Method for quantifying neoplastic follicular regulatory t cells and neoplastic follicular cytotoxic t cells, method for screening neoplastic follicular regulatory t cells and neoplastic follicular cytotoxic t cells, therapeutic composition, and kit

Abstract

The present invention provides a technology useful for predicting prognosis in a follicular lymphoma patient. The present invention is a method for quantifying neoplastic follicular regulatory T cells and neoplastic follicular cytotoxic T cells in T cells collected from a subject, in order to determine the prognosis of follicular lymphoma. The method comprises: a flow cytometry step in which neoplastic follicular regulatory T cells and neoplastic follicular cytotoxic T cells in T cells collected from a subject are detected and counted with flow cytometry using an anti-CD4 antibody, an anti-CD8 antibody, an anti-PD-1 antibody, an anti-CD25 antibody, and an anti-TIM-3 antibody; or a multiplex immunostaining step in which neoplastic follicular regulatory T cells and neoplastic follicular cytotoxic T cells in T cells collected from a subject are detected and counted with multiplex immunostaining using an anti-CD4 antibody, an anti-CD8 antibody, an anti-PD -1 antibody, an anti-FOXP3 antibody, an anti-TCF-1 antibody, and an anti-Granzyme K antibody.

Description

Method for quantifying neoplastic follicle regulatory T cells and neoplastic follicle cytotoxic T cells, method for screening neoplastic follicle regulatory T cells and neoplastic follicle cytotoxic T cells, therapeutic composition, and kit

[0001] The present invention relates to a method for quantifying neoplastic follicle regulatory T cells and neoplastic follicle cytotoxic T cells in T cells collected from a subject, a method for screening neoplastic follicle regulatory T cells and neoplastic follicle cytotoxic T cells from T cells collected from a subject, a therapeutic composition, a kit, a method for assisting in predicting the prognosis of follicular lymphoma, and a method for predicting the prognosis of follicular lymphoma in a subject.This application claims priority based on Japanese Patent Application No. 2023-141172, filed in Japan on August 31, 2023, the contents of which are incorporated herein by reference.

[0002] Follicular lymphoma (FL) is a typical indolent (low-grade) B-cell lymphoma, accounting for 10-20% of all non-Hodgkin's lymphomas. The incidence of FL in Japan has been increasing recently. FL is histopathologically classified as grade 1, 2, 3A, or 3B, with grade 3B usually treated as aggressive (intermediate- to high-grade) lymphoma. Most patients present with lymph node (LN) enlargement, but 70-85% of patients present with advanced clinical stage III or IV disease at diagnosis, with a high incidence of bone marrow infiltration. FL generally has an indolent course and is initially well-responsive to chemotherapy. However, repeated recurrence is common in FL patients, particularly in advanced stages. Analysis after the introduction of rituximab® has shown that approximately 10% of patients undergo histologic transformation to aggressive lymphoma within five years. Data prior to the introduction of rituximab® indicated that the 50% survival time for FL patients was 7-10 years, but recent reports suggest that the 50% survival time exceeds 20 years for patients aged 40 years or younger at the time of diagnosis. The Ann Arbor classification is used for staging FL. Prognostic prediction models specific to FL include the Follicular Lymphoma International Prognostic Index (FLIPI) (Non-Patent Document 1) and FLIPI2 (Non-Patent Document 2). Cases in which disease progression or recurrence occurs within two years of diagnosis (POD24, Non-Patent Document 3) have been reported to have a poor prognosis, but no method for predicting this has been established. Among those diagnosed with FL, those with low tumor burden are actively selected for "watch and wait." Interestingly, when "watchful waiting" is selected, spontaneous regression is observed in approximately 20% of cases, suggesting that the patient is receiving continuous external stimulation from the surrounding microenvironment.

[0003] Philippe Solal-Celigny and 34 others, Follicular Lymphoma International Prognostic Index, Blood, September 1, 2004, Vol. 104, No. 5, pp. 1258-1265; Massimo Federico and 19 others, Follicular Lymphoma International Prognostic Index 2: A New Prognostic Index for Follicular Lymphoma Developed by the International Follicular Lymphoma, Journal of Clinical Oncology, September 20, 2009, Vol. 27, No. 27, pp. 4555-4562; Carla Casulo and 11 others, Early Relapse of Follicular Lymphoma After Rituximab Plus Cyclophosphamide, Doxorubicin, Vincristine, and Prednisone Defines Patients at High Risk for Death: An Analysis From the National LymphoCare Study, Journal of Clinical Oncology, August 10, 2015, Volume 23, Issue 23, p.2516-2522

[0004] The above-mentioned FLIPI and FLIPI2 include age as a factor and primarily reflect overall survival rate, but with improvements in anticancer drug treatment, emphasis is now being placed on recurrence rate (especially early recurrence rate within two years), and the development of a new prognosis prediction system that is in line with the times is desired. Furthermore, the above-mentioned POD24 accurately predicts overall survival rate, but this indicator itself refers to early recurrence, and since it takes 24 months for this factor to be determined, the risk at the time of diagnosis is unknown, and it cannot be used to improve initial treatment.

[0005] The present invention has been made in view of the above circumstances, and an objective of the present invention is to provide a technique useful for predicting the prognosis in patients with follicular lymphoma.

[0006] As a result of intensive research to solve the above-mentioned problems, the present inventors discovered that neoplastic follicle regulatory T cells (T neoplastic follicular regulatory cells: Tnfr) and neoplastic follicle cytotoxic T cells (T neoplastic follicular cytotoxic cells: Tnfc) can be detected in T cells collected from a subject by an antibody immune reaction using a specific antibody combination, and thus completed the present invention. Aspects of the present invention are as follows.

[0007] [1] A method for quantifying neoplastic follicle regulatory T cells (Tnfr) and neoplastic follicle cytotoxic T cells (Tnfc) in T cells collected from a subject for predicting the prognosis of follicular lymphoma, comprising: a flow cytometry step of detecting and counting neoplastic follicle regulatory T cells (Tnfr) and neoplastic follicle cytotoxic T cells (Tnfc) in T cells collected from the subject by flow cytometry using an anti-CD4 antibody, an anti-CD8a antibody, an anti-PD-1 antibody, an anti-CD25 antibody, and an anti-TIM-3 antibody; or [2] A method comprising a multiplex immunostaining step of detecting and counting neoplastic follicle regulatory T cells (Tnfr) and neoplastic follicle cytotoxic T cells (Tnfc) in T cells collected from a subject by a multiplex immunoassay using CD4 K antibody. ＋ PD-1 ＋ CD25 ＋ The cells were detected and counted as tumor follicle regulatory T cells (Tnfr) and CD8a ＋ PD-1 ＋ TIM-3 － [3] The method according to [1], wherein the cells are detected and counted as CD8-positive neoplastic follicle cytotoxic T cells (Tnfc8). ＋ PD-1 ＋ FOXP3 ＋ The cells were detected and counted as tumor follicle regulatory T cells (Tnfr) and CD8 ＋ Granzyme K ＋ PD-1＋ TCF-1 ＋ The cells were detected and counted as CD8-positive tumor follicle cytotoxic T cells (Tnfc8) and CD4 ＋ Granzyme K ＋ PD-1 ＋ TCF-1 ＋ The method according to [1], wherein the cells are detected and counted as CD4-positive tumor follicle cytotoxic T cells (Tnfc4).

