Use of akrb10 inhibitors in the manufacture of improved adoptive gd t cell therapy drugs

Abstract

The application discloses application of an AKR1B10 inhibitor in preparation of a medicine for improving adoptive gamma delta T cell therapy, the AKR1B10 inhibitor is epalrestat, the AKR1B10 inhibitor is used for improving the effect of adoptive gamma delta T cell therapy on a cancer patient, and the cancer is liver cancer. The application can significantly promote proliferation of gamma delta T cells by pretreating the gamma delta T cells cultured in vitro by using epalrestat, and can significantly improve tumor killing ability of the gamma delta T cell therapy.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of immune cell therapy, in particular to an AKR1B10 inhibitor in the preparation of a drug for improving adoptive γδ T cell therapy. BACKGROUND

[0002] Adoptive cell therapy (ACT) is to enhance the body's anti-tumor ability by extracting, modifying and reinfusing immune cells. The core therapy mainly involves chimeric antigen receptor T cells, tumor infiltrating lymphocytes, etc., which has a certain effect on tumor treatment.

[0003] γδ T cells are a unique subset of T lymphocytes, named after their T cell receptors (TCRs) composed of γ and δ chains. Unlike traditional αβ T cells, γδ T cells can directly recognize tumor antigens without relying on major histocompatibility complex (MHC). However, in γδ T cell therapy, γδ T cells are difficult to expand and have poor tumor treatment effect.

[0004] It should be noted that the information disclosed in the above background section is only used to strengthen the understanding of the background of the present disclosure, and therefore can include information that does not constitute prior art known to those of ordinary skill in the art. SUMMARY

[0005] The technical task of the present application is to solve the above problems, and provide an AKR1B10 inhibitor in the preparation of a drug for improving adoptive γδ T cell therapy. The present application can significantly promote the proliferation of γδ T cells by pretreating γδ T cells cultured in vitro with epalrestat, and can significantly improve the tumor killing ability of γδ T cell therapy.

[0006] To achieve the above object, the present application provides the following technical scheme:

[0007] According to one aspect of the present application, an AKR1B10 inhibitor is provided for use in the preparation of a drug for improving adoptive γδ T cell therapy. The AKR1B10 inhibitor is epalrestat, and the AKR1B10 inhibitor is used to improve the effect of adoptive γδ T cell therapy in cancer patients. The cancer is liver cancer.

[0008] In some embodiments, the use is in vitro by co-culturing γδ T cells with the epalrestat.

[0009] In some embodiments, the concentration of the epalrestat is 40-160 nmol / mL.

[0010] In some embodiments, the concentration of the epalrestat is 80 nmol / mL.

[0011] In some embodiments, the time of the co-culture is 24h-48h.

[0012] In some embodiments, the time of the co-culture is 24h.

[0013] In some embodiments, the epalrestat is used to promote proliferation of γδ T cells and / or enhance killing effect of γδ T cells on hepatoma cells.

[0014] In some embodiments, the epalrestat is used to promote differentiation of γδ T cells to γδ T1 direction.

[0015] According to another aspect of the present application, there is also provided use of an AKR1B10 inhibitor in the preparation of a medicament for treating hepatoma.

[0016] In some embodiments, the AKR1B10 inhibitor is epalrestat, the medicament for treating hepatoma comprises γδ T cells treated by the epalrestat, and the medicament for treating hepatoma is used to reduce tumor volume and / or growth rate of hepatoma.

[0017] Compared with the prior art, the present application has the advantages and positive effects that: the present application can significantly promote proliferation of γδ T cells and significantly improve tumor killing ability of γδ T cell therapy by pretreating γδ T cells cultured in vitro with epalrestat, and animal experiment results show that γδ T cells pretreated by epalrestat have better therapeutic effect on hepatoma of mice. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0019] Figure 1 Differential gene expression of γδ T cells infiltrating hepatocellular carcinoma tumor and paracancerous control tissue in the embodiments of the present application is shown;

[0020] Figure 2 Promotion of proliferation of γδ T cells and killing function of γδ T cells on tumor cells by AKR1B10 inhibitor in the embodiments of the present application is shown;

[0021] Figure 3 Promotion of killing function of γδ T cells on tumor cells by AKR1B10 inhibitor in the embodiments of the present application is shown;

[0022] Figure 4The proportion of cytokine secretion in the cytotoxic effect of γδ T cells after inhibition of AKR1B10 activity in the embodiments of the application is shown.

[0023] Figure 5 The AKR1B10 inhibitor significantly improves the therapeutic effect of γδ T cells on mouse liver cancer in the embodiments of the application. DETAILED DESCRIPTION

[0024] In order to enable the above-mentioned purposes, features and advantages of the present application to be more clearly understood, the present application will be further described below with reference to the accompanying drawings and embodiments. It should be noted that the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

[0025] The present application will be further described below with reference to the accompanying drawings and specific embodiments.

