes of P14T cells (i.e. stem cell-like cells) include Tcf7, Id3, Cxcr5 and Ccr7. + CD8 + T cells are T prog This further illustrates that BCL6 is selectively and highly expressed in tumor-infiltrating T cells. prog In cells.

[0135] 5. Further testing of the moment when BCL6 expression was induced was performed as follows: Similar to step 3 of this example, the inventors adoptively transferred CFSE-labeled naive CD45.2 OT-I cells into CD45.1 recipient mice (8 mice) and subcutaneously inoculated E.G7 tumor cells one day later. The method described in step 2 of this example was used for experiments and testing, and then the expression of BCL6 in OT-I cells infiltrating the tumors and TDLN (tumor-draining lymph nodes) of the recipient mice was analyzed.

[0136] The results showed that BCL6 was not significantly expressed in TDLN-infiltrating naive OT-I cells and early-expanded OT-I cells. However, around the 9th day after OT-I cells underwent multiple rounds of expansion, BCL6 expression increased significantly. In addition, around the 9th day after OT-I cells underwent multiple rounds of expansion, TOX, a marker of T cell exhaustion, was significantly expressed in OT-I cells, and BCL6 was selectively expressed in TOX. + High expression in OT-I cells, but not in TOX - TCF1 is not expressed in OT-I cells; however, in TDLN, TCF1 is highly expressed in naive OT-I cells and is continuously expressed in OT-I cells after multiple rounds of proliferation. Furthermore, in TDLN, LY108 is highly expressed in naive OT-I cells, and its expression level further increases in proliferating OT-I cells.

[0137] In summary, in TDLN, early proliferating OT-I cells expressed high levels of TCF1, LY108, and CD62L, but almost no TOX and BCL6. At day 9, OT-I cells differentiated into TOX-depleted + BCL6 + The cells maintained high levels of TCF1 and LY108 expression, while CD62L expression was significantly downregulated. In the tumor, CFSE in tumor-infiltrating OT-I cells was almost completely diluted, indicating that these cells underwent multiple rounds of proliferation in TDLN. Tumor-infiltrating OT-I cells were almost all TOX + Some of them highly express BCL6, TCF1 and LY108 molecules, so this group of cells are tumor-infiltrating T cells. prog In tumor-infiltrating OT-I cells, CD62L expression levels were very low. These results suggest that BCL6 expression is a key factor in the depletion of T cells in tumor models. prog One of the hallmark characteristics of cells.

[0138] 6. Previous studies have shown that T prog cells can differentiate into T term Next, the inventors used a lineage tracing system to further validate BCL6 in tumor models. + CD8 + The differentiation ability of T cells. The specific steps are as follows:

[0139] BCL6 CreERT2x Rosa26 tdTomato Naive CD8 +T cells were adoptively transferred into CD45.1 recipient mice (8 mice), and then E.G7 tumor cells were inoculated one day later. Tamoxifen (T5648; Sigma-Aldrich) was injected intraperitoneally once daily on days 7-9 at a concentration of 0.2 mg / g body weight. Experiments and testing were performed using the method described in step 2 of this example.

[0140] The results showed that on day 11, almost all BCL6 - tdTomato + OT-I cells are LY108 + TIM-3 - T prog cells; however, as the tumor develops and progresses, some OT-I cells differentiate into LY108 - TIM-3 + T term In addition, as the tumor develops and progresses, BCL6 + LY108 + OT-I cells gradually differentiate into LY108 - CX3CR1 + and LY108 - CD39 + T term cells; BCL6 - tdTomato + The proportion of OT-I cells gradually increased, increasing by about 1.5 times from day 11 to day 21. Therefore, these results indicate that BCL6 + CD8 + T cells are true T prog cells, and can continue to differentiate into T term cell.

[0141] Example 2: BCL6 does not regulate the generation of tumor Tprog cells but is essential for their long-term maintenance

[0142] Previous studies have reported that BCL6 is expressed in T cells in a chronic viral infection model. prog Similarly, in the LCMV cl13 virus infection model, loss of BCL6 led to T cell death at day 8. prog The cells almost completely disappeared. Therefore, this example needs to verify the role of BCL6 in the tumor model. The specific steps are as follows:

[0143] 1. Verify the role of BCL6 in tumor-specific CD8 + The specific steps of the action in T cells are as follows:

[0144] Equal numbers of naive WT OT-I cells and BCL6 - / - OT-I cells (BCL6 was knocked out in naive WT OT-I cells using CRISPR-Cas9 technology, and the preparation method of the two OT-I cells is shown in Step 3 of Example 1) were adoptively transferred into CD45.1 recipient mice (8 mice) and inoculated with E.G7 tumor cells 1 day later. The experiment was performed using the method described in Step 2 of Example 1.

[0145] The results showed that the loss of BCL6 significantly enhanced the control of tumor growth by OT-I cells in recipient mice and also led to the induction of TIM-3 in tumor infiltration. + The proportion of cells increased significantly, while LY108 + The proportion of cells was significantly reduced. + The results showed that the loss of BCL6 led to a significant increase in the number of OT-I cells infiltrating the tumor per unit weight, especially T cells. term However, at day 16, E.G7 tumors infiltrated with BCL6 - / - OT-I cells still contain a group of T prog cells, and, per unit weight, tumor-infiltrating T prog The number of T cells was not affected by BCL6 deficiency, indicating that BCL6 plays an important role in the early development of tumors. prog Furthermore, loss of BCL6 leads to the expression of TCF1 in tumor-infiltrating OT-I cells. + T prog The proportion of cells and the expression level of EOMES were significantly decreased, while CX3CR1 + T term The proportion of OT-I cells and the expression level of T-bet were significantly increased. The loss of BCL6 led to a significant increase in the expression levels of IFN-γ, Granzyme B and Ki-67 (biomarkers of T cell effector function and proliferation activity), indicating that the effector function and proliferation activity of OT-I cells were significantly increased. In the chronic LCMV cl13 virus infection model, on day 8, the loss of BCL6 led to a significant increase in the expression of T-bet. prog The number of cells decreased significantly.

[0146] Taken together, these results suggest that BCL6 plays a crucial role in the regulation of CD8 + T cells play different roles in chronic viral infection models. prog The generation of T cells is essential but does not affect T cell proliferation in short-term tumor models.prog The production of cells.

[0147] 2. Verify the expression of BCL6 in T prog It plays an important role in the long-term maintenance of cells. The specific steps are as follows:

[0148] WT OT-I cells and BCL6 were isolated from the E.G7 tumor of the mouse in step 1 of this example using the method of step 3 of example 1. - / - OT-I cells were then adoptively transferred into a second Tcrbd - / - In recipient mice (8 mice), the experiment and detection were performed using the method described in step 2 of Example 1.

[0149] The results showed that the loss of BCL6 led to a significant increase in tumor growth in the second recipient mice. + T prog Cells were successfully maintained in some WT OT-I cells, but not in BCL6 - / - Furthermore, loss of BCL6 led to a significant decrease in the number of tumor- and TDLN-infiltrating OT-I cells, with T prog and T term The number of cells was significantly reduced. The loss of BCL6 resulted in the decrease of Granzyme B per tumor infiltration. + The number of OT-I cells was significantly reduced, and the ability to produce IFN-γ was significantly impaired. Therefore, these results indicate that BCL6 plays a role in T prog Long-term maintenance of cells and tumor antigen-specific CD8 + T cells are essential for long-term anti-tumor function.

[0150] 3. Further investigate the function of BCL6 in the B16-OVA tumor model. The specific steps are as follows:

[0151] The experiment was conducted using the experimental methods of step 1 and step 2 in this example, with the only difference being that the inoculated tumor cells were different, that is, the E.G7 tumor cells in step 1 and step 2 were replaced with B16-OVA tumor cells.

[0152] The results showed that loss of BCL6 resulted in T cell proliferation in tumor-infiltrating OT-I cells at days 14 and 22. prog The proportion of T cells decreased significantly. term The proportion of T cells increased significantly. On day 22, the loss of BCL6 led to prog At the same time, on day 14, the loss of BCL6 did not affect the tumor-infiltrating T progHowever, at day 22, the loss of BCL6 resulted in the prog The number of cells was significantly reduced, indicating that BCL6 prog Furthermore, loss of BCL6 leads to the depletion of tumor-infiltrating T cells. term The number of cells increased significantly on day 14, but decreased significantly on day 22, indicating that although BCL6 deficiency temporarily promoted T term Cell development, the T term The cells can have a strong instantaneous killing ability against early-stage tumors containing fewer tumor cells.