[0008] [4] A method for screening neoplastic follicle regulatory T cells (Tnfr) and neoplastic follicle cytotoxic T cells (Tnfc) from T cells collected from a subject to predict the prognosis of follicular lymphoma, comprising: a flow cytometry step of detecting and counting neoplastic follicle regulatory T cells (Tnfr) and neoplastic follicle cytotoxic T cells (Tnfc) in T cells collected from the subject by flow cytometry using an anti-CD4 antibody, an anti-CD8a antibody, an anti-PD-1 antibody, an anti-CD25 antibody, and an anti-TIM-3 antibody; or [5] A method comprising a multiplex immunostaining step of detecting and counting neoplastic follicle regulatory T cells (Tnfr) and neoplastic follicle cytotoxic T cells (Tnfc) in T cells collected from a subject by a multiplex immunoassay using CD4 K antibody. ＋ PD-1 ＋ CD25 ＋ The cells were detected and counted as tumor follicle regulatory T cells (Tnfr) and CD8a ＋ PD-1 ＋ TIM-3 － [6] The method according to [4], wherein the cells are detected and counted as CD8-positive neoplastic follicle cytotoxic T cells (Tnfc8). ＋ PD-1 ＋ FOXP3 ＋ The cells were detected and counted as tumor follicle regulatory T cells (Tnfr) and CD8 ＋ Granzyme K ＋ PD-1 ＋ TCF-1 ＋The cells were detected and counted as CD8-positive tumor follicle cytotoxic T cells (Tnfc8) and CD4 ＋ Granzyme K ＋ PD-1 ＋ TCF-1 ＋ The method according to [4], wherein the cells are detected and counted as CD4-positive tumor follicle cytotoxic T cells (Tnfc4).

[0009] [7] A therapeutic composition for treating follicular lymphoma, comprising: a flow cytometry step of detecting and counting neoplastic follicle regulatory T cells (Tnfr) and neoplastic follicle cytotoxic T cells (Tnfc) in T cells collected from a subject by flow cytometry using an anti-CD4 antibody, an anti-CD8a antibody, an anti-PD-1 antibody, an anti-CD25 antibody, and an anti-TIM-3 antibody; ＋ PD-1 ＋ CD25 ＋ cells or CD8a ＋ PD-1 ＋ TIM-3 － A therapeutic composition comprising T cells detected as cells.

[0010] [8] A therapeutic composition for treating follicular lymphoma, comprising: a multiplex immunostaining step of detecting and counting neoplastic follicle regulatory T cells and neoplastic follicle cytotoxic T cells in T cells collected from a subject by a multiplex immunoassay using an anti-CD4 antibody, an anti-CD8 antibody, an anti-PD-1 antibody, an anti-FOXP3 antibody, an anti-TCF-1 antibody, and an anti-Granzyme K antibody; ＋ PD-1 ＋ FOXP3 ＋ , CD8 ＋ Granzyme K ＋ PD-1 ＋ TCF-1 ＋ cells or CD4 ＋ Granzyme K ＋ PD-1 ＋ TCF-1 ＋[9] The therapeutic composition according to [7] or [8], wherein the T cells are T cells pre-cultured with IL-21.

[10] The therapeutic composition according to [7] or [8], wherein the T cells are T cells that have been artificially genetically modified.

[0011]

[11] A kit for predicting the prognosis of follicular lymphoma, comprising an antibody set comprising an anti-CD4 antibody, an anti-CD8a antibody, an anti-PD-1 antibody, an anti-CD25 antibody, and an anti-TIM-3 antibody, or an antibody set comprising an anti-CD4 antibody, an anti-CD8 antibody, an anti-PD-1 antibody, an anti-FOXP3 antibody, an anti-TCF-1 antibody, and an anti-Granzyme K antibody.

[12] The kit according to

[11] , for identifying, detecting, or screening neoplastic follicle regulatory T cells (Tnfr) and neoplastic follicle cytotoxic T cells (Tnfc) from a population of T cells collected from a subject.

[0012]

[13] A method for assisting in predicting the prognosis of follicular lymphoma in a subject, the method comprising: a flow cytometry step of detecting and counting neoplastic follicle regulatory T cells (Tnfr) and neoplastic follicle cytotoxic T cells (Tnfc) in T cells collected from the subject by flow cytometry using an anti-CD4 antibody, an anti-CD8a antibody, an anti-PD-1 antibody, an anti-CD25 antibody, and an anti-TIM-3 antibody; and a proportion calculation step of calculating the proportions of neoplastic follicle regulatory T cells (Tnfr), CD4-positive neoplastic follicle cytotoxic T cells (Tnfc4), and CD8-positive neoplastic follicle cytotoxic T cells (Tnfc8) in the T cells collected from the subject.

[14] The method according to

[13] , wherein the proportion of CD8-positive neoplastic follicle cytotoxic T cells (Tnfc8) is calculated based on the results of detecting and counting CD4-positive neoplastic follicle cytotoxic T cells (Tnfc4) in the proportion calculation step.

[15] A method for assisting in predicting the prognosis of follicular lymphoma in a subject, the method comprising: a multiplex immunostaining step of detecting and counting neoplastic follicle regulatory T cells (Tnfr) and neoplastic follicle cytotoxic T cells (Tnfc) in T cells collected from the subject by a multiplex immunoassay using an anti-CD4 antibody, an anti-CD8 antibody, an anti-PD-1 antibody, an anti-FOXP3 antibody, an anti-TCF-1 antibody, and an anti-Granzyme K antibody; and a proportion calculation step of calculating the proportions of neoplastic follicle regulatory T cells (Tnfr), CD4-positive neoplastic follicle cytotoxic T cells (Tnfc4), and CD8-positive neoplastic follicle cytotoxic T cells (Tnfc8) in the T cells collected from the subject.

[0013]

[16] A method for predicting the prognosis of follicular lymphoma in a subject, comprising: a quantification step of quantifying neoplastic follicle regulatory T cells (Tnfr) and neoplastic follicle cytotoxic T cells (Tnfc) in T cells collected from the subject by the method according to any one of [1] to [6]; a proportion calculation step of calculating the proportions of neoplastic follicle regulatory T cells, CD4-positive neoplastic follicle cytotoxic T cells (Tnfc4), and CD8-positive neoplastic follicle cytotoxic T cells (Tnfc8) in the T cells collected from the subject based on the quantification results obtained in the quantification step; and a prognosis evaluation step of evaluating the prognosis of the subject based on the proportions calculated in the proportion calculation step. In the prognosis evaluation step, if the proportions calculated in the proportion calculation step are less than 2.0% for neoplastic follicle regulatory T cells (Tnfr) and less than 2.5% for the sum of the CD4-positive neoplastic follicle cytotoxic T cells and the CD8-positive neoplastic follicle cytotoxic T cells, the prognosis is assessed as poor.

[0014] According to the present invention, a technique useful for predicting the prognosis of patients with follicular lymphoma can be provided.

