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

Abstract

Provided is a technique useful for predicting the prognosis of a patient suffering from follicular lymphoma. The present invention is a method for quantifying neoplastic follicular regulatory T cells and neoplastic follicular killer T cells from among T cells collected from a subject for predicting the prognosis of follicular lymphoma, the method comprising: a flow cytometry step in which the follicular regulatory T cells and neoplastic follicular killer T cells are quantified using an anti-CD4 antibody, an anti-CD8a antibody, an anti-PD-1 antibody, an anti-CD25 antibody, and an anti-TIM-3 antibody; detecting and counting neoplastic follicular regulatory T cells or neoplastic follicular killer T cells in the T cells collected from the subject by a flow cytometry method; the present invention relates to a method for detecting and counting neoplastic follicular regulatory T cells or neoplastic follicular killer T cells from among 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, or a multiple immunostaining step in which neoplastic follicular regulatory T cells or neoplastic follicular killer T cells are detected and counted 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,

Description

Technical Field

[0001] This invention relates to a method for quantifying tumor-follicular regulatory T cells and tumor-follicular cytotoxic T cells in T cells collected from subjects, a method for screening tumor-follicular regulatory T cells and tumor-follicular cytotoxic T cells from T cells collected from subjects, a therapeutic composition, a kit, a method for assisting in the prediction of the prognosis of follicular lymphoma, and a method for predicting the prognosis of follicular lymphoma in subjects.

[0002] 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. Background Technology

[0003] Follicular lymphoma (FL) is a representative indolent (low-grade) B-cell lymphoma, accounting for 10%–20% of all non-Hodgkin lymphomas. The incidence of FL in Japan has recently been increasing. Pathologically, FL is classified into grades 1, 2, 3A, and 3B, but grade 3B is usually treated as an aggressive (intermediate to high-grade) lymphoma. Most patients develop lymph node (LN) enlargement, leading to diagnosis, but at diagnosis, 70–85% of patients are in clinical stage III–IV, with a high probability of bone marrow infiltration.

[0004] Fluorosis (FL) typically progresses slowly and initially responds well to chemotherapy. However, FL patients often experience relapses, a tendency that is pronounced during the progression phase. In analyses following the introduction of rituximab (R), approximately 10% of patients underwent histologic transformation to aggressive lymphoma within 5 years. Prior to R introduction, the 50% survival rate for FL patients was 7–10 years, but in recent reports, among patients under 40 years of age at diagnosis, the 50% survival rate exceeded 20 years.

[0005] The Ann Arbor classification is used for the stage classification of FL. As prognostic prediction models specifically for FL, there are the Follicular Lymphoma International Prognostic Index (FLIPI) (Non-Patent Literature 1) and FLIPI2 (Non-Patent Literature 2).

[0006] Cases with confirmed disease progression or recurrence within 2 years of diagnosis (POD24, Non-Patent Literature 3) were reported as having poor prognoses, but no method for predicting this in advance was established. In cases of low tumor volume following a diagnosis of FL, a highly attentive watch-and-wait approach was actively selected. Interestingly, with this watch-and-wait approach, spontaneous regression was confirmed in approximately 20% of cases, suggesting continuous external stimulation from the surrounding microenvironment.

[0007] Existing technical documents Non-patent literature Non-patent literature 1: Philippe Solal-Celigny, Foreign author 34, Follicular Lymphoma International Prognostic Index, Blood, September 1, 2004, Vol. 104, No. 5, pp. 1258-1265 Non-patent literature 2: Massimo Federico, 19 other authors, 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 Prognostic Factor Project Non-patent literature 3: Carla Casulo, 11 other authors, 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, Vol. 23, No. 23, pp. 2516-2522 Summary of the Invention The problem that the invention aims to solve The aforementioned FLIPI and FLIPI2 factors include age and mainly reflect overall survival. However, due to improvements in anticancer drug treatment, the focus is now on recurrence rate (especially early recurrence rate within 2 years), and there is a desire to develop new prognostic prediction systems that are in line with the times.

[0008] Furthermore, while POD24 accurately predicts overall survival, it is itself an indicator of early relapse. This factor determines that it takes 24 months to develop, so the risk at diagnosis is unclear and is unrelated to improvements in initial treatment.

[0009] The present invention was made in view of the above circumstances, and its subject is to provide a technique useful for predicting the prognosis of patients with follicular lymphoma.

[0010] Methods for solving problems In order to solve the above-mentioned problems, the inventors conducted repeated and in-depth research and found that by using an antibody immune response using a combination of specific antibodies, it is possible to detect tumor follicular regulatory T cells (Tnfr) and tumor follicular cytotoxic T cells (Tnfc) in T cells collected from the subject, thus completing the present invention.

[0011] The present invention is as follows.

[0012] [1] A method for predicting the prognosis of follicular lymphoma, comprising quantifying tumor-follicular regulatory T cells (Tnfr) and tumor-follicular cytotoxic T cells (Tnfc) in T cells collected from subjects, wherein, The method comprises: In the flow cytometry procedure, anti-CD4 antibody, anti-CD8a antibody, anti-PD-1 antibody, anti-CD25 antibody, and anti-TIM-3 antibody are used to detect and count tumor-follicular regulatory T cells (Tnfr) and tumor-follicular cytotoxic T cells (Tnfc) in T cells collected from the subject; or The multiple immunostaining process involves using anti-CD4 antibody, anti-CD8 antibody, anti-PD-1 antibody, anti-FOXP3 antibody, anti-TCF-1 antibody, and anti-Granzyme K antibody to detect and count tumor follicular regulatory T cells (Tnfr) and tumor follicular cytotoxic T cells (Tnfc) in T cells collected from the subject using a multiple immunoassay.

[0013] [2] As described in [1], wherein, in the flow cytometry step, CD4+ PD-1 + CD25 + Cell detection and counting identified tumor-bearing follicular regulatory T cells (Tnfr), with CD8a... + PD-1 + TIM-3 - Cells were detected and counted as CD8-positive tumor follicular cytotoxic T cells (Tnfc8).

[0014] [3] As described in [1], wherein, in the multiple immunostaining process, CD4 + PD-1 + FOXP3 + Cell detection and counting identified tumor-associated follicular regulatory T cells (Tnfr), with CD8... + Granzyme K + PD-1 + TCF-1 + Cell detection and counting identified CD8-positive tumor follicular cytotoxic T cells (Tnfc8), with CD4+... + Granzyme K + PD-1 + TCF-1 + Cells were detected and counted as CD4-positive tumor follicular cytotoxic T cells (Tnfc4).

[0015] [4] A method for predicting the prognosis of follicular lymphoma by screening tumor-follicular regulatory T cells (Tnfr) and tumor-follicular cytotoxic T cells (Tnfc) from T cells collected from subjects, wherein, The method comprises: In the flow cytometry procedure, anti-CD4 antibody, anti-CD8a antibody, anti-PD-1 antibody, anti-CD25 antibody, and anti-TIM-3 antibody are used to detect and count tumor-follicular regulatory T cells (Tnfr) and tumor-follicular cytotoxic T cells (Tnfc) in T cells collected from the subject; or The multiple immunostaining process involves using anti-CD4 antibody, anti-CD8 antibody, anti-PD-1 antibody, anti-FOXP3 antibody, anti-TCF-1 antibody, and anti-Granzyme K antibody to detect and count tumor follicular regulatory T cells (Tnfr) and tumor follicular cytotoxic T cells (Tnfc) in T cells collected from the subject using a multiple immunoassay.

