CN117568280A

CN117568280A - Modified T cell and application thereof

Info

Publication number: CN117568280A
Application number: CN202311280795.1A
Authority: CN
Inventors: 董晨; 孙勤利; 倪凌
Original assignee: Binoji Shanghai Biotechnology Co ltd; Shanghai Jiaotong University; Renji Hospital Shanghai Jiaotong University School of Medicine
Current assignee: Binoji Shanghai Biotechnology Co ltd; Shanghai Jiaotong University; Renji Hospital Shanghai Jiaotong University School of Medicine
Priority date: 2023-09-28
Filing date: 2023-09-28
Publication date: 2024-02-20

Abstract

The invention discloses a T cell. The expression of a given protein in said T cell is altered, said given protein being adapted to regulate the expression of BCL6 in said T cell; which is a kind ofIn which the T cells are CD8 ⁺ T cells and CD4 ⁺ At least one of the T cells, preferably CD8, specifically recognizing tumor cells ⁺ T cells; the given protein comprises at least one selected from the group consisting of: PRDM1 or a family member thereof. The T cells can maintain the Tprog state, enhance the stem property of the T cells and maintain the long-term survival of the T cells, and can improve the killing capacity of the T cells on tumors, thereby being beneficial to developing novel anti-tumor immunotherapy for treating and preventing cancers; alternatively, T cells of the invention may be used without T depletion _prog Promoting T on the premise of cell number _term The T cells have strong instant killing ability to early tumors containing less tumor cells and can be used for treating early cancers.

Description

Modified T cell and application thereof

Technical Field

The invention relates to the field of medicines, in particular to a modified T cell and application thereof.

Background

Immune checkpoint blocking therapies have shown good clinical promise for treating solid tumors, however, due to the severe depletion of T cells, only a fraction of cancer patients can respond to the therapy. Tumor-specific depletion type CD8 ⁺ T cells (Exhausted CD 8) ⁺ T cells，T _ex ) Mainly comprises two sub-populations, namely a stem cell-like T cell sub-population (T _prog ) And terminally differentiated T cell subsets (T _term )。T _prog The cells are in a relatively quiescent state and have a high level of proliferative potential. Although T _term The cell proliferation activity is high, and the cell has a strong tumor killing function, but cannot survive continuously. In addition, T _prog Cells are a major subset of T cells responding to PD-1/PD-L1 blocking therapy and are capable of rapidly proliferating and differentiating into T _term Cells exert potent antitumor effects. Thus T _prog And T _term Cell synergy mediates long-term and direct antitumor effects, but it is unclear how their differentiation is regulated.

During the depletion and differentiation of T cells, transcription factors exert a central regulatory role. NR4A1 is a transcription factor associated with T cell activation and death at T _ex High expression in cells and promote the depletion of T cells and inhibit the function of T cells by inhibiting the function of c-Jun. T in LCMV viral chronic infection and tumor model _ex In cells, sustained antigen stimulation and activation of NFAT induce high levels of TOX, which regulate T cell depletion and maintenance at the transcriptional and epigenetic levels. TCF1 is a CD8 in naive and memory form ⁺ Transcription factors highly expressed in T cells, also T _prog Characteristic molecules of cells.

In addition to transcription factors, cytokines are found in CD8 ⁺ The process of T cell depletion also plays a very important role. Previous studies reported that IL-27 and IL-2 are T-inducing in tumor models _ex Critical cytokines expressed by co-suppressor molecules in cells. Furthermore, IL-27 is able to significantly increase TCF1 in a chronic viral infection model ⁺ T _prog Viability of the cells; IL-27 is capable of significantly enhancing the anti-tumor ability of T cells in tumor models. Type I interferon significantly enhances T by antagonizing TCF1 _term Development of cells. IL-10 and IL-21 significantly enhance T by activating STAT3 signaling pathways _term The development and effector functions of the cells, thereby enhancing the anti-tumor capability of the T cells. However, in tumor models, how cytokines control T _ex Differentiation of cell subsets is not yet clear.

Therefore, research on specific mechanisms of T cell differentiation regulated by transcription factors and cytokines is needed to modify T cells, and support and help for anti-tumor immunotherapy are provided.

Disclosure of Invention

The present invention aims to solve at least one of the technical problems in the related art to some extent. For this reason, the present invention provides an altered T cell and uses thereof, which can improve its stem properties and maintain T _prog Or can be performed without reducing T _prog Promoting T on the premise of cell number _term The development of the state, thereby improving the killing capacity of the tumor, and providing support and help for anti-tumor immunotherapy.

The present invention has been completed based on the following findings by the inventors:

BCL6 is a transcription factor of the BTB/POZ zinc finger family of proteins, and is also a follicular helper CD4 ⁺ T-cells (Follicular helper CD 4) ⁺ Tcells, tfh cells) and germinal center B cells. Previous studies reported that BCL6 was specific for memory CD8 in a mouse model of acute viral infection and protein immunization ⁺ The development of T cells is important. In a model of chronic LCMV viral infection, BCL6 is highly expressed in TCF1 ⁺ CXCR5 ⁺ T _prog Among the cells, such cells are able to enter the B cell follicles of lymphoid tissues.

However, in the intensive studies of the inventors, it was unexpectedly found that BCL6 can be found in tumor-specific CD8 in tumor models ⁺ High expression in T cells. Although tumor tissue generally does not have lymphoid structures, the aggregate region of antigen presenting cells contains a large number of TCF1 ⁺ T _prog And (3) cells. Thus, the inventors speculate that T is tumor-infiltrating _prog T in cell potential and chronic infection model _prog Cells have different mechanisms of production and maintenance.

Based on this, the inventors further studied BCL6 and T in tumor models _prog Relationship between cells. The inventors have found that over-expression of BCL6 in T cells can cause damage to the T cells themselves. Furthermore, the inventors have unexpectedly found that up-regulating BCL6 in T cells does not cause damage to T cells themselves, but also increases T _prog The cell expansion improves the killing capacity of the cell to the tumor. In addition, the inventors have also unexpectedly found that such technical effects cannot be achieved by merely increasing the copy number of the BCL6 encoding gene in T cells, and that such effects can be achieved only by regulating the expression regulatory mechanism of the BCL6 encoding gene.

Further, the inventors found that BCL6 could be upregulated by downregulating PRDM1 expression, and adoptively delivering PRDM1 deleted CD8 in tumor models ⁺ anti-PD-1 for T cell parallel connection can remarkably promote tumor infiltration T _prog The expansion of the cells produces a more effective anti-tumor effect.

Thus, the present inventionIn a first aspect of the invention, the invention provides a T cell. According to an embodiment of the invention, the expression of a given protein in said T cell is altered, said given protein being adapted to regulate the expression of BCL6 in said T cell. The T cells of the invention can sustain T _prog The state can enhance the stem property of T cells and maintain the long-term survival of the T cells, and can improve the killing capacity of the T cells to tumors, thereby being beneficial to developing novel anti-tumor immunotherapy for treating and preventing cancers. Alternatively, T cells of the invention may be used without T depletion _prog Promoting T on the premise of cell number _term The T cells have strong instant killing ability to early tumors containing less tumor cells and can be used for treating early cancers.

In a second aspect of the invention, the invention provides a pharmaceutical composition. According to an embodiment of the invention, the pharmaceutical composition comprises an agent and the T cell according to the first aspect of the invention. The agents are useful for modulating the IL-2-STAT5 pathway and/or the TGF-beta-SMAD2 pathway in order to up-regulate the expression of BCL6 in T cells. From the foregoing, it is clear that the T cells of the first aspect can maintain T _prog The state can enhance the stem property of the T cells and maintain the long-term survival of the T cells, and can improve the killing capacity of the T cells to tumors. Thus, the use of pharmaceutical compositions containing the agents or T cells can be used to sustain and effectively combat tumor immune responses, which can be helpful in developing novel anti-tumor immunotherapy to treat and prevent cancer. Alternatively, it is known that the T cells according to the first aspect can be used without reducing T _prog Promoting T on the premise of cell number _term The T cells have stronger instant killing ability to early tumors containing fewer tumor cells. Thus, the use of pharmaceutical compositions containing the agents or T cells described above can be used to treat early stage cancers.

In a third aspect of the invention, the invention provides the use of a T cell according to the first aspect of the invention or a pharmaceutical composition according to the second aspect of the invention in the manufacture of a medicament for the treatment or prophylaxis of cancer. The T cells or the pharmaceutical composition can effectively kill tumors, and is helpful for developing novel anti-tumor immunotherapy for treating or preventing cancers.

In a fourth aspect of the invention, the invention provides a combination or kit. According to an embodiment of the invention, the combination or kit comprises an immune checkpoint inhibitor and a T cell according to the first aspect of the invention or a pharmaceutical composition according to the second aspect of the invention. According to the embodiment of the invention, the combined application of the two medicaments has higher tumor killing capability, can effectively kill tumor cells, inhibit tumor growth or reduce tumor volume, and can effectively prevent and treat tumors or cancers.

In a fifth aspect of the invention, the invention provides a method of treating cancer. According to an embodiment of the invention, the method comprises: administering to a patient a T cell according to the first aspect of the invention or a pharmaceutical composition according to the second aspect of the invention. From the foregoing, it can be seen that both the T cells of the first aspect and the pharmaceutical composition of the second aspect have a high tumor killing ability, and are effective in killing tumor cells, inhibiting tumor growth, or reducing tumor volume. Thus, the methods of the invention are effective in the treatment and prevention of cancer.

In a sixth aspect of the invention, a method of up-regulating stem cell-like cell content in immune cells is provided. According to an embodiment of the invention, the method comprises: administering to a patient a T cell according to the first aspect of the invention or a pharmaceutical composition according to the second aspect of the invention. From the foregoing, it can be seen that the aforementioned T cells are in a stem cell-like T cell subset (T _prog ) In the stage, the proportion of stem cell-like cells in immune cells can be increased by adding the T cells. Due to T _prog The cells are in a relatively resting state and have high proliferation potential, so that the immune cells obtained by the method have strong tumor cell killing capacity and can effectively treat and prevent cancers.

In a seventh aspect, the present invention provides a method of inhibiting T cell progression to T _term Methods of cell transformation. According to an embodiment of the invention, the method comprises up-regulating the expression of BCL6 in the T cells; the T cells are selected from CD4 ⁺ T cells, CD8 ⁺ At least one of the T cells. From the front, go upModulation of BCL6 protein in T cells maintains T cells in stem cell-like cells (T _prog ) Status, thereby inhibiting T cell to T _term Cell (terminally differentiated T cells) transformation. Thus the method helps to maintain T in T cells _prog The state, the more durable and effective anti-tumor capability is generated.

Drawings

The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:

FIG. 1 is a flow chart showing the expression of BCL6 at the transcriptional and protein level and the expression of BCL6-RFP at CD8 in example 4 ⁺ Expression in T. Wherein the ordinate of the flow chart in FIG. 1 indicates the amount of cells expressing BCL 6-RFP.

FIG. 2 is a graph showing the comparison of the difference in functions of BCL6 and BLIMP1 in example 6 of the present invention. FIG. 2A is a thermal and Wen diagram of DNA binding centered on the binding sites of BCL6 and BLIMP1, FIG. 2B is a Wen diagram of genes binding to BCL6 and BLIMP1, FIG. 2C is BCL6 ⁺ And TIM-3 ⁺ CD8 ⁺ The expression of the characteristic genes of T cells in the transcriptome of PRDM1 over-expressed and control OT-I cells, and FIG. 2D is a graph showing the results of Gene Ontolog (GO) analysis of the biological signaling pathway of 20 before the significance of the enrichment of the genes whose expression is up-and down-regulated by PRDM1 over-expression.

FIG. 3 is a graph showing the comparison of the difference in functions of BCL6 and BLIMP1 in example 6 of the present invention. FIG. 3A is a correlation analysis of the amounts of gene expression regulated by BCL6 and PRDM1 over-expression, FIG. 3B is a Wen diagram of the gene directly regulated by BLIMP1, FIG. 3C is a BLIMP 1-regulated BCL6 ⁺ And TIM-3 ⁺ CD8 ⁺ Wien diagram of T cell signature genes and gene regulatory network diagram centered on BCL6 and BLIMP 1.

FIG. 4 shows the CD8 of BCL6 and BLIMP1 in tumor in example 6 of the present invention ⁺ Results of molecular mechanisms that play a role in the differentiation process of T cells. FIG. 4A is a ChIP-seq and ATAC-seq diagram of the binding of BCL6 and BLIMP1 at a particular gene locus, and FIG. 4B is a ChIP-seq diagram of the binding of BCL6 and BLIMP1 at a particular gene locus.

FIG. 5 shows CD8 in tumor with BCL6 and BLIMP1 of example 6 of the present invention ⁺ Results of antagonism during T cell differentiation. FIG. 5A is a CD8 activated in vitro by a specific gene ⁺ Expression in T cells, FIG. 5B is the expression of LY108 and TIM-3, wherein the abscissa of the flow chart in FIG. 5B indicates the amount of cells expressing TIM-3 and the ordinate indicates the amount of cells expressing LY 108.

FIG. 6 shows the CD8 in tumors of BCL6 and BLIMP1 in example 6 of the present invention ⁺ Results of antagonism during T cell differentiation. FIG. 6A is a graph showing the expression of BCL6-RFP, BLIMP1-EYFP, LY108 and TIM-3 in OT-I cells, wherein the abscissa in FIG. 6A indicates the amount of cells expressing BLIMP1-EYFP and the ordinate indicates the amount of cells expressing BCL6-RFP, LY108 and TIM-3. FIG. 6B is a UMAP diagram showing the expression of a specific gene, and FIG. 6C is a diagram showing the expression of a specific gene in CD8 ⁺ Violin plots of expression levels in T cell subpopulations.

