Application of T cell subsets in the preparation of kits for evaluating B cell depletion therapy

Abstract

This application discloses the application of a T cell subset in the preparation of a kit for evaluating B cell depletion therapy. The T cell subset includes CD4+. + T CM CD4 + T EM CD8 + T CM CD8 + T EM CD20 dim CD4 + T, CD20 dim CD8 + T, CD20 dim CD4 + T CM CD20 dim CD4 + T EM CD20 dim CD8 + T CM CD20 dim CD8 + T EM Th17.1, Th17, Th2, CD4 + T CM Th17.1, CD4 + T CM Th17, CD4 + T CM Th2, CD4 + T EM Th17.1, CD4 + T EM Th17 and CD4 + T EM The kit in this application assesses the depletion effect of B cells by using one or more of the Th2 cytoplasm types.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to the application of a T cell subset in the preparation of a kit for evaluating B cell depletion therapy. Background Technology

[0002] Neuromyelitis optica spectrum disorders (NMOSD) are a rare, immune-mediated inflammatory demyelinating disease of the central nervous system, primarily affecting the optic nerve and spinal cord. They are characterized by high relapse rates and high disability rates, posing a serious threat to human health. B-cell depletion therapy (such as rituximab and oftatumumab) is commonly used to prevent NMOSD relapse. In clinical practice using B-cell depletion therapy to prevent NMOSD relapse, peripheral blood B-cell levels are typically rechecked approximately every 3 months to determine whether to administer B-cell depletion agents.

[0003] In related technologies, peripheral blood B cells and T cells influence each other. Relying solely on monitoring peripheral blood B cell levels cannot fully reflect the immune status of patients with B cell depletion, thus affecting the reliability of the decision to administer B cell depletion agents.

[0004] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of this disclosure, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0005] The technical objective of this application is to address the above-mentioned shortcomings by providing an application of T cell subsets in the preparation of a kit for assessing B cell depletion therapy. This application uses a kit to supplement the detection of the proportion of peripheral blood T cell subsets, assess the T lymphocyte level in patients with neuromyelitis optica spectrum disorder after B cell depletion, effectively supplement peripheral blood B cell monitoring, comprehensively assess the immune status of patients with neuromyelitis optica spectrum disorder, and thus effectively guide medication.

[0006] To achieve the above objectives, this application provides the following technical solution:

[0007] According to one aspect of this application, there is provided the use of a T cell subset in the preparation of a kit for evaluating B cell depletion therapy, said T cell subset including CD4 + T CM CD4 + T EM CD8 + T CM CD8 + T EM CD20 dim CD4 + T, CD20dim CD8 + T, CD20 dim CD4 + T CM CD20 dim CD4 + T EM CD20 dim CD8 + T CM CD20 dim CD8 + T EM Th17.1, Th17, Th2, CD4 + T CM Th17.1, CD4 + T CM Th17, CD4 + T CM Th2, CD4 + T EM Th17.1, CD4 + T EM Th17 and CD4 + T EM One or more of Th2.

[0008] In some embodiments, the T cell subset includes CD4 + T CM CD20 dim CD4 + T, Th17.1, Th17, Th2, CD4 + T CM Th17.1, CD4 + T CM Th17 and CD4 + T CM One or more of Th2.

[0009] In some embodiments, the kit includes antibodies for detecting the T cell subsets.

[0010] In some embodiments, the antibody is a monoclonal antibody.

[0011] In some embodiments, the kit further includes a fluorescently labeled indicator, human peripheral blood erythrocyte lysis buffer, and phosphate buffer.

[0012] In some embodiments, the fluorescent labeling indicator includes PerCP / Cyanine 5.5, Brilliant Violet 510, FITC, APC / Cyanine 7, PE / Cyanine 7, PE, and Brilliant Violet 421.

[0013] In some embodiments, the kit assesses the efficacy of B-cell depletion therapy by detecting the proportion of peripheral blood T-cell subsets in a patient.

[0014] In some embodiments, the peripheral blood T cell subset ratio is CD4. + T CM Cell percentage less than 43.30%, CD20 dim CD4 + The percentages of T cells were less than 1.26%, Th17.1 cells were less than 12.90%, Th17 cells were less than 20.85%, Th2 cells were greater than 44.40%, and CD4+ were also present. + T CM The proportion of Th17.1 cells was less than 17.55%, and CD4+ cells were present. + T CM The proportion of Th17 cells was less than 19.55%, and CD4+ cells were less than 40%. + T CM A Th2 cell proportion greater than 26.25% indicates that B cell depletion therapy is effective. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 The diagrams of each group of flow gate strategies in the embodiments of this application are shown.