[0016] [5] As described in [4], wherein, in the flow cytometry step, CD4 + PD-1+ CD25 + Cell detection and counting identified tumor-bearing follicular regulatory T cells (Tnfr), with CD8a... + PD-1 + TIM-3 - Cells were detected and counted as CD8-positive tumor follicular cytotoxic T cells (Tnfc8).

[0017] [6] As described in [4], wherein, in the multiple immunostaining process, CD4 + PD-1 + FOXP3 + Cell detection and counting identified tumor-associated follicular regulatory T cells (Tnfr), with CD8... + Granzyme K + PD-1 + TCF-1 + Cell detection and counting identified CD8-positive tumor follicular cytotoxic T cells (Tnfc8), with CD4+... + Granzyme K + PD-1 + TCF-1 + Cells were detected and counted as CD4-positive tumor follicular cytotoxic T cells (Tnfc4).

[0018] [7] A therapeutic composition for treating follicular lymphoma, wherein, The therapeutic composition comprises T cells, which are detected and counted by flow cytometry in a process involving the detection and counting of tumor-associated follicular regulatory T cells (Tnfr) and tumor-associated follicular cytotoxic T cells (Tnfc) in T cells collected from a subject using anti-CD4 antibody, anti-CD8a antibody, anti-PD-1 antibody, anti-CD25 antibody, and anti-TIM-3 antibody. The T cells are then identified as CD4+. + PD-1 + CD25 + Cells or CD8a + PD-1 + TIM-3 - cell.

[0019] [8] A therapeutic composition for treating follicular lymphoma, wherein, The therapeutic composition comprises T cells, which are detected and counted as CD4+ T cells in T cells collected from a subject using a multiplex immunoassay process employing anti-CD4 antibody, anti-CD8 antibody, anti-PD-1 antibody, anti-FOXP3 antibody, anti-TCF-1 antibody, and anti-Granzyme K antibody. + PD-1 + FOXP3 + CD8 + Granzyme K + PD-1 + TCF-1 + Cells or CD4 + Granzyme K + PD-1 + TCF-1 + cell.

[0020] [9] The therapeutic composition as described in [7] or [8], wherein the T cells are T cells precultured with IL-21.

[0021]

[10] The therapeutic composition as described in [7] or [8], wherein the T cell is an artificially genetically modified T cell.

[0022]

[11] A kit for predicting the prognosis of follicular lymphoma, wherein, The kit contains: An antibody group containing anti-CD4 antibody, anti-CD8a antibody, anti-PD-1 antibody, anti-CD25 antibody, and anti-TIM-3 antibody; or The antibody group includes anti-CD4 antibody, anti-CD8 antibody, anti-PD-1 antibody, anti-FOXP3 antibody, anti-TCF-1 antibody, and anti-Granzyme K antibody.

[0023]

[12] The kit described in

[11] is used to identify, detect or screen tumor follicular regulatory T cells (Tnfr) and tumor follicular cytotoxic T cells (Tnfc) from a population of T cells collected from an object.

[0024]

[13] A method for predicting the prognosis of follicular lymphoma in auxiliary subjects, wherein, The method comprises: The flow cytometry procedure involves using anti-CD4 antibody, anti-CD8a antibody, anti-PD-1 antibody, anti-CD25 antibody, and anti-TIM-3 antibody to detect and count tumor-follicular regulatory T cells (Tnfr) and tumor-follicular cytotoxic T cells (Tnfc) in T cells collected from the subject. The proportion calculation process involves calculating the proportions of tumor-positive follicular regulatory T cells (Tnfr), CD4-positive tumor-positive follicular cytotoxic T cells (Tnfc4), and CD8-positive tumor-positive follicular cytotoxic T cells (Tnfc8) among the T cells collected from the subject.

[0025]

[14] The method as described in

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

[0026]

[15] A method for predicting the prognosis of follicular lymphoma in auxiliary subjects, The method comprises: The multiplex immunostaining process involves using anti-CD4 antibody, anti-CD8 antibody, anti-PD-1 antibody, anti-FOXP3 antibody, anti-TCF-1 antibody, and anti-Granzyme K antibody to detect and count tumor-follicular regulatory T cells (Tnfr) and tumor-follicular cytotoxic T cells (Tnfc) in T cells collected from the subject via multiplex immunoassay. The proportion calculation process involves calculating the proportions of tumor-positive follicular regulatory T cells (Tnfr), CD4-positive tumor-positive follicular cytotoxic T cells (Tnfc4), and CD8-positive tumor-positive follicular cytotoxic T cells (Tnfc8) among the T cells collected from the subject.

[0027]

[16] A method for predicting the prognosis of follicular lymphoma in subjects, wherein, The method comprises: A quantitative process, wherein the tumor follicular regulatory T cells (Tnfr) and tumor follicular cytotoxic T cells (Tnfc) collected from the subject are quantified by any one of the methods described in [1] to [6]. The proportion calculation step involves calculating, based on the quantitative results obtained in the quantitative step, the proportions of tumor-positive follicular regulatory T cells, CD4-positive tumor-positive follicular cytotoxic T cells (Tnfc4), and CD8-positive tumor-positive follicular cytotoxic T cells (Tnfc8) among the T cells collected from the subject; and In the prognostic evaluation step, the prognosis of the object is evaluated based on the proportion calculated in the proportion calculation step. In the prognostic evaluation process, if the proportion calculated in the proportion calculation process is less than 2.0% for tumor-associated follicular regulatory T cells (Tnfr) and less than 2.5% for the sum of CD4-positive tumor-associated follicular cytotoxic T cells and CD8-positive tumor-associated follicular cytotoxic T cells, the prognosis is considered poor.

[0028] Invention Effects According to the present invention, a technique useful for predicting the prognosis of patients with follicular lymphoma can be provided. Attached Figure Description

[0029] Figure 1 The results of Experiment 1 are shown. The graphs show the results of flow cytometry.

[0030] Figure 2 The results of Experiment Example 2 are shown. The images show the results of multiple immunostaining. a. Multiple immunostaining, b. Detection of Tnfr, c. Detection of Tnfc.

[0031] Figure 3A The results of Experiment 3 are shown. The graph shows the relationship between the relapse rate (vertical axis) and the time period in the follicular lymphoma patient cohort (original cohort and validation cohort 1) based on the proportion of tumor-follicular regulatory T cells (Tnfr) and tumor-follicular cytotoxic T cells (Tnfc).

[0032] Figure 3B The results of Experiment 3 are shown. The graph shows the relationship between relapse rate (vertical axis) and time (horizontal axis) in a cohort of follicular lymphoma patients (validation cohort 2 and the overall cohort) based on the proportion of tumor-follicular regulatory T cells (Tnfr) and tumor-follicular cytotoxic T cells (Tnfc).

