Method for detecting and / or identifying BAFF + DNT cells and application thereof

Abstract

The invention relates to a method for detecting and / or identifying BAFF + DNT cells and application thereof, and belongs to the technical field of cell detection. According to the present invention, the anti BAFF monoclonal antibody is combined with the flow cytometry to characterize the BAFF protein expression efficiency of the DNT cell, and the DNT cell is positioned through the CD3, the CD4 and the CD8 so as to achieve the synchronous detection of the cell activity, the DNT cell grouping and the BAFF positive rate; the kit has the characteristics of high sensitivity, strong specificity, short detection period, less cell sample consumption, low false positive rate, high quality control standardization degree and suitability for GMP production.

Description

Technical Field

[0001] This invention relates to the field of cell detection technology, and more particularly to a method for detecting and / or identifying BAFF. + Methods and applications of DNT cells. Background Technology

[0002] B-cell activating factor (BAFF), also known as B lymphocyte stimulating factor (BLyS), TNF- and APOL-associated leukocyte expression ligand (TALL-1), dendritic cell-derived TNF-like molecule, and tumor necrosis factor ligand superfamily member 13B, is a protein encoded by the TNFSF13B gene. It is primarily produced by macrophages, dendritic cells, and stromal cells, and is crucial for B cell survival, maturation, and antibody production. However, in nature, DNT cells lack the TNFSF13B gene in their genome and therefore cannot express the BAFF protein.

[0003] Current technologies typically employ lentiviral transduction to transform the TNFSF13B gene into DNT cells. However, traditional transformation detection methods, such as Western blotting / qPCR, suffer from significant indirect errors and cannot accurately assess transformation efficiency. Flow cytometry is susceptible to interference from debris, two-cell pairs, or phenotypic drift, and DNT cells (CD3+) also exhibit these issues. + CD4 - CD8 - The proportion of BAFF protein expression in the mixed cell population is very low, making it impossible to accurately identify successfully transformed DNT cells; in addition, the stepwise detection of BAFF protein expression and T cell typing is cumbersome and has large batch-to-batch variations, which cannot meet the needs of standardized quality control and rapid detection. Summary of the Invention

[0004] The purpose of this invention is to provide a method for detecting and / or identifying BAFF. + The method and application of DNT cells aim to solve the problem that existing technologies cannot accurately and rapidly detect BAFF protein expression and T cell typing.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for detecting and / or identifying BAFF. + The DNT cell method includes the following steps:

[0007] (1) Prepare flow cytometry buffer and CAR-DNT system;

[0008] (2) Centrifuge the CAR-DNT cells for 3-10 min, take the cell pellet and mix it with the CAR-DNT system, stain it, and obtain the stained CAR-DNT system;

[0009] (3) Mix the stained CAR-DNT system with 0.5-3 mL of flow cytometry buffer, centrifuge for 3-10 min, and collect the precipitate to obtain CAR-DNT precipitate;

[0010] (4) Mix the CAR-DNT precipitate with 100–500 μL of flow cytometry buffer and perform flow cytometry analysis to obtain BAFF. + DNT cells.

[0011] Preferably, the flow cytometry buffer comprises the following components at the following concentrations: fetal bovine serum 5–30 μL / mL and phosphate buffer 970–995 μL / mL.

[0012] Preferably, the CAR-DNT system described in step (1) comprises the following components in volume fractions: 70-90 parts of flow cytometry buffer, 1-3 parts of CD45-QB500 antibody, 0.5-2 parts of CD3-PE-Cy7 antibody, 1-3 parts of CD4-QB450 antibody, 1-10 parts of CD8-APC-QB710 antibody, 1-5 parts of BAFF-PE antibody, and 1-4 parts of 7-aminoactinomycin D.

[0013] Preferably, the number of CAR-DNT cells in step (2) is 1 to 5 × 10⁻⁶. 5 indivual;

[0014] The centrifugation speed is 300–600 × g;

[0015] The staining temperature is 2–8℃, and the staining time is 20–60 min.