FIG. 7 shows the expression of BLIMP1 in CD8 in example 6 of the present invention ⁺ Results of induced molecular mechanisms in T cells. FIG. 7A is CD8 activated by TIM-3 and BLIMP1-EYFP in vitro ⁺ Expression in T cells, wherein the abscissa in fig. 7A indicates the amount of cells expressing BLIMP1-EYFP and the ordinate indicates the amount of cells expressing TIM-3. FIG. 7B is a CD8 with CD25, TIM-3 and TCF1 activated in vitro ⁺ Expression in T cells, wherein the abscissa in fig. 7B indicates the amount of cells expressing TIM-3 and the ordinate indicates the amount of cells expressing CD 25. FIG. 7C is CD8 activated in vitro by LY108 and Granzyme B ⁺ Expression in T cells, wherein the abscissa in fig. 7C indicates the expression amounts of expression LY108 and Granzyme B, respectively.

FIG. 8 shows the BLIMP1 vs. CD8 in tumors in example 6 of the present invention ⁺ T cell regulatory results. FIG. 8A is the expression of BCL6 and TCF1 in OT-I cells, wherein the abscissa in FIG. 8A indicates the amount of cells expressing CD8 and the ordinate indicates the amounts of cells expressing TCF1, BCL 6. FIG. 8B is the expression of Granzyme B, IFN- γ and TNF- α in OT-I cells, wherein the abscissa in FIG. 8B indicates the amount of cells expressing TNF- α and the ordinate indicates the amount of cells expressing IFN- γ, granzyme B.

FIG. 9 shows the BLIMP1 vs. CD8 in tumors in example 6 of the present invention ⁺ T cell regulatory results. FIG. 9A is the expression of T-BET, TCF1 and BCL6 in OT-I cells, wherein the abscissa in FIG. 9A indicates the amount of cells expressing T-BET and the ordinate indicates the amount of cells expressing TCF1, BCL 6. FIG. 9B is LY108 in tumor-infiltrating OT-I cells ⁺ And TIM-3 ⁺ The ratio of cells, FIG. 9C, is the dilution of CTV in OT-I cells in the spleen, where the abscissa in FIG. 9C indicates the amount of CTV expressing cells.

FIG. 10 is a CD8 of example 6 of the present invention ⁺ Results of expression and function of BLIMP1 during immune response of T cells to endogenous tumor antigens. FIG. 10A is a CD8 showing infiltration of BLIMP1-EYFP in tumors and draining lymph nodes ⁺ Expression in T cells, wherein the abscissa in fig. 10A indicates the amount of cells expressing BLIMP1-EYFP and the ordinate indicates the amount of cells expressing CD 44. FIG. 10B is a CD8 showing tumor infiltration of TCF1 and Granzyme B ⁺ Expression in T cells, wherein the abscissa in fig. 10B indicates the amount of cells expressing BLIMP1-EYFP and the ordinate indicates the amount of cells expressing CD 44.

FIG. 11 is a graph showing the result of controlling the anti-tumor function of T cells by the overexpression of BCL6 in example 7 of the present invention. FIG. 11A shows the growth curve of E.G7 tumors, and FIG. 11B shows the expression of LY108 and TIM-3 in OT-I cells, wherein the abscissa in FIG. 11B indicates the amount of cells expressing TIM-3 and the ordinate indicates the amount of cells expressing LY 108. FIG. 11C is a unit weight of tumor-infiltrating OT-I cells, LY108 ⁺ And TIM-3 ⁺ Number of OT-I cells.

FIG. 12 is a graph showing that anti-PD-1 treatment in example 7 of the present invention was unable to significantly amplify BCL6 overexpressed T _prog Cells and produce a graph of the results of effective tumor control effects. FIG. 12A is a schematic diagram showing adoptive transport of OT-I cells, E.G7 tumor vaccination and anti-PD-1 treatment, and FIG. 12B, C is a growth curve of E.G7 tumor.

FIG. 13 shows that anti-PD-1 treatment in example 7 of the invention is not capable of significantly amplifying BCL6 overexpressed T _prog Cells and produce a graph of the results of effective tumor control effects. FIG. 13A shows the expression of TCF1 and TIM-3 in OT-I cells,wherein the abscissa in FIG. 13A indicates the amount of cells expressing TIM-3 and the ordinate indicates the amount of cells expressing TCF 1. FIG. 13B is a unit E.G7 tumor-infiltrating OT-I cell, TCF1 ⁺ And TIM-3 ⁺ The number of OT-I cells, FIG. 13C Granzyme B ⁺ Proportion of cells, granzyme B per unit weight E.G7 tumor infiltration ⁺ Number of OT-I cells, wherein the abscissa in fig. 13C indicates the amount of cells expressing TIM-3 and the ordinate indicates the amount of cells expressing Granzyme B.

FIG. 14 is a tumor specific CD8 of PRDM1 in example 7 of the present invention ⁺ Results for deletions in T cells in combination with anti-PD-1. 14A is a schematic diagram of adoptive transport of OT-I cells, E.G7 tumor inoculation and anti-PD-1 treatment, FIG. 14B is a growth curve of E.G7 tumor, FIG. 14C is the expression of TCF1 and TIM-3 in OT-I cells, OT-I cells with unit E.G7 tumor infiltration, TCF1 ⁺ And TIM-3 ⁺ Number of OT-I cells, wherein the abscissa in fig. 14C indicates the amount of cells expressing TIM-3 and the ordinate indicates the amount of cells expressing TCF 1.

FIG. 15 is a tumor specific CD8 of PRDM1 in example 7 of the present invention ⁺ Results for deletions in T cells in combination with anti-PD-1. FIG. 15A is Granzyme B ⁺ Proportion of OT-I cells and Granzyme B per tumor infiltration ⁺ Number of OT-I cells, wherein the abscissa in fig. 15A indicates the amount of cells expressing TIM-3 and the ordinate indicates the amount of cells expressing Granzyme B. FIG. 15B is a graph showing IFN-gamma and TNF-alpha expression in OT-I cells, wherein the abscissa in FIG. 15B indicates the amount of cells expressing TNF-alpha and the ordinate indicates the amount of cells expressing IFN-gamma.

Detailed Description

Embodiments of the present invention are described in detail below. The following examples are illustrative only and are not to be construed as limiting the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

It should be noted that the terms "first," "second," and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implying a number of technical features being indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. Further, in the description of the present invention, unless otherwise indicated, the meaning of "a plurality" is two or more.

The terms "comprising," "including," or "comprising" are used herein in an open-ended fashion, i.e., to include what is indicated by the present invention, and not to exclude other aspects.

The term "pharmaceutical composition" as used herein generally refers to unit dosage forms and may be prepared by any of the methods well known in the pharmaceutical arts. All methods include the step of combining the active ingredient (T cells) with a carrier that constitutes one or more accessory ingredients. Generally, the compositions are prepared by uniformly and intimately bringing into association the active compound with liquid carriers, finely divided solid carriers or both.

In this context, the term "pharmaceutically acceptable" ingredients are substances which are suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e. commensurate with a reasonable benefit/risk ratio.

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 undue adverse side effects (such as toxicity, irritation, and allergic response), commensurate with a reasonable benefit/risk ratio. For example, an "effective amount" or "effective dose" may be used, where "effective amount" or "effective dose" refers to an amount that is functionally or actively produced in and acceptable to a human and/or animal.

As used herein, the term "pharmaceutically acceptable excipients" may include any solvent, diluent or other liquid excipient, and the like, suitable for the particular target dosage form. In addition to the extent to which any conventional adjuvant is incompatible with the compounds of the present invention, such as any adverse biological effects produced or interactions with any other component of the pharmaceutically acceptable composition in a deleterious manner, their use is also contemplated by the present invention.

As used herein, the term "administering" refers to introducing a predetermined amount of a substance into a patient by some suitable means. The T cell or pharmaceutical composition of the invention may be administered by any common route, provided that it reaches the desired tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but the invention is not limited to these illustrated modes of administration. Preferably, the T cells or pharmaceutical compositions of the invention are administered by intravenous injection.

In this context, the term "treatment" refers to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the disease or symptoms thereof, and/or may be therapeutic in terms of partially or completely curing the disease and/or adverse effects caused by the disease. As used herein, "treating" encompasses diseases in mammals, particularly humans, including: (a) Preventing the occurrence of a disease or disorder in an individual susceptible to the disease but not yet diagnosed with the disease; (b) inhibiting disease, e.g., arresting disease progression; or (c) alleviating a disease, e.g., alleviating symptoms associated with a disease. As used herein, "treating" or "treatment" encompasses any administration of a T cell or pharmaceutical composition to an individual to treat, cure, alleviate, ameliorate, reduce or inhibit a disease in the individual, including, but not limited to, administration of a medicament comprising a T cell as described herein to an individual in need thereof.

The invention provides a T cell, a pharmaceutical composition, application of the T cell or the pharmaceutical composition in preparing medicines, a combined medicine or a medicine box, a method for treating cancers, a method for up-regulating the content of stem cell-like cells in immune cells and a method for inhibiting the T cell from going to T _term Methods of cell transformation.

T cell

In a first aspect of the invention, the invention provides a T cell. According to an embodiment of the invention, the expression of a given protein in said T cell is altered, said given protein being adapted to regulate the expression of BCL6 in said T cell. The T cells of the invention are maintainableT-holding _prog The state can enhance the stem property of T cells and maintain the long-term survival of the T cells, and can improve the killing capacity of the T cells to tumors, thereby being beneficial to developing novel anti-tumor immunotherapy for treating and preventing cancers. Alternatively, T cells of the invention may be used without T depletion _prog Promoting T on the premise of cell number _term The T cells have strong instant killing ability to early tumors containing less tumor cells and can be used for treating early cancers.

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

in some alternative embodiments of the invention, the T cell is an engineered T cell.

In some alternative embodiments of the invention, the T cells are derived from isolated T cells in a healthy individual or patient. The patient may be, for example, a tumor patient or other disease patient. In a preferred embodiment of the invention, the patient is a tumor patient.

According to an embodiment of the invention, the T cells are selected from CD8 ⁺ T cells and CD4 ⁺ At least one of the T cells, preferably CD8, specifically recognizing tumor cells ⁺ T cells.

In this context, the term "CD 8 specifically recognizing tumor cells ⁺ T cells "positive for CD44 (CD 44 ⁺ )CD8 ⁺ T cells.

Exemplary, in particular embodiments of the invention, CD8 specifically recognizing tumor cells is detected using flow cytometry ⁺ T cells, CD44 ⁺ CD8 ⁺ T cells (CD 44 positive CD 8) ⁺ T cells) are determined to specifically recognize CD8 of tumor cells ⁺ T cells.

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

According to an embodiment of the invention, the given protein is adapted to regulate the expression of BCL6 via the IL-2-STAT5 pathway and/or the TGF-beta-SMAD2 pathway.

According to an embodiment of the invention, the expression of immune checkpoint related proteins in the T cells is down-regulated.

Herein, the term "expression of an immune checkpoint related protein is down-regulated" means that the expression level of the immune checkpoint related protein is reduced. In particular, the expression of immune checkpoint related proteins in the engineered T cells is increased compared to T cells prior to the engineering.

It should be noted that "down-regulating the expression of immune checkpoint related proteins" in the present invention can be achieved by methods conventional in the art, including but not limited to gene silencing or gene editing systems (CRISPR-Cas 9 virus and non-virus, shRNA, siRNA), small molecule inhibitors and antibody drugs (immune checkpoint related protein antibodies).

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

According to an embodiment of the invention, the expression of BCL6 in said T cells is up-regulated.

In this context, the term "the expression of BCL6 is up-regulated" means that the expression level of BCL6 protein in the T cells after engineering is increased compared to the T cells before engineering.

Illustratively, the T cells prior to non-engineering are isolated T cells in a tumor-infiltrating individual; by "up-regulated" expression of BCL6 is meant that the expression level of BCL6 protein in the T cells of the invention is increased compared to isolated T cells.

According to an embodiment of the invention, the expression of the given protein in the T cell is altered in order to inhibit the IL-2-STAT5 pathway and/or activate the TGF-beta-SMAD2 pathway.

According to an embodiment of the invention, 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.

In this context, the term "the expression of a given protein is down-regulated" means that the expression level of the given protein in the T cell after engineering is reduced compared to the T cell before engineering.

Illustratively, the T cells prior to non-engineering are isolated T cells in a tumor-infiltrating individual; by "the expression of a given protein is down-regulated" is meant that the expression level of the given protein in the T cell of the invention is reduced compared to an isolated T cell.

According to an embodiment of the invention, the down-regulation of the expression of the given protein is achieved by at least one of gene silencing, gene editing, small molecule inhibitors and antibody drugs.

In some alternative embodiments of the invention, the small molecule inhibitor comprises at least one of STAT5a/b, STAT3, STAT1, and STAT4 small molecule inhibitors.

In some alternative embodiments of the invention, the antibody drug comprises at least one of blocking antibodies to IL-2, IL-2Rα, IL-2β, and IL-2Rγ.

According to an embodiment of the invention, the gene silencing or gene editing comprises CRISPR-Cas9 virus and at least one of non-virus, TALEN, ZFN, shRNA and siRNA.

According to an embodiment of the invention, the given protein comprises at least one selected from the group consisting of: PRDM1 or a family member thereof.

According to an embodiment of the invention, the PRDM1 gene is knocked out in the T cell.

According to an embodiment of the invention, the PRDM1 gene is knocked down in the T cell.

According to an embodiment of the invention, the expression of BCL6 in the T cells is down-regulated. Thus, the T cells can be used without reducing T _prog Promoting T on the premise of cell number _term The T cells have strong instant killing ability to early tumors containing less tumor cells and can be used for treating early cancers.

In this context, the term "down-regulated" of BCL6 expression means that the expression level of BCL6 protein in the engineered T-cells is reduced compared to the T-cells prior to the engineering.

Illustratively, the T cells prior to non-engineering are isolated T cells in a tumor-infiltrating individual; by "down-regulated" expression of BCL6 is meant that the expression level of BCL6 protein in the T cells of the invention is reduced compared to isolated T cells.