[0033] Figure 4 The results of Experiment Example 3 are shown. The graph shows the relationship between relapse rate (vertical axis) and time period (horizontal axis) in the cohort of patients diagnosed as low-risk follicular lymphoma and the cohort of patients diagnosed as high-risk follicular lymphoma in the FLIPI-based risk classification.

[0034] Figure 5The results of Experiment 4 are shown below. a. Violin diagram of the expression of inhibitory factor genes in follicular regulatory T cells of normal lymph nodes and Tnfr; b. Inhibitory effect of Tnfr on Tfh cell activation; c. Inhibitory effect of Tnfr on Tfh cell differentiation; d. Inhibitory effect of Tnfr on tumor B cell activation; e. Inhibitory effect of Tnfr on tumor B cell survival.

[0035] Figure 6 The results of Experiment 5 are as follows: a. Comparison of gene expression of Tnfc8 and effector CD8 cells; b. Comparison of gene expression of Tnfc8 and depleted CD8 cells; c. Cell division capacity; d. Interferon-γ production capacity; e. TNFα production capacity; f. Inhibitory effect of Tnfc8 on the activation of tumor B cells; g. Killing effect of Tnfc8 on tumor B cells.

[0036] Figure 7 This indicates the results of culturing cells in Experiment 6, where cells were stimulated by T cell receptors (TCR) and in the presence of TGF (transforming growth factor) β1, with and without the addition of IL-21. a. Induction of Tnfr expression by IL-21; b. Induction of Tnfc8 expression by IL-21.

[0037] Figure 8 This indicates the results of culturing cells under TCR stimulation in Experiment 6, with and without the addition of TGFβ1 and IL-21. a. Induction of Tnfr expression by IL-21; b. Induction of Tnfc8 expression by IL-21. Detailed Implementation

[0038] In this invention, when “~” is used to represent a numerical range, the numerical range includes the values ​​before and after “~”.

[0039] In this invention, "subject" refers to a person from whom T cells are collected, preferably a person with follicular lymphoma.

[0040] The embodiments of the present invention will now be described in detail. However, the present invention is not limited to the embodiments described below, and various modifications can be made without departing from the spirit of the present invention.

[0041] [Quantitative Methods] The method of the present invention for quantifying tumor follicular regulatory T cells and tumor follicular cytotoxic T cells in T cells collected from subjects (hereinafter also referred to as the "quantification method") includes a flow cytometry step or a multiplex immunostaining step.

[0042] The flow cytometry and multiple immunostaining procedures described above are performed in vitro.

[0043] <Flow Cytometry Procedure> The flow cytometry procedure involves using an antibody group containing anti-CD4 (cluster of differentiation 4) antibody, anti-CD8a antibody, anti-PD-1 (Programmed Cell Death Protein 1) antibody, anti-CD25 antibody, and anti-TIM-3 (Tcell immunoglobulin domain and mucin domain 3) antibody to detect and count tumor follicular regulatory T cells (Tnfr) and tumor follicular cytotoxic T cells (Tnfc) in T cells collected from the subject by flow cytometry.

[0044] In addition to using the aforementioned antibody group as antibodies for cell detection, flow cytometry can employ methods previously used in this technical field.

[0045] The aforementioned antibodies can be primary antibodies that have been fluorescently labeled, metal-labeled, or DNA-barcode-labeled, or secondary antibodies that have been fluorescently labeled, metal-labeled, or DNA-barcode-labeled and specifically bind to each other.

[0046] Common precursor cells derived from bone marrow differentiate into three functionally distinct T cell types in the thymus. One type expresses γδ receptors that do not recognize peptide antigens presented by MHC (Major Histocompatibility Complex) molecules; the other two types express αβ receptors. During periods of positive and negative selection in the thymus, αβ-type T cells differentiate into CD4 or CD8 single-positive cells based on receptor specificity to MHC. CD4 and CD8 are co-receptors expressed on the T cell surface, binding to the preservation regions of MHC class II and MHC class I molecules, respectively.

[0047] 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 inhibited, leading to immune escape by cancer cells. Anti-PD-1 antibodies bind to PD-1 on T cells, preventing the binding of PD-1 to PD-L1 / PD-L2, thereby blocking the transmission of inhibitory signals, maintaining T cell activation, and restoring anti-tumor effects.

[0048] The CD25 (IL-2 Rα chain, Tac or p55) antigen is a 55 kDa glycoprotein chain and a low-affinity receptor for IL-2 (IL-2 Rα or IL-2 R1). It forms a high-affinity IL-2 R complex with CD12 (IL-2 Rβ chain, p75) and CD132 (IL-2 Rγ chain, p64). It is strongly expressed in activated regulatory CD4-positive T cells but not in resting CD8-positive lymphocytes. However, all activated T cells express CD25 protein. CD25 is also found in B cell subsets (CD20-positive), activated monocytes, macrophages, and T cell clones. A recombinant human / mouse chimeric anti-CD25 monoclonal antibody (IgG1) is known as bailiximab (recombinant).

[0049] TIM-3 (also known as HACR2) is a member of a recently discovered superfamily of molecules containing the T cell immunoglobulin-mucin domain. TIM-3 is an important regulatory molecule in T cell tolerance, playing a crucial role in autoimmunity and T cell exhaustion during chronic viral infections. TIM-3 is expressed in Th1 cells (type 1 helper T cells), macrophages, monocytes, dendritic cells, CD8-positive 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 helper T cell) immunity. TIM3 is a membrane protein with a calculated molecular weight of 33 kDa, consisting of 280 residues.

[0050] The aforementioned antibody group may also include anti-CD3 antibodies. Anti-CD3 antibodies recognize the human CD3 molecule. CD3 is a component of the T cell receptor and is most commonly used as a marker of T cells. As an anti-CD3 antibody, for example, an anti-CD3e antibody that recognizes the ε chain of the human CD3 molecule can be used.

[0051] In the flow cytometry procedure, tumor-associated follicular regulatory T cells (Tnfr) were detected and counted as CD4. + PD-1 + CD25 + cell.

[0052] In the flow cytometry procedure, CD8-positive tumor follicular cytotoxic T cells (Tnfc8) were detected and counted as CD8a. + PD-1 + TIM-3- cell.

[0053] In the flow cytometry procedure, CD4-positive tumor follicular cytotoxic T cells (Tnfc4) are not directly detected and counted. However, there is a strong correlation between the proportion of CD8-positive tumor follicular cytotoxic T cells and CD4-positive tumor follicular cytotoxic T cells, allowing the calculation of the proportion of CD4-positive tumor follicular cytotoxic T cells. Specifically, as shown in the experimental examples described later, a strong positive correlation with a correlation coefficient (Spearman's) of 0.93 was demonstrated in a study of 169 patients.

[0054] <Multiple Immunostaining Procedure> The multiplex immunostaining process involves using anti-CD4 antibody, anti-CD8 antibody, anti-PD-1 antibody, anti-FOXP3 (forkhead box P3) antibody, anti-TCF-1 (T cell factor 1) antibody, and anti-Granzyme K antibody to detect and count tumor follicular regulatory T cells (Tnfr) and tumor follicular cytotoxic T cells (Tnfc) in T cells collected from the subject through multiplex immunoassay.