[0016] Preferably, the centrifugation speed in step (3) is 300 to 600 × g.

[0017] Preferably, the gate logic for the flow cytometry analysis in step (4) includes one or more of the following: FSC-H vs SSC-H gate logic, FSC-H vs FSC-A gate logic, 7-AAD-H vs SSC-H gate logic, CD45-QB500-H vs SSC-H gate logic, CD3-PE-Cy7-H vs SSC-H gate logic, CD4-QB450-H vs CD8-APC-QB710-H gate logic, and BAFF-PE-H vs SSC-H gate logic.

[0018] This invention also provides the method for detecting and / or identifying BAFF. + Application in DNT cells.

[0019] The present invention also provides the application of the method in determining the BAFF positivity rate of DNT cells.

[0020] The present invention has the following technical effects and advantages:

[0021] This invention uses an anti-BAFF monoclonal antibody combined with flow cytometry to characterize the BAFF protein expression efficiency of DNT cells, and uses CD3, CD4 and CD8 to locate DNT cells, thereby specifically detecting the BAFF protein expression efficiency of DNT cells.

[0022] (1) Single tube multiplex detection: Optimize the antibody combination (CD45 / CD3 / CD4 / CD8 / BAFF / 7-AAD) to achieve simultaneous detection of cell viability + DNT cell clustering + BAFF positivity rate, shorten the experimental cycle from 6h to 2h, and reduce cell sample consumption by 70%.

[0023] (2) Ultra-high specificity: based on CD3 + CD4 - CD8 - DNT cells are strictly defined, excluding CD4 cells. + CD8 + T-cell interference; using a >99% negative population shift method to set the positive threshold for each logic gate, combined with 7-AAD screening, the false positive rate is <1%, and the sensitivity reaches 10. 3 BAFF molecules / cells;

[0024] (3) High degree of quality control standardization: The isotype control system ensures that the setting of each gate's logic threshold is not affected by the voltage fluctuation of the flow cytometer, with batch-to-batch CV < 5%; the non-gate standard can collect 10 4 More than one cell, ensuring low abundance of BAFF + Statistical reliability of DNT cells (<1%);

[0025] (4) Adapted to GMP production: The composition of the flow cytometry buffer is clearly defined. The technical solution of this invention is compatible with automated pipetting workstations and supports batch release testing of CAR-DNT cell preparations. Attached Figure Description

[0026] Figure 1 Two-parameter plot of FSC-H vs SSC-H for CAR-DNT mixture;

[0027] Figure 2 The two-parameter plot of FSC-H vs FSC-A for CAR-DNT mixture;

[0028] Figure 3Two-parameter plot of 7-AAD-H vs SSC-H for CAR-DNT mixture;

[0029] Figure 4 Two-parameter plot of CD45-QB500-H vs SSC-H for CAR-DNT mixture;

[0030] Figure 5 Two-parameter plot of CD3-PE-Cy7-H vs SSC-H for CAR-DNT mixture;

[0031] Figure 6 Two-parameter plots of CD4-QB450-H vs CD8-APC-QB710-H for CAR-DNT mixtures;

[0032] Figure 7 Two-parameter plot of BAFF-PE-H vs SSC-H for CAR-DNT mixture;

[0033] Figure 8 The graph shows the two parameters of BAFF-PE-H vs SSC-H for the isotype control mixture. Detailed Implementation

[0034] This invention provides a method for detecting and / or identifying BAFF. + The DNT cell method includes the following steps:

[0035] (1) Prepare flow cytometry buffer, isotype control system and CAR-DNT system;

[0036] (2) Centrifuge CAR-DNT cells for 3 to 10 minutes, preferably 5 minutes, take the cell pellet and mix it with the isotype control system, stain it, and obtain the stained isotype control system.