[0015] FIG. 1 shows the results of Experimental Example 1. The graph shows the results of flow cytometry. FIG. 2 shows the results of Experimental Example 2. The images show the results of multiple immunostaining. a. Multiple immunostaining, b. Tnfr detection, c. Tnfc detection. FIG. 3A shows the results of Experimental Example 3. The graph shows the relationship between the recurrence rate (vertical axis) and the period (horizontal axis) for follicular lymphoma patient cohorts (original cohort and validation cohort 1) according to the cell percentages of neoplastic follicle regulatory T cells (Tnfr) and neoplastic follicle cytotoxic T cells (Tnfc). FIG. 3B shows the results of Experimental Example 3. The graph shows the relationship between the recurrence rate (vertical axis) and the period (horizontal axis) for follicular lymphoma patient cohorts (validation cohort 2 and all cohorts) according to the cell percentages of neoplastic follicle regulatory T cells (Tnfr) and neoplastic follicle cytotoxic T cells (Tnfc). Figure 4 shows the results of Experimental Example 3. The graph shows the relationship between the recurrence rate (vertical axis) and the period (horizontal axis) for a cohort of follicular lymphoma patients judged to be low risk and a cohort of follicular lymphoma patients judged to be high risk according to FLIPI risk classification (original cohort and validation cohort 1). Figure 5 shows the results of Experimental Example 4. a. Violin plot of the expression of inhibitory factor genes in Tnfr and follicular regulatory T cells in normal lymph nodes; b. The inhibitory effect of Tnfr on the activation of Tfh cells; c. The inhibitory effect of Tnfr on the differentiation of Tfh cells; d. The inhibitory effect of Tnfr on the activation of neoplastic B cells; e. The inhibitory effect of Tnfr on the cell survival of neoplastic B cells. Figure 6 shows the results of Experimental Example 5. a. Comparison of gene expression between Tnfc8 and effector CD8 cells; b. Comparison of gene expression between Tnfc8 and exhausted CD8 cells; c. a. Cell division ability; d. Interferon gamma production ability; e. TNF-alpha production ability; f. Activation-inhibitory effect of Tnfc8 on neoplastic B cells; g. Cytocidal effect of Tnfc8 on neoplastic B cells. Figure 7 shows the results of Experimental Example 6, in which each cell was cultured under T cell receptor (TCR) stimulation in the presence of TGF (transforming growth factor) β1, with or without the addition of IL-21. a. Induction of Tnfr phenotype by IL-21; b. Induction of Tnfc8 phenotype by IL-21. Figure 8 shows the results of Experimental Example 6, in which each cell was cultured under TCR stimulation, with or without the addition of TGFβ1 and IL-21.a. Induction of Tnfr phenotype by IL-21; b. Induction of Tnfc8 phenotype by IL-21.

[0016] In the present invention, when a numerical range is expressed using "to" it is intended that the numerical range includes the numerical values ​​before and after "to." In the present invention, the "subject" refers to a human from whom T cells are to be collected, and is preferably a human suffering from follicular lymphoma.

[0017] The following describes in detail the embodiments of the present invention. However, the present invention is not limited to the embodiments described below, and various modifications are possible without departing from the gist of the present invention.

[0018] [Quantification Method] The method of the present invention for quantifying neoplastic follicle regulatory T cells and neoplastic follicle cytotoxic T cells in T cells collected from a subject (hereinafter also simply referred to as the "quantification method") comprises a flow cytometry step or a multiple immunostaining step. The flow cytometry step and the multiple immunostaining step are steps performed in vitro.

[0019] <Flow Cytometry Step> The flow cytometry step is a step of detecting and counting neoplastic follicle regulatory T cells (Tnfr) and neoplastic follicle cytotoxic T cells (Tnfc) in T cells collected from a subject by flow cytometry using an antibody set including an anti-CD4 (cluster of differentiation 4) antibody, an anti-CD8a antibody, an anti-PD-1 (programmed cell death protein 1) antibody, an anti-CD25 antibody, and an anti-TIM-3 (T cell immunoglobulin domain and mucin domain 3) antibody.

[0020] The flow cytometry method can be a method conventionally used in the art, except that the above-mentioned antibody set is used as the antibody for detecting cells.

[0021] The aforementioned antibody may be a primary antibody that is itself fluorescently labeled, metal-labeled, or DNA barcode-labeled, or may be an antibody to which a secondary antibody that is fluorescently labeled, metal-labeled, or DNA barcode-labeled specifically binds.

[0022] Common progenitor cells derived from bone marrow differentiate into three types of T cells with different functions in the thymus. One type expresses γδ receptors, which do not recognize peptide antigens presented by MHC (Major Histocompatibility Complex) molecules, and the other two types express αβ receptors. αβ-type T cells undergo positive and negative selection in the thymus, and differentiate into CD4 or CD8 single-positive cells, depending on the specificity of the receptor for MHC. CD4 and CD8 are coreceptors expressed on the surface of T cells and bind to conserved regions of MHC class II molecules and MHC class I molecules, respectively.

[0023] When PD-1 expressed on activated T cells binds to PD-L1 or PD-L2 expressed on cancer cells or antigen-presenting cells, T cell activation is suppressed, causing the cancer cells to escape the immune system. Anti-PD-1 antibodies bind to PD-1 on T cells and inhibit the binding of PD-1 to PD-L1 / PD-L2, thereby blocking the transmission of inhibitory signals, maintaining T cell activation and restoring antitumor effects.

[0024] The CD25 (IL-2Rα chain, Tac or p55) antigen is a 55-kDa monosaccharide protein and a low-affinity receptor for IL-2 (IL-2Rα or IL-2R1). It forms the high-affinity IL-2R complex with CD12 (IL-2Rβ chain, p75) and CD132 (IL-2Rγ chain, p64). It is highly expressed on activated regulatory CD4+ T cells but not on resting CD8+ lymphocytes. However, all activated T cells express the CD25 protein. CD25 is also found on a subset of B cells (CD20+), activated monocytes, macrophages, and T-cell clones. A recombinant human / mouse chimeric anti-CD25 monoclonal antibody (IgG1) is known as basiliximab (recombinant).

[0025] TIM-3 (also known as HAVCR2) is a recently discovered member of the T cell immunoglobulin-mucin domain-containing molecule superfamily. TIM-3 is a key regulatory molecule in T cell tolerance and plays a crucial role in autoimmunity and T cell exhaustion during chronic viral infection. TIM-3 is expressed on Th1 cells (type 1 T helper cells), macrophages, monocytes, dendritic cells, CD8+ T cells, and other lymphocyte subsets. The ligand for TIM-3 is galectin-9. The TIM-3-galectin-9 pathway negatively regulates Th1 cell (type 1 T helper cell) immunity. TIM3 is a membrane protein consisting of 280 residues with a calculated molecular weight of 33 kDa.

[0026] The antibody set described above may further include an anti-CD3 antibody. The anti-CD3 antibody recognizes the human CD3 molecule. CD3 is a component of the T cell receptor and is most commonly used as a T cell marker. As the anti-CD3 antibody, for example, an anti-CD3e antibody that recognizes the ε chain of the human CD3 molecule may be used.

[0027] In the flow cytometry step, tumor follicle regulatory T cells (Tnfr) were detected by CD4 ＋ PD-1 ＋ CD25 ＋ They are detected and counted as cells.

[0028] In the flow cytometry step, CD8-positive tumor follicle cytotoxic T cells (Tnfc8) were detected by CD8a ＋ PD-1 ＋ TIM-3 － They are detected and counted as cells.

[0029] In the flow cytometry step, CD4-positive neoplastic follicle cytotoxic T cells (Tnfc4) are not directly detected or counted, but there is a strong correlation between the proportions of CD8-positive neoplastic follicle cytotoxic T cells and CD4-positive neoplastic follicle cytotoxic T cells, making it possible to calculate the proportion of CD4-positive neoplastic follicle cytotoxic T cells. Specifically, as shown in the experimental example described below, a strong positive correlation was observed in a study of 169 patients, with a correlation coefficient (Spearman's) of 0.93.

[0030] <Multiple Immunostaining Step> The multiple immunostaining step is a step of detecting and counting neoplastic follicle regulatory T cells (Tnfr) and neoplastic follicle cytotoxic T cells (Tnfc) in T cells collected from a subject by multiple immunoassay using an anti-CD4 antibody, an anti-CD8 antibody, an anti-PD-1 antibody, an anti-FOXP3 (forkhead box P3) antibody, an anti-TCF-1 (T cell factor 1) antibody, and an anti-Granzyme K antibody.

[0031] The multiplex immunostaining method can be a method conventionally used in the art, except that the above-mentioned antibody set is used as the antibodies for detecting cells.

[0032] The anti-CD4 antibody and anti-PD-1 antibody are as described above.

[0033] The aforementioned antibody may be a primary antibody that is itself fluorescently labeled, metal-labeled, or DNA barcode-labeled, or may be an antibody to which a secondary antibody that is fluorescently labeled, metal-labeled, or DNA barcode-labeled specifically binds.