[0055] In addition to using the aforementioned antibody group as antibodies for cell detection, multiplex immunostaining methods can employ methods previously used in this technical field.

[0056] Anti-CD4 antibodies and anti-PD-1 antibodies are as described above.

[0057] The aforementioned antibodies can be primary antibodies that have been fluorescently labeled, metal-labeled, or DNA-barcode-labeled, or secondary antibodies that have been fluorescently labeled, metal-labeled, or DNA-barcode-labeled and specifically bind to each other.

[0058] Transcription factor T cytokine 1 (TCF-1) has the same downstream effects as the WNT / -catenin signaling pathway and is essential for the production of T cells in the thymus.

[0059] FOXP3 is a 50-55 kDa transcription factor, also known as IPEX, JM2, Forkhead box P3, and Scurfin.

[0060] In the multiplex immunostaining process, tumor-associated follicular regulatory T cells (Tnfr) were detected and counted as CD4. + PD-1 + FOXP3 + cell.

[0061] In the multiple immunostaining process, CD8-positive tumor follicular cytotoxic T cells (Tnfc8) were detected and counted as CD8. + Granzyme K + PD-1 + TCF-1 + cell.

[0062] In the multiple immunostaining process, CD4-positive tumor follicular cytotoxic T cells (Tnfc4) were detected and counted as CD4+. + Granzyme K + PD-1 + TCF-1 + cell.

[0063] [Filtering Method] The method of the present invention for screening tumor follicular regulatory T cells (Tnfr) and tumor follicular cytotoxic T cells (Tnfc) from T cells collected from subjects (hereinafter also referred to as the "screening method") includes a flow cytometry procedure or a multiplex immunostaining procedure.

[0064] The flow cytometry and multiple immunostaining procedures are the same as those described in the quantitative methods above.

[0065] In the flow cytometry procedure, tumor-associated follicular regulatory T cells (Tnfr) were screened for CD4+. + PD-1 + CD25 + Cells, CD8-positive tumor follicular cytotoxic T cells (Tnfc8) were selected for CD8a + PD-1 + TIM-3 - cell.

[0066] In the multiple immunostaining process, tumor follicular regulatory T cells (Tnfr) were screened for CD4. + PD-1 + FOXP3 + Cells, CD8-positive tumor follicular cytotoxic T cells (Tnfc8) were selected as CD8 cells. + Granzyme K + PD-1 + TCF-1 + Cells, CD4-positive tumor follicular cytotoxic T cells (Tnfc4) were selected as CD4 cells. + Granzyme K + PD-1 + TCF-1 + cell.

[0067] [T cells] One embodiment of the T cells of the present invention comprises T cells collected from a subject, wherein, in a flow cytometry step using anti-CD4 antibody, anti-CD8 antibody, anti-PD-1 antibody, anti-CD25 antibody, and anti-TIM-3 antibody to detect and count tumor-forming follicular regulatory T cells (Tnfr) and tumor-forming follicular cytotoxic T cells (Tnfc) in the T cells collected from the subject, the T cells are detected as CD4... + PD-1 + CD25 + Cells or CD8a + PD-1 + TIM-3 - cell.

[0068] The flow cytometry procedure is the same as the aforementioned flow cytometry procedure.

[0069] Another embodiment of the T cells of the present invention is a type of T cells collected from a subject. In a multiplex immunostaining process using anti-CD4 antibody, anti-CD8 antibody, anti-PD-1 antibody, anti-FOXP3 antibody, anti-TCF-1 antibody, and anti-Granzyme K antibody to detect and count tumor-follicular regulatory T cells (Tnfr) and tumor-follicular cytotoxic T cells (Tnfc) in the T cells collected from the subject, the T cells are detected as CD4... + PD-1 + FOXP3 + CD8 + Granzyme K + PD-1 + TCF-1 + Cells or CD4 + Granzyme K + PD-1 + TCF-1 + cell.

[0070] The multiple immunostaining procedure is the same as the aforementioned multiple immunostaining procedure.

[0071] The T cells described in this embodiment can be used to treat follicular lymphoma.

[0072] As shown in Experiments 5-7 described later, Tnfr exhibits both activation-inhibiting and differentiation-inhibiting effects on follicular helper T cells (Tfh) within normal lymph nodes. Furthermore, Tnfc demonstrates both activation-inhibiting and killing effects on tumor-like B cells, which are cancer cells in follicular lymphoma.

[0073] Furthermore, Tnfr exhibits hyperexpression of repressive genes such as IL2RA, LAG3, and IL10, while Tnfc highly expresses genes that promote migration and anchorage to follicular structures, namely CXCL13, CXCR5, and BCL6, and also highly expresses the TCF7 gene, which maintains cellular immaturity.

[0074] Furthermore, through the cytokine IL-21, regulatory T cells can induce Tnfr (high expression of CXCR5 and BCL6), and naive CD8 cells can induce Tnfc8 cells (high expression of CXCR5, CCR7, Granzyme M, and TCF1). That is, the T cells in this embodiment are preferably T cells pre-cultured in IL-21. Based on this cell induction method, by modifying T cell genes for therapeutic purposes and adding the characteristics of Tnfr and Tnfc cells, improved therapeutic effects are expected.

[0075] Tumor-derived follicular regulatory T cells (Tnfr) and tumor-derived follicular cytotoxic T cells (Tnfc) extracted from the patient's T cells can be used to develop treatments for direct therapy.

[0076] One embodiment of the T cell of the present invention may be obtained by further artificially genetically modifying the T cell of one embodiment or another embodiment described above.

[0077] In this invention, "artificial gene modification" refers to modifying the function of T cells by introducing genes into T cells or editing the genes of T cells. As "artificial gene modification" in this invention, it can be modification of the genes inherent in the T cells themselves, modification of the genes inherent in the T cells themselves, modification by introducing foreign genes, or modification by combining two or more of these methods, preferably modification by introducing foreign genes.

[0078] As methods of artificial gene modification, any previously known methods or methods based on them can be cited without particular limitation. For example, methods using viral vectors, myosin methods, calcium phosphate methods, microinjection methods, particle gun methods, DEAE-dextran methods, electroporation methods, cationic lipid methods, and T cell gene editing (genome editing) using site-specific nucleases (large-scale nucleases, zinc finger nucleases, TALEN, PPR, CRISPR-Cas, etc.) can be cited.

[0079] As a method of artificial gene modification, one of these methods can be used alone, or two or more can be combined.

[0080] The gene that alters the function of T cells, i.e. the target gene that is modified through artificial genetic engineering, is preferably selected from at least one of the following groups: a gene encoding a protein expressed on the surface of T cells and a fusion protein containing at least one intracellular signal transduction domain, and a gene encoding a protein expressed on the surface of T cells and a fusion protein containing at least one simultaneous stimulation domain and at least one intracellular signal transduction domain. More preferably, it is a gene encoding an antigen receptor. More preferably, it is selected from at least one of the following groups: a gene encoding a T cell receptor (TCR) and a gene encoding a chimeric antigen receptor (CAR). Particularly preferred is a gene encoding a chimeric antigen receptor (CAR).