[0153] 4. Further verify the role of BCL6 in TDLN prog TDLN-infiltrating T cells prog The cells were identified as memory CD8 + T cells can continuously migrate into the tumor and exert a sustained anti-tumor effect. prog BCL6 is also highly expressed in T cells in TDLN. prog BCL6 is expressed in TDLN cells, but their differentiation and number are not affected by BCL6 deficiency. Therefore, it is urgent to verify the role of BCL6 in TDLN models. The specific steps are as follows:

[0154] WT OT-I cells and BCL6 were isolated from the TDLN of the mice described in step 1 of this example using the method of step 3 of example 1. - / - LY108 + TIM-3 - OT-I cells were then adoptively transferred in equal numbers into recipients of CD4 + Tcrbd on T cells - / - The recipient mice (8 mice) were inoculated with E.G7 tumor cells one day later, and then the recipient mice were tested and detected using the method described in step 2 of Example 1 on day 16.

[0155] The results showed that in these secondary recipient mice, BCL6 was persistently expressed in tumor- and TDLN-infiltrating T cells. prog BCL6 deficiency significantly impaired the ability of OT-I cells to control tumors in secondary recipient mice. BCL6 deficiency led to the inhibition of tumor-infiltrating TCF1 + T prog The cells could not be maintained, and the number of tumor-infiltrating OT-I cells was significantly reduced, among which T prog and T term However, in TDLN, BCL6 deficiency did not affect TCF1 + T progFurthermore, although the loss of BCL6 resulted in the decrease of tumor-infiltrating Granzyme B + The proportion of OT-I cells increased significantly, but their cell number decreased significantly. In addition, the loss of BCL6 significantly impaired the ability of tumor-infiltrating OT-I cells to secrete IFN-γ, but did not affect the ability to secrete TNF-α. These results demonstrate that BCL6 plays a role in T-cell proliferation and differentiation in TDLN. prog BCL6 deficiency does not affect T cells in TDLN. prog Cell production and maintenance.

[0156] 5. Further verification of BCL6 in CD8 + T cells play a role in the immune response against endogenous tumor antigens. The specific steps are as follows:

[0157] from CD45.1WT mice and CD45.2BCL6fl / fl Cd8aCre WT CD8 + T cells and BCL6 - / - naive CD8 + T cells (naive WT CD8 + Knockout of BCL6 in T cells using CRISPR-Cas9 is called BCL6 - / - ), then WT CD8 + T cells and BCL6 - / - naive CD8 + T cells and CD4 + T cells were simultaneously adoptively transferred into Tcrbd - / - Recipient mice (8 mice) were then subcutaneously inoculated with B16BL6, EL4, HEPA1-6 and MC38 tumor cells one day later, and the experiment and detection were performed using the method described in step 2 of Example 1.

[0158] The results showed that on day 15, the inventors first detected the expression of BCL6 in these donor CD8 + Expression levels in T cells. And BCL6 is significantly expressed in CD8 + T cells, and the expression of Granzyme B is mutually exclusive. However, in tumors, almost all CD8 + TILs differentiate into TCF1 - Granzyme B + T term cells and do not express BCL6. Loss of BCL6 leads to tumor-infiltrating CD8 + TCF1 in T cells +Granzyme B - T prog The proportion of cell subpopulations was significantly reduced, while TCF1 - Granzyme B + T term These results indicate that the proportion of CD8 + In the immune response of T cells to endogenous tumor antigens, the loss of BCL6 significantly promotes T prog cells differentiate into T term cell.

[0159] Example 3: BCL6 transcriptional regulation of CD8 in tumors + T cell differentiation

[0160] 1. Systematic study of BCL6 regulation of CD8 + The molecular mechanism of T cells. The specific steps are as follows:

[0161] The inventors used the method described in step 2 of Example 1 to obtain isolated CD8 + T cells were then activated in vitro using a flat-bottom plate conjugated with anti-CD3 (BioXCell, clone 145-2C11, 5 μg / ml) and anti-CD28 (BioXCell, clone 37.51, 1 μg / ml) antibodies to obtain in vitro activated CD8 + Next, BCL6-HA (NM_001348026.1) was cloned into a retroviral vector expressing IRES-GFP. Using the empty retroviral vector as a control vector, the retroviral plasmid and the pCL-ECO packaging plasmid were first transfected into the Phoenix cell line using calcium transfection. After 48 hours, the viral supernatant was collected and used to infect in vitro activated CD8 T cells. + T cells, obtained in vitro activated CD8 overexpressing BCL6-HA + T cells were then collected. 1×10 7 Left and right in vitro activated CD8 overexpressing BCL6-HA + T cells were then cross-linked and fixed with paraformaldehyde at a final concentration of 1%. ChIP experiments were performed according to the instructions of ActiveMotif's ChIP assay kit (53035). The obtained DNA fragments were sent to BGI genomics for library construction and high-throughput sequencing to obtain in vitro activated CD8 overexpressing BCL6-HA. +ChIP-seq data of T cells. Sequencing data were analyzed using bowtie2, picardMarkDuplicates, MACS2, ChIPseeker, bedtoolsintersect, Deeptools, computeMatrix and HOMER to obtain in vitro activated CD8 overexpressing BCL6-HA + Genome-wide binding results for T cells.

[0162] The results showed that BCL6 has similar sequence binding characteristics in different cell types, and in CD8 + In T cells, BCL6 significantly bound to 5949 DNA sites and 2931 genes. KEGG analysis results showed that BCL6-bound genes were significantly enriched in multiple T cell-related biological pathways, including T cell differentiation, activation, proliferation and adhesion. In addition, 42.58% and 39.89% of BCL6-bound DNA sites were distributed in introns and distal gene intergenic regions, and only 12.96% of BCL6 binding sites were distributed in promoter regions. Consistently, more than 70% of BCL6 binding sites were distributed in regions more than 10kb away from the transcription start site (TSS). These results indicate that BCL6 may tend to bind to gene intergenic regions and intronic regions to regulate gene expression.

[0163] 2. Verify the expression of BCL6 in CD8 + Regulatory role in the transcriptome of T cells. The specific steps are as follows:

[0164] First, the method described in step 3 of Example 1 was used to obtain isolated CD45.1 OT-I cells. Next, BCL6 (NM_001348026.1) was cloned into a retroviral vector expressing IRES-GFP and the method described in step 1 of this example was used to obtain CD45.1 OT-I cells overexpressing BCL6. The inventors co-adoptively transferred CD45.1 OT-I cells overexpressing BCL6 and control CD45.2 OT-I cells into Tcrbd cells inoculated with E.G7 tumors. - / -Eight recipient mice were transplanted. Eight days later, the inventors minced the E.G7 tumors with scissors and transferred them to DMEM supplemented with 1 mg / ml collagenase D and 10 U / ml DNase I. The cells were digested at 37°C for 40 minutes. Density gradient centrifugation with a Percoll gradient (GE Healthcare) was then performed to isolate and purify the donor OT-I cells. The OT-I cell transcriptome library and high-throughput sequencing were sent to BGI Genomics. The cDNA library was sequenced using an Illumina HiSeq 4000 from BGI Genomics, generating RNA-seq data for BCL6. The sequenced data were analyzed using GSEA using TrimGalorev 0.4.4, Bowtie 2, and FeatureCounts software.

[0165] GSEA results showed that overexpression of BCL6 could induce OT-I cells to express significant BCL6 + TIM-3 - T prog Transcriptome characteristics of cells. To further identify genes whose expression is directly regulated by BCL6, the inventors integrated and analyzed the ChIP-seq data and RNA-seq data of BCL6, and obtained 180 genes whose expression is directly induced by BCL6 and 156 genes whose expression is directly inhibited by BCL6. In addition, 49 genes whose expression is directly induced by BCL6 are BCL6 + T prog Cell characteristic genes (Id3, Cxcr5, Tgfbr3, Ccr6, etc.), 49 BCL6-inhibited genes are TIM-3 + T term These results indicate that BCL6 can induce T cells to express characteristic genes (PRDM1, Havcr2, Cxcr6, Gzmb, etc.). prog Expression of cell-specific genes and inhibition of T term Regulating the expression of cell signature genes to regulate CD8 + Differentiation of T cells.

[0166] 3. The inventors further compared the functional differences between BCL6 and TCF1. The details are as follows:

[0167] (1) Analysis of the differences in genes bound by BCL6 and TCF1. First, by comparing the ChIP-seq experimental results of BCL6 obtained in step 1 of this example with the ChIP-seq experimental results of TCF1 reported in previous studies, the inventors found that BCL6 and TCF1 have very different DNA binding site specificities. However, these two transcription factors bind to a large number of the same genes, and more than half of the genes bound by BCL6 are also bound by TCF1, which indicates that BCL6 and TCF1 may synergistically regulate the expression of target genes. Therefore, it is urgent to identify the genes whose expression is regulated by TCF1.