[0026] I. Experimental animals, instruments and reagents

[0027] 1. The mice used in the embodiments of the present application are C57 / BL6JNifdc (C57), male, 6-8 week old mice, weighing 20-25 g. The mice are purchased from China Food and Drug Inspection Research Institute (Daxing) with SPF (Specific pathogen free, SPF) level experimental animal room. All animal experiments involving mice are in accordance with the relevant regulations of the Tianjin Medical University General Hospital Animal Ethics and Use Committee.

[0028] 2. Mainly used reagents

[0029]

[0030] II. Preparation of experimental reagents

[0031] 1. Preparation of gradient dehydrated alcohol.

[0032] Take an appropriate amount of anhydrous ethanol, and add distilled water in different proportions in sequence to prepare 95% concentration alcohol solution, 90% concentration alcohol solution, 80% concentration alcohol solution and 70% concentration alcohol solution. Put the prepared alcohol solutions of different concentrations into glass jars for tissue soaking and dehydration for wax preparation.

[0033] 2. Preparation of 1x PBS phosphate buffer.

[0034] Pour the PBS powder into a 1000 mL volumetric flask, and make up to 1000 mL with distilled water. After the PBS powder is completely dissolved, perform high-pressure steam sterilization treatment for subsequent cell culture experiments.

[0035] 3. Preparation of 1640 complete culture medium.

[0036] The Australia fetal bovine serum was incubated in a constant temperature water bath at 56°C for 30 min for antibody inactivation. The 1640 complete culture medium containing 10% fetal bovine serum (1640 medium 45 mL + 5 mL inactivated Australia fetal bovine serum + 500 μL penicillin-streptomycin) was prepared for the culture of mouse γδ T cells and Hepa1-6 cells.

[0037] 4. Preparation of IL-2 cytokine.

[0038] 2 μg of IL-2 powder was placed in a centrifuge, centrifuged at 10,000 rpm for 3 min, and the powder on the bottle cap and bottle body was centrifuged to the bottom of the bottle. 100 μL of 1640 culture medium containing 10% FBS was added to dissolve the dry powder, and the mixture was shaken well. The concentration of IL-2 was 20 ng / μL, which was the storage solution. Then, a working solution with a concentration of 2 ng / μL was prepared for subsequent experiments.

[0039] 5. Preparation of AKR1B10 inhibitor (epalrestat).

[0040] 10 mg of epalrestat dry powder was placed in a centrifuge, centrifuged at 10,000 rpm for 3 min, and the powder on the bottle cap and bottle body was centrifuged to the bottom of the bottle. 978.4 μL of DMSO solution was added to dissolve the dry powder, and the mixture was shaken well. The concentration of epalrestat was 3.2×10 4 nmol / mL, which was the working solution and was stored in a -80°C refrigerator.

[0041] III. Experimental methods

[0042] 1. Isolation, culture and identification of mouse spleen γδ T cells.

[0043] ① UC7 antibody (10 μg / mL) was diluted in 1640 culture medium, 100 μL per well was added to a 48-well plate, and incubated in a 37°C cell incubator for 2 h. Before use, wash once with 1640 culture medium to remove free UC7 antibody.

[0044] ② The mouse was sacrificed, the spleen was separated, and the single cell suspension was prepared by grinding in a clean bench.

[0045] ③ The obtained single cell suspension was transferred to a 15 mL centrifuge tube, centrifuged at 1500 rpm / min for 10 min, and the supernatant was discarded. 5-6 mL of red blood cell lysis solution was added to the cell precipitate, and the mixture was shaken well and placed in a 4°C refrigerator for lysis for 15 min.

[0046] After lysis is complete, add 10 mL PBS to stop lysis, centrifuge at 1500 rpm / min for 10 min. Discard the supernatant, resuspend the cell pellet with 10 mL PBS and perform cell counting.

[0047] V. Centrifuge the cell suspension at 1500 rpm / min for 10 min, discard the supernatant, resuspend the cells with 1640 complete medium, adjust the cell concentration to 2.5 x 10 6 / mL, add IL-2 cytokine to a final concentration of 2 ng / mL and CD28 cytokine to a final concentration of 1 μg / mL. Add 0.5 mL cell suspension per well to the 48-well plate precoated with UC7 antibody in step I, and incubate in a 37°C, 5% CO2 cell incubator.

[0048] VI. After 24 h of incubation, transfer the cells to a 48-well plate without antibody coating and continue incubation.

[0049] VII. After 48 h of incubation, observe the growth and activation of γδ T cells under an inverted microscope. The activated cells are large, have tentacles and are irregular in shape.

[0050] VIII. After 7 days of incubation, randomly collect 1-2 wells of cells from the culture plate, wash thoroughly and stain with flow antibodies (FITC anti-mice CD3 and PE-Cy7 anti-mice TCR γδ). Analyze the expression of double-positive cells for surface antigens CD3 and TCR γδ using a flow cytometer. Cells with a purity of >60% can be used for subsequent experiments and sorting.

[0051] 2. Immunomagnetic bead sorting of mouse TCR γ / δ T cells +

[0052] Use the TCRg / d T Cell Isolation kit, as follows:

[0053] 1) Magnetic labeling of non-T cells (negative selection).

[0054] I. Determine the number of lymphocytes.

[0055] II. Centrifuge the cells at 300 x g for 10 min, completely remove the supernatant.