[0037] (3) Centrifuge the CAR-DNT cells for 3 to 10 minutes, preferably 5 minutes, take the cell pellet and mix it with the CAR-DNT system, stain it, and obtain the stained CAR-DNT system.

[0038] (4) Mix the staining isotype control system and the staining CAR-DNT system with 0.5-3 mL of flow cytometry buffer, preferably 1 mL; centrifuge for 3-10 min, preferably 5 min, and take the precipitate to obtain the isotype control precipitate and the CAR-DNT precipitate;

[0039] (5) Mix the isotype control precipitate and CAR-DNT precipitate with 100–500 μL of flow cytometry buffer, preferably 200 μL, and perform flow cytometry analysis to obtain BAFF. + DNT cells.

[0040] In this invention, the flow cytometry buffer comprises the following components at the following concentrations: fetal bovine serum (FBS) 5-30 μL / mL, preferably 10 μL / mL; phosphate buffer (PBS) 970-995 μL / mL, preferably 990 μL / mL.

[0041] In this invention, the isotype control system described in step (1) comprises the following components in volume fractions: 70-90 parts of flow cytometry buffer, preferably 85 parts; 1-3 parts of CD45-QB500 antibody, preferably 2 parts; 0.5-2 parts of CD3-PE-Cy7 antibody, preferably 1 part; 1-3 parts of CD4-QB450 antibody, preferably 2 parts; 1-10 parts of CD8-APC-QB710 antibody, preferably 6 parts; 1-5 parts of IgG-PE antibody, preferably 2 parts; and 1-4 parts of 7-aminoactinomycin D (7-AAD), preferably 2 parts.

[0042] In this invention, the CAR-DNT system described in step (1) comprises the following components in volume fractions: 70-90 parts of flow cytometry buffer, preferably 85 parts; 1-3 parts of CD45-QB500 antibody, preferably 2 parts; 0.5-2 parts of CD3-PE-Cy7 antibody, preferably 1 part; 1-3 parts of CD4-QB450 antibody, preferably 2 parts; 1-10 parts of CD8-APC-QB710 antibody, preferably 6 parts; 1-5 parts of BAFF-PE antibody, preferably 2 parts; and 1-4 parts of 7-aminoactinomycin D (7-AAD), preferably 2 parts.

[0043] In this invention, the number of CAR-DNT cells in steps (2) to (3) is independently 1 to 5 × 10⁻⁶. 5 1, preferably 2×10 5 indivual;

[0044] The centrifugation speed is 300-600×g, preferably 500×g;

[0045] The dyeing temperature is 2-8℃, preferably 4℃; the dyeing time is 20-60 min, preferably 30 min.

[0046] In this invention, the centrifugation speed in step (4) is 300 to 600 × g, preferably 500 × g.

[0047] In this invention, the gate logic for flow cytometry analysis in step (5) includes one or more of the following: FSC-H vs SSC-H gate logic, FSC-H vs FSC-A gate logic, 7-AAD-H vs SSC-H gate logic, CD45-QB500-H vs SSC-H gate logic, CD3-PE-Cy7-H vs SSC-H gate logic, CD4-QB450-H vs CD8-APC-QB710-H gate logic, BAFF-PE-H vs SSC-H gate logic, and non-designated gate standards.

[0048] The FSC-H vs SSC-H gate logic is used for the initial screening of CAR-DNT cells by forward scattering / side scattering (FSC / SSC).

[0049] The FSC-H vs FSC-A gate logic is used for single-cell screening in CAR-DNT cells;

[0050] The 7-AAD-H vs SSC-H gate logic is used for live cell screening in single cells;

[0051] The CD45-QB500-H vs SSC-H gate logic is used for lymphocyte screening in live cells;

[0052] The CD3-PE-Cy7-H vs SSC-H gate logic is used for T cell screening in lymphocytes;

[0053] The CD4-QB450-H vs CD8-APC-QB710-H gate logic is used for DNT cell screening in T cells;

[0054] The BAFF-PE-H vs SSC-H gate logic is used for BAFF positivity rate detection and / or BAFF in DNT cells. + DNT cell identification;

[0055] The non-gate standard is used for quality control.