[0168] Furthermore, the inventors integrated and analyzed previously reported ChIP-seq data for TCF1 and RNA-seq data from P14 cells overexpressing Tcf7 in mice infected with LCMV cl13 virus, and obtained 224 genes whose expression was directly induced by TCF1 and 294 genes whose expression was directly repressed by TCF1. Then, by integrating and analyzing the genes whose expression was directly regulated by BCL6 and TCF1, the inventors identified 19 genes whose expression was commonly upregulated by BCL6 and TCF1 (Cxcr5, Xcl1, Ikzf2, etc.) and 20 genes whose expression was commonly repressed by BCL6 and TCF1 (PRDM1, Gzmb, Prf1, Entpd1, Cxcr6, etc.).

[0169] (2) Further comparison of BCL6 and TCF1 in maintaining T prog The specific steps are as follows:

[0170] First, the inventors used the method described in step 3 of Example 1 to obtain isolated OT-I cells. Next, the inventors used the method in step 1 of this example to obtain in vitro activated OT-I cells. Subsequently, the inventors cloned BCL6 (NM_001348026.1) and Tcf7 (NM_001313981.1) into a retroviral vector expressing IRES-GFP and used the method in step 1 of this example to obtain in vitro activated CD45.1 OT-I cells that overexpressed BCL6 and Tcf7 (also known as TCF1). Next, the above-mentioned OT-I cells overexpressing BCL6 and Tcf7 and the CD45.2 control group OT-I cells were co-adoptively transferred into Tcrbd cells inoculated with E.G7 tumors. - / - In recipient mice (8 mice), the experiment was performed using the method described in step 2 of Example 1 and the results were analyzed.

[0171] The results showed that after 8 days, overexpression of BCL6 or Tcf7 led to the proliferation of LY108 cells in OT-I cells. + T prog The proportion of cells increased significantly, TIM-3 + T termThe proportion of cells decreased significantly, indicating that these two transcription factors play an important role in maintaining T prog The overexpression of BCL6 and Tcf7 promotes their expression in E.G7 tumor-infiltrating OT-I cells. In addition, the overexpression of BCL6 leads to a significant decrease in the expression levels of molecules such as BLIMP1, T-BET, CD25 and CD39, while the expression levels of EOMES and TOX are significantly increased. By analyzing the proportion of tumor-infiltrating OT-I cells, this example found that the overexpression of BCL6 leads to a decrease of about 4 times in the number of OT-I cells in tumors and TDLNs. At the same time, the expression of Ki-67 in tumor-infiltrating OT-I cells is significantly increased. + The proportion of cells also decreased significantly. + T prog cells decreased by about 1.71 times, while T term However, overexpression of Tcf7 did not significantly change the number of tumor-infiltrating OT-I cells, among which T prog The number of cells increased significantly, and T term These results together indicate that BCL6 and TCF1 have similar functions in maintaining T cells. prog Characteristic capabilities.

[0172] (3) Further compare the ability of BCL6 and TCF1 to regulate cell proliferation. The specific steps are as follows:

[0173] The inventors used CTV to label the OT-I cells overexpressing BCL6 or Tcf7 obtained in step 3 (2) of the above example and the OT-I cells of the control group, and then adoptively transferred them into C57BL / 6 recipient mice (8 mice). The experiment was carried out using the method described in step 2 of Example 1 and the results were analyzed.

[0174] The results showed that after 3 days, up to 70-80% of BCL6-overexpressing cells in the spleen still retained CTV signals, while in control OT-I cells, the CTV signals were almost completely diluted. However, only 20% of Tcf7-overexpressing OT-I cells retained CTV signals, a significantly lower proportion than in BCL6-overexpressing OT-I cells. Furthermore, GO analysis revealed that more than half of the top 20 most significant signaling pathways enriched for genes inhibited by BCL6 were related to cell proliferation, whereas few pathways were enriched for genes inhibited by TCF7. This suggests that BCL6 has a significant inhibitory effect on cell proliferation, while TCF1 does not. These results suggest that BCL6 and TCF1 have different abilities to regulate cell proliferation.

[0175] (4) Further compare the functions of BCL6 and TCF1 in T cell differentiation and survival. The specific steps are as follows:

[0176] First, in Tcf7-deficient CD45.2OT-I cells (abbreviated as Tcf7 - / - OT-I cells) overexpressed BCL6, and then Tcf7 overexpressing BCL6 - / - OT-I cells and CD45.1WT OT-I cells were adoptively transferred into CD45.1 recipient mice (8 mice) 7 days after inoculation with E.G7 tumors. Experiments and detection were performed using the method described in step 2 of Example 1.

[0177] At the same time, the inventors used the method in step 3 (2) of Example 3 to obtain BCL6 - / - Tcf7 was overexpressed in OT-I cells, and then BCL6 overexpressing Tcf7 was transfected into - / - OT-I cells and CD45.1WT OT-I cells were adoptively transferred into CD45.1 recipient mice (8 mice) inoculated with E.G7, and the experiment was performed using the method described in step 2 of Example 1, and then analyzed 12 days later.

[0178] The results showed that after 5 days, BCL6 was expressed in Tcf7 in E.G7 tumors and TDLN. - / - Overexpression in OT-I cells can still significantly increase the expression of LY108 + TIM-3 - T prog The ratio of cells and reduced LY108 - TIM-3 + T term This indicates that under the condition of Tcf7 deficiency, overexpression of BCL6 can still significantly inhibit T prog cells differentiate into T term At the same time, due to the strong inhibitory effect of BCL6 on cell proliferation, overexpression of BCL6 leads to the downregulation of Tcf7 in tumors and TDLNs. - / - The number of OT-I cells was significantly decreased.

[0179] Tcf7 in BCL6 - / - Overexpression of LY108 in OT-I cells did not significantly alter the expression of LY108 in tumors and TDLNs. + TIM-3 - T prog and LY108 - TIM-3 + T term This indicates that under the condition of BCL6 deficiency, overexpression of Tcf7 cannot significantly inhibit T termIn addition, Tcf7 overexpression leads to the expression of BCL6 in tumors and TDLNs. - / - The number of OT-I cells increased significantly, indicating that TCF1 significantly improved the survival of OT-I cells in a BCL6-independent manner.

[0180] Furthermore, the inventors analyzed the ChIP-seq data of BCL6 and TCF1 obtained in step 3(2) of this example. The results showed that TCF1 significantly bound to the promoter region of BCL6, but BCL6 did not bind to the gene locus of Tcf7. In addition, BCL6 significantly bound to T term The gene locus of Havcr2, a characteristic gene of T, but TCF1 does not bind to T term These results indicate that BCL6 acts as a downstream molecule of TCF1 and significantly inhibits T prog cells differentiate into T term cells, but does not regulate cell viability.

[0181] Furthermore, the inventors analyzed the RNA-seq data of BCL6 obtained in step 3 of this example. Overexpression of BCL6 resulted in a significant decrease in the expression of Gzmb (abbreviation of Granzyme B), Prf1 and Fasl at the transcriptional level. In addition, overexpression of BCL6 resulted in a significant decrease in the expression of Granzyme B in tumor infiltration. + The proportion of OT-I cells was significantly reduced, which also led to the term The expression level of Granzyme B in cells was significantly reduced. However, overexpression of BCL6 did not affect the expression level of IFN-γ, but slightly enhanced the expression level of TNF-α. In addition, overexpression of BCL6 led to the inhibition of Caspase-3 + The cell ratio was significantly reduced, indicating that the apoptosis level of OT-I cells was significantly reduced.

[0182] In summary, as a downstream molecule of TCF1, BCL6 transcriptionally inhibits T prog cells differentiate into T term In addition, BCL6 significantly inhibited the proliferation of T cells, while TCF1 did not.

[0183] Example 4: TGF-β / SMAD2 signaling pathway induces CD8 + BCL6 expression in T cells

[0184] 1. Identification and regulation of CD8 + The signaling pathway of BCL6 expression in T cells is as follows: The inventors used the method described in step 2 of Example 1 to obtain isolated CD8 +T cells were then activated in vitro using a flat-bottom plate conjugated with anti-CD3 (BioXCell, clone 145-2C11, 5 μg / ml) and anti-CD28 (BioXCell, clone 37.51, 1 μg / ml) antibodies to obtain in vitro activated BCL6-RFPCD8 + T cells were stimulated by adding IL-2, IL-6, IL-10, IL-12, IL-12, anti-IL-2, anti-IFN-γ and TGF-β. Three days later, CD8 + T cells, CD8 by flow cytometry + The expression of BCL6 at the protein level was determined by the RFP fluorescence intensity of T cells, and the expression of BCL6 at the transcriptional level was detected by fluorescence real-time quantitative PCR. The results showed that anti-IL-2 and TGF-β could significantly inhibit the expression of BCL6 in CD8 T cells. + High expression of BCL6 at the RNA and protein levels was induced in T cells, as shown in Figure 1.