[0081] That is, as an embodiment of the T cell of the present invention, it is preferable to express a surface protein encoded by a target gene that has been artificially genetically modified on its surface. Such a genetically modified T cell is more preferably selected from at least one of the group consisting of TCR-T cells that express a T cell receptor (TCR) encoded by a gene that has been artificially genetically modified and CAR-T cells that express a chimeric antigen receptor (CAR), and is even more preferably CAR-T cells.

[0082] [Therapeutic Composition] One embodiment of the therapeutic composition of the present invention is a therapeutic composition for treating follicular lymphoma, comprising T cells, wherein the T cells are detected as CD4+ in a flow cytometry step of detecting and counting tumor-related follicular regulatory T cells and tumor-related follicular cytotoxic T cells in T cells collected from a subject using anti-CD4 antibody, anti-CD8a antibody, anti-PD-1 antibody, anti-CD25 antibody, and anti-TIM-3 antibody. + PD-1 + CD25 + Cells or CD8a + PD-1 + TIM-3 - cell.

[0083] The T cells described herein are the same as those in this embodiment.

[0084] Alternatively, another embodiment of the therapeutic composition of the present invention is a therapeutic composition for treating follicular lymphoma, comprising T cells, wherein the T cells are detected as CD4+ in a multiplex immunostaining process using anti-CD4 antibody, anti-CD8 antibody, anti-PD-1 antibody, anti-FOXP3 antibody, anti-TCF-1 antibody, and anti-Granzyme K antibody to detect and count tumor-related follicular regulatory T cells and tumor-related follicular cytotoxic T cells collected from a subject by a multiplex immunoassay. + PD-1+ FOXP3 + CD8 + Granzyme K + PD-1 + TCF-1 + Cells or CD4 + Granzyme K + PD-1 + TCF-1 + cell.

[0085] The T cells described herein are the same as those in this embodiment.

[0086] The therapeutic composition of this embodiment may include T cells obtained by further culturing the T cells of this embodiment to proliferate, or by concentrating them through centrifugation or other methods, or by appropriately combining these methods. In addition to the T cells of this embodiment, it may also include known culture media, cell washing solutions, excipients, and additives.

[0087] The T cells of this embodiment can be used to manufacture a therapeutic composition for treating follicular lymphoma.

[0088] The therapeutic composition of this embodiment can be prepared into an injection or the like by conventional methods together with various additives, and used to manufacture a therapeutic agent for follicular lymphoma.

[0089] In one embodiment, the present invention can be a treatment method for patients with follicular lymphoma using the T cells or therapeutic composition of this embodiment. As a treatment method, in addition to using the T cells or therapeutic composition of this embodiment, methods conventional in the art can be employed. For example, it can 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.

[0090] CAR-T cell therapy using the T cells or therapeutic compositions of this embodiment can be a treatment method for patients with acute B-lymphoblastic leukemia, diffuse large B-cell lymphoma, multiple myeloma, chronic lymphocytic leukemia, and acute myeloid leukemia.

[0091] TCR-T cell therapy using the T cells or therapeutic compositions of this embodiment can 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, etc.

[0092] TIL therapy using T cells or therapeutic compositions according to this embodiment can be a treatment method for patients with malignant melanoma, cervical cancer, etc.

[0093] That is, the present invention may include the following methods.

[0094] A treatment method for patients with follicular lymphoma, wherein, in a flow cytometry procedure using anti-CD4 antibodies, anti-CD8 antibodies, anti-PD-1 antibodies, anti-CD25 antibodies, and anti-TIM-3 antibodies, tumor-follicular regulatory T cells (Tnfr) and tumor-follicular cytotoxic T cells (Tnfc) collected from the subject are detected and counted using CD4 antibodies. + PD-1 + CD25 + Cells or CD8a + PD-1 + TIM-3 - T cells of the cell.

[0095] Alternatively, a treatment for patients with follicular lymphoma, wherein, in a multiplex immunostaining process using anti-CD4 antibodies, anti-CD8 antibodies, anti-PD-1 antibodies, anti-FOXP3 antibodies, anti-TCF-1 antibodies, and anti-Granzyme K antibodies to detect and count tumor-follicular regulatory T cells (Tnfr) and tumor-follicular cytotoxic T cells (Tnfc) in T cells collected from the subject, CD4 antibodies are used. + PD-1 + FOXP3 + CD8 + Granzyme K + PD-1 + TCF-1 + Cells or CD4 + Granzyme K + PD-1 + TCF-1 + T cells of the cell.

[0096] Alternatively, a treatment for patients with follicular lymphoma, wherein, in a flow cytometry procedure using anti-CD4 antibodies, anti-CD8 antibodies, anti-PD-1 antibodies, anti-CD25 antibodies, and anti-TIM-3 antibodies to detect and count tumor-follicular regulatory T cells (Tnfr) and tumor-follicular cytotoxic T cells (Tnfc) in T cells collected from the subject, the flow cytometry procedure uses antibodies detected as CD4. + PD-1 + CD25 + Cells or CD8a + PD-1 + TIM-3 - A therapeutic composition for T cells.

[0097] Alternatively, a treatment for patients with follicular lymphoma, wherein, in a multiplex immunostaining process using anti-CD4 antibodies, anti-CD8 antibodies, anti-PD-1 antibodies, anti-FOXP3 antibodies, anti-TCF-1 antibodies, and anti-Granzyme K antibodies to detect and count tumor-follicular regulatory T cells (Tnfr) and tumor-follicular cytotoxic T cells (Tnfc) in T cells collected from the subject, the immunostaining agent includes CD4 antibodies. + PD-1 + FOXP3 + CD8 + Granzyme K + PD-1 + TCF-1 + Cells or CD4 + Granzyme K + PD-1 + TCF-1 + A therapeutic composition for T cells.

[0098] [Reagent test kit] The kit of the present invention comprises: an antibody group (first antibody group) comprising anti-CD4 antibody, anti-CD8a antibody, anti-PD-1 antibody, anti-CD25 antibody, and anti-TIM-3 antibody; or an antibody group (second antibody group) comprising anti-CD4 antibody, anti-CD8 antibody, anti-PD-1 antibody, anti-FOXP3 antibody, anti-TCF-1 antibody, and anti-Granzyme K antibody.

[0099] Both the aforementioned first antibody group and second antibody group may further include anti-CD3 antibody or anti-CD3e antibody.

[0100] The kit of the present invention can be used to detect and count tumor follicular regulatory T cells (Tnfr) and tumor follicular cytotoxic T cells (Tnfc) by flow cytometry using the first antibody group.

[0101] The kit of the present invention can be used to detect and count tumor follicular regulatory T cells (Tnfr) and tumor follicular cytotoxic T cells (Tnfc) using the second antibody group via multiple immunostaining methods.

[0102] The kit of the present invention may also include other components. For example, it may also include experimental protocols, etc.