According to an embodiment of the invention, the expression of the given protein in the T cell is altered in order to activate the IL-2-STAT5 pathway and/or to inhibit the TGF-beta-SMAD2 pathway.

According to an embodiment of the invention, the given protein comprises at least one downstream protein on the IL-2-STAT5 pathway, and the expression of the given protein is up-regulated.

In this context, the term "the expression of a given protein is up-regulated" means that the expression level of the given protein in the T cell after engineering is increased compared to the T cell before engineering.

Illustratively, the T cells prior to non-engineering are isolated T cells in a tumor-infiltrating individual; by "the expression of a given protein is up-regulated" is meant that the expression level of the given protein in the T cell of the invention is increased compared to an isolated T cell.

According to an embodiment of the invention, the up-regulation of the expression of the given protein is achieved by at least one of gene transfection, promoter enhancers and co-expression factors.

In some alternative embodiments of the invention, the promoter enhancer comprises at least one of a CMV promoter enhancer, an EF1 alpha promoter enhancer, a CD2 promoter enhancer, and an IL-2 promoter enhancer.

In some alternative embodiments of the invention, the accessory expression factor comprises at least one of a T cell activating factor, NF-. Kappa. B, CD28 costimulatory factor, and IL-2 cytokine.

According to an embodiment of the invention, the gene transfection is achieved by at least one of liposomes, calcium phosphate and PEI.

According to an embodiment of the invention, the PRDM1 gene is up-regulated in the T cells.

Pharmaceutical composition

In a second aspect of the invention, the invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprisesAn agent for modulating the IL-2-STAT5 pathway and/or the TGF-beta-SMAD2 pathway, so as to modulate BCL6 expression in T cells; or a T cell according to the first aspect of the invention. From the foregoing, it is clear that the T cells of the first aspect can maintain T _prog The state can enhance the stem property of the T cells and maintain the long-term survival of the T cells, and can improve the killing capacity of the T cells to tumors. Thus, the use of the pharmaceutical composition containing the T cells can permanently and effectively prevent an antitumor immune response, and contribute to the development of a novel antitumor immunotherapy for the treatment and prevention of cancer. Alternatively, it is known that the T cells according to the first aspect can be used without reducing T _prog Promoting T on the premise of cell number _term The T cells have stronger instant killing ability to early tumors containing fewer tumor cells. Thus, the use of pharmaceutical compositions containing the agents or T cells described above can be used to treat early stage cancers.

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

According to an embodiment of the invention, the agent inhibits the IL-2-STAT5 pathway by down-regulating the expression of a given protein comprising at least one selected from the group consisting of: PRDM1 or a family member thereof.

According to an embodiment of the invention, the agent comprises at least one of a small molecule inhibitor, an antibody drug, CRISPR-Cas9 virus and non-virus, TALEN, ZFN, shRNA and siRNA.

According to embodiments of the invention, the agent is used to activate the TGF-beta-SMAD2 pathway, the agent comprising a TGF-beta-SMAD2 pathway activator and/or a TGF-beta-SMAD3 pathway activator.

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

According to an embodiment of the invention, the agent activates the IL-2-STAT5 pathway by up-regulating the expression of a given protein comprising at least one selected from the group consisting of: PRDM1 or a family member thereof.

According to an embodiment of the invention, the agent comprises at least one of liposomes, calcium phosphate and PEI.

According to embodiments of the invention, the agent is used to inhibit the TGF-beta-SMAD2 pathway, the agent comprising a TGF-beta-SMAD2 pathway inhibitor and/or a TGF-beta-SMAD3 pathway inhibitor.

According to an embodiment of the present invention, the pharmaceutical composition further comprises: immune checkpoint blockers.

In some alternative embodiments of the invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

In some alternative embodiments of the invention, the pharmaceutical composition is a combination or kit.

It should be noted that the immune checkpoint blocker in the present invention should be understood in a broad sense, including but not limited to pharmaceutical agents, therapeutic methods, etc., as long as blocking of immune checkpoints can be achieved, which are all within the scope of the present invention.

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

In some alternative embodiments of the invention, the immune checkpoint blocker may be further achieved by methods including, but not limited to, tyrosine kinase inhibitors, co-stimulatory molecule monoclonal antibodies, epigenetic therapeutic drugs, chemotherapeutic drugs, radiation therapy formulations, vaccines, PDCD1 knockout therapies, PD1, PDL1 antibodies, or viral vector-based gene knockout approaches.

In some alternative embodiments of the invention, the immune checkpoint blocker further comprises a CAR-T, CAR-NK, tumor vaccine, oncolytic virus vaccine, TLR7/8 agonist, anti-CD 47 or IL-2 receptor agonist.

According to an embodiment of the invention, the immune checkpoint blocker is formulated for simultaneous administration with the T cells or for separate administration.

Use of T cells or pharmaceutical compositions in the preparation of a medicament

According to an embodiment of the invention, the cancer comprises at least one selected from the group consisting of: stomach cancer, lung cancer, pancreatic cancer, liver cancer, breast cancer, cervical cancer, skin cancer, prostate cancer, melanoma, thyroid cancer, uterine fibroids, lymphatic cancer, esophageal cancer, intestinal cancer, bone marrow cancer, nasal cancer, bone cancer, head and neck cancer, oral cancer, renal cancer, kaposi's sarcoma.

Combination or kit

Methods of treating cancer

In a fifth aspect of the invention, the invention provides a method of treating cancer. According to an embodiment of the invention, the method comprises: administering to a patient a T cell according to the first aspect of the invention and a pharmaceutical composition according to the second aspect of the invention. From the foregoing, it can be seen that both the T cells of the first aspect and the pharmaceutical composition of the second aspect have a high tumor killing ability, and are effective in killing tumor cells, inhibiting tumor growth, or reducing tumor volume. Thus, the methods of the invention are effective in the treatment and prevention of cancer.

According to an embodiment of the invention, further comprising administering at least one of a PD-1 antibody, a PD-L1 antibody, a CTLA-4 antibody, a LAG-3 antibody, a TIM-3 antibody, a TIGIT antibody, and a VISTA antibody to the patient simultaneously or sequentially.

The effective amount of the active ingredient (T cells, and at least one of PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, LAG-3 antibody, TIM-3 antibody, TIGIT antibody, VISTA antibody) described herein may vary depending on the mode of administration, the severity of the disease to be treated, and the like. The selection of the preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life etc.; the severity of the disease to be treated in the patient, the weight of the patient, the immune status of the patient, the route of administration, etc. For example, separate doses may be administered several times per day, or the dose may be proportionally reduced, as dictated by the urgent need for the treatment of the condition.

Method for up-regulating stem cell-like cell content in immune cells

In a sixth aspect of the invention, a method of up-regulating stem cell-like cell content in immune cells is provided. According to an embodiment of the invention, the method comprises the following steps: administering to a patient a T cell according to the first aspect of the invention or a pharmaceutical composition according to the second aspect; the expression of BCL6 in the T cells is up-regulated. From the foregoing, it can be seen that the aforementioned T cells are in a stem cell-like T cell subset (T _prog ) In the stage, the proportion of stem cell-like cells in immune cells can be increased by adding the T cells. Due to T _prog The cells are in a relatively resting state and have high proliferation potential, so that the immune cells obtained by the method have strong tumor cell killing capability and can effectively treat and prevent cancers or tumors according to the embodiment of the invention.

Inhibition of T cell to T _term Method for cell transformation

In a seventh aspect of the invention, the invention provides a method of inhibiting T cell transformation of a T cell into a terminally differentiated T cell. According to an embodiment of the invention, the method comprises up-regulating the expression of BCL6 in the T cells; the T cells are selected from CD8 ⁺ T cells and CD4 ⁺ At least one of the T cells. From the foregoing, it was found that up-regulation of BCL6 protein in T cells can maintain T cells in stem cell-like cells (T _prog ) Status, thereby inhibiting T cell to T _term Cell (terminally differentiated T cells) transformation. Thus, according to embodiments of the invention, the method helps to maintain T in T cells _prog The state, the more durable and effective anti-tumor capability is generated.

According to an embodiment of the invention, said up-regulating the expression of BCL6 in said T cells is achieved by down-regulating the expression of a given protein in said T cells, said given protein being used to inhibit the IL-2-STAT5 pathway; or said up-regulating expression of BCL6 in said T cells is achieved by contacting said T cells with an agent for activating the TGF-beta-SMAD2 pathway.

According to embodiments of the invention, the agent comprises a TGF-beta-SMAD2 pathway activator, a TGF-beta-SMAD3 pathway activator.

According to an embodiment of the invention, said down-regulating the expression level of a given protein in said T-cells is achieved by at least one of gene silencing or gene editing, small molecule inhibitors and antibody drugs.

According to an embodiment of the invention, said up-regulating the expression of BCL6 in said T cells is achieved by knocking out the PRDM1 gene in said T cells.

In some alternative embodiments of the invention, said knocking out the PRDM1 gene in said T cell is accomplished by at least one of CRISPR-Cas9, shRNA and siRNA.

Illustratively, in a specific embodiment of the invention, PRDM1 is knocked out in OT-I cells by using CRISPR-Cas9 technology, and T cells from which PRDM1 is knocked out are combined with anti-PD-1 to detect the killing capacity of E.G7 tumors of a CD45.1 receptor mouse.

The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Example 1: BCL6 selectively highly expressed in tumor stem cell-like CD8 ⁺ In T cells

Previous studies reported that BCL6 was selectively expressed in stem cell-like CD8 in a mouse chronic viral infection model ⁺ T cells. Therefore, this example demonstrates the expression of BCL6 in tumor models. The method comprises the following specific steps:

1. first, single cell RNA-seq data from patients with cervical cancer and melanoma were subjected to dimension reduction and cluster analysis (GEO No. GSE103322 and GSE 120575) with dimension reduction and cluster analysis software of Seurat (version 3.2.2), find Neighborsand, find Clustersfunction and Feature Plotfunction. Second, CD8 was identified by the expression of CD3E, CD8B, CD and FOXP3 molecules ⁺ T cells, two major cell populations are obtained. Stem cell-like TCF1 with remarkably high expression of mouse B16-GP33 tumor infiltration by one cell population ⁺ Characteristic genes of P14 cells, including TCF7, CCR7, IL7R, and the like, are thus identified as T _prog A cell subpopulation; while another cell subset significantly highly expresses terminally differentiated TCF1 infiltrated by the mouse B16-GP33 tumor ^- Characteristic genes of P14 cells, including GZMB, PRF1, HAVCR2, PRDM1 (encoding BLIMP1 protein), PDCD1 and TOX, etc., are thus identified as T _term A cell subpopulation. By T of _term Cell subsets and T _prog Analysis of the expression level of BCL6 in cell subsets revealed that BCL6 expression was significantly enriched in T in head and neck cancer and melanoma patients _prog In cells.

2. The expression level of BCL6 was further verified in a mouse tumor model as follows:

the C57BL/6 mice were divided into two groups of 8 mice. Both groups of mice were inoculated subcutaneously with E.G7 tumor cells and B16-OVA tumor cells, respectively. On day 21 after inoculation, takeE.G7 and B16-OVA tumors, tumor draining lymph nodes and spleen of mice were developed. The isolated CD8 was obtained by obtaining tumor-infiltrating lymphocytes by density gradient centrifugation with 30% and 70% Percoll (GE) solutions and single cell suspensions by grinding tumor draining lymph nodes and spleen, respectively ⁺ T cells. Naive forms were then detected by flow cytometry (CD 44 ^- ) Is CD8 of the outer circumference of (C) ⁺ T cells, post-activation (CD 44) ⁺ ) Is CD8 of the outer circumference of (C) ⁺ T cells and tumor-infiltrating PD-1 ^- CD8 ⁺ T cells and PD-1 ⁺ CD8 ⁺ Expression of BCL6 in T cells. As a result, it was found that, on day 21 after inoculation, E.G7 and B16-OVA tumors infiltrated PD-1 ⁺ CD8 of (C) ⁺ Significant expression of BCL6 in T cells, however tumor-infiltrating PD-1 ^- CD8 of (C) ⁺ CD8 of naive forms (T cells that leave thymus after T cell maturation have not encountered antigen) in T cells, spleen and lymph nodes ⁺ T cells and activated CD8 ⁺ T cells, there is little significant BCL6 expression.

3. Further analysis of BCL6 on tumor antigen specific CD8 ⁺ Expression levels in T cells are as follows:

will specifically recognize OVA _257-264 Is adoptively transferred to C57BL/6 receptor mice (8 mice) and then vaccinated with e.g7 tumor cells after 1 day. Spleen and lymph nodes were then removed from CD45.1 OT-I mice and then used with Dynabeads ^TM FlowComp ^TM Separation and purification of CD8 by using Mouse CD8 Kit (Invitrogen) kit ⁺ T cells. CD25 is then conjugated with a flow cytometric antibody ^- CD44low CD62L high naive OT-I cells were labeled, and then the naive OT-I cells were sorted by FACSaria (BD) flow cytometer to obtain isolated naive OT-I cells, which were then tested and examined as described in step 2 of this example. As a result, it was found that BCL6 was continuously highly expressed in E.G7 tumor-infiltrated OT-I cells from day 11 to day 20. Furthermore, BCL6 is selectively expressed at T _prog In cells, but not expressed in T _term In cells.