[0034] The transcription factor T cell factor 1 (TCF-1) is essential for T cell development in the thymus, as well as a downstream effector of WNT / -catenin signaling.

[0035] The FOXP3 molecule is a 50-55 kD transcription factor, also known as IPEX, JM2, Forkhead box P3, and Scurfin.

[0036] In the multiple immunostaining step, tumor follicle regulatory T cells (Tnfr) were detected by CD4 ＋ PD-1＋ FOXP3 ＋ They are detected and counted as cells.

[0037] In the multiple immunostaining step, CD8-positive tumor follicle cytotoxic T cells (Tnfc8) were detected by CD8 ＋ Granzyme K ＋ PD-1 ＋ TCF-1 ＋ They are detected and counted as cells.

[0038] In the multiple immunostaining step, CD4-positive tumor follicle cytotoxic T cells (Tnfc4) were detected by CD4 ＋ Granzyme K ＋ PD-1 ＋ TCF-1 ＋ They are detected and counted as cells.

[0039] [Screening Method] The method of the present invention for screening neoplastic follicle regulatory T cells (Tnfr) and neoplastic follicle cytotoxic T cells (Tnfc) from T cells collected from a subject (hereinafter, also simply referred to as the "screening method") includes a flow cytometry step or a multiple immunostaining step. The flow cytometry step and the multiple immunostaining step are the same as the above-mentioned quantification method.

[0040] In the flow cytometry step, tumor follicle regulatory T cells (Tnfr) were detected by CD4 ＋ PD-1 ＋ CD25 ＋ CD8-positive tumor follicle cytotoxic T cells (Tnfc8) were screened as CD8a ＋ PD-1 ＋ TIM-3 － The cells are screened.

[0041] In the multiple immunostaining step, tumor follicle regulatory T cells (Tnfr) were detected by CD4 ＋ PD-1 ＋ FOXP3 ＋ CD8-positive tumor follicle cytotoxic T cells (Tnfc8) were screened as CD8 ＋ Granzyme K ＋ PD-1 ＋ TCF-1＋ CD4-positive tumor follicular cytotoxic T cells (Tnfc4) were screened as CD4 ＋ Granzyme K ＋ PD-1 ＋ TCF-1 ＋ The cells are screened.

[0042] [T Cells] One embodiment of the T cells of the present invention is T cells collected from a subject, wherein the T cells are CD4, CD8, PD-1, CD25, and TIM-3 antibodies, and a flow cytometry step is performed to detect and count neoplastic follicle regulatory T cells (Tnfr) and neoplastic follicle cytotoxic T cells (Tnfc) in the T cells collected from the subject by flow cytometry. ＋ PD-1 ＋ CD25 ＋ cells or CD8a ＋ PD-1 ＋ TIM-3 － The cells detected are T cells. The flow cytometry process is the same as the flow cytometry process described above.

[0043] Another embodiment of the T cells of the present invention is a method for detecting and counting neoplastic follicle regulatory T cells (Tnfr) and neoplastic follicle cytotoxic T cells (Tnfc) in T cells collected from a subject by a multiplex immunoassay using an anti-CD4 antibody, an anti-CD8 antibody, an anti-PD-1 antibody, an anti-FOXP3 antibody, an anti-TCF-1 antibody, and an anti-Granzyme K antibody in a multiplex immunostaining step. ＋ PD-1 ＋ FOXP3 ＋ , CD8 ＋ Granzyme K ＋ PD-1 ＋ TCF-1 ＋ cells or CD4 ＋ Granzyme K ＋ PD-1 ＋ TCF-1 ＋ The multiple immunostaining step is the same as the multiple immunostaining step described above.

[0044] The T cells of this embodiment can be used to treat follicular lymphoma. As shown in Experimental Examples 5 to 7 described below, Tnfr has an inhibitory effect on the activation and differentiation of follicular helper T cells (Tfh) in normal lymph nodes. Furthermore, Tnfc has an inhibitory effect on the activation and cytocidal effect of neoplastic B cells, which are cancer cells in follicular lymphoma. Furthermore, Tnfr enhances the expression of inhibitory genes such as IL2RA, LAG3, and IL10, while Tnfc highly expresses CXCL13, CXCR5, and BCL6 genes, which are factors that promote migration and settlement in follicular structures, and also highly expresses the TCF7 gene, which maintains the juvenile state of cells. Furthermore, the cytokine IL-21 can induce Tnfr from regulatory T cells (high expression of CXCR5 and BCL6), and can induce Tnfc8 cells from naive CD8 cells (high expression of CXCR5, CCR7, Granzyme M, and TCF1). That is, the T cells of this embodiment are preferably T cells pre-cultured with IL-21. This cell induction method can be used to modify T cell gene modification therapy, and adding the characteristics of Tnfr or Tnfc cells is expected to improve therapeutic efficacy.

[0045] Neoplastic follicle regulatory T cells (Tnfr) and neoplastic follicle cytotoxic T cells (Tnfc) extracted from patient T cells can be used to develop therapeutics for use as direct intervention.

[0046] In one embodiment of the T cell of the present invention, the T cell of the above-mentioned embodiment or the T cell of the above-mentioned other embodiment may be further artificially genetically modified.

[0047] In the present invention, "artificial genetic modification" means modifying the function of a T cell by introducing a gene into or editing the gene of a T cell. The "artificial genetic modification" in the present invention may be a modification of a gene carried by a T cell, a deletion of a gene carried by a T cell, a modification in which an exogenous gene is introduced, or a combination of two or more of these, and is preferably a modification in which an exogenous gene is introduced.

[0048] Methods for artificial gene modification include, but are not limited to, conventionally known methods and methods based thereon. For example, methods using viral vectors, agroinfection, calcium phosphate, microinjection, particle gun, DEAE-dextran, electroporation, cationic lipid, and gene editing (genome editing) of T cells using site-specific nucleases (meganucleases, zinc finger nucleases, TALEN, PPR, CRISPR-Cas, etc.). The artificial gene modification method may be one of these methods alone or a combination of two or more of them.

[0049] The gene that alters the function of a T cell, i.e., the gene to be altered by artificial genetic modification, is preferably at least one selected from the group consisting of genes encoding a fusion protein comprising a protein expressed on the surface of a T cell and at least one intracellular signaling domain, and genes encoding a fusion protein comprising a protein expressed on the surface of a T cell, at least one costimulatory domain, and at least one intracellular signaling domain, more preferably a gene encoding an antigen receptor, even more preferably at least one selected from the group consisting of genes encoding a T cell receptor (TCR) and genes encoding a chimeric antigen receptor (CAR), and particularly preferably a gene encoding a chimeric antigen receptor (CAR).

[0050] That is, in one embodiment of the T cells of the present invention, it is preferable that the T cells express on their surface a surface protein encoded by a gene of interest that has been modified by artificial genetic modification. Such genetically modified T cells are more preferably at least one type selected from the group consisting of TCR-T cells that express a T cell receptor (TCR) encoded by a gene that has been modified by artificial genetic modification, and CAR-T cells that express a chimeric antigen receptor (CAR), and are even more preferably CAR-T cells.

[0051] [Therapeutic Composition] One embodiment of the therapeutic composition of the present invention is a therapeutic composition for treating follicular lymphoma, and comprises: an anti-CD4 antibody, an anti-CD8a antibody, an anti-PD-1 antibody, an anti-CD25 antibody, and an anti-TIM-3 antibody; a flow cytometry step for detecting and counting neoplastic follicle regulatory T cells and neoplastic follicle cytotoxic T cells in T cells collected from a subject by flow cytometry; ＋ PD-1 ＋ CD25 ＋ cells or CD8a ＋ PD-1 ＋ TIM-3 － The therapeutic composition includes T cells detected as T cells. The T cells are the same as the T cells of this embodiment.