[0017] Figure 2 The comparison results of B lymphocyte and T lymphocyte subsets in each group in the embodiments of this application are shown.

[0018] Figure 3 This document illustrates the levels of T cells and CD20 in each group after B cell depletion in embodiments of this application. dim Comparison results of T cell and Th cell subsets.

[0019] Figure 4 This embodiment of the application shows T cells and CD20 after B cell depletion. dim T cell and Th cell subsets and CD20 + Correlation analysis of B cells.

[0020] Figure 5 This application illustrates the T cells after B cell depletion in an embodiment of the present application. CM T EM CD20dim ROC curves of T cells and Th cell subsets. Detailed Implementation

[0021] To better understand the above-mentioned objectives, features, and advantages of this application, the application will be further described below with reference to the accompanying drawings and embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of this application can be combined with each other.

[0022] The present application will be further described below with reference to the accompanying drawings and specific embodiments.

[0023] This application proposes that monitoring peripheral blood T cell levels, such as central memory T cells (T cells), can improve T cell count. CM Effector memory T cells (T cells) EM ), central memory T cells expressing CD20 (CD20 dim T CM ), effector memory T cells expressing CD20 (CD20 dim T EM ) and CD4-positive T helper cells (CD4 + T helpercells, CD4 + Th) effectively supplements peripheral blood B cell monitoring, comprehensively assessing the activation status of peripheral blood B cells and T cells in the immune system.

[0024] The erythrocyte lysis buffer used in this embodiment was purchased from BD Biosciences (catalog number 349202); PerCP / Cyanine 5.5-CD3 was purchased from BioLegend (catalog number 300430); Brilliant Violet 510™- CD4 was purchased from BioLegend (item number 317444); FITC-CD20 was purchased from BioLegend (item number 302304); APC / Cyanine7-CD8 was purchased from BioLegend (item number 344714); PE / Cyanine7-CD45RA was purchased from BioLegend (item number 304126); PE-CCR7 was purchased from BioLegend (item number 353204); FITC-CXCR3 was purchased from BioLegend (item number 353704); APC / Cyanine7-CCR6 was purchased from BioLegend (item number 353432); Brilliant Violet 421™-CCR7 was purchased from BioLegend (item number 353208).

[0025] I. Subject Screening

[0026] Subject selection criteria: Patients with neuromyelitis optica spectrum disorder (NMOSD) who visited the Department of Neurology at Tianjin Medical University General Hospital between January 2024 and February 2025 and did not receive highly effective disease-modifying therapy were included.

[0027] All patients met the 2015 revised diagnostic criteria for NMOSD, as follows:

[0028] (1). Patients who are positive for AQP4-IgG only need to meet at least one core clinical feature to be diagnosed with NMOSD, while other possible causes (such as MS, infection, etc.) must be excluded.

[0029] (2). Patients with negative or unknown AQP4-IgG status must meet at least two core clinical features, including one of optic neuritis, long segmental myelitis or extreme zone syndrome, and must also meet the criteria of multiple spatial involvement (≥2 different sites), meet the requirements of additional MRI, and exclude other diseases.

[0030] This also includes NMOSD patients who received anti-CD20 therapy (aCD20) at the Department of Neurology, Tianjin Medical University General Hospital between January 2024 and February 2025. The initial treatment regimen is as follows:

[0031] (1) Rituximab 100mg intravenously, once a week for 3 consecutive weeks;

[0032] (2) At weeks 0, 1, and 2, 20 mg of olfatumab was administered subcutaneously. Peripheral blood B-cell levels were measured every three months, and when peripheral blood B-cells were ≥1%, 100 mg of rituximab or 20 mg of olfatumab was infused. All enrolled NMOSD patients had received CD20 monoclonal antibody therapy for at least 6 months.

[0033] A total of 18 NMOSD patients who did not receive highly effective disease-modifying therapy were included, including 0 males and 18 females, aged 28-79 years. A total of 38 NMOSD patients treated with CD20 monoclonal antibodies were included, including 4 males and 34 females, aged 24-78 years. Among the CD20 monoclonal antibody-treated patients, 34 received rituximab and 4 received olfatumumab. Forty healthy controls (HC) from the Tianjin Medical University General Hospital Health Examination Center were also included during the same period, including 3 males and 37 females, aged 24-68 years. This part of the study protocol has been approved by the institution's ethics committee.