4. Is thatFurther detection of BCL6 ⁺ CD8 ⁺ Transcriptome characterization of T cells the inventors isolated infiltrated BCL6-RFP from E.G7 tumors of BCL6-RFP (BCL 6 tandem red fluorescent protein) reporter mice ⁺ TIM-3 ^- Cell (BCL 6) ⁺ CD8 ⁺ T) and BCL6-RFP ^- TIM-3 ⁺ Cells (TIM-3 is T) _term Marker molecules of cells), using Magzolreagent (Magen) from CD8 ⁺ Total RNA is extracted from T cells. The transcriptome was pooled and high throughput sequenced to BGI Genomics. The cDNA library was sequenced by Illumina Hiseq4000 from BGI Genomics. The sequenced data were analyzed using Trim galorev0.4.4, boot 2 and feature Counts. The differentially expressed genes were screened with DESeq2 according to the FDR adjusted p-value0.05 standard. The results showed that BCL6 ⁺ CD8 ⁺ T cell transcriptome high expression B16-GP33 tumor infiltration TCF1 ⁺ Characteristic genes of P14T cells (i.e., stem cell-like cells) include Tcf7, id3, cxcr5, and Ccr7, among others. Thus, BCL6 ⁺ CD8 ⁺ T cells are T _prog Cells, further demonstrating that BCL6 is selectively and highly expressed in T of tumor infiltration _prog In cells.

5. Further detecting the moment when the expression of BCL6 is induced, the specific steps are 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 inoculated e.g7 tumor cells subcutaneously after one day, performed experiments and assays using the method described in step 2 of this example, and analyzed BCL6 expression in recipient mice' tumors and TDLN (tumor draining lymph node) -infiltrated OT-I cells.

The results showed that BCL6 was not significantly expressed in TDLN-infiltrated naive OT-I cells and early-expanded OT-I cells, but BCL6 expression was significantly elevated around day 9 after the OT-I cells underwent multiple rounds of expansion; in addition, on day 9 or so after the OT-I cells underwent multiple rounds of expansion, the T-cell depleted marker molecule TOX was significantly expressed in the OT-I cells, and BCL6 was selectively expressed in TOX ⁺ High level expression in OT-I cells, but in TOX ^- Not expressed in OT-I cells; in addition, atIn TDLN, TCF1 is expressed at high levels in naive OT-I cells, and TCF1 is expressed continuously in OT-I cells after undergoing multiple rounds of proliferation. Furthermore, in TDLN, LY108 is highly expressed in naive OT-I cells, and its expression level is further increased in the OT-I cells after proliferation.

In conclusion, in TDLN, early proliferating OT-I cells had high levels of expression of TCF1, LY108 and CD62L, but little expression of TOX and BCL 6. On day 9, OT-I cells differentiated into depleted TOX ⁺ BCL6 ⁺ Cells also maintained high levels of expression of TCF1 and LY108, while significant downregulation of expression levels of CD62L occurred. In tumors, CFSE in tumor-infiltrated 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 population of cells is T tumor-infiltrating _prog And (3) cells. In tumor-infiltrating OT-I cells, CD62L expression levels were low. Taken together, these results demonstrate that in tumor models, BCL6 expression is depleted T _prog One of the marker characteristics of cells.

6. Previous studies have shown that T using adoptive transport experiments _prog Cells can differentiate into T _term And (3) cells. Next, the inventors further validated BCL6 in tumor models using lineage tracing systems ⁺ CD8 ⁺ T cell differentiation ability. The method comprises the following specific steps:

BCL6 ^CreERT2x Rosa26 ^tdTomato Naive CD8 of OT-I mice ⁺ T cells were adoptively delivered to CD45.1 receptor mice (8 mice) and then inoculated with e.g7 tumor cells after 1 day and injected intraperitoneally once daily at a concentration of 0.2mg Tamoxifen (T5648; sigma-Aldrich) per gram of body weight on days 7-9 for experiments and detection using the procedure described in step 2 of this example.

The results show that on day 11, almost all BCL6 ^- tdTomato ⁺ OT-I cells were LY108 ⁺ TIM-3 ^- T of (2) _prog A cell; however, as the tumor progresses, part of the OT-I cells differentiate into LY108 ^- TIM-3 ⁺ T _term And (3) cells. In addition, as the tumor develops and progresses, BCL6 ⁺ LY108 ⁺ Progressive differentiation of OT-I cells into LY108 ^- CX3CR1 ⁺ And LY108 ^- CD39 ⁺ T _term A cell; BCL6 ^- tdTomato ⁺ The proportion of OT-I cells gradually increased, increasing by about 1.5-fold from day 11 to day 21. Thus, these results may indicate that BCL6 in tumors ⁺ CD8 ⁺ T cells are true T _prog Cells and can be continuously differentiated into T _term And (3) cells.

Example 2: BCL6 does not regulate the production of tumor Tprog cells, but is essential in its long-term maintenance

Previous studies reported that BCL6 was at T in a chronic viral infection model _prog Are essential in the development of cells. Similarly, on day 8, in LCMV cl13 virus infection model, deletion of BCL6 resulted in T _prog The cells almost completely disappeared. Therefore, this example demonstrates the role of BCL6 in tumor models. The method comprises the following specific steps:

1. verification of BCL6 on tumor-specific CD8 ⁺ The role in T cells is as follows:

OT-I cells and BCL6 of the same number of naive WT ^-/- BCL6 was knocked out by CRISPR-Cas9 technique in the OT-I cells of naive WT, and the preparation of both OT-I cells was performed as described in example 1, step 3) by adoptively delivering them to CD45.1 receptor mice (8 mice), respectively, and inoculating e.g7 tumor cells after 1 day, and experiments were performed as described in example 1, step 2.

The results show that the absence of BCL6 significantly enhances the control of tumor growth in the recipient mice by OT-I cells, while leading to TIM-3 tumor infiltration ⁺ The proportion of cells was significantly increased, while LY108 ⁺ The proportion of cells is significantly reduced. The number of OT-I cells per unit weight of tumor infiltration was judged for CD8 ⁺ One of the important criteria for T cell antitumor capacity, the inventors therefore compared the number of OT-I cells per unit weight of tumor infiltration. The results show that the absence of BCL6 results in a unit weight of tumor-infiltrating OT-I cellsIs significantly increased in number, especially T _term Number of cells. However, on day 16, e.g7 tumor infiltrated BCL6 ^-/- OT-I cells still contain a population of T _prog Cells, and, per unit weight of tumor-infiltrated T _prog The number of cells was not affected by the absence of BCL6, suggesting that BCL6 was responsible for T in the early stages of tumor development _prog The production of cells is not necessary. Furthermore, the absence of BCL6 leads to TCF1 in tumor-infiltrating OT-I cells ⁺ T _prog The proportion of cells and the expression level of EOMES were significantly reduced, whereas CX3CR1 ⁺ T _term The proportion of cells and the expression level of T-bet are significantly increased. The absence of BCL6 resulted in a significant increase in the expression levels of IFN- γ, granzyme B and Ki-67 (a biomarker of effector function and proliferative activity of T cells), indicating a significant increase in effector function and proliferative activity of OT-I cells. Whereas on day 8, in the chronic LCMV cl13 virus infection model, the deletion of BCL6 resulted in T _prog The number of cells is significantly reduced.

Taken together, these results demonstrate that BCL6 is CD8 in tumor and chronic viral infection models ⁺ T cells play a different role in the chronic viral infection model T _prog Cell production is indispensable but does not affect T in short-term tumor models _prog And (3) generating cells.

2. Verify BCL6 at T _prog Plays an important role in the long-term maintenance of cells. The method comprises the following specific steps:

the OT-I cells and BCL6 of WT were isolated from E.G7 tumors of mice in step 1 of this example by the method of step 3 of example 1 ^-/- Is then adoptively transferred to the second E.G7 tumor-vaccinated Tcrbd ^-/- In recipient mice (8 mice), experiments and assays were performed using the methods described in example 1, step 2.

The results show that the deletion of BCL6 results in a significant increase in tumor growth rate in the second recipient mice. In tumors and TDLN, TCF1 ⁺ T _prog Cells were successfully maintained in part of WT OT-I cells but were not present in BCL6 ^-/- OT-I cellsIs a kind of medium. Furthermore, the absence of BCL6 results in a significant reduction in the number of tumor and TDLN-infiltrated OT-I cells, where T _prog And T _term The number of cells was significantly reduced. Deletion of BCL6 results in Granzyme B, a unit tumor infiltration ⁺ The number of OT-I cells is significantly reduced, leading to a significant impairment of IFN-gamma production. Thus, these results indicate that BCL6 is at T _prog Long-term maintenance of cells and tumor antigen-specific CD8 ⁺ T cells are essential for their long-term antitumor function.

3. The function of BCL6 was further studied in a B16-OVA tumor model. The method comprises the following specific steps:

the experimental methods of step 1 and step 2 in this example were used to perform the experiments, with the difference that the inoculated tumor cells were different, i.e., the E.G7 tumor cells in step 1 and step 2 were replaced with B16-OVA tumor cells.

The results show that at days 14 and 22, the absence of BCL6 resulted in T in tumor-infiltrating OT-I cells _prog Cell proportion is significantly reduced, while T _term The proportion of cells increased significantly, and at day 22, the absence of BCL6 resulted in T _prog The subpopulations completely disappeared. Meanwhile, on day 14, the absence of BCL6 did not affect T of tumor infiltration _prog Number of cells; however, on day 22, the deletion of BCL6 resulted in T _prog The number of cells was significantly reduced, indicating that BCL6 was at T _prog Is essential for the maintenance of cells. Furthermore, the absence of BCL6 leads to T of tumor infiltration _term The number of cells increased significantly on day 14, but decreased significantly on day 22, suggesting that the loss of BCL6, although transiently promoting T _term Development of cells, T _term The cells can have stronger instant killing ability to early tumors containing fewer tumor cells.

4. Further validation of T of BCL6 in TDLN _prog Cells address functions in secondary tumor challenges. T of TDLN infiltration _prog Cells were identified as memory CD8 ⁺ T cells can migrate into tumors continuously and exert a sustained antitumor effect. T except for tumor infiltration _prog Cells, BCL6, also highly expressed T in TDLN _prog CellsBut their differentiation and number are not affected by the BCL6 deficiency. Therefore, there is a need to verify the role of BCL6 in TDLN models. The method comprises the following specific steps:

WT OT-I cells and BCL6 were isolated from TDLN of the mice described in step 1 of this example by the method of step 3 of example 1 ^-/- LY108 ⁺ TIM-3 ^- OT-I cells, then adoptively delivered in equal amounts to CD4 reception ⁺ Tcrbd of T cells ^-/- In recipient mice (8 mice) and after one day were vaccinated with e.g7 tumor cells, then on day 16 the recipient mice were tested and examined using the method described in example 1, step 2.

The results show that in these secondary receptor mice BCL6 is continuously expressed in tumor and TDLN infiltrated T _prog In cells. The absence of BCL6 results in a significant impairment of the ability of OT-I cells to control tumors in secondary recipient mice. Deletion of BCL6 results in TCF1 for tumor infiltration ⁺ T _prog Failure of the cells to maintain and a significant reduction in the number of tumor-infiltrating OT-I cells, wherein T _prog And T _term The number of cells was significantly reduced. However, in TDLN, the deletion of BCL6 does not affect TCF1 ⁺ T _prog Ratio and number of cells. Furthermore, although deletion of BCL6 resulted in Granzyme B with tumor infiltration ⁺ The proportion of OT-I cells increases significantly, but their cell number decreases significantly. In addition, the absence of BCL6 results in significantly impaired ability of tumor-infiltrating OT-I cells to secrete IFN- γ, but does not affect the ability to secrete TNF- α. These results demonstrate that BCL 6T in TDLN _prog Cells are indispensable in dealing with secondary tumor challenges, however, the absence of BCL6 does not affect T in TDLN _prog Cell production and maintenance.

5. Further validation of BCL6 on CD8 ⁺ T cells should play a role in the immune response against endogenous tumor antigens. The method comprises the following specific steps:

from CD45.1WT mice and CD45.2 BCL6 fl/fl ^Cd8aCre The WT CD8 was isolated and purified from mice by the method of example 1, step 2, respectively ⁺ T cells and BCL6 ^-/- Naive CD8 ⁺ T cells (CD 8 in naive WT) ⁺ T cellKnocking out BCL6 using CRISPR-Cas9 is referred to as BCL6 ^-/- ) WT CD8 was then removed ⁺ T cells and BCL6 ^-/- Naive CD8 ⁺ T cells and CD4, respectively ⁺ Simultaneous adoptive transport of T cells to Tcrbd ^-/- In recipient mice (8 mice), B16BL6, EL4, HEPA1-6 and MC38 tumor cells were then inoculated subcutaneously after 1 day, and experiments and assays were performed using the method described in example 1, step 2.

The results show that on day 15, the inventors first examined BCL6 in these donor CDs 8 ⁺ Expression levels in T cells. And BCL6 significantly expressed CD8 in TDLN ⁺ In T cells and exhibits mutual exclusion with the expression of Granzyme B. However, in tumors, almost all CD8 ⁺ TILs all differentiate into TCF1 ^- Granzyme B ⁺ T _term Cells, and does not express BCL6. Deletion of BCL6 results in tumor infiltration of CD8 ⁺ TCF1 in T cells ⁺ Granzyme B ^- T _prog The proportion of cell subsets was significantly reduced, whereas TCF1 ^- Granzyme B ⁺ T _term The proportion of cell subsets increases significantly. These results demonstrate that in CD8 ⁺ In the immune response process of T cells to endogenous tumor antigen stimulation, the deletion of BCL6 significantly promotes T _prog Differentiation of cells into T _term And (3) cells.