[0056] III. Resuspend the cell pellet with 450 μL buffer per 10 8 total cells.

[0057] IV. Add 50 μL biotinylated antibody per 10 8 ​Add 50 μL of Non-T Cell Depletion Cocktail (containing antibodies to mouse CD45R and CD11b) per 10

[0058] v. Mix well and incubate at 4°C for 25 minutes (with shaking for 2-3 times in between).

[0059] vi. Wash the cells by adding 10-20x the volume of buffer, centrifuge at 300 x g for 10 minutes at 4°C and completely remove the supernatant.

[0060] vii. Resuspend the cells in 500 μL of buffer and perform magnetic separation using a LD column (Miltenyi Biotec, Germany) per 10 8 cells.

[0061] viii. Apply the cell suspension to the LD column and place the LD column in the magnetic field of a MACS separator. All non-T cells (including B cells, neutrophils, monocytes / macrophages) will be retained in the column by the magnetic beads and the flow-through will mainly contain T cells.

[0062] ix. Collect the flow-through from the LD column and wash the column twice with 1 mL of buffer and pool the eluate.

[0063] 2) TCR γ / δ + Magnetic labeling of T cells (positive selection)

[0064] i. Centrifuge the pooled cell suspension and collect the cells at 300 x g for 10 minutes.

[0065] ii. Resuspend the cell pellet in 450 μL of buffer.

[0066] iii. Add 50 μL of magnetic beads coupled to biotinylated mouse TCR γ / δ monoclonal antibody, mix well and incubate at 4°C for 25 minutes (with shaking for 2-3 times in between).

[0067] iv. Wash the cells by adding 5 mL of buffer, centrifuge at 300 x g for 10 minutes at 4°C and completely remove the supernatant.

[0068] v. Resuspend the pellet in 500 μL of buffer and subsequently enrich for TCR γ / δ + T cells by magnetic separation using a MS column (Miltenyi Biotec, Germany).

[0069] vi. Apply the cell suspension to the MS column, mix well and place the MS column in the magnetic field of a MACS separator. Allow the buffer to flow out and the TCR γ / δ +T is retained on the MS column by magnetic field due to capture by biotin-labeled antibody.

[0070] 7. The column is washed with 500 μL buffer 3 times. Once the column reservoir is empty, perform the washing step by adding buffer 3 times.

[0071] 8. The column is removed from the separator and placed on an appropriate collection tube. Add 1 mL buffer and use the plunger provided with the column to flush the cells adsorbed to the magnetic beads out of the column.

[0072] 9. The purified cells are cultured and expanded in a 48-well plate.

[0073] 10. After 24 h, 1 well of cells is randomly selected and stained with flow cytometry antibodies and analyzed for surface antigen CD3 and TCR γδ double positive cell expression using a flow cytometer. Cells with a purity of >90% can be used for subsequent experimental analysis.

[0074] 3. Co-culture experiment of γδ T cells and tumor cells

[0075] 1. Observe the growth of γδ T cells under a microscope. When the positive rate reaches more than 90% as identified by flow cytometry, subsequent research can be performed. After the cells are collected and washed, the cells are counted and a co-culture system of γδ T cells and Hepa1-6 cells (a mouse hepatoma cell line purchased from the ATCC Cell Resource Center) is established in a 96-well plate.

[0076] 2. Different groups of Hepa1-6 cells and γδ T cells are co-cultured at an E﹕T (effector to target) ratio of 1:10. 10 4 cells / well of Hepa1-6 cells and 10 5 cells / well of γδ T cells. The Hepa1-6 cells are plated 12 h in advance, and after the Hepa1-6 cells are completely adherent, the γδ T cells are added for co-culture.

[0077] 3. The specific groups of the experiment are as follows: a control group (Hepa1-6); an Epalrestat-treated Hepa1-6 cell group (Hepa1-6+Epalrestat); a mixed co-culture group of γδ T cells and Hepa1-6 cells (Hepa1-6+γδ T); and an Epalrestat-treated mixed co-culture group of γδ T cells and Hepa1-6 cells (Hepa1-6+γδ T+Epalrestat). After 24 h or 48 h of co-culture, subsequent detection is performed.

[0078] 4. Effect of exogenous Epalrestat on γδ T cell proliferation and tumor killing function.

[0079] 1) CCK-8 detection of γδ T cell proliferation

[0080] ① Take γδ T cells from a 37°C, 5% CO2 incubator, resuspend and centrifuge for cell counting. Cells are inoculated in a 96-well plate at 1 x 10 5 cells / well, with 100 μL of culture medium per well; appropriate PBS is added to the edge wells to prevent the influence of liquid evaporation on the experiment.

[0081] ② Use different drug concentrations (40 nmol / mL, 80 nmol / mL, 160 nmol / mL) of Epalrestat to treat γδ T cells, set 24 h and 48 h detection plates according to different detection times, and set up experimental groups (culture medium containing cells, drugs and CCK-8 reagent), negative control groups (culture medium containing cells and drugs, CCK-8 reagent) and blank control groups (culture medium without cells, CCK-8 reagent) for each plate, wherein the negative control group is denoted as γδ T, and the experimental groups are denoted as γδ T+Epalrestat (40 nmol / mL), γδ T+Epalrestat (80 nmol / mL) and γδ T+Epalrestat (160 nmol / mL), respectively, with 3 replicate wells for each group. Place the 96-well plate in a 37°C, 5% CO2 incubator for further incubation.