[0052] Alternatively, another embodiment of the therapeutic composition of the present invention is a therapeutic composition for treating follicular lymphoma, comprising: a CD4 antibody, an anti-CD8 antibody, an anti-PD-1 antibody, an anti-FOXP3 antibody, an anti-TCF-1 antibody, and an anti-Granzyme K antibody in a multiplex immunostaining step of detecting and counting neoplastic follicle regulatory T cells and neoplastic follicle cytotoxic T cells in T cells collected from a subject by a multiplex immunoassay method using an anti-CD4 antibody, an anti-CD8 antibody, an anti-PD-1 antibody, an anti-FOXP3 antibody, an anti-TCF-1 antibody, and an anti-Granzyme K antibody; ＋ PD-1 ＋ FOXP3 ＋ , CD8 ＋ Granzyme K ＋ PD-1 ＋ TCF-1 ＋ cells or CD4 ＋ Granzyme K ＋ PD-1 ＋ TCF-1 ＋ The therapeutic composition includes T cells detected as T cells. The T cells are the same as the T cells of this embodiment.

[0053] The therapeutic composition of this embodiment may contain T cells obtained by further culturing and expanding the T cells of this embodiment, or by concentrating the T cells by centrifugation or the like, or by an appropriate combination of these methods, and may contain, in addition to the T cells of this embodiment, known culture media, cell washing solutions, excipients, additives, and the like.

[0054] The T cells of this embodiment can be used to produce a therapeutic composition for treating follicular lymphoma. The therapeutic composition of this embodiment can be formulated into an injection or the like by a standard method together with various additives, and can be used to produce a therapeutic agent for follicular lymphoma.

[0055] In one embodiment, the present invention may be a method for treating a patient with follicular lymphoma using the T cells or therapeutic composition of this embodiment. The treatment method may be a method conventionally used in the technical field, except that the T cells or therapeutic composition of this embodiment is used. For example, the treatment method may be chimeric antigen receptor-T cell therapy (CAR-T cell therapy) using the T cells or therapeutic composition of this embodiment, T cell receptor (TCR)-T cell therapy (TCR-T cell therapy) using the T cells or therapeutic composition of this embodiment, or tumor-infiltrating T cell therapy (TIL therapy) using the T cells or therapeutic composition of this embodiment.

[0056] CAR-T cell therapy using the T cells or therapeutic composition of the present embodiment may be a treatment method for patients with B-cell acute lymphoblastic leukemia, diffuse large B-cell lymphoma, multiple myeloma, chronic lymphocytic leukemia, acute myeloid leukemia, or the like.

[0057] TCR-T cell therapy using the T cells or therapeutic composition of the present embodiment may be a treatment method for patients with malignant melanoma, ovarian cancer, non-small cell lung cancer, esophageal cancer, colorectal cancer, synovial sarcoma, myeloma, osteosarcoma, malignant mesothelioma, adult T-cell leukemia / lymphoma, myelodysplastic syndrome, acute myeloid leukemia, or the like.

[0058] TIL therapy using the T cells or therapeutic composition of this embodiment may be a treatment method for patients with malignant melanoma, cervical cancer, and the like.

[0059] That is, the present invention may include the following aspects: A method for treating a patient with follicular lymphoma, comprising: detecting and counting neoplastic follicle regulatory T cells (Tnfr) and neoplastic follicle cytotoxic T cells (Tnfc) in T cells collected from a subject by flow cytometry using an anti-CD4 antibody, an anti-CD8 antibody, an anti-PD-1 antibody, an anti-CD25 antibody, and an anti-TIM-3 antibody; ＋ PD-1 ＋ CD25 ＋ cells or CD8a ＋ PD-1 ＋ TIM-3 － The method uses T cells detected as the cells.

[0060] Alternatively, a method for treating a patient with follicular lymphoma, comprising: a multiplex immunostaining step of detecting and counting neoplastic follicle regulatory T cells (Tnfr) and neoplastic follicle cytotoxic T cells (Tnfc) in T cells collected from a subject by a multiplex immunoassay using an anti-CD4 antibody, an anti-CD8 antibody, an anti-PD-1 antibody, an anti-FOXP3 antibody, an anti-TCF-1 antibody, and an anti-Granzyme K antibody; ＋ PD-1 ＋ FOXP3 ＋ , CD8 ＋ Granzyme K ＋ PD-1 ＋ TCF-1 ＋ cells or CD4 ＋ Granzyme K ＋ PD-1 ＋ TCF-1 ＋ The method uses T cells detected as the cells.

[0061] Alternatively, a method for treating a patient with follicular lymphoma, comprising: detecting and counting neoplastic follicle regulatory T cells (Tnfr) and neoplastic follicle cytotoxic T cells (Tnfc) in T cells collected from a subject by flow cytometry using an anti-CD4 antibody, an anti-CD8 antibody, an anti-PD-1 antibody, an anti-CD25 antibody, and an anti-TIM-3 antibody; ＋ PD-1 ＋ CD25 ＋ cells or CD8a＋ PD-1 ＋ TIM-3 － A method using a therapeutic composition comprising T cells detected as the cells.

[0062] Alternatively, a method for treating a patient with follicular lymphoma, comprising: a multiplex immunostaining step of detecting and counting neoplastic follicle regulatory T cells (Tnfr) and neoplastic follicle cytotoxic T cells (Tnfc) in T cells collected from a subject by a multiplex immunoassay using an anti-CD4 antibody, an anti-CD8 antibody, an anti-PD-1 antibody, an anti-FOXP3 antibody, an anti-TCF-1 antibody, and an anti-Granzyme K antibody; ＋ PD-1 ＋ FOXP3 ＋ , CD8 ＋ Granzyme K ＋ PD-1 ＋ TCF-1 ＋ cells or CD4 ＋ Granzyme K ＋ PD-1 ＋ TCF-1 ＋ A method using a therapeutic composition comprising T cells detected as the cells.

[0063] [Kit] The kit of the present invention comprises an antibody set (first antibody set) comprising an anti-CD4 antibody, an anti-CD8a antibody, an anti-PD-1 antibody, an anti-CD25 antibody, and an anti-TIM-3 antibody, or an antibody set (second antibody set) comprising an anti-CD4 antibody, an anti-CD8 antibody, an anti-PD-1 antibody, an anti-FOXP3 antibody, an anti-TCF-1 antibody, and an anti-Granzyme K antibody. Both the first antibody set and the second antibody set described above may further comprise an anti-CD3 antibody or an anti-CD3e antibody.

[0064] The kit of the present invention can detect and count neoplastic follicle regulatory T cells (Tnfr) and neoplastic follicle cytotoxic T cells (Tnfc) by flow cytometry using the first antibody set.

[0065] The kit of the present invention can detect and count neoplastic follicle regulatory T cells (Tnfr) and neoplastic follicle cytotoxic T cells (Tnfc) by a multiplex immunostaining method using the second antibody set.

[0066] The kit of the present invention may further include other components, such as an experimental protocol.

[0067] The kit of the present invention can be used to diagnose or predict the prognosis of follicular lymphoma.

[0068] The kits of the present invention can be used to identify, detect, or screen for neoplastic follicle regulatory T cells (Tnfr) and neoplastic follicle cytocidal T cells (Tnfc) from a population of T cells collected from a subject.

[0069] In one embodiment, the present invention provides the first set of antibodies and the second set of antibodies for predicting the prognosis of follicular lymphoma.

[0070] In one embodiment, the present invention provides use of the first set of antibodies and the second set of antibodies for producing a kit for predicting the prognosis of follicular lymphoma.