Example 3: CD8 in BCL6 transcriptional regulated tumors ⁺ Differentiation of T cells

1. Systematic study of BCL6 modulation of CD8 ⁺ Molecular mechanisms of T cells. The method comprises the following specific steps:

the inventors obtained isolated CD8 by the method described in example 1, step 2 ⁺ T cells. Next, in vitro activation was performed with a flat-bottom plate conjugated with anti-CD3 (BioXcell, clone145-2C11,5. Mu.g/ml) and anti-CD28 (BioXcell, clone37.51, 1. Mu.g/ml) antibodies to obtain in vitro activated CD8 ⁺ T cells. BCL6-HA (nm_ 001348026.1) was then cloned into a retroviral vector expressing IRES-GFP. Taking empty vector of retrovirus as control vector, transfecting retrovirus plasmid and pCL-ECO packaging plasmid into Phoenix cell line by calcium transfer method, collecting virus supernatant after 48 hr,and infecting in vitro activated CD8 with the viral supernatant ⁺ T cells, obtaining in vitro activated CD8 overexpressing BCL6-HA ⁺ T cells. Then collect 1X 10 ⁷ Left and right in vitro activated CD8 over expressing 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 the Active Motif's ChIP assay kit (53035). The obtained DNA fragment is sent to BGI genemics company for library construction and high-flux sequencing to obtain in vitro activated CD8 of over-expressed BCL6-HA ⁺ ChIP-seq data for T cells. Sequencing data were analyzed using bowtie2, picard MarkDuplicates, MACS2, chusseeker, bedtool sintersect, deeptools, computeMatrix and HOMER to obtain in vitro activated CD8 overexpressing BCL6-HA ⁺ Genome-wide binding results for T cells.

The results show that BCL6 HAs similar sequence binding characteristics in different cell types and that CD8 is overexpressed in BCL6-HA ⁺ BCL6 binds significantly to 5949 DNA sites and 2931 genes in T cells. KEGG analysis shows that BCL 6-binding genes are significantly enriched in multiple T cell-associated biological pathways, including T cell differentiation, activation, proliferation, adhesion, and the like. Furthermore, 42.58% and 39.89% of the BCL6 binding DNA sites are distributed over the introns and distal gene spacer regions, and only 12.96% of the BCL6 binding sites are distributed over the promoter region. Consistently, more than 70% of BCL6 binding sites are distributed over a region more than 10kb from the Transcription Start Site (TSS). These results indicate that BCL6 may be prone to bind to the intergenic and intronic regions to regulate gene expression.

2. Verifying BCL6 at CD8 ⁺ Regulatory effects in the transcriptome of T cells. The method comprises the following specific steps:

first, isolated CD45.1OT-I cells were obtained by the method described in step 3 of example 1, followed by cloning BCL6 (NM-001348026.1) into a retroviral vector expressing IRES-GFP and obtaining CD45.1OT-I cells over-expressing BCL6 by the method described in step 1 of this example. The inventors adoptively delivered CD45.1OT-I cells overexpressing BCL6 and control CD45.2 OT-I cells together to Tc vaccinated with E.G7 tumor rbd ^-/- Recipient mice (8 mice). After 8 days, the inventors cut E.G7 tumor with scissors, transferred to DMEM digest with 1mg/ml collageaseD and 10U/ml DNaseI added, digested for 40 min at 37℃and then subjected to density gradient centrifugation with Percollgradient (GE Healthcare) solution, and isolated and purified to obtain donor OT-I cells. The construction of the OT-I cell transcriptome and high throughput sequencing were carried out by BGI Genomics. The cDNA library was sequenced by Illumina Hiseq4000 from BGI Genomics, inc., to obtain sequencing data for RNA-seq of BCL 6. The sequenced data were analyzed for GSEA using TrimGluorev0.4.4, bowtie2, featureContents, etc. software.

GSEA results show that over-expression of BCL6 can induce OT-I cells to show remarkable BCL6 ⁺ TIM-3 ^- T _prog Transcriptome characteristics of cells. To further identify genes whose expression was directly regulated by BCL6, the inventors integrated and analyzed ChIP-seq data and RNA-seq data of BCL6, and obtained 180 genes whose expression was directly induced by BCL6 and 156 genes whose expression was directly inhibited by BCL6, respectively. In addition, the 49 genes directly inducing expression of BCL6 are BCL6 ⁺ T _prog Characteristic genes of cells (Id 3, cxcr5, tgfbr3, ccr6, etc.), and the genes for inhibiting expression of 49 BCL6 are TIM-3 ⁺ T _term Characteristic genes of cells (PRDM 1, havcr2, cxcr6, gzmb, etc.). These results demonstrate that BCL6 is induced by T _prog Expression of cell-specific genes and inhibition of T _term Expression of cellular signature genes to regulate CD8 ⁺ Differentiation of T cells.

3. The inventors further compared the functional differences of BCL6 and TCF 1. The method comprises the following steps:

(1) The difference between BCL6 and TCF1 binding genes was analyzed. First, the inventors found that BCL6 and TCF1 have very different DNA binding site specificities by comparing the ChIP-seq experimental results of BCL6 obtained in step 1 of this example with those of TCF1 reported in the previous study. However, these two transcription factors bind together to a large number of identical genes, and more than half of the BCL 6-bound genes are bound simultaneously by TCF1, suggesting that BCL6 and TCF1 may synergistically regulate target gene expression. Thus, there is a need to identify genes that regulate expression of TCF 1.

Furthermore, the inventors have integrally analyzed the ChIP-seq data of TCF1 reported in the previous literature and the RNA-seq data of P14 cells overexpressed by TCF7 in LCMV cl13 virus infected mice, and obtained 224 genes directly induced to express TCF1 and 294 genes directly inhibiting expression of TCF1, respectively. Then, by integrating analysis of genes whose BCL6 and TCF1 directly regulate expression, the inventors obtained 19 genes whose expression was co-upregulated (Cxcr 5, xcl1, ikzf2, etc.), and 20 genes whose expression was co-repressed (PRDM 1, gzmb, prf1, entpd1, cxcr6, etc.).

(2) Further comparison of BCL6 and TCF1 maintenance T _prog Capability of the feature. The method comprises the following specific steps:

first, the inventors obtained isolated OT-I cells using the method described in step 3 of example 1, followed by in vitro activated OT-I cells using the method in step 1 of this example, and subsequently cloned BCL6 (NM-001348026.1) and Tcf7 (NM-001313981.1) into a retroviral vector expressing IRES-GFP and obtained in vitro activated CD45.1 OT-I cells over-expressing BCL6 and Tcf7 (also known as TCF 1) using the method in step 1 of this example. Next, the above-mentioned OT-I cells overexpressing BCL6 and Tcf7 and the OT-I cells of the CD45.2 control group were adoptively delivered together to Tcrbd vaccinated with E.G7 tumor ^-/- In the recipient mice (8 mice), experiments were performed and the results were analyzed by the method described in step 2 of example 1.

The results show that after 8 days, overexpression of BCL6 or Tcf7 resulted in LY108 in OT-I cells ⁺ T _prog The proportion of cells was significantly increased, TIM-3 ⁺ T _term The proportion of cells was significantly reduced, indicating that these two transcription factors are maintaining T _prog The characteristics of the cells play a similar role. In addition, overexpression of BCL6 or Tcf7 mutually promotes their expression in OT-I cells infiltrated by e.g7 tumors. In addition, overexpression of BCL6 resulted in a significant decrease in expression levels of BLIMP1, T-BET, CD25 and CD39 molecules, while EOMES and TOX were significantly increased. By analyzing the proportion of tumor-infiltrated OT-I cells, this example found that overexpression of BCL6 resulted in a reduction in the number of OT-I cells in tumors and TDLNs by about 4-fold, as Ki-67 for tumor infiltration ⁺ The proportion of cells also decreases significantly. Wherein LY108 ⁺ T _prog Cells were reduced by approximately 1.71-fold, while T _term The cells were then reduced by a factor of about 5.54. However, overexpression of Tcf7 did not significantly alter the number of tumor-infiltrating OT-I cells, where T _prog The number of cells is significantly increased, while T _term The number of cells is significantly reduced. Together, these results demonstrate that BCL6 and TCF1 have similar maintenance T _prog Capability of the feature.

(3) BCL6 and TCF1 were further compared for their ability to regulate cell proliferation. The method comprises the following specific steps:

the inventors labeled the above-described OT-I cells overexpressing BCL6 or Tcf7 obtained in step 3 (2) of this example with CTV and OT-I cells of the control group, followed by adoptive transfer into C57BL/6 receptor mice (8 mice) together, and conducted experiments and analysis of the results using the method described in step 2 of example 1.

The results showed that after 3 days, up to 70-80% of BCL6 overexpressing cells remained CTV signaling in the spleen, whereas CTV signaling was almost completely diluted in the control OT-I cells. However, only 20% of OT-I cells overexpressed by Tcf7 retained CTV signaling, a proportion significantly lower than BCL6 overexpressed OT-I cells. Furthermore, GO analysis results show that more than half of the first 20 signal pathways with the highest significance of BCL6 inhibition of expressed genes are involved in cell proliferation, but that TCF7 inhibition of expressed genes are rarely involved in cell proliferation, indicating that BCL6 has significant inhibition of cell proliferation, whereas TCF1 does not. Together, these results demonstrate that BCL6 and TCF1 have different capacities to regulate cell proliferation.

(4) The functions of BCL6 and TCF1 in T cell differentiation and survival were further compared. The method comprises the following specific steps:

CD45.2 OT-I cells deleted first in Tcf7 (abbreviated as Tcf7 ^-/- OT-I cells) over-express BCL6 and then over-express Tcf7 of BCL6 ^-/- The OT-I cells and CD45.1 WT OT-I cells were adoptively transferred together to CD45.1 receptor mice (8 mice) 7 days after E.G7 tumor inoculation, and harvestedExperiments and assays were performed as described in example 1, step 2.

Meanwhile, the inventors utilized the method in example 3, step 3 (2), to control the reaction rate in BCL6 ^-/- Overexpression of Tcf7 in OT-I cells followed by overexpression of BCL6 of Tcf7 ^-/- The OT-I cells and CD45.1 WT OT-I cells were adoptively transferred together into E.G7-vaccinated CD45.1 receptor mice (8 mice), and experiments were performed as described in example 1, step 2, and then analyzed after 12 days.

The results showed that after 5 days, in E.G7 tumors and TDLN, BCL6 was found to be in Tcf7 ^-/- Overexpression in OT-I cells still significantly increases LY108 ⁺ TIM-3 ^- T _prog Cell ratio and decrease LY108 ^- TIM-3 ⁺ T _term The ratio of cells, which indicates that under the condition of Tcf7 deficiency, the overexpression of BCL6 can still significantly inhibit T _prog Differentiation of cells into T _term And (3) cells. Meanwhile, due to the strong inhibition of cell proliferation by BCL6, overexpression of BCL6 leads to tumor and Tcf7 in TDLN ^-/- The number of OT-I cells was significantly reduced.

Tcf7 at BCL6 ^-/- Overexpression in OT-I cells did not significantly alter LY108 in tumor and TDLN ⁺ TIM-3 ^- T _prog And LY108 ^- TIM-3 ⁺ T _term Proportion of cells. This suggests that overexpression of Tcf7 does not significantly inhibit T under BCL 6-deleted conditions _term Differentiation of cells. Furthermore, overexpression of Tcf7 results in tumors and BCL6 in TDLN ^-/- The number of OT-I cells increased significantly, suggesting that TCF1 significantly increased survival of OT-I cells by a BCL6 independent manner.

Further, the inventors analyzed ChIP-seq data of BCL6 and TCF1 obtained in step (2) of this example. The results show that TCF1 binds significantly to the promoter region of BCL6, but BCL6 does not bind to the gene locus of TCF 7. Furthermore, BCL6 binds significantly to T _term The gene locus of the characteristic gene Havcr2 of (2), but TCF1 is not bound to T _term The gene locus of the characteristic gene Havcr 2. These results demonstrate that BCL6 acts as a downstream molecule of TCF1, significantly inhibiting T _prog Differentiation of cells into T _term Cells, but do not regulate the viability of the cells.

Further, the inventors analyzed the RNA-seq data of BCL6 obtained in step 3 of this example, and overexpression of BCL6 resulted in a significant decrease in expression of Gzmb (abbreviation of Granzyme B), prf1 and fast at the transcriptional level. Furthermore, overexpression of BCL6 resulted in Granzyme B with tumor infiltration ⁺ The proportion of OT-I cells is significantly reduced, leading to T _term The expression level of Granzyme B in the 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 resulted in Caspase-3 ⁺ The cell proportion was significantly reduced, indicating that the apoptosis level of OT-I cells was significantly reduced.

Taken together, BCL6 transcription inhibits T in tumors as a downstream molecule of TCF1 _prog Differentiation of cells into T _term And (3) cells. In addition, BCL6 significantly inhibited T cell proliferation, whereas TCF1 did not.

Example 4: TGF-beta/SMAD 2 signaling pathway induces CD8 ⁺ Expression of BCL6 in T cells

1. Identification of regulatory CD8 ⁺ Signal pathway of BCL6 expression in T cells. The method comprises the following specific steps: the inventors obtained isolated CD8 by the method described in example 1, step 2 ⁺ T cells were then activated in vitro with a flat-bottomed plate conjugated with anti-CD3 (BioXcell, clone145-2C11,5. Mu.g/ml) and anti-CD28 (BioXcell, clone37.51, 1. Mu.g/ml) antibodies to obtain in vitro activated BCL6-RFPCD8 ⁺ T cells are stimulated by simultaneous addition of IL-2, IL-6, IL-10, IL-12, anti-IL-2, anti-IFN-gamma and TGF-beta, respectively. Three days later, in vitro activated CD8 was collected ⁺ T cell, detection of CD8 by flow cytometry ⁺ The RFP fluorescence intensity of the T cells judges the expression condition of BCL6 at the protein level, and the expression condition of BCL6 at the transcription level is detected by fluorescent real-time quantitative PCR. The results show that anti-IL-2 and TGF-beta can be significantly in CD8 ⁺ High expression of BCL6 at RNA and protein levels was induced in T cells as shown in figure 1.

2. Further validating the regulation of BCL6 expression by TGF- β signals. Specific stepsThe method comprises the following steps: in vitro activation and stimulation of Tgfbr2 with anti-CD3/28 and TGF-beta ^-/- CD8 ⁺ T cells were tested by the method of step 1 of this example. The results show that the deletion of Tgfbr2 results in TGF- β being unable to induce high expression of BCL6 at RNA and protein levels. In addition, activated CD8 in vitro ⁺ In T cells, stimulation of TGF- β significantly inhibited expression of T-bet, but did not affect expression of TCF 1.