[0185] 2. Further verify the regulation of TGF-β signaling on BCL6 expression. The specific steps are as follows: Activate and stimulate Tgfbr2 in vitro with anti-CD3 / 28 and TGF-β - / - CD8 + T cells were tested using the method described in step 1 of this example. The results showed that the absence of Tgfbr2 resulted in TGF-β being unable to induce high expression of BCL6 at the RNA and protein levels. In addition, CD8 + In T cells, TGF-β stimulation significantly inhibited the expression of T-bet but did not affect the expression of TCF1.

[0186] 3. Previous studies have reported that TGF-β regulates T in a mouse model of chronic viral infection. prog Therefore, this example needs to verify the role of TGF-β signaling pathway in regulating BCL6 expression and CD8 + The specific steps are as follows:

[0187] (1) Equal numbers of naive CD45.2WT and Tgfbr2 - / - OT-I cells were adoptively transferred into CD45.1 recipient mice, and then E.G7 tumor cells were inoculated one day later. Experiments and tests were performed using the method in step 3 of Example 1. The results showed that the loss of Tgfbr2 significantly enhanced the tumor control effect of OT-I cells. The loss of Tgfbr2 led to the expression of BCL6 in OT-I cells. + TCF1 + TIM-3- T prog The proportion of cells was significantly decreased, while BCL6 - TCF1 - TIM-3 + T term In addition, the loss of Tgfbr2 led to a significant increase in the number of OT-I cells infiltrating the tumor per unit weight, among which T term The number of cells increased significantly, and T prog The number of cells did not change significantly, and the loss of Tgfbr2 led to the expression of IFN-γ + and Granzyme B + The proportion of cells infiltrating Granzyme B cells was significantly increased, and Tgfbr2 deficiency resulted in a significant increase in the proportion of cells infiltrating Granzyme B cells. + The number of OT-I cells increased significantly, leading to the expression of Ki-67 + The proportion of proliferating cells increased significantly, indicating that the TGF-β signaling pathway significantly inhibited tumor-infiltrating T cells. term cell development, but for T prog There was no significant effect on the production of tumor-infiltrating T cells, and TGF-β signaling had no significant effect on the production of tumor-infiltrating T cells. term It has a significant inhibitory effect on the effector function and proliferation activity of cells.

[0188] 4. Verify the effect of TGF-β signaling on T cell differentiation during long-term tumor progression. The specific steps are as follows:

[0189] (1) The inventors first activated WT and Tgfbr2 in vitro - / - OT-I cells were co-transferred into CD45.1 recipient mice 8 days after inoculation with E.G7 tumors. Experiments and tests were performed using the method in step 3 of Example 1. The recipient mice were then analyzed on days 16 and 24. The results showed that Tgfbr2 deficiency led to the expression of BCL6 + TCF1 + T prog The proportion of cells was significantly reduced, while TCF1 - TIM-3 + T term In addition, from day 16 to day 24, the loss of Tgfbr2 led to a significant increase in the proportion of T cells. prog Different from previous reports, in the LCMV cl13 virus infection model, a portion of LY108 cells remained in the absence of Tgfbr2. + TIM-3 - T progThe cells persisted at day 100 of infection. Furthermore, in the E.G7 tumor model, Tgfbr2 deletion did not significantly affect the proliferation of LY108 cells in TDLN. + TIM-3 - T prog The proportion of cells.

[0190] (2) Next, the tumor-infiltrating OT-I cells obtained in step 4 (1) of this example were counted using flow cytometry to obtain the number of OT-I cells in the tumor. The results showed that the loss of Tgfbr2 led to a significant increase in the number of tumor-infiltrating OT-I cells on day 16. However, on day 24, the loss of Tgfbr2 led to a significant decrease in the number of OT-I cells. prog and T term The numbers of cells increased significantly, but their numbers decreased significantly on the 24th day.

[0191] (3) Further, the inventors analyzed the WT and Tgfbr2 - / - Transcriptome sequencing data of P14 cells (GEO number: GSE209577) were obtained for WT and Tgfbr2 - / - Differentially expressed genes in P14 cells, as WT and Tgfbr2 - / - GSEA was then used to compare the expression of characteristic genes in P14 cells between WT and Tgfbr2 - / - The characteristic expression genes of P14 cells are expressed in BCL6 + and TIM-3 + CD8 + GSEA analysis showed that the characteristic genes of WT P14 cells in the LCMV cl13 model were significantly enriched in BCL6 + CD8 + In the T cell transcriptome, Tgfbr2 - / - The characteristic genes of P14 cells were significantly enriched in TIM-3 + CD8 + These results indicate that in the early stages of tumor development, TGF-β signaling significantly inhibits the proliferation and T cell proliferation of tumor-infiltrating T cells. prog cell differentiation, but it has no effect on T prog Indispensable for the long-term maintenance of cells.

[0192] 5. Previous research results have shown that the phenotypes of Tgfbr2 deficiency and BCL6 deficiency are consistent. The TGF-β signaling pathway may regulate T cell differentiation, proliferation, and effector function by regulating BCL6 expression. Therefore, this example will verify that overexpression of BCL6 can rescue the phenotype of Tgfbr2 deficiency. The specific steps are as follows:

[0193] The same number of BCL6-overexpressing CD45.2Tgfbr2 - / - OT-I cells, Tgfbr2 - / - OT-I cells and WT OT-I cells were adoptively transferred into CD45.1 recipient mice inoculated with E.G7 tumors, and then analyzed on day 12. The experiment and detection were performed using the method in step 3 of Example 1. The results showed that overexpression of BCL6 successfully rescued Tgfbr2 - / - LY108 in OT-I cells + T prog Furthermore, overexpression of BCL6 resulted in a decrease in the number of tumor-infiltrating Tgfb2 - / - The number of OT-I cells was significantly reduced and significantly less than that of WT and Tgfbr2 - / - The number of OT-I cells. Among them, BCL6 is expressed in Tgfbr2 - / - Overexpression of LY108 in OT-I cells leads to + T prog The number of cells returned to that of WT OT-I cells. prog The number of cells, but resulted in TIM-3 + T term In addition, overexpression of BCL6 also led to the + Tgfbr2 - / - The number of OT-I cells was significantly reduced. These results indicate that BCL6 is downstream of the TGF-β signaling pathway and that TGF-β promotes T cells in tumors through BCL6. prog Long-term maintenance of cells.

[0194] 6. Verify the molecular mechanism by which the TGF-β signaling pathway regulates BCL6 expression. The specific steps are as follows:

[0195] (1) By analyzing the ChIP-seq data of SMAD2 in ESCs and EBs cells (GEO number: GSE125116), the results showed that SMAD2 significantly bound to the promoter region of the BCL6 gene.

[0196] (2) Verify whether SMAD2 significantly binds to the promoter region of the BCL6 gene in CD8 +The inventors first collected 1x10 7 In vitro activated CD8 + T cells were then cross-linked and fixed with paraformaldehyde at a final concentration of 1%. ChIP experiments were performed according to the instructions of ActiveMotif's ChIP assay kit (53035). For the ChIP experiment of SMAD2, in vitro activated CD8 + T cells were stimulated with 2 ng / ml hTGF-β for 30 minutes before cross-linking and fixation, DNA was extracted, and the obtained DNA was detected by real-time quantitative PCR. + In T cells, SAMD2 significantly binds to the promoter region of the BCL6 gene, but not to its exon region, and this binding is not observed in SAMD2-deficient CD8 T cells. + SAMD2 loss leads to almost complete loss of TGF-β-stimulated CD8 + The expression of BCL6 in T cells was significantly reduced; SMAD2 also significantly bound to the promoter region of the Tcf7 gene, but the loss of SAMD2 had no effect on TGF-β-stimulated CD8 + The expression of Tcf7 in T cells was not significantly affected, but the expression level of T-bet was significantly increased. These results indicate that the TGF-β-SMAD2 signaling pathway significantly increases the expression of Tcf7 in T cells. + The transcriptional activity of BCL6 in T cells may be achieved through SMAD2 binding to the promoter region of the BCL6 gene.