[0082] ③ After 24 h and 48 h of treatment, take the 96-well plates of each group from the 37°C, 5% CO2 incubator, add 10 μL of CCK-8 reagent to each well, and incubate at 37°C for 2 h. Use a microplate reader to detect the OD value of each well at 450 nm absorbance.

[0083] ④ Calculation formula: proliferation rate = (experimental group - blank control group) / (negative control group - blank control group).

[0084] 2) CCK-8 detection of γδ T cell killing of Hepa1-6 cells.

[0085] ① Take the expanded γδ T cells from a 37°C, 5% CO2 incubator, resuspend and centrifuge for cell counting. γδ T cells (10 5 cells / well) and mouse liver cancer cells Hepa1-6 (10 4 cells / well) are co-cultured in a 96-well culture plate, with a culture system of 200 μL, and appropriate PBS is added to the edge wells of the culture plate to prevent the influence of liquid evaporation on the experiment.

[0086] ② According to different detection times, set 24 h and 48 h detection plates, and set up a control group (Hepa1-6) for each plate; process Hepa1-6 cells with Epalrestat (Hepa1-6+Epalrestat); mix γδ T cells with Hepa1-6 cells for co-culture (Hepa1-6+γδ T); mix γδ T cells treated with Epalrestat with Hepa1-6 cells for co-culture (Hepa1-6+γδ T+Epalrestat). Set 3 replicate wells for each group. Place the 96-well plates in a 37°C, 5% CO2 incubator for continued culture.

[0087] ③ After 24 h and 48 h of treatment, remove the 96-well plates of each group from the 37°C, 5% CO2 incubator, wash the γδ T cells according to the manufacturer's instructions, and add 10 μL of CCK-8 reagent to each well. Incubate at 37°C for 2 h. Use a microplate reader to detect the OD value of each well at 450 nm absorbance.

[0088] 3) Flow cytometry detection of γδ T cell proliferation

[0089] ① Set up a blank control group and an Epalrestat-treated γδ T cell group according to experimental needs, with 3 replicate wells for each group. According to different detection times, set 24 h and 48 h treatment groups. Here we use CD3 and TCR γδ to label γδ T cells, and use ki67 as the γδ T cell proliferation index.

[0090] ② Collect the γδ T cells in each group system with 1.5 mL EP tubes, add an appropriate amount of PBS to wash the cells and centrifuge.

[0091] ③ First, perform γδ T cell surface staining. Add 100 μL of PBS to each group for resuspension, and add FITC-CD3 (0.5 μL) and PE-Cy7-TCR γδ (1 μL) antibodies to each group for staining. Mix well with a shaker, incubate at room temperature for 30 min, and avoid light.

[0092] ④ Add 1 mL of PBS to each tube for resuspension, centrifuge at 1500 rpm / min for 10 min, and discard the supernatant after centrifugation.

[0093] ⑤ Add 200 μL of prepared Fix membrane-breaking reagent to each tube, mix well by blowing, incubate at 4°C for 1 h, and avoid light. Perform punching.

[0094] ⑥ Centrifuge at 1500 rpm / min for 10 min, discard the supernatant, and add 200 μL of 1x Buffer to each tube for washing.

[0095] ⑦ 1500 rpm / min, 10 min, discard supernatant, add APC-ki67 flow antibody 1 μL in each group of tube, incubate at room temperature for 30 min in the dark.

[0096] ⑧ Add 200 μL washing Buffer to each tube, wash at 1500 rpm / min for 10 min, discard supernatant, add 200 μL paraformaldehyde to each tube for fixation, and detect by flow cytometry.

[0097] 4) Immunofluorescence detection of γδ T cell killing of Hepa1-6 cells

[0098] ① Thaw 10 mM CFSE stock solution, and transfer Hepa1-6 cells to a 15 mL centrifuge tube.

[0099] ② Wash Hepa1-6 cells in 1×PBS to remove any residual serum proteins, and repeat step 2.

[0100] ③ Resuspend cells in 1-3×10 7 Cells / mL in 1×PBS as a single cell suspension. Note: Avoid staining in buffers containing azide, serum, or proteins, as these substances bind to free dyes and can interfere with cell staining.

[0101] ④ Add CFSE stock solution to the Hepa1-6 cell suspension to achieve a final concentration of 5 μM, and incubate at 37°C in a water bath for 10-15 minutes.

[0102] ⑤ Add 9 volumes of 1×PBS to the Hepa1-6 cells, centrifuge at 120×g for 10 minutes, and pour off the supernatant.

[0103] ⑥ Add 10 mL complete medium containing 10% FBS, repeat the centrifugation step. Pour off the supernatant.

[0104] ⑦ Resuspend the Hepa1-6 cells in complete medium, and perform cell culture.