[0071] [Method for Assisting Prognosis Prediction] One embodiment of the method for assisting in prognosis prediction of follicular lymphoma in a subject of the present invention (hereinafter also simply referred to as the "method for assisting prognosis prediction") comprises a flow cytometry step of detecting and enumerating neoplastic follicle regulatory T cells (Tnfr) and neoplastic follicle cytotoxic T cells (Tnfc) in T cells collected from the subject by flow cytometry using an anti-CD4 antibody, an anti-CD8a antibody, an anti-PD-1 antibody, an anti-CD25 antibody, and an anti-TIM-3 antibody, and a proportion calculation step of calculating the proportions of neoplastic follicle regulatory T cells (Tnfr), CD4-positive neoplastic follicle cytotoxic T cells (Tnfc4), and CD8-positive neoplastic follicle cytotoxic T cells (Tnfc8) in the T cells collected from the subject. The flow cytometry step and the proportion calculation step are performed in vitro.

[0072] In the proportion calculation step, it is preferable to calculate the proportion of CD8-positive neoplastic follicle cytotoxic T cells (Tnfc8) based on the results of detecting and counting CD4-positive neoplastic follicle cytotoxic T cells (Tnfc4).

[0073] Another embodiment of the method of assisting in prognosis prediction of the present invention is a method for assisting in predicting the prognosis of follicular lymphoma in a subject, comprising: a multiplex immunostaining step of detecting and enumerating neoplastic follicle regulatory T cells (Tnfr) and neoplastic follicle cytotoxic T cells (Tnfc) in T cells collected from the subject by multiplex immunoassay using an anti-CD4 antibody, an anti-CD8 antibody, an anti-PD-1 antibody, an anti-FOXP3 antibody, an anti-TCF-1 antibody, and an anti-Granzyme K antibody; and a proportion calculation step of calculating the proportions of neoplastic follicle regulatory T cells (Tnfr), CD4-positive neoplastic follicle cytotoxic T cells (Tnfc4), and CD8-positive neoplastic follicle cytotoxic T cells (Tnfc8) in the T cells collected from the subject.

[0074] [Method for predicting prognosis] The method for predicting the prognosis of follicular lymphoma in a subject of the present invention comprises, after the above-described method for assisting in prognosis prediction, a prognosis evaluation step of evaluating the prognosis of the subject based on the proportions of neoplastic follicle regulatory T cells (Tnfr), CD4-positive neoplastic follicle cytotoxic T cells (Tnfc4), and CD8-positive neoplastic follicle cytotoxic T cells (Tnfc8) in T cells collected from the subject. In the prognosis evaluation step, if the proportions of neoplastic follicle regulatory T cells (Tnfr), CD4-positive neoplastic follicle cytotoxic T cells (Tnfc4), and CD8-positive neoplastic follicle cytotoxic T cells (Tnfc8) among T cells collected from a subject are less than 2.0% for Tnfr and less than 2.5% for Tnfc (total of Tnfc4 and Tnfc8), the patient is evaluated as having a high risk (poor prognosis), whereas if Tnfr is 2.0% or more and Tnfc is 2.5% or more, the patient is evaluated as having a low risk (not being considered to have a poor prognosis). The prognosis evaluation step is a step performed in vitro.

[0075] Alternatively, the method of the present invention for predicting the prognosis of follicular lymphoma in a subject comprises: a quantification step of quantifying neoplastic follicle regulatory T cells (Tnfr) and neoplastic follicle cytotoxic T cells (Tnfc) in T cells collected from the subject by the above-mentioned quantification method; a proportion calculation step of calculating the proportions of neoplastic follicle regulatory T cells (Tnfr), CD4-positive neoplastic follicle cytotoxic T cells (Tnfc4), and CD8-positive neoplastic follicle cytotoxic T cells (Tnfc8) in the T cells collected from the subject based on the quantification results obtained in the quantification step; and a prognosis evaluation step of evaluating the prognosis of the subject based on the proportions calculated in the proportion calculation step, In the prognosis evaluation step, if the proportions calculated in the proportion calculation step are less than 2.0% for the neoplastic follicle regulatory T cells and less than 2.5% for the sum of the CD4-positive neoplastic follicle cytotoxic T cells and the CD8-positive neoplastic follicle cytotoxic T cells, the prognosis is evaluated as poor, and if Tnfr is 2.0% or more and Tnfc is 2.5% or more, the patient is evaluated as having a low risk (the prognosis cannot be said to be poor).

[0076] Furthermore, by combining it with existing indices such as the conventional FLIPI or FLIPI2, more accurate prognosis predictions can be made, and it is also possible to extract subjects (patients) with extremely good prognosis or extremely poor prognosis.

[0077] In one embodiment, the present invention may further include another step after the prognosis evaluation step of the method for predicting prognosis of this embodiment. Such another step may include a step of treating a patient with follicular lymphoma based on the results of the prognosis prediction. When the method for predicting prognosis of this embodiment further includes a treatment step in addition to the prognosis evaluation step, the method for predicting prognosis of this embodiment may be a method for treating a patient with follicular lymphoma.

[0078] In the treatment step, a treatment policy for follicular lymphoma can be determined based on the evaluation results of the method for predicting prognosis of the present embodiment. Treatment methods for follicular lymphoma patients evaluated as having a poor prognosis include the same treatment methods as those for follicular lymphoma patients described above.

[0079] The present invention will be described in more detail below with reference to experimental examples, but the present invention is not limited to the examples described below.

[0080] Experimental Example 1: Flow cytometry experiment A flow cytometry method similar to that typically used in clinical testing was used. Clinical lymphoma samples were processed into single cells, and then flow cytometry was performed using anti-CD3 antibody, anti-CD4 antibody, anti-CD8a antibody, anti-PD-1 antibody, anti-CD25 antibody, and anti-TIM-3 antibody.

[0081] CD4-positive cells and CD8-positive cells were detected and counted from T cells collected from patients (Figure 1). ＋ (CD3 ＋ )PD-1 ＋ CD25 ＋ As a group, tumor follicle regulatory T cells (Tnfr) were detected. ＋ PD-1 ＋ TIM-3 － As a population, CD8-positive tumor follicle cytotoxic T cells (Tnfc8) were detected, and the proportion of these cells in the total T cells (CD3-positive cells or CD4-positive cells and CD8-positive cells) could be calculated.

[0082] Experimental Example 2: Experiment using multiple immunostaining method A method similar to the multiple immunostaining method typically used in clinical testing was used. Formalin-fixed, paraffin-embedded clinical lymphoma samples were subjected to multiple immunostaining using an anti-CD3e antibody, anti-CD4 antibody, anti-CD8 antibody, anti-PD-1 antibody, anti-FOXP3 antibody, anti-TCF-1 antibody, and anti-Granzyme K antibody with a PhenoCycler-Fusion system (AKOYA).

[0083] The antibodies were prepared according to the protocol recommended by AKOYA, and the experiment was carried out (Figure 2: a. Multiple immunostaining). ＋ PD-1 ＋ FOXP3 ＋ As a group, tumor follicle regulatory T cells (Tnfr) were detected (Fig. 2: b. Detection of Tnfr). ＋ Granzyme K ＋ PD-1 ＋TCF-1 ＋ or CD4 ＋ Granzyme K ＋ PD-1 ＋ TCF-1 ＋ As a group, tumor follicle cytotoxic T cells (Tnfc) were detectable (Fig. 2: c. Detection of Tnfc).

[0084] [Experimental Example 3] Specific example of prognosis prediction A specific example of stratification of the time to recurrence in follicular lymphoma patients based on the proportion of neoplastic follicle regulatory T cells (Tnfr) and neoplastic follicle cytotoxic T cells (Tnfc) calculated by multiplex immunostaining using the PhenoCycler-Fusion system (AKOYA) is shown (Figures 3A and 3B).