[0197] Example 5: IL-2 / STAT5 signaling pathway inhibits CD8 + BCL6 expression in T cells and antagonism of TGF-β signaling pathway

[0198] Previous studies have reported that IL-2 signaling pathway significantly inhibits the expression of BCL6 in Tfh cells and has an adverse effect on effector CD8 T cells in acute viral infection and immune models. + The differentiation of T cells is very important. Therefore, this example needs to verify the role of IL-2 and TGF-β signaling pathways in CD8 + Interactions in T cells.

[0199] 1. First, WT and Il2ra-deficient CD8 + Naive CD8 T cells were activated using a flat-bottom plate containing anti-CD3 (5 μg / ml) and anti-CD28 (1 μg / ml) antibodies. + T cells were stimulated for three days by adding IL-2. After three days, CD8 +The expression level of BCL6 protein in T cells was measured, and the expression of BCL6 gene at the transcriptional level was detected by fluorescence real-time quantitative PCR. - / - CD8 + The expression levels of RNA and protein in T cells were significantly increased. - / - or anti-IL2-treated CD8 + In T cells, the expression level of TCF1 was significantly increased, while the expression level of T-bet was significantly decreased.

[0200] 2. Verify that IL-2 regulated genes are in BCL6 + and TIM-3 + CD8 + Expression levels in the transcriptome of T cells.

[0201] (1) We first analyzed the two previously reported IL-2-related CD8 + The RNA-seq data of T cells (GEO number: GSE58081 and GSE39110) were then analyzed using GSEA. The results showed that CD8 + Signature genes highly expressed in T cells were significantly overexpressed in BCL6 + CD8 + T cells, while in vitro IL-2 treated CD8 + The characteristic genes highly expressed in T cells were significantly overexpressed in TIM-3 + CD8 + IL-2 inhibits the expression of genes and BCL6 in T cells + CD8 + Genes highly expressed in T cells, as well as genes induced by IL-2 and TIM-3 + CD8 + There are significant positive correlations between the differentially expressed genes in T cells. prog The expression levels of the characteristic genes (Tcf7, Id3, Slamf6 and etc.) of T cells were significantly inhibited by IL-2 treatment in vivo, while term The expression levels of the cell's characteristic genes (PRDM1, Id2, Havcr2 and etc.) were significantly upregulated by IL-2 treatment in vivo.

[0202] 3. Further verify the role of IL-2 signaling in vivo.

[0203] (1) First, the expression level of CD25 was detected, and the tumor-infiltrating CD8+ The expression of BCL6 protein in T cells. The results showed that in E.G7 tumors, CD25 was specifically expressed in TIM-3 + T term In addition, although T prog The cells expressed very low levels of CD25, but they were the main T cell subset producing IL-2, which was consistent with previous results.

[0204] (2) Further explore the role of IL-2 signaling in CD8 + The inventors injected equal amounts of naive CD45.2WT, Il2ra + / - and Il2ra - / - OT-I cells were adoptively transferred into CD45.1 recipient mice and inoculated with E.G7 tumor cells one day later. Experiments and tests were performed using the method in step 3 of Example 1. The results showed that after 18 days, the loss of Il2ra caused the OT-I cells infiltrating the E.G7 tumor to almost maintain BCL6 + TCF1 + T prog cell status, leading to TIM-3 + T term In addition, the loss of Il2ra led to a significant decrease in the proportion and number of tumor-infiltrating OT-I cells, among which T term The number of cells decreased by about 21 times, while T prog The number of cells decreased by only about 3 times, which shows that the loss of Il2ra significantly impairs T term cell development, but for T prog In addition, the ability of Il2ra-deficient OT-I cells to control tumors was significantly impaired. At the same time, the ability of Il2ra-deficient OT-I cells to produce IFN-γ and Granzyme B and the expression level of KI-67 were significantly reduced, indicating that their proliferation and effector functions were significantly impaired. These results indicate that the IL-2 signaling pathway plays a role in inhibiting the expression of BCL6 and promoting T cell proliferation in tumor models. term Plays a very important role in cell development.

[0205] 4. Verify the molecular mechanism by which IL-2 inhibits BCL6 expression. The specific steps are as follows:

[0206] (1) We first analyzed the previously reported CD8 +ChIP-seq data of STAT5 in T cells (GEO number: GSE192386) showed that STAT5 significantly bound to the promoter region of the BCL6 gene, but not to the Tcf7 gene site.

[0207] (2) To further verify that STAT5 significantly binds to the promoter region of the BCL6 gene, the inventors used in vitro IL-2-treated CD8 + ChIP experiments of STAT5 were performed on T cells. The experimental method was similar to that in Example 4, step 6 (2), except that 10 U / ml IL-2 was used instead of 2 ng / ml hTGF-β for 30 minutes. ChIP-qPCR results showed that STAT5 significantly bound to the promoter region of the BCL6 gene, but not to the exon region of the BCL6 gene. In addition, the addition of exogenous IL-2 significantly enhanced the binding of STAT5 protein to the promoter region of the BCL6 gene. CD8 T cells treated with STAT5 inhibitors in vitro + In T cells, BCL6 expression was significantly increased at the transcriptional and protein levels. In addition, TCF1 expression was significantly increased, while T-BET expression was significantly decreased. These results suggest that the IL-2-STAT5 signaling pathway significantly inhibits BCL6 gene transcription, possibly through STAT5 binding to the BCL6 promoter region.

[0208] 5. Further verify the antagonistic effect of IL-2-STAT5 and TGF-β-SMAD2.

[0209] (1) We first examined the CD8 + Expression of BCL6 in T cells was determined by in vitro activation of naive CD8 T cells using a flat-bottom plate containing anti-CD3 (5 μg / ml) and anti-CD28 (1 μg / ml) antibodies. + T cells were stimulated for three days by adding IL-2 and TGF-β. After three days, CD8 + The expression level of BCL6 protein in T cells was measured, and the expression of the BCL6 gene at the transcriptional level was detected by fluorescent real-time quantitative PCR. The results showed that TGF-β-induced BCL6 expression at the transcriptional and protein levels was significantly inhibited by IL-2. In addition, TGF-β significantly antagonized IL-2-induced T-BET expression, but was unable to restore IL-2-inhibited TCF1 expression.

[0210] (2) Next, the published CD8 +Transcriptome sequencing data of T cells (GEO number: GSE125471) were then used to compare the expression of CD8 T cells treated with IL-2 or TGF-β using Pearson analysis. + Pearson analysis showed that CD8 T cells treated with IL-2 or TGF-β in vitro + The transcriptome of T cells showed a significant negative correlation in gene expression.

[0211] (3) GSEA analysis was performed on the RNA-seq results in step 2 of this example. The results of GSEA analysis showed that in mice infected with LCMV cl13, the characteristic genes in WT P14 cells were significantly highly expressed in OT-I cells not treated with IL-2, while Tgfbr2 - / - The characteristic genes of P14 cells were significantly overexpressed in OT-I cells treated with IL-2. + In T cells, STAT5 and SMAD2 bind to the same promoter site of the BCL6 gene, but produce opposite transcriptional regulatory effects; the amount of STAT5 binding to the BCL6 gene promoter site is significantly increased in CD8 T cells treated with TGF-β in vitro. + T cells decreased significantly, and at the same time, the binding amount of SMAD2 at the BCL6 gene promoter site was significantly decreased in IL-2 treated CD8 + T cells were significantly decreased in vitro by TGF-β-treated CD8 + In T cells, the secretion of IL-2 and the expression level of CD25 decreased significantly, indicating that TGF-β may indirectly regulate the expression of BCL6 by inhibiting the IL-2 signaling pathway.

[0212] Example 6: BCL6 and BLIMP1 in CD8 + Plays an antagonistic role in the differentiation of T cells

[0213] BLIMP1 (encoded by PRDM1) is one of the downstream effector proteins of the IL-2-STAT5 pathway. In GCB cells and Tfh cells, BLIMP1 significantly inhibits the expression of BCL6 and antagonizes the function of BCL6. In the acute infection model, BLIMP1 significantly inhibits the expression of memory CD8 + The formation of T cells; in a chronic viral infection model, BLIMP1 significantly inhibits the development of Tfc cells. Therefore, this example needs to verify the expression and function of BLIMP1 in a tumor model. The specific steps are as follows:

[0214] 1. Compare the functional differences between BCL6 and BLIMP1 in tumor models. The specific steps are as follows:

[0215] (1) First, the inventors analyzed the + ChIP-seq data of BLIMP1-Biotin in T cells (GEO number: GSE79339) were obtained, and the expression of BLIMP1 in mouse CD8 + Binding genes in the T cell genome. By comparing the ChIP-seq data for BCL6 obtained in step 1 of Example 3 with the ChIP-seq data for BLIMP1 obtained in this example, the inventors found that BCL6 and BLIMP1 have significantly different binding profiles, as shown in Figure 2A. However, these two transcription factors bind to a large number of the same target genes, with more than half of the genes bound by BCL6 also bound by BLIMP1, as shown in Figure 2B.