[0105] ⑧ Mix expanded γδ T cells (2×10 6 cells / well) with CFSE (5 μΜ / mL, labeled Hepa1-6 cells (2×10 5 cells / well) in a 48-well plate, with a total volume of 500 μL, in two groups, with three replicates in each group, for 12 h.

[0106] ⑨ Group 1: Collect the supernatant, stain the cells in the supernatant with 7-AAD, and measure the fluorescence staining of CFSE+Hepa1-6 cells using a fluorescence inverted microscope.

[0107] 10. Group two: DAPI was used to restain the cell nucleus, and the cell slide was photographed under a fluorescence inverted microscope to observe the killing effect of γδ T cells on Hepa1-6 cells. Image J software (version 1.52a) was used to quantify the positive cells by counting the number of cells in one high-power field.

[0108] 5. Flow cytometry was used to detect the effect of exogenous epalrestat on the secretion of cytokines by γδ T cells

[0109] 1) γδ T cells, IFN-γ, and IL-17 staining

[0110] ① According to the experimental requirements, set up a blank control group (-Epalrestat) and an epalrestat-treated γδ T cell group (+Epalrestat), with 3 replicates in each group. Set up 24 h and 48 h treatment groups according to different detection times.

[0111] ② Add cytokine stimulants at a ratio of 1:500 to each experimental group, and incubate in a 37°C incubator for 6 h. Collect γδ T cells from each group using 1.5 mL EP tubes, centrifuge, and then wash once with an appropriate amount of PBS.

[0112] ③ First, perform γδ T cell surface staining by adding 100 μL PBS to each group and resuspending the cells, while adding corresponding FITC-CD3 (0.5 μL) and PE-Cy7-TCR γδ (1 μL) antibodies to each group for staining. Mix well using a shaker, and incubate at room temperature for 30 min in the dark.

[0113] ④ Add 1 mL PBS to each tube and mix well by blowing, then centrifuge at 1500 rpm / min for 10 min, and discard the supernatant.

[0114] ⑤ Add 200 μL of prepared Fix membrane-breaking reagent to each tube, mix well by blowing, and incubate at 4°C in the dark for 1 h.

[0115] ⑥ Centrifuge at 1500 rpm / min for 10 min, discard the supernatant, and add 200 μL of 1×Buffer to each tube for washing.

[0116] ⑦ Centrifuge at 1500 rpm / min for 10 min, discard the supernatant, and add 1 μL of APC-IFN-γ and PE-IL-17 flow cytometry antibodies to each group, and incubate at room temperature in the dark for 30 min.

[0117] ⑧ Add 200 μL of Buffer to each tube, centrifuge at 1500 rpm / min for 10 min, discard the supernatant, add 200 μL of paraformaldehyde to each tube for fixation, and perform flow cytometry detection.

[0118] 2) γδ T cells, GZMB, PRF1 staining

[0119] ① First, γδ T cell surface staining was performed, 100 μL PBS was added to each group for resuspension, and corresponding FITC-CD3 (0.5 μL) and PE-Cy7-TCR γδ (1 μL) antibodies were added to each group for staining, mixed on a shaker, incubated at room temperature for 30 min in the dark.

[0120] ② Add 1 mL PBS to each tube and mix well by blowing, 1500 rpm / min, 10 min, discard the supernatant.

[0121] ③ Add 200 μL of prepared Fix membrane-breaking reagent to each tube, mix well by blowing, and incubate at 4°C in the dark for 1 h.

[0122] ④ 1500 rpm / min, 10 min, discard the supernatant, add 200 μL of 1×Buffer to each tube.

[0123] ⑤ 1500 rpm / min, 10 min, discard the supernatant, add 1 μL of APC-PRF1 and PE-GZMB flow cytometry antibodies to each group, incubate at room temperature in the dark for 30 min.

[0124] ⑥ Add 200 μL of Buffer to each tube, 1500 rpm / min, 10 min, discard the supernatant, add 200 μL of paraformaldehyde to each tube, and detect by flow cytometry.

[0125] 6. Establishment of mouse liver cancer tumor model and experimental grouping

[0126] First, adjust the concentration of Hepa1-6 cells to 5×10 7 / mL, and inject 100 μL of cell suspension subcutaneously into the right axillary wall of each C57BL / 6 mouse. On the fifth day after planting, a clear tumor nodule appeared in the right axillary wall, and the nodule was hard. The size of the nodule was measured with a vernier caliper. Each mouse was injected with a total of 1×10 6 treated or untreated γδ T cells, repeated once after 7 days, and the change in tumor size was measured every other day.

[0127] The experiment was divided into 3 groups:

[0128] ① PBS treatment control group (Hepa1-6).

[0129] ② γδ T cell treatment group (Hepa1-6+γδ T).

[0130] ③ The γδ T cell treatment group treated with Epalrestat (Hepa1-6 + γδ T + Epalrestat).