[0056] In this invention, the BAFF + The positive determination of DNT cells was performed using the >99% negative population shift method.

[0057] This invention also provides the method for detecting and / or identifying BAFF. + Application in DNT cells.

[0058] The present invention also provides the application of the method in determining the BAFF positivity rate of DNT cells.

[0059] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0060] In the test samples of this invention, the CAR-DNT cells were obtained from Shanghai Bailian Biomedical Technology Co., Ltd.

[0061] In the reagents of this invention, FBS (catalog number: La822500) was purchased from Ling'an Biotechnology (Shanghai) Co., Ltd., PBS (catalog number: C10010500BT) was purchased from Gibco, USA, and 7-AAD (catalog number: 420404), CD3-PE-Cy7 antibody (catalog number: 300316, clone number: HIT3a), BAFF-PE antibody (catalog number: 318606, clone number: T7241), IgG- PE antibody (catalog number: 400113, clone number: MOPC-21) was purchased from Biolegend, Inc., USA; CD4-QB450 antibody (catalog number: S0031, clone number: RPA-T4), CD8-APC-QB710 antibody (catalog number: S0377, clone number: HIT8a), and CD45-QB500 antibody (catalog number: S0165, clone number: HI30) were purchased from Suzhou Weidu Biotechnology Co., Ltd.

[0062] In the apparatus of this invention, the Advanteon flow cytometer was purchased from Agilent Technologies, Inc., USA.

[0063] Example 1: BAFF + DNT cell detection method

[0064] (1) Preparation of flow cytometry buffer: Mix 10 μL LBS and 990 μL PBS to obtain flow cytometry buffer;

[0065] Preparation of the isotype control system: Mix 85 μL of flow cytometry buffer, 2 μL of CD45-QB500 antibody, 1 μL of CD3-PE-Cy7 antibody, 2 μL of CD4-QB450 antibody, 6 μL of CD8-APC-QB710 antibody, 2 μL of IgG-PE antibody, and 2 μL of 7-AAD to obtain the isotype control system;

[0066] Preparation of the CAR-DNT system: Mix 85 μL of flow cytometry buffer, 2 μL of CD45-QB500 antibody, 1 μL of CD3-PE-Cy7 antibody, 2 μL of CD4-QB450 antibody, 6 μL of CD8-APC-QB710 antibody, 2 μL of BAFF-PE antibody, and 2 μL of 7-AAD to obtain the CAR-DNT system;

[0067] (2) Take 2×10 5CAR-DNT cells were centrifuged at 500×g for 5 min, and the cell pellet was mixed with 100 μL of isotype control system. The mixture was then stained at 4℃ for 30 min to obtain the staining isotype control system. Simultaneously, 2×10⁶ CAR-DNT cells were... 5 One CAR-DNT cell was centrifuged at 500×g for 5 min, and the cell pellet was mixed with 100 μL of CAR-DNT system and stained at 4℃ for 30 min to obtain the stained CAR-DNT system.

[0068] (3) Mix the staining isotype control system and the staining CAR-DNT system with 1 mL of flow cytometry buffer, and centrifuge at 500×g for 5 min to obtain the corresponding isotype control precipitate and CAR-DNT precipitate;

[0069] (4) The isotype control precipitate and CAR-DNT precipitate were mixed with 200 μL of flow cytometry buffer to obtain the corresponding isotype control mixture and CAR-DNT mixture. The CAR-DNT mixture was used for flow cytometry analysis of FSC-H vs SSC-H gate logic, FSC-H vs FSC-A gate logic, 7-AAD-H vs SSC-H gate logic, CD45-QB500-H vs SSC-H gate logic, CD3-PE-Cy7-H vs SSC-H gate logic, CD4-QB450-H vs CD8-APC-QB710-H gate logic, and BAFF-PE-H vs SSC-H gate logic. The analysis was performed using the method described by Wong et al. (Wong DP, Roy NK, Zhang K, et al. ABAFFligand-based CAR-T cell targeting three receptors and multiple B cell cancers[J]. Nat The >99% negative population shift method for BAFF, disclosed in Commun, 2022, 13(1):217, is used. + The positive result of DNT cells yielded BAFF. + DNT cells; the isotype control mixture is used for non-gated standard analysis and quality control in flow cytometry. When the number of single cells screened in CAR-DNT cells is ≥10,000, it indicates that the cell quality is good.