[0085] The time to recurrence was significantly longer in patients with a high percentage of both Tnfr and Tnfc cells, and particularly longer in patients with a high percentage of both types of cells, and shorter in patients with a low percentage of both types. This result was confirmed in three independent patient cohorts (Figure 3A, "Original Cohort," "Validation Cohort 1," and Figure 3B, "Validation Cohort 2"). This prognostic prediction was also independent of FLIPI, and its effectiveness was confirmed in patient cohorts classified as low risk (FLIPI low risk) and high risk (FLIPI high risk) by FLIPI risk classification (Figure 4).

[0086] [Experimental Example 4] (1) The expression of inhibitory factor genes in follicular regulatory T cells of Tnfr and normal lymph nodes was compared using single-cell RNA sequencing data. Figure 5(a) shows a violin plot of the expression of inhibitory factor genes in follicular regulatory T cells of Tnfr and normal lymph nodes. It was shown that the expression of inhibitory genes was enhanced in Tnfr.

[0087] (2) We investigated the inhibitory effects of Tnfr cells on Tfh cell activation and differentiation. Tfh cells actively interact with neoplastic B cells, which are cancer cells in follicular lymphoma, promoting lymphoma progression. Co-culture of Tfh and Tnfr cells under T cell receptor (TCR) stimulation demonstrated a decrease in the percentage of CD25-positive activated Tfh cells compared to Tfh cells cultured alone (Figure 5: b. Inhibitory effect of Tnfr on Tfh cell activation), and a decrease in Tfh cell division activity as detected by cell tracking reagents (Figure 5: c. Inhibitory effect of Tnfr on Tfh cell differentiation).

[0088] (3) We investigated the inhibitory effect of Tnfr on the activation of neoplastic B cells and the inhibitory effect of Tnfr on the cell survival of neoplastic B cells. Neoplastic B cells alone, co-culture of neoplastic B cells with Tfh cells, and co-culture of neoplastic B cells, Tfh cells, and Tnfr were performed. Compared to the culture of neoplastic B cells alone, the co-culture of neoplastic B cells with Tfh cells demonstrated a greater activation effect on neoplastic B cells ( Figure 5: d. Inhibitory effect of Tnfr on the activation of neoplastic B cells) and a greater effect on the cell survival of neoplastic B cells ( Figure 5: e. Inhibitory effect of Tnfr on the cell survival of neoplastic B cells). Furthermore, co-culture of neoplastic B cells, Tfh cells, and Tnfr cells showed a decrease in the percentage of cells positive for the activation marker CD86 of neoplastic B cells, i.e., a weakening of the activating effect of Tfh cells on neoplastic B cells (Fig. 5: d), and an increase in the percentage of cells positive for the cell death marker Annexin V of neoplastic B cells, i.e., a weakening of the cell survival-promoting effect of Tfh cells on neoplastic B cells (Fig. 5: e).

[0089] [Experimental Example 5] (1) Comparative analysis of gene expression was performed between Tnfc8 and effector CD8 T cells (effector CD8 cells), or between Tnfc8 and exhausted CD8 T cells (exhausted CD8 cells). Gene expression information between Tnfc8 and effector CD8 cells, or between Tnfc8 and exhausted CD8 cells, was extracted and analyzed comparatively, and genes showing differential expression were plotted in a volcano plot ( Figure 6 : a. Comparison of gene expression between Tnfc8 and effector CD8 cells; b. Comparison of gene expression between Tnfc8 and exhausted CD8 cells). Tnfc8 highly expressed CXCL13, CXCR5, and BCL6 genes, which promote migration and settlement into follicular structures, and also highly expressed TCF7, which maintains blastocytic phenotype. Compared to exhausted CD8 cells, Tnfc8 showed reduced expression of exhaustion markers LAG3 and HAVCR2, indicating their blastocytic phenotype.

[0090] (2) We verified cell immaturity by measuring cell division activity. Using a cell tracking reagent to trace cell division, we stimulated T cell receptors (TCRs) to induce cell division, and found that Tnfc8 cells maintained a higher cell division activity than exhausted CD8 cells (Figure 6: c. Cell division activity).

[0091] (3) Cytokine production, an index of cytotoxicity, was measured. Cells were stimulated with PMA (phorbol myristate acetate) / ionomycin, and the production of interferon gamma (Fig. 6: d) and tumor necrosis factor (TNF) alpha (Fig. 6: e) was evaluated. It was found that the production was higher than that of naive CD8 cells.

[0092] (4) We investigated the activation-inhibitory and cytocidal effects of Tnfc8 on neoplastic B cells. Neoplastic B cells, which are cancer cells in follicular lymphoma, were cocultured alone, cocultured with Tnfc8, cocultured with effector CD8 cells, and cocultured with exhausted CD8 cells. Coculture with Tnfc8 significantly inhibited the activation of neoplastic B cells, although not as strongly as with effector CD8 cells ( Figure 6 , f. Inhibitory effect of Tnfc8 on neoplastic B cells), and promoted cell death ( Figure 6 , g. Cytocidal effect of Tnfc8 on neoplastic B cells).

[0093] [Experimental Example 6] (1)a. Experiment on Tnfr induction from regulatory T cells by cytokine IL-21. Human lymph node regulatory T cells were cultured under TCR stimulation in the presence of transforming growth factor (TGF) β1, with or without the addition of IL-21. The presence of IL-21 induced high expression of CXCR5, a characteristic of Tnfr (Figure 7: a. Induction of Tnfr phenotype by IL-21). Furthermore, human lymph node regulatory T cells were cultured under TCR stimulation in the presence or absence of TGFβ1 and IL-21. The presence of IL-21 induced high expression of CXCR5 and BCL6, characteristic of Tnfr (Figure 8: a. Induction of Tnfr phenotype by IL-21).

[0094] (2) Experimental study on the induction of Tnfc8 expression from naive CD8 cells by the cytokine IL-21. Naive CD8 cells from human lymph nodes were cultured under TCR stimulation in the presence of TGFβ1, with or without the addition of IL-21. The presence of IL-21 induced high expression of CXCR5 (top) and CCR7 (bottom), which are characteristic of Tnfc (Figure 7: b. Induction of Tnfc8 phenotype by IL-21). Furthermore, naive CD8 cells from human lymph nodes were cultured under TCR stimulation in the presence or absence of TGFβ1 and IL-21. The presence of IL-21 induced high expression of Granzyme M and TCF1, which are characteristic of Tnfc (Figure 8: b. Induction of Tnfc8 phenotype by IL-21).

[0095] This cell induction method is expected to improve the therapeutic effect by adding modifications to the T cell gene modification therapy, which is currently undergoing research, and adding the characteristics of Tnfr and Tnfc cells.

[0096] While preferred embodiments of the present invention have been described and illustrated, it should be understood that these are exemplary of the present invention and should not be considered limiting. Additions, omissions, substitutions, and other modifications can be made without departing from the spirit or scope of the present invention. Accordingly, the present invention is not to be deemed limited by the foregoing description, but is limited only by the appended claims.

[0097] By implementing the present invention, it is possible to more accurately predict the prognosis of follicular lymphoma patients at the time of initial diagnosis. It is also expected that the present invention will be useful in developing a method for directly treating follicular regulatory T cells (Tnfr) and cytotoxic T cells (Tnfc) extracted from neoplastic follicles.

Claims

1. A method for quantifying neoplastic follicle regulatory T cells and neoplastic follicle cytotoxic T cells in T cells collected from a subject to predict the prognosis of follicular lymphoma, comprising: a flow cytometry step of detecting and counting neoplastic follicle regulatory T cells and neoplastic follicle cytotoxic T cells in T cells collected from the subject by a flow cytometry method using an anti-CD4 antibody, an anti-CD8a antibody, an anti-PD-1 antibody, an anti-CD25 antibody, and an anti-TIM-3 antibody; or a multiplexed immunostaining step of detecting and counting neoplastic follicle regulatory T cells and neoplastic follicle cytotoxic T cells in T cells collected from the subject by a multiplexed immunoassay method using an anti-CD4 antibody, an anti-CD8 antibody, an anti-PD-1 antibody, an anti-FOXP3 antibody, an anti-TCF-1 antibody, and an anti-Granzyme K antibody.