[0103] The kit of the present invention can be used to diagnose or predict the prognosis of follicular lymphoma.

[0104] The kit of the present invention can be used to identify, detect or screen tumor follicular regulatory T cells (Tnfr) and tumor follicular cytotoxic T cells (Tnfc) from a population of T cells collected from a subject.

[0105] In one embodiment, the present invention provides a first antibody group and a second antibody group for predicting the prognosis of follicular lymphoma.

[0106] In one embodiment, the present invention provides the use of the first antibody group and the second antibody group in the preparation of a kit for predicting the prognosis of follicular lymphoma.

[0107] [Methods to assist in prognosis prediction] One embodiment of a method for assisting in predicting the prognosis of follicular lymphoma in the subject of the present invention (hereinafter also simply referred to as "method for assisting in prognosis prediction") comprises: a flow cytometry step, in which anti-CD4 antibody, anti-CD8a antibody, anti-PD-1 antibody, anti-CD25 antibody, and anti-TIM-3 antibody are used to detect and count tumor-positive follicular regulatory T cells (Tnfr) and tumor-positive follicular cytotoxic T cells (Tnfc) in T cells collected from the subject by flow cytometry; and a proportion calculation step, in which the proportions of tumor-positive follicular regulatory T cells (Tnfr), CD4-positive tumor-positive follicular cytotoxic T cells (Tnfc4), and CD8-positive tumor-positive follicular cytotoxic T cells (Tnfc8) in T cells collected from the subject are calculated.

[0108] The flow cytometry and proportion calculation procedures described above are performed in vitro.

[0109] Preferably, in the ratio calculation step, the ratio of CD8-positive tumor follicular cytotoxic T cells (Tnfc8) is calculated based on the results of detection and counting of CD4-positive tumor follicular cytotoxic T cells (Tnfc4).

[0110] Another embodiment of the method for assisting in prognostic 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, in which anti-CD4 antibody, anti-CD8 antibody, anti-PD-1 antibody, anti-FOXP3 antibody, anti-TCF-1 antibody, and anti-Granzyme K antibody are used to detect and count tumor-positive follicular regulatory T cells (Tnfr) and tumor-positive follicular cytotoxic T cells (Tnfc) in T cells collected from the subject by a multiplex immunoassay; and a proportion calculation step, in which the proportions of tumor-positive follicular regulatory T cells (Tnfr), CD4-positive tumor-positive follicular cytotoxic T cells (Tnfc4), and CD8-positive tumor-positive follicular cytotoxic T cells (Tnfc8) in T cells collected from the subject are calculated.

[0111] [Methods for prognostic prediction] The method for predicting the prognosis of follicular lymphoma in the present invention includes a prognostic evaluation step following the aforementioned auxiliary prognostic prediction method. In this prognostic evaluation step, the prognosis of the subject is evaluated based on the proportions of tumor-positive follicular regulatory T cells (Tnfr), CD4-positive tumor-positive follicular cytotoxic T cells (Tnfc4), and CD8-positive tumor-positive follicular cytotoxic T cells (Tnfc8) among T cells collected from the subject.

[0112] In the prognostic evaluation process, if the proportions of tumor-follicular regulatory T cells (Tnfr), CD4-positive tumor-follicular cytotoxic T cells (Tnfc4), and CD8-positive tumor-follicular cytotoxic T cells (Tnfc8) collected from the subject are Tnfr less than 2.0% and Tnfc (the sum of Tnfc4 and Tnfc8) less than 2.5%, the patient is evaluated as having a high risk (poor prognosis). If Tnfr is greater than 2.0% and Tnfc is greater than 2.5%, the patient is evaluated as having a low risk (not considered a poor prognosis).

[0113] The above-mentioned prognostic evaluation procedure is performed in vitro.

[0114] Alternatively, the method for predicting the prognosis of follicular lymphoma in the subject according to the present invention comprises: a quantification step, in which the number of tumor-positive follicular regulatory T cells (Tnfr) and tumor-positive follicular cytotoxic T cells (Tnfc) in T cells collected from the subject is quantified using the aforementioned quantification method; a proportion calculation step, in which the proportion of tumor-positive follicular regulatory T cells (Tnfr), CD4-positive tumor-positive follicular cytotoxic T cells (Tnfc4), and CD8-positive tumor-positive follicular cytotoxic T cells (Tnfc8) in T cells collected from the subject is calculated based on the quantification result obtained in the quantification step; and a prognostic evaluation step, in which the prognosis of the subject is evaluated based on the proportions calculated in the proportion calculation step. In the prognostic evaluation process, if the proportion calculated in the proportion calculation process is less than 2.0% for tumor-related follicular regulatory T cells and less than 2.5% for the sum of CD4-positive tumor-related follicular cytotoxic T cells and CD8-positive tumor-related follicular cytotoxic T cells, then the prognosis is considered poor. If Tnfr is greater than 2.0% and Tnfc is greater than 2.5%, then the patient risk is considered low (not considered a poor prognosis).

[0115] Furthermore, by combining it with existing indicators such as FLIPI or FLIPI2, more accurate prognostic predictions can be made, and it is also possible to identify patients with extremely good or extremely poor prognoses.

[0116] In one embodiment, the present invention may further include other steps after the prognostic evaluation step in the method for predicting prognosis of this embodiment. Examples of other steps include treating a patient with follicular lymphoma based on the results of the prognostic prediction. Where a treatment step is included in addition to the prognostic evaluation step, the method for predicting prognosis of this embodiment may be a treatment method for a patient with follicular lymphoma.

[0117] During the treatment process, the treatment strategy for follicular lymphoma can be determined based on the evaluation results of the prognosis prediction method of this embodiment. As a treatment method for patients with follicular lymphoma evaluated as having a poor prognosis, the same method as the aforementioned treatment method for patients with follicular lymphoma is given.

[0118] Example The present invention will now be described in more detail through experimental examples. However, the present invention is not limited to the embodiments described below.

[0119] [Experimental Example 1] Experiment based on flow cytometry method The same method as flow cytometry, which is usually performed as a clinical examination, is used.

[0120] After processing clinical lymphoma samples into single cells, 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.

[0121] T cells collected from the patient were analyzed and counted to identify and count CD4-positive and CD8-positive cells. Figure 1 ).

[0122] As CD4 + (CD3) + PD-1 + CD25 + The population was able to detect tumor-related follicular regulatory T cells (Tnfr). Additionally, as a CD8 group... + PD-1 + TIM-3 - The population was able to detect CD8-positive tumor follicular cytotoxic T cells (Tnfc8). The proportion of these cells in all T cells (CD3-positive cells or CD4-positive and CD8-positive cells) could be calculated.

[0123] [Experimental Example 2] Experiment based on multiplex immunostaining Use the same method as the multiple immunostaining method that is usually performed as a clinical examination.

[0124] For formalin-fixed paraffin-embedded samples of clinical lymphoma, multiplex immunostaining based on the PhenoCycler-Fusion system (AKOYA) was performed using 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.