3. Previous studies reported that TGF- β regulates T in a chronic viral infection model in mice _prog Differentiation and maintenance of cells. Therefore, this example demonstrates that TGF-beta signaling pathway regulates BCL6 expression and CD8 in tumor models ⁺ Role in T cell differentiation. The method comprises the following specific steps:

(1) The same number of naive CD45.2 WT and Tgfbr2 ^-/- OT-I cells were adoptively transferred to CD45.1 receptor mice, respectively, and then inoculated with e.g7 tumor cells after 1 day, and experiments and detection were performed using the procedure of step 3 of example 1. The results show that the absence of Tgfbr2 significantly enhances the tumor controlling effect of OT-I cells. Deletion of Tgfbr2 results in BCL6 in OT-I cells ⁺ TCF1 ⁺ TIM-3 ^- T _prog The proportion of cells is significantly reduced, while BCL6 ^- TCF1 ^- TIM-3 ⁺ T _term The proportion of cells increases significantly. Furthermore, the absence of Tgfbr2 results in a significant increase in the number of OT-I cells per unit weight of tumor infiltration, wherein T _term The number of cells is significantly increased, while T _prog The number of cells was not significantly changed, and the deletion of Tgfbr2 resulted in IFN-gamma ⁺ And Granzyme B ⁺ The proportion of cells was significantly increased and the absence of Tgfbr2 resulted in Granzyme B, a unit tumor infiltration ⁺ The number of OT-I cells increased significantly and resulted in Ki-67 ⁺ The proportion of proliferating cells is significantly increased, and this result indicates that the TGF-beta signaling pathway significantly inhibits T of tumor infiltration _term Development of cells, but for T _prog The production of cells was not significantly affected and TGF-beta signaling was T-cell on tumor infiltration _term The effector function and proliferation activity of the cells have remarkable inhibition effect.

4. The effect of TGF- β signaling on T cell differentiation during long-term tumor progression was validated. The method comprises the following specific steps:

(1) The inventors first activated WT and Tgfbr2 in vitro ^-/- OT-I cells were co-adoptively delivered to CD45.1 recipient mice 8 days after e.g7 tumor inoculation, and the recipient mice were assayed at 16 and 24 days using the procedure of step 3 of example 1. The results show that at days 16 and 24, the deletion of Tgfbr2 resulted in BCL6 ⁺ TCF1 ⁺ T _prog The proportion of cells was significantly reduced, while TCF1 ^- TIM-3 ⁺ T _term The proportion of cells increases significantly. In addition, the deletion of Tgfbr2 resulted in T during the course of from day 16 to day 24 _prog The cell subpopulation gradually disappeared. Unlike previous reports, in LCMV cl13 virus infection model, there is still a portion of LY108 in the absence of Tgfbr2 ⁺ TIM-3 ^- T _prog Cells persist on day 100 of infection. Furthermore, in the E.G7 tumor model, the deletion of Tgfbr2 did not significantly affect LY108 in TDLN ⁺ TIM-3 ^- T _prog Proportion of cells.

(2) Next, the OT-I cells infiltrated by the tumor obtained in step 4 (1) of this example were counted by flow cytometry to obtain the number of OT-I cells in the tumor, and the result showed that the deletion of Tgfbr2 resulted in a significant increase in the number of OT-I cells infiltrated by the tumor on day 16, whereas the deletion of Tgfbr2 resulted in a significant decrease in the number of OT-I cells on day 24. Wherein the deletion of Tgfbr2 results in T on day 16 _prog And T _term The number of cells increased significantly, but their number decreased significantly at day 24.

(3) Further, the inventors analyzed WT and Tgfbr2 in the disclosed chronic LCMV clone13 virus infection model ^-/- Transcriptome sequencing data of P14 cells (GEO No. GSE 209577) to obtain WT and Tgfbr2 ^-/- Differential expression genes of P14 cells as WT and Tgfbr2 ^-/- The characteristic expression genes of P14 cells. WT and Tgfbr2 were then compared using GSEA ^-/- Characteristic expression genes of P14 cells in BCL6 ⁺ And TIM-3 ⁺ CD8 ⁺ Expression in the transcriptome of T cells.GSEA analysis results show that the characteristic genes of WT P14 cells in LCMV cl13 model are obviously enriched in BCL6 ⁺ CD8 ⁺ In the transcriptome of T cells, tgfbr2 ^-/- The characteristic genes of the P14 cells are obviously enriched in TIM-3 ⁺ CD8 ⁺ In the transcriptome of T cells. These results demonstrate that TGF- β signaling significantly inhibits proliferation and T of tumor infiltrating T cells in early stages of tumorigenesis and progression _prog Differentiation of cells, but for T _prog The long-term maintenance of cells is indispensable.

5. Previous studies have shown that Tgfbr2 deletion and BCL6 deletion are phenotypically consistent, and that TGF- β signaling pathways may regulate T cell differentiation, proliferation and effector function by regulating BCL6 expression. Thus, next, this example demonstrates that the phenotype of Tgfbr2 deletion is rescued by BCL6 overexpression. The method comprises the following specific steps:

CD45.2Tgfbr2 overexpressed by the same amount of BCL6 ^-/- OT-I cells, tgfbr2 ^-/- OT-I cells and WT OT-I cells were adoptively transferred to CD45.1 receptor mice vaccinated with E.G7 tumor, respectively, and then analyzed on day 12, and experiments and detection were performed using the procedure of step 3 of example 1. The results showed that overexpression of BCL6 successfully rescued Tgfbr2 ^-/- LY108 in OT-I cells ⁺ T _prog Cell ratio and expression of TCF 1. In addition, overexpression of BCL6 leads to Tgfb2 for tumor infiltration ^-/- The number of OT-I cells was significantly reduced and significantly less than WT and Tgfbr2 ^-/- Number of OT-I cells. Wherein BCL6 is Tgfbr2 ^-/- Overexpression in OT-I cells results in LY108 ⁺ T _prog Cell numbers reverted to T in WT OT-I cells _prog Cell count, but resulted in TIM-3 ⁺ T _term The number of cells is significantly reduced. In addition, overexpression of BCL6 also resulted in Granzyme B ⁺ Tgfbr2 of (F) ^-/- The number of OT-I cells was significantly reduced. These results indicate that BCL6 is downstream of TGF- β signaling pathway, TGF- β promotes T in tumors through BCL6 _prog Long-term maintenance of cells.

6. The molecular mechanism by which TGF-beta signaling pathways regulate BCL6 expression was validated. The method comprises the following specific steps:

(1) ChIP-seq data (GEO number: GSE 125116) in ESCs and EBs cells were analyzed by SMAD 2. The results show that SMAD2 binds significantly to the promoter region of BCL6 gene.

(2) Verification of whether the promoter region of the BCL6 gene to which SMAD2 binds significantly is present in CD8 ⁺ T cells. The inventors first collected 1x10 ⁷ Left and right 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 the Active Motif's ChIP assay kit (53035). For ChIP experiments with SMAD2, CD8 activated in vitro ⁺ T cells were stimulated with 2ng/ml hTGF-. Beta.for 30 min before cross-linking and immobilization, DNA was extracted and the resulting DNA was detected by real-time quantitative PCR. The results show that in CD8 ⁺ In T cells, SAMD2 binds significantly to the promoter region of BCL6 gene, but not to its exon region, and this binds to SAMD2 deleted CD8 ⁺ Almost complete disappearance in T cells; deletion of SAMD2 results in TGF-beta stimulated CD8 ⁺ The expression of BCL6 in T cells is significantly reduced; SMAD2 also binds significantly to the promoter region of the Tcf7 gene, but the deletion of SAMD2 is for TGF- β stimulated CD8 ⁺ Expression of Tcf7 in T cells did not have a significant effect, but resulted in a significant increase in the expression level of T-bet. These results indicate that the TGF- β -SMAD2 signaling pathway significantly increases CD8 ⁺ Transcriptional activity of BCL6 in T cells is likely achieved by SMAD2 binding to the promoter region of BCL6 gene.

Example 5: inhibition of CD8 by IL-2/STAT5 signaling pathway ⁺ Expression of BCL6 in T cells and antagonism of TGF-beta signaling pathway

Previous studies reported that the IL-2 signaling pathway significantly inhibited BCL6 expression in Tfh cells and was responsible for the effect on CD8 in acute viral infection and immune models ⁺ Differentiation of T cells is important. Thus, this example demonstrates that IL-2 and TGF-beta signaling pathways are in CD8 ⁺ Interactions in T cells.

1. First by culturing WT and Il2ra deleted CD8 in vitro ⁺ T cells, activation of naive CD8 with flat bottom plates conjugated with anti-CD3 (5. Mu.g/ml) and anti-CD28 (1. Mu.g/ml) antibodies ⁺ T cells were stimulated three days with the addition of IL-2. Three days later, CD8 was detected using flow cytometry ⁺ The expression level of BCL6 protein in T cells, and simultaneously, the expression condition of BCL6 gene at the transcription level is detected by utilizing fluorescence real-time quantitative PCR. The results showed that BCL6 cultured Il2ra in vitro ^-/- CD8 ⁺ The expression levels of RNA and protein in T cells are significantly increased. In addition, il2ra cultured in vitro ^-/- Or anti-IL2 treated CD8 ⁺ In T cells, the expression level of TCF1 is significantly increased, while the expression level of T-bet is significantly decreased.

2. Verification of IL-2 regulated Gene in BCL6 ⁺ And TIM-3 ⁺ CD8 ⁺ Expression levels in the transcriptome of T cells.

(1) First two IL-2 related CD8 previously reported were analyzed ⁺ RNA-seq data of T cells (GEO No. GSE58081 and GSE 39110) were then analyzed using GSEA. The results show that anti-IL-2 treated CD8 in vitro ⁺ The characteristic gene highly expressed in T cells is remarkably highly expressed in BCL6 ⁺ CD8 ⁺ CD8 treated with IL-2 in T cells in vitro ⁺ The characteristic gene highly expressed in T cells is remarkably highly expressed in TIM-3 ⁺ CD8 ⁺ T cells; IL-2 repression expressed gene and BCL6 ⁺ CD8 ⁺ Genes highly expressed in T cells, IL-2-induced expression genes and TIM-3 ⁺ CD8 ⁺ There is a significant positive correlation between the highly expressed differential genes in T cells, respectively. Wherein T is _prog Expression levels of the characteristic genes (Tcf 7, id3, slamf6 and etc.) of the cells were significantly inhibited by treatment with IL-2 in vivo, while T _term The expression levels of the characteristic genes of the cells (PRDM 1, id2, havcr2 and etc.) were significantly up-regulated by the treatment with IL-2 in vivo.

3. The role of IL-2 signaling in vivo was further verified.

(1) First, the expression level of CD25 was detected, and tumor infiltration of CD8 was detected by flow cytometry in mice (8 mice) 12 days after inoculation with E.G7 tumor ⁺ Expression of BCL6 protein in T cells. The results show that in E.G7 tumors, CD25 is specifically expressed in TIM-3 ⁺ T _term On the cells. Furthermore, although T in E.G7 tumors _prog The CD25 expression levels of cells were low, but they were the major T cell subset producing IL-2, consistent with previous results.

(2) Further investigation of IL-2 Signal at CD8 ⁺ The inventors have identified an equivalent amount of naive CD45.2 WT, il2ra ^+/- And Il2ra ^-/- OT-I cells were adoptively transferred to CD45.1 receptor mice, and after 1 day, E.G7 tumor cells were inoculated, and experiments and detection were performed using the method of step 3 of example 1. The results show that, after 18 days, deletion of Il2ra resulted in the OT-I cells infiltrated by e.g7 tumor to remain almost at BCL6 ⁺ TCF1 ⁺ T _prog Cell status leading to TIM-3 ⁺ T _term The proportion of cells and the expression level of T-bet decreased significantly. Furthermore, the absence of Il2ra results in a significant decrease in the proportion and number of tumor-infiltrating OT-I cells, where T _term The number of cells was reduced by about 21-fold, while T _prog The number of cells decreased only about 3-fold, which indicates that the deletion of Il2ra significantly impairs T _term Development of cells, but for T _prog The effect of the cells is relatively small. In addition, the ability of Il2 ra-deleted OT-I cells to control tumors was significantly impaired, while the ability of Il2 ra-deleted OT-I cells to produce IFN- γ and Granzyme B, as well as the expression level of KI-67, was significantly reduced, indicating that their proliferation and effector functions were significantly impaired. These results demonstrate that IL-2 signaling pathway is inhibiting BCL6 expression and promoting T in tumor models _term Plays a very important role in the development of cells.

4. The molecular mechanism by which IL-2 inhibits BCL6 expression was validated. The method comprises the following specific steps:

(1) First, previously reported CD8 was analyzed ⁺ ChIP-seq data for STAT5 in T cells (GEO number: GSE 192386). The results show that STAT5 binds significantly to the promoter region of BCL6 gene, but not to the gene locus of Tcf 7.

(2) To further verify that STAT5 binds significantly to the promoter region of BCL6 gene, the inventors utilized in vitro IL-2 treated CD8 ⁺ T cell STAT5ChIP experiments, experimental methods refer to example 4, step 6 (2), except that 10U/ml IL-2 instead of 2ng/ml hTGF-beta was used for 30 minutes. ChIP-qPCR results showed that STAT5 binds significantly to the promoter region of BCL6 gene, but not to the exon region of BCL6 gene. Furthermore, the addition of exogenous IL-2 significantly enhanced binding of STAT5 protein to the BCL6 gene promoter region. CD8 treated with STAT5 inhibitors in vitro ⁺ In T cells, BCL6 expression at the transcriptional and protein levels was significantly elevated, in addition, TCF1 expression levels were significantly elevated, while T-BET expression levels were significantly reduced. Together, these results demonstrate that the IL-2-STAT5 signaling pathway significantly inhibits transcription of the BCL6 gene, which may be achieved by binding of STAT5 at the BCL6 promoter region.