[0070] Experimental Example 1: Initial screening of CAR-DNT cells using FSC / SSC

[0071] On an Advanteon flow cytometer, FSC-H vs SSC-H gate logic was configured to exclude CAR-DNT cell debris (FSC). low / SSC low), delineating the CAR-DNT cell master population (FSC) mid / SSC mid The result is as follows Figure 1 As shown.

[0072] The results showed that the main population of CAR-DNT cells accounted for 88.11%.

[0073] Experiment Example 2: Single-cell screening in CAR-DNT cells

[0074] On an Advanteon flow cytometer, FSC-H vs FSC-A gate logic was configured to exclude aggregates of CAR-DNT cells, ensuring that only single cells participated in event analysis. The results are as follows: Figure 2 As shown.

[0075] The results showed that, after excluding the adhesion cells of CAR-DNT cells, single cells accounted for 97.66% of CAR-DNT cells.

[0076] Experiment Example 3: Live Cell Screening in Single Cells

[0077] On an Advanteon flow cytometer, the 7-AAD-H vs SSC-H gate logic was set up, and the presence of live cells in a single cell was confirmed using 7-AAD. The results are as follows: Figure 3 As shown.

[0078] The results showed that, after excluding dead cells in single cells, live cells accounted for 98.90% of the single cells.

[0079] Experiment Example 4: Screening of Lymphocytes from Live Cells

[0080] On an Advanteon flow cytometer, CD45-QB500-H vs SSC-H gate logic was set up, and CD45-QB500 antibody was used to confirm the presence of CD45 in live cells. + Lymphocytes (Lym) were present, and the results were as follows: Figure 4 As shown.

[0081] The results showed that excluding CD45 - After non-lymphocytes, CD45 + Lymphocytes account for 99.40% of living cells.

[0082] Experiment Example 5: Screening of T cells in lymphocytes

[0083] On an Advanteon flow cytometer, CD3-PE-Cy7-H vs SSC-H gate logic was set up, and CD45 was confirmed using CD3-PE-Cy7 antibody. + CD3 in lymphocytes+ T cells were present, and the results were as follows: Figure 5 As shown.

[0084] The results showed that excluding CD3 - After non-T cells, CD3 + T cells at CD45 + The proportion of lymphocytes was 99.53%.

[0085] Experiment Example 6: Screening of DNT cells in T cells

[0086] On an Advanteon flow cytometer, CD4-QB450-H vs CD8-APC-QB710-H gate logic was set up, and CD3 was confirmed using CD4-QB450 antibody and CD8-APC-QB710 antibody. + CD4 in T cells - CD8 - DNT cells were present, and the results were as follows Figure 6 As shown.

[0087] The results showed that CD4 - CD8 - DNT cells in CD3 + The proportion of T cells was 69.78%.

[0088] Experimental Example 7: Determination of BAFF Positive Rate in DNT Cells

[0089] Setting up BAFF-PE-H vs SSC-H gate logic on an Advanteon flow cytometer and confirming CD4 using BAFF-PE antibody. - CD8 - BAFF in DNT cells + DNT cells were present, and BAFF was analyzed using the >99% negative population shift method disclosed by Wong et al. in the literature (Wong DP, Roy NK, Zhang K, et al. A BAFF ligand-based CAR-T cell targeting three receptors and multiple B cell cancers[J]. Nat Commun, 2022, 13(1):217). + The positive result of DNT cells was as follows: Figure 7 As shown.