[0125] Antibodies were prepared and experiments were conducted according to the protocol recommended by AKOYA. Figure 2 a. Multiple immunostaining). As CD4 + PD-1 + FOXP3 + The population was able to detect tumor-associated follicular regulatory T cells (Tnfr). Figure 2 (b.Tnfr detection). Additionally, as a CD8... + Granzyme K + PD-1 + TCF-1 + or CD4 + Granzyme K + PD-1 +The population was able to detect tumor-derived follicular cytotoxic T cells (Tnfc). Figure 2 (c. TNFC detection).

[0126] [Experimental Example 3] Specific Examples of Prognostic Prediction This illustration shows specific examples of stratification in follicular lymphoma patients up to relapse, based on the ratio of tumor-follicular regulatory T cells (Tnfr) and tumor-follicular cytotoxic T cells (Tnfc) calculated using multiple immunostaining with the PhenoCycler-Fusion system (AKOYA). Figure 3A , Figure 3B ).

[0127] Cases with high proportions of both Tnfr and Tnfc cells showed a significantly longer time to relapse; cases with high proportions of both cells had a particularly long time to relapse, while cases with low proportions of both cells had a particularly short time. These results were observed in three independent patient cohorts ( Figure 3A The "original queue", "verification queue 1" and Figure 3B The results were confirmed in “Validation Cohort 2”. Furthermore, this prognostic prediction was also independent of FLIPI, and its effectiveness was confirmed in both the FLIPI-based risk classification of patients as low risk (FLIPI low risk) and high risk (FLIPI high risk). Figure 4 ).

[0128] [Experiment Example 4] (1) Compare the expression of inhibitory factor genes in Tnfr and follicular regulatory T cells of normal lymph nodes using single-cell RNA sequencing data.

[0129] Figure 5 Figure a shows a violin plot illustrating the expression of repressive gene in follicular regulatory T cells of Tnfr and normal lymph nodes. Figure 2 shows enhanced repressive gene expression in Tnfr.

[0130] (2) The activation inhibition effect and differentiation inhibition effect of Tnfr cells on Tfh cells were verified.

[0131] Tfh cells actively interact with cancerous B cells in follicular lymphoma, thereby promoting lymphoma progression. Co-culturing Tfh and Tnfr cells with T cell receptor (TCR) stimulation showed a reduced proportion of CD25-positive activated Tfh cells compared to Tfh culture alone. Figure 5 b. The inhibitory effect of Tnfr on the activation of Tfh cells), and the reduction in the mitotic activity of Tfh cells captured by cell tracking reagents ( Figure 5 c. Tnfr's inhibitory effect on Tfh cell differentiation.

[0132] (3) Verify the activation inhibition effect of Tnfr on tumor B cells and the cell survival inhibition effect of Tnfr on tumor B cells.

[0133] Cultures of tumor-bacterial B cells alone, co-culture of tumor-bacterial B cells and Tfh cells, and co-culture of tumor-bacterial B cells, Tfh cells, and Tnfr cells were performed. Compared with culture of tumor-bacterial B cells alone, the co-culture of tumor-bacterial B cells and Tfh cells confirmed the activation effect of tumor-bacterial B cells. Figure 5 d.Tnfr's inhibitory effect on the activation of tumor B cells and its effect on promoting the survival of tumor B cells ( Figure 5 (e., the inhibitory effect of Tnfr on the cell survival of tumor B cells). Further co-culture of tumor B cells, Tfh cells, and Tnfr showed a decrease in the proportion of CD86-positive cells, the activation marker of tumor B cells, indicating a weakening of the activation effect of Tfh cells on tumor B cells. Figure 5 :d), and the increase in the proportion of Annexin V positive cells, a cell death marker of tumor B cells, indicating a weakening effect of Tfh cells on promoting cell survival of tumor B cells ( Figure 5 :e).

[0134] [Experiment Example 5] (1) Comparative analysis of gene expression between Tnfc8 and effector CD8 T cells (effector CD8 cells), or between Tnfc8 and exhausted CD8 T cells (exhausted CD8 cells). Gene expression information of Tnfc8 and effector CD8 cells, or between Tnfc8 and exhausted CD8 cells, was extracted, compared, and genes showing differential expression were displayed in a volcano plot ( Figure 6 a. Comparison of gene expression in Tnfc8 and effector CD8 cells; b. Comparison of gene expression in Tnfc8 and exhausted CD8 cells. Tnfc8 cells highly express CXCL13, CXCR5, and BCL6 genes, which promote migration and anchorage to follicular structures, and also highly express TCF7, a gene that maintains cellular immaturity. Compared to exhausted CD8 cells, the expression of LAG3 and HACR2, markers of exhaustion, is reduced, indicating cellular immaturity.

[0135] (2) Cell immaturity was verified by cell division capacity assay.

[0136] Using cell-tracking reagents that monitor cell division, T cell receptors (TCRs) were stimulated to promote cell division. It was found that Tnfc8 cells maintained a higher cell division capacity compared to exhausted CD8 cells. Figure 6 c. Cell division capacity.

[0137] (3) The ability to produce cytokines as an indicator of lethality was determined.

[0138] Cells were stimulated with PMA (phorbol myristate acetate) / ionomycin to evaluate interferon-γ (… Figure 6 :d) and TNF (tumor necrosis factor) α ( Figure 6 The production capacity of :e) indicates that it maintains a high production capacity compared to the initial CD8 cells.

[0139] (4) The inhibitory effect and killing effect of Tnfc8 on tumor B cells were verified.

[0140] Cultures of tumor-bearing B cells (cancer cells in follicular lymphoma), co-culture of tumor-bearing B cells and Tnfc8, co-culture of tumor-bearing B cells and effector CD8 cells, and co-culture of tumor-bearing B cells and exhausted CD8 cells, while not as effective as effector CD8 cells, showed that co-culture with Tnfc8 significantly inhibited the activation of tumor-bearing B cells. Figure 6 f.Tnfc8's inhibitory effect on the activation of tumor B cells), and the promotion of cell death ( Figure 6 (g. Tnfc8's killing effect on tumor B cells).

[0141] [Experiment Example 6] (1) a. Tnfr induction experiment based on cytokine IL-21 from regulatory T cells.

[0142] Regulatory T cells from human lymph nodes were cultured under TCR stimulation and in the presence of TGF (transforming growth factor) β1, with and without the addition of IL-21. In the presence of IL-21, high expression of CXCR5, a characteristic of Tnfr, was induced. Figure 7 a. Induction of Tnfr expression system based on IL-21. Additionally, regulatory T cells from human lymph nodes were cultured with and without TGFβ1 and IL-21 after TCR stimulation. In the presence of IL-21, high expression of CXCR5 and BCL6, characteristic of Tnfr, was induced. Figure 8 a. Induction of Tnfr expression system based on IL-21.

[0143] (2) Tnfc8 induction experiment from naïve CD8 cells based on cytokine IL-21.