5. Antagonism of IL-2-STAT5 and TGF-beta-SMAD 2 was further demonstrated.

(1) CD8 treated with IL-2 and TGF-beta in combination in vitro was first tested ⁺ Expression of BCL6 in T cells in vitro activation of naive CD8 with anti-CD3 (5. Mu.g/ml) and anti-CD28 (1. Mu.g/ml) antibody-conjugated flat-bottomed plates ⁺ T cells were stimulated three days with the addition of IL-2 and TGF- β. Three days later, CD8 was detected using flow cytometry ⁺ The expression level of BCL6 protein in T cells, and simultaneously, the expression condition of BCL6 gene at the transcription level is detected by utilizing fluorescence real-time quantitative PCR. The results show that TGF-beta induced expression of BCL6 at the transcriptional and protein levels is significantly inhibited by IL-2. Furthermore, TGF-. Beta.significantly antagonizes the induction of T-BET expression by IL-2, but fails to restore the expression of IL-2-inhibited TCF 1.

(2) Next, the disclosed CD8 treated with IL-2 or TGF-beta in vitro was analyzed ⁺ Transcriptome sequencing data of T cells (GEO number: GSE 125471) and then comparing IL-2 or TGF-beta treated CD8 using Pearson analysis ⁺ Correlation of gene expression profiles of T cells. Pearson analysis showed that CD8 treated with IL-2 or TGF-beta in vitro ⁺ The transcriptome of T cells exhibits a significant negative correlation of gene expression.

(3) The RNA-seq results in step 2 of this example were subjected to GSEA analysis, and the results of GSEA analysis showed that, in LCMV cl13 infected mice,the characteristic gene in WT P14 cells was significantly highly expressed in OT-I cells not treated with IL-2, while Tgfbr2 ^-/- The characteristic gene of the P14 cell is remarkably high expressed in the OT-I cell treated by IL-2. In CD8 ⁺ In T cells STAT5 and SMAD2 bind to the same promoter site of BCL6 gene, but produce opposite transcriptional regulatory effects; binding of STAT5 to BCL6 Gene promoter site in vitro TGF- β treated CD8 ⁺ Significantly reduced in T cells, while SMAD2 binding at the BCL6 gene promoter site was found to be in IL-2 treated CD8 ⁺ Significant decrease in T cells; CD8 treated with TGF-beta in vitro ⁺ In T cells, IL-2 secretion and CD25 expression levels were significantly reduced, suggesting that TGF- β may indirectly regulate BCL6 expression by inhibiting the IL-2 signaling pathway.

Example 6: BCL6 and BLIMP1 CD8 in tumors ⁺ Antagonistic action during T cell differentiation

BLIMP1 (encoded by PRDM 1) is one of the effector proteins downstream of the IL-2-STAT5 pathway, and BLIMP1 significantly inhibits the expression of BCL6 and antagonizes the function of BCL6 in GCB cells and Tfh cells; in the acute infection model, BLIMP1 significantly inhibited memory CD8 ⁺ Formation of T cells; in the chronic viral infection model, BLIMP1 significantly inhibited the development of Tfc cells. Therefore, this example demonstrates the expression and function of BLIMP1 in tumor models. The method comprises the following specific steps:

1. functional differences in tumor models were compared between BCL6 and BLIMP 1. The method comprises the following specific steps:

(1) First, the inventors analyzed the presence of CD8 ⁺ ChIP-seq data of BLIMP1-Biotin in T cells (GEO number: GSE 79339) gave CD8 of BLIMP1 in mice ⁺ Binding genes in the genome of T cells. The inventors found that the two molecules BCL6 and BLIMP1 have significantly different binding spectra by comparing the ChIP-seq data of BCL6 obtained in step 1 of example 3 with the ChIP-seq data of BLIMP1 obtained in this example, as shown in fig. 2A. However, these two transcription factors bind together to a large number of identical target genes, with more than half of the BCL 6-bound genes being bound simultaneously by BLIMP1, as shown in fig. 2B.

(2) Next, the inventors isolated and purified co-adoptively delivered PRDM1 (encoding the BLIMP1 protein) over-expressed and control OT-I cells from e.g7 tumors, then obtained RNA-seq data over-expressed by PRDM1 (except that PRDM1 (nm_ 007548.4) was cloned into IRES-BFP expressing retroviral vector instead of BCL6 and TCF 1) by the method described in example 3, step 2, and GSEA analysis was performed on the RNA-seq data.

GSEA results showed that overexpression of PRDM1 resulted in OT-I cells exhibiting significant BCL6 ^- TIM-3 ⁺ T _term Transcriptome characteristics of the cells are shown in FIG. 2C. PRDM1 overexpression induces significant enrichment of differentially expressed genes in multiple biosignal pathways, including T cell activation, differentiation, adhesion, etc., as shown in figure 2D. And BCL6 and PRDM1 overexpression induced a significant negative correlation of differentially expressed common genes, as shown in figure 3A. Together, these data demonstrate that BCL6 and BLIMP1 may reciprocally regulate the expression of a range of common downstream genes in their specific ways.

The inventors further analyzed the BLIMP1 ChIP-seq data and PRDM1 over-expressed RNA-seq data obtained in example step 1 by integration to obtain 133 genes directly induced to be expressed by BLIMP1 and 132 genes directly inhibited to be expressed by BLIMP1, respectively, as shown in fig. 3B. Among the 27 genes whose BLIMP1 direct induction was expressed were TIM-3 ⁺ T _term Characteristic genes of cells (Havcr 2, cd244a, il2ra, cd38, etc.), 41 genes for direct inhibition of expression of BLIMP1 are BCL6 ⁺ T _prog The characteristic genes of the cells (Tcf 7, id3, cxcr5, ccr7, etc.), as shown in fig. 3C. Thus, BCL6 and BLIMP1 are regulatory T _prog And T _term Key transcription factors for cell differentiation.

2. Verification of BCL6 and BLIMP1 CD8 in tumor model ⁺ Molecular mechanisms that play a role in the differentiation process of T cells.

First, the inventors analyzed the ChIP-seq data and ATAC-seq data (GEO number: GSE 122713) of BCL6 obtained in step 1 of this example. ChIP-seq results show that at CD8 ⁺ In T cells, BCL6 binds directly to multiple sites of PRDM1 gene, distributed over introns andremote intergenic regulatory regions.

BCL6 and BLIMP1 are co-bound to T _prog The characteristic genes Il7r, sell and Ccr7 of the cells are located at different sites and reciprocally regulate their expression.

The ATAC-seq results show that the above gene binding sites are in CD8 tumor infiltration ⁺ The T cells were open as shown in fig. 4A and 4B. These results demonstrate that BCL6 and BLIMP1 may co-regulate expression of a target gene in a variety of ways, including binding directly to different sites of the same gene, competitively binding to the same site of the gene, or inducing expression of a downstream gene indirectly by inhibiting expression of the other. In addition, although BCL6 and BLIMP1 bind directly to multiple sites of Ifng gene (Interferon-gamma gene), they do not regulate expression of Ifng gene, as shown in fig. 3C and 4B.

3. Further validation of BCL6 and BLIMP1 in CD8 in tumors ⁺ Antagonism is exerted during T cell differentiation.

(1) First, the inventors obtained in vitro activated CD8 simultaneously overexpressing BCL6 and PRDM1 by the method of example 3, step 1 ⁺ T cells and uses Magzolreagent (Magen) from CD8 ⁺ Total RNA is extracted from T cells. The cDNA was then obtained using the M-MLV reverse transcription kit (Invitrogen). Then, the expression level of the specific gene was measured by SYBR real-time kit (Bio-Rad Laboratories). The results show that BCL6 and PRDM1 reciprocally regulate transcription of Tcf7, cxcr5, id3, havcr2, etc., genes, as shown in fig. 5A.

(2) Next, the inventors obtained OT-I cells in which BCL6 and PRDM1 were simultaneously overexpressed by the method of step 3 (2) of example 3, and co-adoptively delivered the OT-I cells in which BCL6 and PRDM1 were simultaneously overexpressed and the OT-I cells of the control group to Tcrbd vaccinated with E.G7 tumor ^-/- In recipient mice (8 mice), experiments were performed by the method described in example 1, step 2. The results show that the overexpression of BCL6 and PRDM1 reciprocally regulates T _prog And T _term The ratio of cells is shown in FIG. 5B. These results demonstrate that T is specific for tumors _prog And T _term During the development of cells, BCL6 and BLIMP1 exert reciprocal regulationActing as a medicine.

(3) The inventors prepared naive CD45.2 BLIMP1-EYFP OT-I cells by the method described in step 3 (2) of example 3 and adoptively transferred the naive CD45.2 BLIMP1-EYFP OT-I cells into CD45.1 receptor mice (8 mice), and then inoculated E.G7 tumor cells after 1 day, and conducted experiments using the method described in step 2 of example 1, the experimental procedure being shown in FIG. 6A.

The results show that BLIMP1 significantly highly expresses tumor-infiltrating OT-I cells after 11 days and 20 days. Furthermore, BLIMP1-EYFP is specifically expressed in TIM-3 ⁺ T _term In cells, but not expressed in LY108 ⁺ T _prog In cells. Furthermore, PRDM1 is significantly highly expressed in CD8, which is infiltrated in tumors, in the results of single cell RNA-seq of head and neck cancer and melanoma patients ⁺ In T cells, and expression of PRDM1 and TCF7, BCL6 are mutually exclusive, as shown in figure 6B, C.

4. Verification of expression of BLIMP1 at CD8 ⁺ Induced molecular mechanisms in T cells. Previous studies reported that BLIMP1 is a downstream transcription factor of the IL-2 signaling pathway, and therefore the inventors first activated the naive BLIMP1-EYFPCD8 described in step 3 (3) of this example with anti-CD3/28 in vitro by performing the method of step 3 1 ⁺ T cells, and culturing WT CD8 in vitro by the method of example 1, step 2 ⁺ T cells and PRDM1 ^-/- CD8 ⁺ T cells were continuously stimulated with IL-2 for 6 days and then BLIMP1-EYFPCD8 was detected by flow cytometry ⁺ Expression level of BLIMP1 in T, and CD8 obtained without IL-2 ⁺ T cells, hereafter abbreviated as control CD8 ⁺ T cells.

As shown in FIG. 7A, BLIMP1 expressed little CD8 at 3 days of activation first ⁺ T cells. But on day 6 of IL-2 sustained stimulation at TIM-3 ⁺ CD8 ⁺ BLIMP1-EYFP is significantly highly expressed in T cells. CD8 without IL-2 stimulation ⁺ In T cells, BLIMP1-EYFP was not significantly expressed. At the same time, over 60% of CD8 under continued IL-2 stimulation ⁺ Differentiation of T cells into TIM-3 ⁺ A cell; CD8 without stimulation of IL-2 ⁺ Of T cells, only less than 10% of CD8 ⁺ T cells express TIM-3.

Second, CD8 deleted in PRDM1 ⁺ In T cells, CD25 high TIM-3 could not be induced without sustained stimulation with IL-2 ⁺ Cell production, while unable to inhibit TCF1 expression, as shown in fig. 7B. In addition, and IL-2 stimulated WT CD8 ⁺ PRDM1 stimulated in IL-2 compared to T cells ^-/- CD8 ⁺ In T cells, the expression level of Granzyme B was significantly reduced, while the expression level of LY108 was significantly increased, as shown in fig. 7C.

Taken together, the results demonstrate that sustained IL-2 stimulation significantly induced TIM-3 under in vitro culture conditions ⁺ CD8 ⁺ Production of T cells and expression of BLIMP1. And IL-2 vs TIM-3 ⁺ CD8 ⁺ Induction of T cell differentiation and inhibition of TCF1 expression is dependent on downstream BLIMP1.

5. Verification of BLIMP1 vs. CD8 in tumor ⁺ Regulatory action of T cells. The method comprises the following specific steps:

the inventors have performed the following procedures on the naive WT and PRDM1 described in step 4 of this example ^-/- OT-I cells were co-adoptively delivered to Tcrbd in a 1:1 ratio ^-/- In recipient mice (8 mice), E.G7 tumor cells were then inoculated after 1 day, and experiments were performed and the experimental results were analyzed using the method described in example 1, step 2.

The results show that 16 days later, PRDM1 lacks LY108 ⁺ T _prog Proportion of cells and BCL6 ⁺ And TCF1 ⁺ The proportion of cells was significantly increased, whereas TIM-3 ⁺ T _term The proportion of cells was significantly reduced, as shown in fig. 8A. Furthermore, the deletion of PRDM1 resulted in a significant decrease in the capacity of Granzyme B production, but the expression level of IFN- γ was not significantly affected, whereas the expression level of TNF- α appeared to be significantly increased, as shown in fig. 8B. LY108 in OT-I cells with PRDM1 overexpression leading to tumor infiltration ⁺ T _prog Proportion of cells and TCF1 ⁺ And BCL6 ⁺ The proportion of cells was significantly reduced, whereas TIM-3 ⁺ T _term The proportion of cells is significantly increased, and the absence of PRDM1 has no significant effect on the number of tumor infiltrating OT-I cells, e.gFig. 9A shows the same. Wherein T is _prog The number of cells is significantly increased, while T _term The number of cells is significantly reduced. Overexpression of PRDM1 results in a slight decrease in the number of tumor-infiltrating OT-I cells, wherein T _prog Cell number is significantly reduced, while T _term The number of cells increased significantly as shown in fig. 9B. In addition, the in vivo proliferation results showed that at day 2 and day 3, PRDM1 overexpression did not significantly affect CTV signaling in OT-I cells, as shown in fig. 9C, demonstrating that BLIMP1 did not significantly regulate proliferation of cells.

6. Verify at CD8 ⁺ T cells cope with the expression and function of BLIMP1 during the immune response of endogenous tumor antigens. The method comprises the following specific steps:

(1) The inventors first vaccinated BLIMP1-EYFP mice with B16BL6, EL4, HEPA1-6 and MC38 tumors, followed by obtaining isolated CD8 using the method described in example 1 step 2 ⁺ T cells were subjected to flow-through experiments and then analyzed for the experimental results on day 15.