[0131] 7. Tumor model monitoring and sampling

[0132] The mice were observed daily for changes in diet and activity, and for changes such as suppuration, tumor growth or regression at the implantation site. When a palpable tumor nodule appeared in the right axillary of the mice on the fifth day after implantation, the measurement of tumor size was started, and the measurement was performed every two days. The tumor formation of the mice was recorded, and a tumor growth curve was drawn. The longest diameter and the maximum transverse diameter perpendicular to the longest diameter of the tumor were measured using a vernier caliper, and the tumor volume was calculated according to the formula V (mm 3 ) = (length x width 2 ) / 2. After 19 days of inoculation, the mice were euthanized by injection of an overdose of 1% sodium pentobarbital, and the tumor size of the mice in each group was measured. The tumor tissue was removed, weighed, and photographed for data retention. The tumor tissue was placed in a 15 mL centrifuge tube and stored in a -80°C freezer and 4% paraformaldehyde for standby use.

[0133] 8. HE staining

[0134] The implanted tumor models of the mice in each group were subjected to HE staining to evaluate cell infiltration. The staining mainly included three steps: embedding the wax block, slicing the tissue, and staining and mounting the slice, and the details are as follows.

[0135] 1) Embedding paraffin:

[0136] ① The obtained specimen tissue block was taken out from the specimen bottle using forceps, and the tissue block was trimmed using a sterilized blade to make the edges flat.

[0137] ② The specimen tissue blocks of each group were placed in disposable embedding boxes, and the embedding boxes were labeled on the outside with a pencil. Then, the embedding boxes containing the tissue blocks were placed in a dehydration jar for gradient dehydration. The specific process and time are as follows:

[0138]

[0139] ③ The paraffin embedding machine was turned on in advance, an appropriate amount of paraffin block was added to the wax jar, the temperature was set to 65°C, and the embedding boxes that had completed dehydration were first placed in the left wax jar and soaked for 30 min. Then, the embedding boxes were moved to the right wax jar and soaked for 90 min.

[0140] ④ The embedding boxes were taken out from the wax jar, the tissue blocks in the embedding boxes were taken out using forceps, the position of the tissue blocks was adjusted using forceps, and the order of the tissue blocks of each group was recorded. Melted paraffin was poured into the embedding boxes, and the boxes were covered.

[0141] ⑤ The embedding boxes were placed on the freezing table to wait for the paraffin to cool and solidify, and the wax block was trimmed.

[0142] 2) Tissue sectioning

[0143] ① Put the tissue block into ice to pre-cool, so that the texture becomes hard, which is beneficial for sectioning.

[0144] ② Put the trimmed tissue block into the slot of the microtome, install the knife, adjust the plane of the block and the distance between the block and the knife, and section at 5 μm thickness for continuous sectioning.

[0145] ③ Carefully pick up the paraffin tissue section with tweezers and put it into a 50℃ water bath, so that the paraffin tissue section is completely unfolded in water.

[0146] ④ Separate the connected tissue sections with tweezers, carefully pick up the tissue sections with a non-stick slide, and mark them.

[0147] ⑤ Place the tissue sections in a slide rack and dry them in a 65℃ oven for 4 h, then store them at room temperature.

[0148] 3) HE staining

[0149] ① Put the tissue sections into a 65℃ oven for 2 h.

[0150] ② Then put the tissue sections into a glass jar for deparaffinization and hydration, ensuring that the tissue is completely soaked, and the specific process and time are as follows:

[0151]

[0152] ③ After deparaffinization and hydration, wash the sections with 1×PBS three times on a shaker for 5 min each time.

[0153] ④ Add hematoxylin staining solution to the tissue, stain for 30 s, then stop, soak in distilled water for 5 min, and observe the staining of the tissue cell nucleus under a stereomicroscope.

[0154] ⑤ Add eosin staining solution, stain for 50 s, then stop, soak in distilled water for 5 min, and observe the staining of the tissue cytoplasm under a stereomicroscope.

[0155] ⑥ Quickly soak the tissue sections, differentiate after soaking in hydrochloric acid alcohol, rinse with tap water for 5 min, then return to blue with 1% ammonia water.

[0156] ⑦ Put the tissue sections through 70%, 80%, and 95% alcohol jars and anhydrous ethanol jar in sequence, then soak in xylene No. 1 and No. 2 jars for 5 min each.

[0157] ⑧ Take out the slide from the jar, seal it with neutral gum, dry it in a fume hood, then scan the section and take photos for observation.

[0158] 9. Statistical methods

[0159] The measurement data is expressed as mean ± standard deviation. The t-test is used for comparison between two groups of samples, and analysis of variance is used for comparison between more than three groups of samples. Statistical analysis and statistical chart production use GraphPad Prism (version 8.0). P values < 0.05 are considered statistically significant (P < 0.05*, P < 0.01**, P < 0.001***). Immunofluorescence images are analyzed and calculated using Image J software (version 1.52a). HE and immunohistochemical images are analyzed and calculated using PRECICE Iviewer software (version 3.3.4).

[0160] 10. Experimental results:

[0161] (1) Differential gene expression of γδ T cells infiltrating hepatocellular carcinoma (HCC)

[0162] The GSE38476 dataset was derived from fresh surgical specimens of 10 pairs of HCC patient tumor tissues and corresponding para-cancer tissues (NCBI-GEO database). After lymphocytes were isolated from the specimens, γδ T cells infiltrating HCC tumor tissues and γδ T cells infiltrating para-cancer liver tissues were separated and purified by TCRγ / δ purification kit (Miltenyi Biotec, Germany), and transcriptome sequencing analysis was performed.