[0034] II. Peripheral blood flow cytometry gate strategy analysis and comparison of B cell and T cell subsets.

[0035] Blood samples were collected after the patient signed an informed consent form. All samples were taken from fasting venous EDTA-anticoagulated peripheral blood in the morning. The specific steps are as follows:

[0036] (a) Labeling: 200 μl of peripheral blood was collected from each sample and added to two flow cytometry tubes (100 μl / tube), labeled as sample tube 1 and sample tube 2.

[0037] (b) Red blood cell lysis: Add 1 ml of red blood cell lysis buffer, vortex, and lyse at room temperature for 10 minutes to obtain a single cell suspension.

[0038] (c) Washing: Add 1 ml of phosphate buffer solution to terminate the lysis, vortex, place in a centrifuge, set the parameters to 2000 rpm, centrifuge for 5 minutes, and discard the supernatant.

[0039] (d) Staining: Add 1 μl of flow cytometry antibody sequentially according to the following staining protocol;

[0040] Sample tube 1: PerCP / Cyanine 5.5 labeled anti-human CD3 antibody, Brilliant Violet 510 labeled anti-human CD4 antibody, FITC labeled anti-human CD20 antibody, APC / Cyanine 7 labeled anti-human CD8 antibody, PE / Cyanine 7 labeled anti-human CD45RA antibody, PE labeled anti-human CCR7 antibody;

[0041] Sample tube 2: PerCP / Cyanine 5.5 labeled anti-human CD3 antibody, Brilliant Violet 510 labeled anti-human CD4 antibody, FITC labeled anti-human CXCR3 antibody, APC / Cyanine 7 labeled anti-human CCR6 antibody, PE / Cyanine 7 labeled anti-human CD45RA antibody, and Brilliant Violet 421 labeled anti-human CCR7 antibody. Vortex and stain at room temperature for 30 minutes.

[0042] (e) Filtration and instrumentation: After washing twice, discard the supernatant, add 400 μl of phosphate buffer solution to each flow cytometer, vortex, filter, and analyze and record using a FACS Aria III flow cytometer.

[0043] Statistical methods: GraphPad Prism 6 software was used for statistical analysis and graphing. The Mann-Whitney U test was used to compare two independent sample measurement data. A p-value < 0.05 was considered statistically significant, expressed as *P < 0.05, **P < 0.01, and ***P < 0.001, respectively.

[0044] Figure 1 This document illustrates the flow gate strategy diagrams for each group in the embodiments of this application. Figure 1 A represents the main gate strategy for T and B cells. Figure 1 B represents T cells that express CD20. Figure 1 C represents CD4-positive T helper cells.

[0045] like Figure 1 As shown in Figure A, the main gate strategy for T and B cells is CD20. + B cells, CD3 + T cells, CD4 + T cells, CD8 + T cells, naïve T cells (T cells) N Central memory T cells (T cells) CM Effector memory T cells (T cells) EM ) and terminally differentiated effector memory T cells (T cells) EMRA ).

[0046] like Figure 1 As shown in B, T cells expressing CD20 include CD20. dim CD4 + T cells and CD20 dim CD8 + T cells.

[0047] like Figure 1 As shown in C, CD4-positive T helper cells (CD4+) + T helper cells, CD4 + Th), including CD4 + Th17.1(CCR6 + CXCR3 + (A mixed subpopulation possessing characteristics of both Th17 and Th1), CD4 + Th17 (CCR6 + CXCR3 − CD4 + Th2(CCR6 − CXCR3 − ) and Th1 (CCR6 − CXCR3 + ).

[0048] Figure 2This document illustrates the comparison results of B lymphocyte and T lymphocyte subsets in each group in the embodiments of this application, wherein... Figure 2 A is CD20 + B cells, CD3 + T cells, CD4 + T cells, CD8 + The percentage of T cells in peripheral blood mononuclear cells. Figure 2 B is T EMRA T N T CM T EM In CD4 + Percentage in T cells, Figure 2 C is T EMRA T N T CM T EM In CD8 + Percentage in T cells, Figure 2 D is CD20 dim CD3 + T cells, CD20 dim CD4 + T cells, CD20 dim CD8 + The percentage of T cells in total T cells. Figure 2 E is T EMRA T N T CM T EM In CD20 dim CD4 + Percentage in T cells, Figure 2 F is T EMRA T N T CM T EM In CD20 dim CD8 + Percentage in T cells, Figure 2 G represents Th1, Th17.1, Th17, and Th2 in CD4. + Percentage in T cells, Figure 2 H represents Th1, Th17.1, Th17, and Th2 in CD4. + T CM Percentage in cells, Figure 2 I represents Th1, Th17.1, Th17, and Th2 in CD4. + T EMPercentage in cells. Untreated patients with NMOSD (UNT), n=18; healthy controls (HC), n=40.