2. In the flow cytometry step, CD4 ＋ PD-1 ＋ CD25 ＋ The cells were detected and counted as tumor follicular regulatory T cells, and CD8a ＋ PD-1 ＋ TIM-3 － The method of claim 1, wherein the cells are detected and enumerated as CD8 positive neoplastic follicular cytocidal T cells.

3. In the multiple immunostaining step, CD4 ＋ PD-1 ＋ FOXP3 ＋ The cells were detected and counted as neoplastic follicular regulatory T cells, and CD8 ＋ Granzyme K ＋ PD-1 ＋ TCF-1 ＋ The cells were detected and counted as CD8 positive tumor follicular cytocidal T cells and CD4 ＋ Granzyme K ＋ PD-1 ＋ TCF-1 ＋ The method of claim 1, wherein the cells are detected and enumerated as CD4 positive neoplastic follicular cytocidal T cells.

4. A method for screening neoplastic follicle regulatory T cells and neoplastic follicle cytotoxic T cells from T cells collected from a subject to predict the prognosis of follicular lymphoma, comprising: a flow cytometry step of detecting and counting neoplastic follicle regulatory T cells and neoplastic follicle cytotoxic T cells in T cells collected from the subject by a flow cytometry method using an anti-CD4 antibody, an anti-CD8a antibody, an anti-PD-1 antibody, an anti-CD25 antibody, and an anti-TIM-3 antibody; or a multiplexed immunostaining step of detecting and counting neoplastic follicle regulatory T cells and neoplastic follicle cytotoxic T cells in T cells collected from the subject by a multiplexed immunoassay method using an anti-CD4 antibody, an anti-CD8 antibody, an anti-PD-1 antibody, an anti-FOXP3 antibody, an anti-TCF-1 antibody, and an anti-Granzyme K antibody.

5. In the flow cytometry step, CD4 ＋ PD-1 ＋ CD25 ＋ The cells were detected and counted as tumor follicular regulatory T cells, and CD8a ＋ PD-1 ＋ TIM-3 － The method of claim 4, wherein the cells are detected and enumerated as CD8 positive neoplastic follicular cytocidal T cells.

6. In the multiple immunostaining step, CD4 ＋ PD-1 ＋ FOXP3 ＋ The cells were detected and counted as neoplastic follicular regulatory T cells, and CD8 ＋ Granzyme K ＋ PD-1 ＋ TCF-1 ＋ The cells were detected and counted as CD8 positive tumor follicular cytocidal T cells and CD4 ＋ Granzyme K ＋ PD-1 ＋ TCF-1 ＋ The method of claim 4, wherein the cells are detected and enumerated as CD4 positive neoplastic follicular cytocidal T cells.

7. A therapeutic composition for treating follicular lymphoma, comprising: a flow cytometry step of detecting and counting neoplastic follicle regulatory T cells and neoplastic follicle cytotoxic T cells in T cells collected from a subject by flow cytometry using an anti-CD4 antibody, an anti-CD8a antibody, an anti-PD-1 antibody, an anti-CD25 antibody, and an anti-TIM-3 antibody; ＋ PD-1 ＋ CD25 ＋ Cells or CD8a ＋ PD-1 ＋ TIM-3 － A therapeutic composition comprising a T cell detected as a cell.

8. A therapeutic composition for treating follicular lymphoma, comprising: a multiple immunostaining step of detecting and counting neoplastic follicle regulatory T cells and neoplastic follicle cytotoxic T cells in T cells collected from a subject by a multiple immunoassay using an anti-CD4 antibody, an anti-CD8 antibody, an anti-PD-1 antibody, an anti-FOXP3 antibody, an anti-TCF-1 antibody, and an anti-Granzyme K antibody; ＋ PD-1 ＋ FOXP3 ＋ , CD8 ＋ Granzyme K ＋ PD-1 ＋ TCF-1 ＋ Cells or CD4 ＋ Granzyme K ＋ PD-1 ＋ TCF-1 ＋ A therapeutic composition comprising a T cell detected as a cell.

9. The therapeutic composition according to claim 7 or 8, wherein the T cells are T cells precultured with IL-21.

10. The therapeutic composition according to claim 7 or 8, wherein the T cells are artificially genetically modified T cells.

11. A kit for predicting the prognosis of follicular lymphoma, comprising an antibody set comprising an anti-CD4 antibody, an anti-CD8a antibody, an anti-PD-1 antibody, an anti-CD25 antibody, and an anti-TIM-3 antibody, or an antibody set comprising an anti-CD4 antibody, an anti-CD8 antibody, an anti-PD-1 antibody, an anti-FOXP3 antibody, an anti-TCF-1 antibody, and an anti-Granzyme K antibody.

12. The kit of claim 11 for identifying, detecting or screening neoplastic follicle regulatory T cells and neoplastic follicle cytotoxic T cells from a population of T cells taken from a subject.

13. A method for assisting in predicting the prognosis of follicular lymphoma in a subject, comprising: a flow cytometry step of detecting and counting neoplastic follicle regulatory T cells and neoplastic follicle cytotoxic T cells in T cells collected from the subject by flow cytometry using an anti-CD4 antibody, an anti-CD8a antibody, an anti-PD-1 antibody, an anti-CD25 antibody, and an anti-TIM-3 antibody; and a proportion calculation step of calculating the proportions of neoplastic follicle regulatory T cells, CD4-positive neoplastic follicle cytotoxic T cells, and CD8-positive neoplastic follicle cytotoxic T cells in the T cells collected from the subject.

14. The method according to claim 13, wherein in the percentage calculation step, the percentage of CD8-positive neoplastic follicle cytotoxic T cells is calculated based on the results of detecting and counting CD4-positive neoplastic follicle cytotoxic T cells.

15. A method for assisting in predicting the prognosis of follicular lymphoma in a subject, comprising: a multiplex immunostaining step of detecting and counting neoplastic follicle regulatory T cells and neoplastic follicle cytotoxic T cells in T cells collected from the subject by a multiplex immunoassay using an anti-CD4 antibody, an anti-CD8 antibody, an anti-PD-1 antibody, an anti-FOXP3 antibody, an anti-TCF-1 antibody, and an anti-Granzyme K antibody; and a ratio calculation step of calculating the ratios of neoplastic follicle regulatory T cells, CD4-positive neoplastic follicle cytotoxic T cells, and CD8-positive neoplastic follicle cytotoxic T cells in the T cells collected from the subject.

16. A method for predicting the prognosis of follicular lymphoma in a subject, comprising: a quantification step of quantifying neoplastic follicle regulatory T cells and neoplastic follicle cytocidal T cells in T cells collected from the subject by the method according to any one of claims 1 to 6; a proportion calculation step of calculating the proportions of neoplastic follicle regulatory T cells, CD4-positive neoplastic follicle cytocidal T cells, and CD8-positive neoplastic follicle cytocidal T cells in the T cells collected from the subject based on the quantification results obtained in the quantification step; and a prognosis evaluation step of evaluating the prognosis of the subject based on the proportions calculated in the proportion calculation step, wherein in the prognosis evaluation step, if the proportions calculated in the proportion calculation step are less than 2.0% for neoplastic follicle regulatory T cells and less than 2.5% for the sum of the CD4-positive neoplastic follicle cytocidal T cells and the CD8-positive neoplastic follicle cytocidal T cells, the prognosis is evaluated as poor.

Images