[0163] After obtaining the original data of the GSE38476 dataset from the NCBI-GEO database, the original data was preprocessed by background correction, data filtering, and quality control. A total of 16058 gene expression matrices were obtained. The Limma loading package in R language statistical software was used to analyze the differential expression genes of the gene expression matrix of the GSE38476 dataset (| Fold Change |>2; FDR <0.05 as the screening standard). Transcriptome sequencing of γδ T cells infiltrating tumor tissues and γδ T cells infiltrating para-cancer tissues, Figure 1 The differential gene expression of γδ T cells infiltrating hepatocellular carcinoma tumor and para-cancer control tissues in the embodiments of the present application is shown, wherein, Figure 1 A is a heat map of differentially expressed genes, Figure 1 B is a volcano plot of differentially expressed genes. As Figure 1 shown, a total of 392 differentially expressed genes were found between the two groups (Fold Change≥2, P <0.001). Among them, 257 genes were significantly up-regulated in tumor-infiltrating γδ T cells, and 135 genes were significantly down-regulated (Fold Change≤-2, P <0.001). Figure 1A). Among them, the expression of AKR1B10 is the most significant, the expression level of AKR1B10 in tumor infiltrating γδ T cells is 27.119 times that of the expression level of γδ T cell infiltrating adjacent control tissue, and the expression level of AKR1B15 in tumor infiltrating γδ T cells is the second, which is 20.366 times that of the expression level of γδ T cell infiltrating adjacent control tissue Figure 1 B).

[0164] (2) Inhibition of AKR1B10 activity significantly promotes γδ T cell proliferation and killing of tumor cells

[0165] Figure 2 The AKR1B10 inhibitor in the embodiment of the application promotes the proliferation of γδ T cells and the killing function of tumor cells, wherein, Figure 2 A is the cell proliferation of each group after 24 hours of co-culture, Figure 2 B is the cell proliferation of each group after 48 hours of co-culture, Figure 2 C is the proportion of surviving Hepa1-6 (OD 450nm) of each group after 24h of co-culture, Figure 2 D is the proportion of surviving Hepa1-6 (OD 450nm) of each group after 48h of co-culture.

[0166] Epalrestat is an aldehyde reductase inhibitor developed for clinical use in diabetes, which can effectively inhibit the activity of AKR1B10. In order to determine the biological role of AKR1B10 in γδ T cells, we expanded and cultured mouse γδ T cells in vitro, constructed an epalrestat drug concentration gradient, and observed the proliferation of γδ T cells after inhibition of AKR1B10 activity by CCK8 experiment. As shown in Figure 2 A-2B, after inhibition of AKR1B10 activity (pre-treatment of γδ T cells with epalrestat), the proliferation rate of γδ T cells increased significantly and was dose-dependent.

[0167] In order to explore the effect of AKR1B10 on the tumor killing function of γδ T cells, we cultured and expanded mouse hepatoma cell line Hepa1-6 and mouse γδ T cells in vitro respectively, and co-cultured them at an effector to target ratio of 10:1. After 24 or 48 hours, the supernatant and suspended γδ T cells were removed, and the number of surviving Hepa1-6 was determined by CCK8 experiment to reflect the killing ability of γδ T cells to tumor cells. The fewer the surviving Hepa1-6, the stronger the killing effect of γδ T cells on it. As shown in Figure 2 C-2D, after inhibition of AKR1B10 activity (pre-treatment of γδ T cells with epalrestat), the killing ability of γδ T cells to hepatoma cells was significantly enhanced.

[0168] Meanwhile, in order to more intuitively observe the killing of γδ T cells to tumor cells, before co-culture, CFSE-labeled Hepa1-6 cells were used, and 7AAD and Dapi staining were performed on the supernatant and cell slide of the cells after co-culture, and the survival and death of Hepa1-6 were observed by fluorescence microscopy. Figure 3 The AKR1B10 inhibitor in the embodiment of the application promotes the killing function of γδ T cells to tumor cells, wherein, Figure 3 A is a diagram of the survival of Hepa1-6 observed by fluorescence microscopy in each group, Figure 3 B is the CFSE + Dapi + Hepa1-6 cell number column chart, Figure 3 C is a diagram of the death of Hepa1-6 observed by fluorescence microscopy, Figure 3 D is the CFSE + 7AAD + Hepa1-6 cell number column chart. As Figure 3 shown, Figure 3 A-B shows the growth of Hepa1-6 cells on the cell slide after 24 hours of co-culture, Figure 3 C-D shows the death of Hepa1-6 cells in the supernatant after co-culture. It can be found that compared with the control group, after 24 hours of co-culture of Hepa1-6 and γδ T cells, the number of surviving cells on the slide is significantly reduced, and the number of dead cells in the supernatant is significantly increased, proving that γδ T cells can effectively kill mouse Hepa1-6 cells. After adding the AKR1B10 inhibitor, compared with the co-culture group without adding the inhibitor, the number of surviving cells on the slide is further reduced, and at the same time, the number of dead cells in the supernatant is also significantly increased, proving that inhibiting the activity of AKR1B10 can significantly improve the killing ability of γδ T cells to hepatoma cells.