[0216] (2) Next, the inventors isolated and purified the co-adoptively delivered PRDM1 (encoding BLIMP1 protein) overexpressing and control OT-I cells from E.G7 tumors, and then obtained RNA-seq data of PRDM1 overexpression by the method described in step 2 of Example 3 (the difference was that PRDM1 (NM_007548.4) was cloned into a retroviral vector expressing IRES-BFP instead of overexpressing BCL6 and TCF1), and the RNA-seq data were analyzed by GSEA.

[0217] GSEA results showed that overexpression of PRDM1 led to a significant expression of BCL6 in OT-I cells. - TIM-3 + T term The transcriptome characteristics of the cells are shown in Figure 2C. Genes differentially expressed by PRDM1 overexpression were significantly enriched in multiple biological signaling pathways, including T cell activation, differentiation, and adhesion, as shown in Figure 2D. Furthermore, the common genes differentially expressed by BCL6 and PRDM1 overexpression showed a significant negative correlation, as shown in Figure 3A. These data collectively suggest that BCL6 and BLIMP1 may mutually regulate the expression of a series of common downstream genes through their specific pathways.

[0218] The inventors further integrated and analyzed the BLIMP1 ChIP-seq data obtained in step 1 of the example and the PRDM1 overexpression RNA-seq data, and obtained 133 genes whose expression was directly induced by BLIMP1 and 132 genes whose expression was directly inhibited by BLIMP1, as shown in Figure 3B. Among them, 27 genes whose expression was directly induced by BLIMP1 were TIM-3. + T term Cell characteristic genes (Havcr2, Cd244a, Il2ra, Cd38, etc.), 41 genes whose expression is directly inhibited by BLIMP1 are BCL6+ T prog The characteristic genes of T cells (Tcf7, Id3, Cxcr5, Ccr7, etc.) are shown in Figure 3C. Therefore, BCL6 and BLIMP1 are the key regulators of T prog and T term Key transcription factors for cell differentiation.

[0219] 2. Verify the expression of BCL6 and BLIMP1 in CD8 tumor models + Molecular mechanisms that play a role in T cell differentiation.

[0220] First, the inventors analyzed the ChIP-seq data and ATAC-seq data (GEO number: GSE122713) of BCL6 obtained in step 1 of this example. The ChIP-seq results showed that in CD8 + In T cells, BCL6 directly binds to multiple sites on the PRDM1 gene, distributed within introns and distal intergenic regulatory regions.

[0221] BCL6 and BLIMP1 co-bind to T prog The cell's characteristic genes Il7r, Sell and Ccr7 are located at different sites and regulate their expression in an inverse manner.

[0222] ATAC-seq results showed that the above gene binding sites were found in tumor-infiltrating CD8 + It is open in T cells, as shown in Figures 4A and 4B. These results suggest that BCL6 and BLIMP1 may co-regulate target gene expression in multiple ways, including directly binding to different sites on the same gene, competitively binding to the same site on the gene, or indirectly inducing downstream gene expression by inhibiting the expression of each other. In addition, although BCL6 and BLIMP1 directly bind to multiple sites on the Ifng gene (interferon-gamma gene), they do not regulate Ifng gene expression, as shown in Figures 3C and 4B.

[0223] 3. Further verify the expression of BCL6 and BLIMP1 in CD8 + It plays an antagonistic role in the differentiation of T cells.

[0224] (1) First, the inventors obtained in vitro activated CD8 T cells that simultaneously overexpressed BCL6 and PRDM1 by the method of step 1 of Example 3. + T cells, and using Magzolreagent (Magen) from CD8 +Total RNA was extracted from T cells. cDNA was then obtained using the M-MLV reverse transcription kit (Invitrogen). The expression of specific genes was then measured using a SYBR real-time kit (Bio-Rad Laboratories). The results showed that BCL6 and PRDM1 reciprocally regulated the transcription of genes such as Tcf7, Cxcr5, Id3, and Havcr2, as shown in Figure 5A.

[0225] (2) Secondly, the inventors obtained OT-I cells that overexpressed both BCL6 and PRDM1 by the method of step 3 (2) of Example 3, and adopted the OT-I cells that overexpressed both BCL6 and PRDM1 and the OT-I cells of the control group to be co-transferred into the Tcrbd - / - The experiment was performed in recipient mice (8 mice) by the method described in step 2 of Example 1. The results showed that overexpression of BCL6 and PRDM1 mutually regulated T prog and T term These results demonstrate that in tumor-specific T prog and T term During cell development, BCL6 and BLIMP1 play mutually opposing regulatory roles.

[0226] (3) The inventors prepared naive CD45.2BLIMP1-EYFP OT-I cells by the method described in step 3(2) of Example 3, and adoptively transferred the naive CD45.2BLIMP1-EYFP OT-I cells into CD45.1 recipient mice (8 mice). Then, E.G7 tumor cells were inoculated one day later. The experiment was performed using the method described in step 2 of Example 1. The experimental process is shown in Figure 6A.

[0227] The results showed that BLIMP1 was significantly overexpressed in tumor-infiltrating OT-I cells after 11 and 20 days. In addition, BLIMP1-EYFP was specifically expressed in TIM-3 + T term cells, but not in LY108 + T prog Furthermore, in the results of single-cell RNA-seq of head and neck cancer and melanoma patients, PRDM1 was significantly overexpressed in tumor-infiltrating CD8 + In T cells, the expression of PRDM1 was mutually exclusive with that of TCF7 and BCL6, as shown in Figures 6B and C.

[0228] 4. Verify the expression of BLIMP1 in CD8 +The molecular mechanism of induction in T cells. Previous studies have reported that BLIMP1 is a downstream transcription factor of the IL-2 signaling pathway. Therefore, the inventors first activated the naive BLIMP1-EYFP CD8 T cells described in step 3 (3) of this example in vitro with anti-CD3 / 28 by implementing the method of step 3 (1). + T cells, and cultured in vitro WT CD8 T cells by the method of step 2 of Example 1 + T cells and PRDM1 - / - CD8 + T cells were stimulated with IL-2 for 6 days, and then BLIMP1-EYFP CD8 + The expression level of BLIMP1 in T cells was detected in the control group, which was CD8 T cells without IL-2 addition. + T cells, hereinafter referred to as control CD8 + T cells.

[0229] The detection results are shown in Figure 7A. First, BLIMP1 is almost not expressed in CD8 + However, on the 6th day of continuous IL-2 stimulation, TIM-3 + CD8 + BLIMP1-EYFP was significantly overexpressed in T cells. + There was no significant expression of BLIMP1-EYFP in T cells. At the same time, under continuous IL-2 stimulation, more than 60% of CD8 + T cell differentiation into TIM-3 + cells; in the absence of IL-2 stimulation, CD8 + Less than 10% of T cells are CD8 + T cells express TIM-3.

[0230] Secondly, in the absence of PRDM1, CD8 + In T cells, CD25high TIM-3 was not induced regardless of the presence or absence of continuous IL-2 stimulation. + The production of cells was not inhibited, and the expression of TCF1 was not suppressed, as shown in Figure 7B. + Compared with PRDM1 in IL-2 stimulated T cells, - / - CD8 + In T cells, the expression level of Granzyme B was significantly decreased, while the expression level of LY108 was significantly increased, as shown in Figure 7C.

[0231] The above results show that under in vitro culture conditions, continuous IL-2 stimulation significantly induced TIM-3 +CD8 + The generation of T cells and the expression of BLIMP1. In addition, IL-2 affects TIM-3 + CD8 + The induction of T cell differentiation and the inhibition of TCF1 expression are dependent on the downstream BLIMP1.

[0232] 5. Verify the effect of BLIMP1 on CD8 + The regulatory role of T cells. The specific steps are as follows:

[0233] The inventors used the naive WT and PRDM1 - / - OT-I cells were adoptively transferred into Tcrbd cells at a 1:1 ratio. - / - The recipient mice (8 mice) were then inoculated with E.G7 tumor cells one day later. The experiment was performed using the method described in step 2 of Example 1 and the experimental results were analyzed.