The results show that in TDLN, a small fraction of CD44 ⁺ CD8 ⁺ T cells express BLIMP1-EYFP; whereas in tumors, more than 1/3 of CD44 ⁺ CD8 ⁺ T cells highly expressed BLIMP1-EYFP (BLIMP 1 tandem yellow fluorescent protein) as shown in FIG. 10A.

(2) This example describes the juvenile WT CD8 of step 4 of this example ⁺ T cells and PRDM1 ^-/- CD8 ⁺ T cells and CD4 ⁺ T cells are commonly adoptively delivered to Tcrbd ^-/- In recipient mice (8 mice) and after 1 day tumor cells were inoculated, experiments were performed and the experimental results were analyzed using the method described in example 1, step 2.

The results show that after 15 days, the deletion of PRDM1 resulted in CD8 tumor infiltration ⁺ TCF1 in T cells ⁺ Granzyme B ^- T _prog The proportion of cells was significantly increased, whereas TCF1 ^- Granzyme B ⁺ T _term The proportion of cells was significantly reduced, as shown in fig. 10B. These results demonstrate that BLIMP1 is T-tumour infiltrating under stimulation with endogenous tumour antigen _term The development of the cells is important.

Taken together, these results demonstrate that BLIMP1 significantly inhibits T in tumor models _prog Maintenance of cells and promotion of T _term Differentiation of cells.

Example 7: PRDM1 in tumor-specific CD8 ⁺ Deletions in T cells significantly enhance the therapeutic effects of tumor control

In cancer patients, T _prog The number of cells and better clinical prognosis are positively correlated with responsiveness to immune checkpoint blocking therapy. Increasing T using overexpression of Tcf7 _prog The number of cells can significantly increase CD8 ⁺ Proliferation of T cells following PD-1/PD-L1 blocking therapy and therapeutic immune treatment results in better tumor control effects. Thus, considering the important role of BCL6 and BLIMP1 on T cell differentiation, it is highly desirable to verify whether BCL6 overexpression and deletion of PRDM1 would enhance the therapeutic effect of anti-PD-1, by the following steps:

1. the regulation and control of the anti-tumor function of the T cells by the BCL6 overexpression are verified, and the specific steps are as follows:

the inventors adoptively delivered equal amounts of BCL6 overexpressed OT-I cells or control OT-I cells (preparation of both OT-I cells see example 3, step 3 (2)) to Tcrbd vaccinated with e.g7 tumor, respectively ^-/- In the recipient mice (8 mice), experiments and assays were performed using the methods described in example 1, step 2, with the results shown in FIG. 11A.

The results show that overexpression of BCL6 results in significant impairment of the anti-tumor effect of OT-I cells, LY108 ⁺ T _prog The proportion of cells was significantly increased, whereas TIM-3 ⁺ T _term The proportion of cells was significantly reduced, as shown in fig. 11B. Furthermore, overexpression of BCL6 results in a significant reduction in the number of tumor-infiltrating OT-I cells, wherein T _prog And T _term The number of cells was significantly reduced as shown in fig. 11C.

2. Verifying whether anti-PD-1 treatment was able to significantly amplify BCL6 overexpressed T _prog Cells, producing an effective tumor control effect. The method comprises the following specific steps:

equal amounts of in vitro activated BCL6 overexpressed OT-I cells and control OT-I cells (see example 3, step 3 (2)) were individually adoptively transferred to CD45.1 receptor mice (8 mice) vaccinated with e.g7 tumors for 7 days, and anti-PD-1 antibody was injected every 2-3 days for 3 total injections (schematic representation is shown in fig. 12A) starting on day 3 after adoptive transfer, and experiments and assays were performed using the methods described in example 1, step 2.

The results show that although anti-PD-1 was able to slightly enhance the ability of BCL6 overexpressed OT-I cells to control tumor growth, the rate of tumor growth was still faster than in recipient mice receiving control OT-I cells, as shown in FIGS. 12B, 12C. By analyzing tumor-infiltrating donor OT-I cells, this example found that most BCL6 overexpressed OT-I cells remained at LY108 after anti-PD-1 treatment ⁺ T _prog The state of the cells is shown in FIG. 13A. Also, the number of BCL6 overexpressing OT-I cells per tumor infiltration, including T _prog And T _term The number of cells did not increase significantly after anti-PD-1 treatment, as shown in fig. 13B. Furthermore, overexpression of BCL6 resulted in Granzyme B of unit tumor infiltration ⁺ The number of OT-I cells was significantly reduced and did not significantly increase after anti-PD-1 treatment, as shown in fig. 13C. Therefore, the treatment of anti-PD-1 cannot effectively promote the expansion and differentiation of OT-I cells overexpressed by BCL6, and thus cannot induce more effective antitumor effects. Thus, in tumor models, although overexpression of BCL6 is effective in maintaining T _prog The characteristics of cells, however, the anti-tumor effect cannot be effectively improved and the combined effect with anti-PD-1 cannot be produced because the anti-tumor effect is remarkably inhibited by the anti-tumor effect.

3. Verification of PRDM1 on tumor-specific CD8 ⁺ The deletion in T cells regulates the anti-tumor function of the T cells, and the specific steps are as follows:

the inventors have equally activated CD45.2 WT and PRDM1 in vitro ^-/- OT-I cells (see example 3, step 3 (2), for preparation of two OT-I cells, except that PRDM1 was knocked out using CRISPR-Cas9 instead of over-expressing BCL6 and TCF 1) were adoptively delivered to E.G7 tumor vaccinated CD45.1 receptor mice (8 Mice) experiments and tests were performed using the method described in example 1, step 2.

The results show that PRDM1 is in tumor specific CD8 ⁺ Deletions in T cells significantly promote T _prog Expansion of the cells produces better tumor control effects.

4. Further validation of PRDM1 on tumor specific CD8 ⁺ Effect of deletions in T cells in combination with anti-PD-1. The method comprises the following specific steps:

the inventors have equally activated CD45.2 WT and PRDM1 in vitro ^-/- OT-I cells (see example 3, step 3 (2), for preparation of two OT-I cells), except that PRDM1 was knocked out using CRISPR-Cas9 instead of over-expressing BCL6 and TCF 1) were adoptively delivered to E.G7 tumor-vaccinated CD45.1 receptor mice (8 mice), respectively, and then anti-PD-1 antibody was intraperitoneally injected 3 times from 3 days later (schematic view is shown in FIG. 14A), and experiments and assays were performed using the method described in example 1, step 2.

The results show that the deletion of PRDM1 and the treatment of anti-PD-1 can produce a more effective effect of tumor control and is significantly superior to the effect of adoptive transport of WT OT-I cells and anti-PD-1 combination, as shown in fig. 14B. By analyzing tumor-infiltrating OT-I cells, the inventors found that deletion of PRDM1 significantly increased TCF1 ⁺ T _prog Cell proportion and significantly increase TCF1 per tumor infiltration ⁺ T _prog Cell number, and treatment with anti-PD-1 further increased TCF1 per tumor infiltration ⁺ T _prog Number of cells. Furthermore, even if deletion of PRDM1 results in T _term The proportion and number of cells are significantly reduced, but treatment with anti-PD-1 significantly increases T per tumor infiltration _term Cell number and up to WT OT-I group number, as shown in fig. 14C. In addition, treatment with anti-PD-1 also significantly increased Granzyme B per tumor infiltration ⁺ PRDM1 ^-/- The number of OT-I cells is shown in FIG. 15A. Furthermore, the combination of deletion of PRDM1 and anti-PD-1 significantly increased the ability of OT-I cells to produce IFN-gamma and TNF-alpha, as shown in FIG. 15B. Thus, PRDM1 is in tumor-specific CD8 ⁺ Deletions in T cells significantly promote T _prog Cell expansionIncrease and significantly enhance their response to anti-PD-1, resulting in better tumor control effects.

In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.

While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Claims

1. A T cell, wherein the expression of a given protein in the T cell is altered, the given protein being adapted to regulate the expression of BCL6 in the T cell.

2. The T cell of claim 1, wherein the T cell is selected from the group consisting of CD8 ⁺ T cells and CD4 ⁺ At least one of the T cells, preferably CD8, specifically recognizing tumor cells ⁺ T cells;

optionally, the T cell is at least one of a CAR-T cell, a TIL cell, and a TCR-T cell;

optionally, the given protein is suitable for modulating BCL6 expression via the IL-2-STAT5 pathway and/or the TGF-beta-SMAD2 pathway;

optionally, expression of an immune checkpoint related protein in the T cell is down-regulated;

optionally, the immune checkpoint related protein comprises at least one of PD-1, CTLA-4, LAG-3, TIM-3, TIGIT and VISTA.

3. The T cell of claim 1 or 2, wherein the expression of BCL6 in the T cell is up-regulated;

Optionally, expression of the given protein in the T cell is altered so as to inhibit the IL-2-STAT5 pathway and/or activate the TGF-beta-SMAD2 pathway;

optionally, the given protein comprises at least one downstream protein on the IL-2-STAT5 pathway, and expression of the given protein is down-regulated;

optionally, the down-regulation of the expression of the given protein is achieved by at least one of gene silencing, gene editing, small molecule inhibitors, and antibody drugs;

optionally, the gene silencing or gene editing comprises at least one of CRISPR-Cas9 virus and non-virus, TALEN, ZFN, shRNA, and siRNA;

optionally, the given protein comprises at least one selected from the group consisting of: PRDM1 or a family member thereof;

optionally, the PRDM1 gene is knocked out in the T cell.

4. The T cell of claim 1 or 2, wherein the expression of BCL6 in the T cell is down-regulated;

optionally, expression of the given protein in the T cell is altered so as to activate the IL-2-STAT5 pathway and/or inhibit the TGF-beta-SMAD2 pathway;

optionally, the given protein comprises at least one downstream protein on the IL-2-STAT5 pathway, and expression of the given protein is up-regulated;

Optionally, the up-regulation of the expression of the given protein is achieved by at least one of gene transfection, promoter enhancers and co-expression factors;

optionally, the gene transfection is achieved by at least one of liposomes, calcium phosphate and PEI;

optionally, the PRDM1 gene is upregulated in the T cell.

5. A pharmaceutical composition comprising:

an agent for modulating the IL-2-STAT5 pathway and/or the TGF-beta-SMAD2 pathway, so as to modulate BCL6 expression in T cells; or alternatively

The T cell according to any one of claims 1 to 4.

6. The pharmaceutical composition of claim 5, wherein the agent is for inhibiting the IL-2-STAT5 pathway;

optionally, the agent inhibits the IL-2-STAT5 pathway by down-regulating expression of a given protein comprising at least one selected from the group consisting of: PRDM1 or a family member thereof;

optionally, the agent comprises at least one of a small molecule inhibitor, an antibody drug, a CRISPR-Cas9 virus, a non-virus, TALEN, ZFN, shRNA, and an siRNA;

Optionally, the agent is for activating a TGF-beta-SMAD2 pathway, the agent comprising a TGF-beta-SMAD2 pathway activator and/or a TGF-beta-SMAD3 pathway activator.

7. The pharmaceutical composition of claim 5, wherein the agent is for activating the IL-2-STAT5 pathway;

optionally, the agent activates the IL-2-STAT5 pathway by upregulating expression of a given protein comprising at least one selected from the group consisting of: PRDM1 or a family member thereof;

optionally, the agent comprises at least one of liposomes, calcium phosphate, and PEI;

optionally, the agent is for inhibiting the TGF-beta-SMAD2 pathway, the agent comprising a TGF-beta-SMAD2 pathway inhibitor and/or a TGF-beta-SMAD3 pathway inhibitor.

8. The pharmaceutical composition of claim 6, wherein the pharmaceutical composition further comprises: immune checkpoint blockers;

optionally, the immune checkpoint blocker comprises at least one of a PD-1 antibody, a PD-L1 antibody, a CTLA-4 antibody, a LAG-3 antibody, a TIM-3 antibody, a TIGIT antibody, and a VISTA antibody;

optionally, the immune checkpoint blocker is formulated for simultaneous administration with the T cells or separately.

9. Use of a T cell according to any one of claims 1 to 4 or a pharmaceutical composition according to any one of claims 5 to 8 in the manufacture of a medicament for the treatment or prevention of cancer;

optionally, the cancer comprises at least one selected from the group consisting of:

stomach cancer, lung cancer, pancreatic cancer, liver cancer, breast cancer, cervical cancer, skin cancer, prostate cancer, melanoma, thyroid cancer, uterine fibroids, lymphatic cancer, esophageal cancer, intestinal cancer, bone marrow cancer, nasal cancer, bone cancer, head and neck cancer, oral cancer, renal cancer, kaposi's sarcoma.

10. A combination or kit comprising:

immune checkpoint inhibitors; and

the T cell of any one of claims 1 to 4 or the pharmaceutical composition of any one of claims 5 to 8.

11. Inhibition of T cell to T _term A method of cell transformation comprising:

upregulating BCL6 expression in said T cells;

the T cells are selected from CD8 ⁺ T cells and CD4 ⁺ At least one of the T cells.

12. The method of claim 11, wherein said up-regulating expression of BCL6 in said T cells is achieved by down-regulating expression of a given protein in said T cells, said given protein being used to inhibit the IL-2-STAT5 pathway; or alternatively

Said upregulating BCL6 expression in said T cells is achieved by contacting said T cells with an agent for activating the TGF-beta-SMAD2 pathway;

optionally, the agent comprises a TGF-beta-SMAD2 pathway activator and/or a TGF-beta-SMAD3 pathway activator;

optionally, said down-regulating expression of a given protein in said T cell is achieved by at least one of gene silencing or gene editing, a small molecule inhibitor, and an antibody drug;

optionally, said upregulating BCL6 expression in said T cells is achieved by knocking out the PRDM1 gene in said T cells.