[0090] The results showed that CD4 - CD8 - BAFF cells in DNT cells + DNT cells were present, and the BAFF positivity rate was 36.84%.

[0091] Comparative Example 1: Quality Control

[0092] Using an Advanteon flow cytometer, a non-gating standard was employed, and the positive threshold for each gate logic was determined using an isotype control mixture. The >99% negative population shift method, as described by Wong et al. in the literature (Wong DP, Roy NK, Zhang K, et al. ABAFF ligand-based CAR-T cell targeting three receptors and multiple B cell cancers[J]. Nat Commun, 2022, 13(1):217), was used to perform BAFF analysis. + The positive result of DNT cells was as follows: Figure 8 As shown.

[0093] The results showed that a total of 56,425 single cells were collected without the gated standard, indicating that the cell quality was good. The BAFF positivity rate in the isotype control mixture was 0.96%, meaning that the threshold positivity rate of each gate logic was <1%, indicating that the method of the present invention has strong system adaptability.

[0094] As can be seen from the above embodiments, the present invention provides a method for detecting and / or identifying BAFF. + The method for detecting DNT cells and its application. The method of this invention can simultaneously detect cell viability, DNT cell clustering, and BAFF positivity rate, and has the advantages of high sensitivity, strong specificity, short detection cycle, low cell sample consumption, low false positive rate, high degree of quality control standardization, and compatibility with GMP production.

[0095] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting and / or identifying BAFF + The method using DNT cells is characterized by... Includes the following steps: (1) Prepare flow cytometry buffer and CAR-DNT system; (2) Centrifuge the CAR-DNT cells for 3-10 min, take the cell pellet and mix it with the CAR-DNT system, stain it, and obtain the stained CAR-DNT system; (3) Mix the stained CAR-DNT system with 0.5-3 mL of flow cytometry buffer, centrifuge for 3-10 min, and collect the precipitate to obtain CAR-DNT precipitate; (4) Mix the CAR-DNT precipitate with 100–500 μL of flow cytometry buffer and perform flow cytometry analysis to obtain BAFF. + DNT cells.

2. The method according to claim 1, characterized in that, The flow cytometry buffer comprises the following components at the following concentrations: fetal bovine serum 5–30 μL / mL and phosphate buffer 970–995 μL / mL.

3. The method according to claim 1, characterized in that, The CAR-DNT system described in step (1) comprises the following components in volume fractions: 70-90 parts of flow cytometry buffer, 1-3 parts of CD45-QB500 antibody, 0.5-2 parts of CD3-PE-Cy7 antibody, 1-3 parts of CD4-QB450 antibody, 1-10 parts of CD8-APC-QB710 antibody, 1-5 parts of BAFF-PE antibody, and 1-4 parts of 7-aminoactinomycin D.

4. The method according to claim 1, characterized in that, The number of CAR-DNT cells in step (2) is 1 to 5 × 10⁻⁶. 5 indivual; The centrifugation speed is 300–600 × g; The staining temperature is 2–8℃, and the staining time is 20–60 min.

5. The method according to claim 1, characterized in that, The centrifugation speed in step (3) is 300 to 600 × g.

6. The method according to claim 1, characterized in that, The gate logic for flow cytometry analysis in step (4) includes one or more of the following: FSC-H vs SSC-H gate logic, FSC-H vs FSC-A gate logic, 7-AAD-H vs SSC-H gate logic, CD45-QB500-H vs SSC-H gate logic, CD3-PE-Cy7-H vs SSC-H gate logic, CD4-QB450-H vs CD8-APC-QB710-H gate logic, and BAFF-PE-H vs SSC-H gate logic.

7. The method according to any one of claims 1 to 6 for detecting and / or identifying BAFF + Application in DNT cells.

8. The application of the method according to any one of claims 1 to 6 in determining the BAFF positivity rate of DNT cells.

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