[0144] Human lymph node naïve CD8 cells were cultured under TCR stimulation in the presence of TGFβ1, with and without IL-21. The presence of IL-21 induced high expression of CXCR5 (top) and CCR7 (bottom), characteristic of TnFc. Figure 7 b. Induction of Tnfc8 expression system based on IL-21. Additionally, naive CD8 cells from human lymph nodes were cultured with and without TGFβ1 and IL-21 after TCR stimulation. In the presence of IL-21, high expression of Granzyme M and TCF1, characteristic of Tnfc, was induced. Figure 8 b. Induction of Tnfc8 expression system based on IL-21.

[0145] Based on this cell induction method, by modifying the T-cell gene editing therapy currently under investigation, adding the characteristics of Tnfr and Tnfc cells, it is expected to improve the therapeutic effect.

[0146] The preferred embodiments of the present invention have been described and illustrated above. However, it should be understood that these embodiments are illustrative of the 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 invention. Therefore, the present invention is not considered to be limited by the foregoing description, but only by the appended claims.

[0147] Industrial availability By implementing this invention, it is possible to more accurately predict the prognosis of patients with follicular lymphoma at the time of initial diagnosis.

[0148] In addition, it is hoped that tumor-derived follicular regulatory T cells (Tnfr) and tumor-derived follicular cytotoxic T cells (Tnfc) can be extracted for the development of treatment methods for direct therapy.

Claims

1. A method for quantifying tumor follicular regulatory T cells and tumor follicular killer T cells, which is a method for predicting the prognosis of follicular lymphoma, for quantifying tumor follicular regulatory T cells and tumor follicular killer T cells in T cells collected from a subject, wherein the method comprises: a flow cytometry procedure in which tumor follicular regulatory T cells and tumor follicular killer T cells in T cells collected from a subject are detected and counted 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 multiple immunostaining procedure in which tumor follicular regulatory T cells and tumor follicular killer T cells in T cells collected from a subject are detected and counted by a multiple immunodetection 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. The method according to claim 1, wherein 3. The method according to claim 1, wherein 4. A method for screening tumor follicular regulatory T cells and tumor follicular killer T cells, which is a method for predicting the prognosis of follicular lymphoma, for screening tumor follicular regulatory T cells and tumor follicular killer T cells from T cells collected from a subject, wherein the method comprises: a flow cytometry procedure in which tumor follicular regulatory T cells and tumor follicular killer T cells in T cells collected from a subject are detected and counted 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 multiple immunostaining procedure in which tumor follicular regulatory T cells and tumor follicular killer T cells in T cells collected from a subject are detected and counted by a multiple immunodetection 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. The method according to claim 4, wherein In the flow cytometry procedure, CD4 + PD-1 + CD25 + Cells were detected and counted as tumor follicular regulatory T cells, CD8a + PD-1 + TIM-3 - Cells were detected and counted as CD8 positive tumor follicular killer T cells.

6. The method according to claim 4, wherein CD4 + PD-1 + FOXP3 + CD8 + Granzyme K + PD-1 + TCF-1 + CD4 + Granzyme K + PD-1 + TCF-1 + CD4 7. A therapeutic composition for treating follicular lymphoma, wherein 8. A therapeutic composition for treating follicular lymphoma, wherein 9. The therapeutic composition according to claim 7 or 8, wherein the T cells are T cells that have been pre-cultured with IL-21.

10. The therapeutic composition according to claim 7 or 8, wherein In the flow cytometry procedure, CD4 + PD-1 + CD25 + Cells were detected and counted as tumor follicular regulatory T cells, CD8a + PD-1 + TIM-3 - Cells were detected and counted as CD8 positive tumor follicular killer T cells. the T cells are T cells that have been artificially genetically modified. CD4 + PD-1 + FOXP3 + cells were detected and counted as CD4 positive tumor follicular regulatory T cells. + Granzyme K + PD-1 + TCF-1 + cells were detected and counted as CD8 positive tumor follicular killer T cells. + Granzyme K + PD-1 + TCF-1 + cells were detected and counted as CD4 positive tumor follicular killer T cells.

11. A kit for predicting the prognosis of follicular lymphoma, wherein The therapeutic composition comprises T cells, which are detected as CD4 + PD-1 + CD25 + cells or CD8a + PD-1 + TIM-3 - cells. the kit comprises: The therapeutic composition comprises T cells, which are detected as CD4 + PD-1 + FOXP3 + , CD8 + Granzyme K + PD-1 + TCF-1 + cells or CD4 + Granzyme K + PD-1 + TCF-1 + cells in a multiplex immunostaining procedure for detecting and counting tumorous follicular regulatory T cells and tumorous follicular killer T cells in T cells collected from a subject using anti-CD4 antibodies, anti-CD8 antibodies, anti-PD-1 antibodies, anti-FOXP3 antibodies, anti-TCF-1 antibodies and anti-Granzyme K antibodies. ​ ​ ​ ​ ​ ​ an antibody group 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 group 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 claim 11, wherein the kit is used for identifying, detecting, or screening the tumor follicular regulatory T cells and the tumor follicular killer T cells from the population of T cells collected from the subject.

13. A method for assisting prediction of prognosis of follicular lymphoma in a subject, wherein the method comprises: a flow cytometry procedure in which tumor follicular regulatory T cells and tumor follicular killer T cells in T cells collected from the subject are detected and counted 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; and a ratio calculation procedure in which a ratio of the tumor follicular regulatory T cells, CD4-positive tumor follicular killer T cells, and CD8-positive tumor follicular killer T cells in the T cells collected from the subject is calculated.

14. The method according to claim 13, wherein in the ratio calculation procedure, the ratio of the CD8-positive tumor follicular killer T cells is calculated based on a result of detecting and counting the CD4-positive tumor follicular killer T cells.

15. A method for assisting prediction of prognosis of follicular lymphoma in a subject, wherein the method comprises: a multiplexed immunostaining procedure in which tumor follicular regulatory T cells and tumor follicular killer T cells in T cells collected from the subject are detected and counted by a multiplexed 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 procedure in which a ratio of the tumor follicular regulatory T cells, CD4-positive tumor follicular killer T cells, and CD8-positive tumor follicular killer T cells in the T cells collected from the subject is calculated.

16. A method for performing prediction of prognosis of follicular lymphoma in a subject, wherein the method comprises: a quantification procedure in which tumor follicular regulatory T cells and tumor follicular killer T cells in T cells collected from the subject are quantified by the method according to any one of claims 1 to 6; a ratio calculation procedure in which a ratio of the tumor follicular regulatory T cells, CD4-positive tumor follicular killer T cells, and CD8-positive tumor follicular killer T cells in the T cells collected from the subject is calculated based on a quantification result obtained in the quantification procedure; and and a ratio calculation procedure in which a ratio of the tumor follicular regulatory T cells, CD4-positive tumor follicular killer T cells, and CD8-positive tumor follicular killer T cells in the T cells collected from the subject is calculated. a prognosis evaluation process in which a prognosis of the subject is evaluated on the basis of the ratio calculated in the ratio calculation process, in the prognosis evaluation process, a prognosis is evaluated as poor when the ratio calculated in the ratio calculation process is less than 2.0% for the tumor follicular regulatory T cells and less than 2.5% for the total of the CD4-positive tumor follicular killer T cells and the CD8-positive tumor follicular killer T cells.

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