[0234] The results showed that after 16 days, the loss of PRDM1 induced the + T prog The proportion of cells and BCL6 + and TCF1 + The proportion of cells increased significantly, while TIM-3 + T term The proportion of cells was significantly reduced, as shown in Figure 8A. In addition, the loss of PRDM1 resulted in a significant decrease in the production of Granzyme B, but the expression level of IFN-γ was not significantly affected, while the expression level of TNF-α was significantly increased, as shown in Figure 8B. PRDM1 overexpression resulted in the inhibition of LY108 in tumor-infiltrating OT-I cells. + T prog The proportion of cells and TCF1 + and BCL6 + The proportion of cells was significantly reduced, while TIM-3 + T term The proportion of cells in the T-cell matrix increased significantly, and the loss of PRDM1 had no significant effect on the number of tumor-infiltrating OT-I cells, as shown in Figure 9A. prog The number of cells increased significantly, and T term Overexpression of PRDM1 resulted in a slight decrease in the number of tumor-infiltrating OT-I cells, among which T prog The number of cells decreased significantly, and T termThe number of cells increased significantly, as shown in Figure 9B. In addition, in vivo proliferation results showed that overexpression of PRDM1 did not significantly affect the CTV signal in OT-I cells on days 2 and 3, as shown in Figure 9C, indicating that BLIMP1 has no significant regulatory effect on cell proliferation.

[0235] 6. Verify in CD8 + The expression and function of BLIMP1 during the immune response of T cells to endogenous tumor antigens. The specific steps are as follows:

[0236] (1) The inventors first inoculated B16BL6, EL4, HEPA1-6 and MC38 tumors into BLIMP1-EYFP mice, and then used the method described in step 2 of Example 1 to obtain isolated CD8 + T cells were analyzed by flow cytometry and the results on day 15 were analyzed.

[0237] The results showed that in TDLN, a small number of CD44 + CD8 + T cells expressed BLIMP1-EYFP; in tumors, more than 1 / 3 of CD44 + CD8 + T cells highly expressed BLIMP1-EYFP (BLIMP1 tandem yellow fluorescent protein), as shown in FIG10A .

[0238] (2) In this example, the naive WT CD8 + T cells and PRDM1 - / - CD8 + T cells and CD4 + T cells were adoptively delivered into Tcrbd - / - Recipient mice (8 mice) were inoculated with tumor cells one day later, and the experiment was performed using the method described in step 2 of Example 1 and the experimental results were analyzed.

[0239] The results showed that after 15 days, the loss of PRDM1 led to the + TCF1 in T cells + Granzyme B - T prog The proportion of cells increased significantly, while TCF1 - Granzyme B + T term The proportion of cells in the tumor was significantly reduced, as shown in Figure 10B. These results indicate that under the stimulation of endogenous tumor antigens, BLIMP1 is effective in regulating the expression of tumor-infiltrating T cells. term Cell development is very important.

[0240] Taken together, these results indicate that BLIMP1 significantly inhibits T prog maintenance of cells and promote T term Cell differentiation.

[0241] Example 7: PRDM1 in tumor-specific CD8 + Deletion of T cells significantly enhances therapeutic efficacy in tumor control

[0242] In cancer patients, T prog The number of T cells is positively correlated with better clinical prognosis and responsiveness to immune checkpoint blockade therapy. prog The number of cells can significantly increase CD8 + T cell proliferation after PD-1 / PD-L1 blockade and therapeutic immunotherapy results in better tumor control. Therefore, considering the important role of BCL6 and BLIMP1 in T cell differentiation, it is urgent to verify whether BCL6 overexpression and PRDM1 deficiency can enhance the therapeutic effect of anti-PD-1. The specific steps are as follows:

[0243] 1. Verify that BCL6 overexpression regulates the anti-tumor function of T cells. The specific steps are as follows:

[0244] The inventors adopted the same amount of BCL6-overexpressing OT-I cells or control OT-I cells (the preparation method of the two OT-I cells is shown in step 3 (2) of embodiment 3) and transferred them into Tcrbd cells inoculated with E.G7 tumor. - / - In recipient mice (8 mice), the experiment and detection were performed using the method described in step 2 of Example 1. The detection results are shown in FIG11A .

[0245] The results showed that overexpression of BCL6 significantly impaired the anti-tumor effect of OT-I cells. + T prog The proportion of cells increased significantly, while TIM-3 + T term The proportion of cells was significantly reduced, as shown in Figure 11B. In addition, overexpression of BCL6 led to a significant decrease in the number of tumor-infiltrating OT-I cells, among which T prog and T term The number of cells was significantly decreased, as shown in FIG11C .

[0246] 2. Verify whether anti-PD-1 treatment can significantly amplify BCL6-overexpressing T cells. prog cells, producing effective tumor control effects. The specific steps are as follows:

[0247] The inventors adoptively transferred equal amounts of in vitro activated BCL6-overexpressing OT-I cells and control OT-I cells (for the preparation method of the two OT-I cells, see Implementation 3, Step 3 (2)) into CD45.1 recipient mice (8 mice) inoculated with E.G7 tumors for 7 days, and injected anti-PD-1 antibodies once every 2-3 days starting from the 3rd day after adoptive transfer, for a total of 3 injections (schematic diagram shown in Figure 12A), and performed experiments and detection using the method described in Example 1, Step 2.

[0248] The results showed that although anti-PD-1 could slightly enhance the ability of BCL6-overexpressing OT-I cells to control tumor growth, the tumor growth rate was still faster than that of the recipient mice receiving the control group OT-I cells, as shown in Figures 12B and 12C. By analyzing the tumor-infiltrating donor OT-I cells, this example found that after anti-PD-1 treatment, most BCL6-overexpressing OT-I cells still maintained LY108 + T prog The status of cells is shown in Figure 13A. In addition, the number of BCL6-overexpressing OT-I cells infiltrating the unit tumor, including T prog and T term The number of cells did not increase significantly after anti-PD-1 treatment, as shown in Figure 13B. In addition, overexpression of BCL6 resulted in a decrease in the number of Granzyme B cells per tumor infiltration. + The number of OT-I cells decreased significantly and did not increase significantly after anti-PD-1 treatment, as shown in Figure 13C. Therefore, anti-PD-1 treatment cannot effectively promote the expansion and differentiation of BCL6-overexpressing OT-I cells, and therefore cannot induce a more effective anti-tumor effect. Therefore, in the tumor model, although BCL6 overexpression can effectively maintain T prog However, due to its significant inhibition of T cell proliferation and cytotoxicity, it cannot effectively improve the anti-tumor effect, nor can it produce a combined effect with anti-PD-1.

[0249] 3. Verify the role of PRDM1 in tumor-specific CD8 + The specific steps of the regulation of T cell anti-tumor function by the deletion of T cells are as follows:

[0250] The inventors used equal amounts of in vitro activated CD45.2WT and PRDM1 - / -OT-I cells (for the preparation method of the two OT-I cells, see step 3 (2) of Example 3, the difference is that CRISPR-Cas9 is used to knock out PRDM1 instead of overexpressing BCL6 and TCF1) were adoptively transferred into CD45.1 recipient mice (8 mice) inoculated with E.G7 tumors, and experiments and detection were performed using the method described in step 2 of Example 1.

[0251] The results showed that PRDM1 is expressed in tumor-specific CD8 + Deletion of T cells significantly promotes T prog Cell expansion leads to better tumor control.

[0252] 4. Further validation of PRDM1 in tumor-specific CD8 + The effect of T cell deficiency combined with anti-PD-1. The specific steps are as follows:

[0253] The inventors used equal amounts of in vitro activated CD45.2WT and PRDM1 - / - OT-I cells (for the preparation method of two types of OT-I cells, see step 3 (2) of Example 3, the difference is that CRISPR-Cas9 is used to knock out PRDM1 instead of overexpressing BCL6 and TCF1) were adoptively transferred into CD45.1 recipient mice (8 mice) inoculated with E.G7 tumors, and then anti-PD-1 antibodies were intraperitoneally injected starting from 3 days later, for a total of 3 injections (schematic diagram shown in Figure 14A), and experiments and detection were performed using the method described in step 2 of Example 1.

[0254] The results showed that the loss of PRDM1 and anti-PD-1 treatment can produce more effective tumor control effects, and are significantly better than the effect of adoptive delivery of WT OT-I cells and anti-PD-1 combined, as shown in Figure 14B. By analyzing tumor-infiltrating OT-I cells, the inventors found that the loss of PRDM1 significantly increased TCF1 + T prog The proportion of cells and the TCF1 infiltrating unit tumors were significantly increased + T prog The number of cells infiltrating TCF1 cells per unit cell was further increased by anti-PD-1 treatment. + T prog Furthermore, even though the loss of PRDM1 leads to the term The proportion and number of cells were significantly reduced, but anti-PD-1 treatment significantly increased the number of T cells infiltrating per tumor. term The number of cells increased significantly and reached that of the WT OT-I group, as shown in Figure 14C. In addition, anti-PD-1 treatment also significantly increased the number of Granzyme B per tumor infiltration.+ PRDM1 - / - 15A. In addition, the combination of PRDM1 deficiency and anti-PD-1 significantly increased the ability of OT-I cells to produce IFN-γ and TNF-α, as shown in Figure 15B. + Deletion of T cells significantly promotes T prog The expansion of cells and significantly enhanced their response to anti-PD-1 resulted in better tumor control.