[0049] like Figure 2 As shown, the specific results of comparing B lymphocyte and T lymphocyte subsets in the HC (healthy controls) and UNT (untreated patients with NMOSD, UNT) groups are as follows:

[0050] (a) UNT group CD20 + The proportion of B cells was significantly higher than that of HC cells;

[0051] (b) UNT group CD8 + T EM The proportion of cells was significantly lower than that of HC;

[0052] (c) The proportion of Th17.1 cells in the UNT group was significantly higher than that in the HC group;

[0053] (d) UNT group CD4 + T CM The proportion of Th17 cells was significantly higher than that of HC cells;

[0054] (e)UNT group CD4 + T EM The proportion of Th17 cells was significantly higher than that of HC cells;

[0055] (f) UNT group CD4 + T CM The proportion of Th1 cells was significantly lower than that of HC cells;

[0056] (g) UNT group CD4 + T EM The proportion of Th1 cells was significantly lower than that of HC cells.

[0057] III. After B cell depletion, T cells and CD20 dim Changes occur in T cell and Th cell subsets.

[0058] Figure 3 This document illustrates the levels of T cells and CD20 in each group after B cell depletion in embodiments of this application. dimComparison results of T cell and Th cell subsets. Untreated patients with NMOSD (UNT), n=18; NMOSD patients treated with anti-CD20 monoclonal antibody (aCD20), n=38; High (≥1% peripheral blood B cells), n=21; Low (<1% peripheral blood B cells), n=17.

[0059] like Figure 3 As shown, B cell depletion therapy can indeed effectively delete B cells. Simultaneously, B cell depletion therapy indirectly reduces CD4+. + T CM CD4 + T EM CD8 + T CM CD8 + T EM CD20 dim CD4 + T CM CD20 dim CD4 + T EM CD20 dim CD8 + T CM CD20 dim CD8 + T EM CD4 + T CM Th17.1 / Th17 / Th2 and CD4 + T EM Th17.1 has changed dynamically. The specific results are as follows:

[0060] (a) aCD20 group CD20 + The proportion of B cells was significantly lower in the UNT group;

[0061] (b) aCD20 set CD4 + T CM The proportion of cells in the aCD20 group was significantly lower than that in the UNT group; CD4 in the aCD20 group was significantly lower. + T EM The proportion of cells was significantly higher in the High and Low subgroups than in the UNT group; this change was confirmed in the High and Low subgroup analyses.

[0062] (c)aCD20 group CD8 + T CM The proportion of cells was significantly lower in the Low group than in the UNT group; CD8 in the Low group + T EM The proportion of cells was significantly higher in the high group than in the high group;

[0063] (d)aCD20 group CD20 dim CD4 + The proportion of T cells was significantly lower in the CD20 group than in the UNT group; CD20 in the CD20 group dim CD8 + The proportion of T cells was significantly lower in the UNT group than in the UNT group;

[0064] (e)aCD20 group CD20 dim CD4 + T CM The proportion of cells was significantly lower in the Low group than in the UNT group; CD20 in the Low group was significantly lower. dim CD4 + T EM The proportion of cells was significantly higher in the High and Low subgroups than in the UNT group; the latter was confirmed in the High and Low subgroup analyses.

[0065] (f)aCD20 group CD20 dim CD8 + T CM The proportion of cells was significantly lower in the Low group than in the UNT group; CD20 in the Low group was significantly lower. dim CD8 + T EM The proportion of cells was significantly higher in the High group than in the High group;

[0066] (g)aCD20 group CD4 + T CM Th17.1 and CD4 + T CM The proportion of Th17 cells was significantly lower in the UNT group; and this was confirmed in the High and Low subgroup analyses.

[0067] (h)aCD20 group CD4 + T CM The proportion of Th2 cells was significantly higher in the Th2 group than in the UNT group; this change was confirmed in the High and Low subgroup analyses.

[0068] (i)aCD20 group CD4 + T EM The proportion of Th17.1 cells was significantly lower than that of the UNT group.

[0069] Figure 4 This embodiment of the application shows T cells and CD20 after B cell depletion. dim T cell and Th cell subsets and CD20 + Correlation analysis of B cells.