[0169] (3) Inhibition of AKR1B10 activity changes the cytokine expression profile of γδ T cells

[0170] After expanding and culturing mouse γδ T cells in vitro, AKR1B10 inhibitor (80 nmol / mL Epalrestat) was added for 24 hours of continuous culture, then PMA, Ionomycin and Golgi-Plug were added for 4-6h stimulation, and then the cells were collected for cytokine staining by flow cytometry, and the change of the cytokine expression profile of γδ T cells was detected by flow cytometry. Figure 4 The proportion of cytokine secretion of cytotoxic effect in γδ T cells after inhibition of AKR1B10 activity in the embodiment of the application is shown. As Figure 4As shown, after inhibiting AKR1B10 activity, cytokines exhibiting cytotoxic effects in γδ T cells, including interferon-γ (IFN-γ), are reduced. + γδ Tcells, perforin (Prf-1) + γδ T cells), granzyme B (GZMB) + γδ T cells) expression levels were significantly increased, while interleukin-17 (IL-17) expression levels were significantly increased. + No significant changes were observed in the expression levels of γδ T cells. These results confirm that inhibiting AKR1B10 activity can promote the transformation of γδ T cells into γδ T1 (IFN-γ) cells, which have anti-tumor and anti-infection functions. + ) directional differentiation.

[0171] (4) Inhibition of AKR1B10 activity significantly improved the therapeutic effect of γδ T cells on mouse liver cancer.

[0172] To investigate the therapeutic effect of γδ T cells on mouse liver tumors after inhibiting AKR1B10 activity, we cultured mouse hepatocellular carcinoma cells Hepa1-6 (derived from C57BL / 6 mice). A mouse subcutaneous xenograft model of hepatocellular carcinoma was established using C57BL / 6 wild-type mice. Each mouse received a subcutaneous injection of 5 × 10⁵ γδ T cells into the axilla. 6 Cells were observed to form a clearly visible tumor mass after 5 days. Starting from day 5, treatment was administered via tail vein infusion of cultured and purified γδ T cells or γδ T cells treated with the AKR1B10 inhibitor (80 nmol / mL Epalrestat) for 24 hours, at a dose of 1×10⁻⁶. 6 γδ T cells were administered to each mouse, while the control group received only an equal volume of saline. Treatment was repeated one week later (day 12 post-tumor inoculation). The length, width, and height of the tumor were measured every other day using calipers to calculate tumor volume and plot tumor growth curves. On day 19 post-inoculation, mice were sacrificed, subcutaneous tumors were isolated, and pathological analysis was performed.

[0173] Figure 5 The embodiments of this application demonstrate that the AKR1B10 inhibitor significantly improves the therapeutic effect of γδ T cells on mouse liver cancer, wherein... Figure 5 A shows photographs of tumor tissue in each group. Figure 5 B represents the tumor growth curves of each group of mice. Figure 5 C is a statistical graph of tumor quality in each group of mice. Figure 5 D represents the HE staining results of mouse tumors in each group.

[0174] like Figure 5As shown, the tumor size and growth rate of the simple γδ T cell treatment group (γδ T) and the AKR1B10 inhibitor treated γδ T cell treatment group (γδ T+Epalrestat) were significantly lower than the control group (Control) injected with only normal saline, indicating that the adoptive γδ T cell therapy had a good therapeutic effect on mouse liver cancer. At the same time, the tumor size and growth rate of the AKR1B10 inhibitor treated γδ T cell treatment group were significantly lower than the simple γδ T cell treatment group, indicating that after inhibiting the activity of AKR1B10, the killing effect of γδ T cells on mouse liver tumors was significantly enhanced.

[0175] The skilled in the art of the technical field can easily implement the present application through the above specific embodiments. However, it should be understood that the present application is not limited to the above specific embodiments. On the basis of the disclosed embodiments, the skilled in the art of the technical field can arbitrarily combine different technical features to implement different technical solutions.​

Claims

1. Use of epalrestat in the manufacture of a medicament for improving the effect of adoptive gd T cell therapy for liver cancer patients, characterized in that, The application is in vitro by co-culturing γδ T cells with the eptastatin.

2. Use according to claim 1, characterized in that, The concentration of the eptastatin is 40-160 nmol / mL.

3. Use according to claim 2, characterized in that, The concentration of the eptastatin is 80 nmol / mL.

4. Use according to claim 1, characterized in that, The co-culturing time is 24 h-48 h.

5. Use according to claim 4, characterized in that, The co-culturing time is 24 h.

6. Use according to any one of claims 1 to 5, characterized in that, The eptastatin is used for promoting γδ T cell proliferation and / or enhancing the killing effect of γδ T cells on liver cancer cells.

7. The use according to any one of claims 1 to 5, characterized in that, The eptastatin is used for promoting γδ T cells to differentiate in the direction of γδ T1.

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