[0255] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0256] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A T cell, characterized in that The expression of a given protein in the T cells is altered, and the given protein is suitable for regulating the expression of BCL6 in the T cells.

2. The T cell according to claim 1, characterized in that The T cells are CD8 + T cells and CD4 + At least one of the T cells is preferably a CD8 T cell that specifically recognizes tumor cells. + T cells.

3. The T cell according to claim 1 or 2, characterized in that The given protein is suitable for regulating the expression of BCL6 via the IL-2-STAT5 pathway and / or the TGF-beta-SMAD2 pathway.

4. The T cell according to claim 1 or 2, characterized in that The expression of BCL6 was upregulated in the T cells.

5. The T cell according to claim 1 or 2, characterized in that The expression of the given protein in the T cells is altered so as to inhibit the IL-2-STAT5 pathway and / or activate the TGF-beta-SMAD2 pathway.

6. The T cell according to claim 1 or 2, characterized in that The given protein includes at least one downstream protein on the IL-2-STAT5 pathway, and the expression of the given protein is downregulated.

7. The T cell according to claim 1 or 2, characterized in that The given protein includes at least one selected from the following: PRDM1 or its homologous family members.

8. The T cell according to claim 6, characterized in that The downregulation of the expression of the given protein is achieved by at least one of gene silencing, gene editing, small molecule inhibitors, and antibody drugs.

9. The T cell according to claim 8, characterized in that The gene silencing or gene editing includes at least one of CRISPR-Cas9 virus and non-virus, TALEN, ZFN, shRNA, and siRNA.

10. The T cell according to claim 7, characterized in that The PRDM1 gene is knocked out in the T cells.

11. The T cell according to claim 1 or 2, characterized in that The expression of BCL6 was downregulated in the T cells.

12. The T cell according to claim 1 or 2, characterized in that The expression of the given protein in the T cells is altered so as to activate the IL-2-STAT5 pathway and / or inhibit the TGF-beta-SMAD2 pathway.

13. The T cell according to claim 1 or 2, characterized in that The given protein includes at least one downstream protein on the IL-2-STAT5 pathway, and the expression of the given protein is upregulated.

14. The T cell according to claim 1 or 2, characterized in that The given protein includes at least one selected from the following: PRDM1 or its homologous family members.

15. The T cell according to claim 13, characterized in that The up-regulation of the expression of the given protein is achieved by at least one of gene transfection, promoter enhancer, and auxiliary expression factor.

16. The T cell according to claim 15, characterized in that The gene transfection is achieved by at least one of liposome, calcium phosphate and PEI.

17. The T cell according to claim 14, characterized in that The PRDM1 gene is upregulated in the T cells.

18. The T cell according to claim 1 or 2, characterized in that The T cell is at least one of a CAR-T cell, a TIL cell and a TCR-T cell.

19. The T cell according to claim 1 or 2, characterized in that The expression of immune checkpoint-related proteins in the T cells was downregulated.

20. The T cell according to claim 19, characterized in that The immune checkpoint-related proteins include at least one of PD-1, CTLA-4, LAG-3, TIM-3, TIGIT and VISTA.

21. A pharmaceutical composition, characterized in that include: An agent for regulating the IL-2-STAT5 pathway and / or the TGF-beta-SMAD2 pathway so as to regulate the expression of BCL6 in T cells; or a T cell according to any one of claims 1 to 20.

22. The pharmaceutical composition according to claim 21, characterized in that The reagent is used to inhibit the IL-2-STAT5 pathway.

23. The pharmaceutical composition according to claim 21, characterized in that The agent inhibits the IL-2-STAT5 pathway by downregulating the expression of a given protein, wherein the given protein comprises at least one selected from the following: PRDM1 or a member of its homologous family.

24. The pharmaceutical composition according to claim 21, characterized in that The reagent includes at least one of small molecule inhibitors, antibody drugs, CRISPR-Cas9 viruses and non-viruses, TALEN, ZFN, shRNA, and siRNA.

25. The pharmaceutical composition according to claim 21, characterized in that The reagent is used to activate the TGF-beta-SMAD2 pathway, and the reagent includes a TGF-beta-SMAD2 pathway activator and a TGF-beta-SMAD3 pathway activator.

26. The pharmaceutical composition according to claim 21, characterized in that The reagent is used to activate the IL-2-STAT5 pathway.

27. The pharmaceutical composition according to claim 21, characterized in that The agent activates the IL-2-STAT5 pathway by upregulating the expression of a given protein, wherein the given protein comprises at least one selected from the following: PRDM1 or a member of its homologous family.

28. The pharmaceutical composition according to claim 21, characterized in that The reagent includes at least one of liposome, calcium phosphate and PEI.

29. The pharmaceutical composition according to claim 21, characterized in that The reagent is used to inhibit the TGF-beta-SMAD2 pathway, and the reagent includes a TGF-beta-SMAD2 pathway inhibitor and a TGF-beta-SMAD3 pathway inhibitor.

30. The pharmaceutical composition according to claim 21, characterized in that Further including: Immune checkpoint blockade.

31. The pharmaceutical composition according to claim 30, characterized in that The immune checkpoint blocker includes at least one of PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, LAG-3 antibody, TIM-3 antibody, TIGIT antibody and VISTA antibody.

32. The pharmaceutical composition according to claim 30, wherein the immune checkpoint blocker is formulated to be suitable for simultaneous administration with the T cells or separate administration.

33. Use of the T cell according to any one of claims 1 to 20 or the pharmaceutical composition according to any one of claims 21 to 32 in the preparation of a drug for treating or preventing cancer.

34. The use according to claim 33, wherein the cancer comprises at least one selected from the group consisting of gastric cancer, lung cancer, pancreatic cancer, liver cancer, breast cancer, cervical cancer, skin cancer, prostate cancer, melanoma, thyroid cancer, uterine fibroids, lymphoma, esophageal cancer, intestinal cancer, bone marrow cancer, nasal cancer, bone cancer, head and neck cancer, oral cancer, kidney cancer, and Kaposi's sarcoma.

35. A drug combination, characterized in that include: Immune checkpoint inhibitors; and The T cell according to any one of claims 1 to 20 or the pharmaceutical composition according to any one of claims 21 to 32.

36. A method for treating cancer, characterized in that: include: Administering the T cell according to any one of claims 1 to 20 or the pharmaceutical composition according to any one of claims 21 to 32 to a patient.

37. The method according to claim 36, characterized in that The method further comprises: administering at least one of PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, LAG-3 antibody, TIM-3 antibody, TIGIT antibody, and VISTA antibody to the patient simultaneously or sequentially.

38. A method for upregulating the content of stem-like cells in immune cells, characterized in that: include: Administering the T cell according to any one of claims 1 to 20 or the pharmaceutical composition according to any one of claims 21 to 32 to a patient; The expression of BCL6 was upregulated in the T cells.

39. A method for inhibiting T cells from term A method for cell transformation, characterized in that include: Upregulating the expression of BCL6 in the T cells; The T cells include CD4 + T cells, CD8 + T cells.

40. The method according to claim 39, characterized in that The upregulation of BCL6 expression in the T cells is achieved by downregulating the expression of a given protein in the T cells, wherein the given protein is suitable for regulating the expression of BCL6 through the IL-2-STAT5 pathway; or the upregulation of BCL6 expression in the T cells is achieved by contacting the T cells with an agent, wherein the agent is used to activate the TGF-beta-SMAD2 pathway.

41. The method according to claim 40, characterized in that The reagents include a TGF-beta-SMAD2 pathway activator and a TGF-beta-SMAD3 pathway activator.

42. The method according to claim 40, characterized in that The given protein includes at least one selected from the following: PRDM1 or its homologous family members.

43. The method according to claim 40, characterized in that The downregulation of the expression of a given protein in the T cells is achieved by at least one of gene silencing or gene editing, small molecule inhibitors and antibody drugs.

44. The method according to claim 43, characterized in that The gene silencing or gene editing includes at least one of CRISPR-Cas9 virus and non-virus, TALEN, ZFN, shRNA, and siRNA.

45. The method according to claim 39, characterized in that The upregulation of BCL6 expression in the T cells is achieved by knocking out the PRDM1 gene in the T cells.

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