[0070] like Figure 4 As shown, after B cell depletion, T cells and CD20... dim T cell and Th cell subsets and CD20+ B cells showed some correlation. Specific results are as follows:

[0071] (a)CD4 + T CM CD8 + T CM CD20 dim CD4 + T CM CD20 dim CD8 + T CM CD4 + T CM Th17.1 / Th17 and CD20 + Positive correlation with B cells;

[0072] (b)CD4 + T EM CD20 dim CD4 + T EM CD4 + T CM Th2 and CD20 + B cells are negatively correlated.

[0073] IV. Specific T cell subset levels to assess the sensitivity and specificity of B cell depletion therapy.

[0074] Figure 5 This application illustrates the T cells after B cell depletion in an embodiment of the present application. CM T EM CD20 dim ROC curves of T cells and Th cell subsets.

[0075] like Figure 5 As shown, the levels of specific T cell subsets were measured to assess the sensitivity and specificity of B cell depletion therapy. The specific results are as follows:

[0076] (a)CD20 dim CD4 + T cells exhibit high sensitivity and specificity, with an AUC of 0.739.

[0077] (b)CD4 + T CM The cells exhibited high sensitivity and specificity, with an AUC of 0.739.

[0078] (c) Th17.1, Th17 and Th2 cells showed high sensitivity and specificity, with AUCs of 0.803, 0.736 and 0.713, respectively;

[0079] (d)CD4 +T CM Th17.1, CD4 + T CM Th17 and CD4 + T CM Th2 cells exhibited high sensitivity and specificity, with AUCs of 0.772, 0.739, and 0.781, respectively.

[0080] In this embodiment of the application, peripheral blood CD4 + T CM Cell percentage less than 43.30%, CD20 dim CD4 + The proportion of T cells was less than 1.26%, the proportion of Th17.1 cells was less than 12.90%, the proportion of Th17 cells was less than 20.85%, the proportion of Th2 cells was greater than 44.40%, and CD4+ was present. + T CM The proportion of Th17.1 cells was less than 17.55%, and CD4+ cells were present. + T CM The proportion of Th17 cells was less than 19.55%, and CD4... + T CM The proportion of Th2 cells is greater than 26.25%, indicating that B cells are basically depleted and the NMOSD patient is in a good immune state, making treatment likely to be effective.

[0081] Through the above specific embodiments, those skilled in the art can easily implement this application. However, it should be understood that this application is not limited to the specific embodiments described above. Based on the disclosed embodiments, those skilled in the art can arbitrarily combine different technical features to achieve different technical solutions.

Claims

1. The application of T cell subsets in the preparation of a kit for evaluating B cell depletion therapy, characterized in that, The T cell subset is CD4. + T CM CD4 + T EM CD8 + T CM CD8 + T EM CD20 dim CD4 + T, CD20 dim CD8 + T, CD20 dim CD4 + T CM CD20 dim CD4 + T EM CD20 dim CD8 + T CM CD20 dim CD8 + T EM Th17.1, Th17, Th2, CD4 + T CM Th17.1, CD4 + T CM Th17, CD4 + T CM Th2, CD4 + T EM Th17.1, CD4 + T EM Th17 and CD4 + T EM One or more of Th2; The kit assesses the efficacy of B-cell depletion therapy by detecting the proportion of peripheral blood T-cell subsets in patients. When using the following T cell subsets to assess the efficacy of B cell depletion therapy, the peripheral blood T cell subset ratio is CD4. + T CM Cell percentage less than 43.30%, CD20 dim CD4 + The percentages of T cells were less than 1.26%, Th17.1 cells were less than 12.90%, Th17 cells were less than 20.85%, Th2 cells were greater than 44.40%, and CD4+ were also present. + T CM The proportion of Th17.1 cells was less than 17.55%, and CD4+ cells were present. + T CM The proportion of Th17 cells was less than 19.55%, and CD4+ cells were less than 40%. + T CM When the proportion of Th2 cells is greater than 26.25% in one or more of these groups, it indicates that B-cell depletion therapy is effective.

2. The application according to claim 1, characterized in that, The kit includes antibodies for detecting the T cell subsets.

3. The application according to claim 2, characterized in that, The antibody is a monoclonal antibody.

4. The application according to claim 1, characterized in that, The kit also includes a fluorescently labeled indicator, human peripheral blood erythrocyte lysis buffer, and phosphate buffer.

5. The application according to claim 4, characterized in that, The fluorescent labeling indicators include PerCP / Cyanine 5.5, Brilliant Violet 510, FITC, APC / Cyanine 7, PE / Cyanine 7, PE, and Brilliant Violet 421.

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