Application of ZNF683 as immunosuppression target in regulation of T cell immune tolerance induction

Abstract

The present application relates to the use of ZNF683 as a target in the diagnosis or therapeutic prognosis monitoring, and treatment of diseases related to the regulation of T cell immune tolerance. In particular, the present application studies the regulatory effect of ZNF683 on human primary T cells, and tests the feasibility of ZNF683 as a target for the prognosis and treatment of GVHD after clinical transplantation.

Description

Technical Field

[0001] This invention relates to the field of gene function and application, and in particular to the use of ZNF683 as a target in the diagnosis or treatment prognosis monitoring of diseases related to T cell immune tolerance regulation, as well as in treatment. Background Technology

[0002] Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a treatment for various hematological diseases, such as aggressive hematological malignancies and myelodysplastic syndromes. On one hand, the remodeled immune system after allo-HSCT plays a role in graft-versus-leukemia effects (GVL), preventing opportunistic bacterial and viral infections, and providing strong immune protection for the recipient. On the other hand, the presence of allogeneic reactive T cells after allo-HSCT may lead to acute and chronic graft-versus-host disease. Domestic and international studies show that 30%-60% of patients develop grade II-IV acute graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation, 14% suffer from severe (grade III-IV) acute graft-versus-host disease, and the incidence of chronic GVHD is as high as 80% (Malard F, Huang XJ, Sim JP Y. Treatment and unmet needs in steroid-refractory acute graft-versus-host disease[J]. Leukemia, 2020, (5): 1229-40. Blazar BR, Murphy WJ, Abedi M. Advances in graft-versus-host disease biology and therapy[J]. Nat Rev Immunol, 2012, 12(6): 443-58. Ferrara JL, Levine JE, Reddy P, et al. Graft-versus-host disease[J].Lancet,2009,373(9674):1550-61).

[0003] Genetically modified hematopoietic stem cell transplantation (GVHD) is a clinical syndrome caused by the interaction between allogeneic donor cells and recipient tissues. Its physiological process is primarily mediated by donor T cells and inflammatory cytokines, leading to multi-organ and tissue damage in the recipient. The key to preventing and treating GVHD is inducing T cell tolerance after allo-HSCT. Therefore, exploring the key regulatory molecules that induce T cell immune tolerance after allogeneic hematopoietic stem cell transplantation, enabling timely diagnosis and monitoring of GVHD and appropriate treatment, and even assessing patient risk before the onset of GVHD to achieve "early warning and prediction," is a crucial scientific issue for improving GVHD diagnosis and treatment strategies.

[0004] In haploidentical stem cell transplantation (haplo-HSCT), incomplete HLA matching leads to a stronger allogeneic reaction between donor and host cells, increasing the likelihood of genetic disease (GVHD) in recipients and severely impacting patient prognosis and quality of life. Therefore, it is crucial to explore the key regulatory molecules that induce immune tolerance after haploidentical hematopoietic stem cell transplantation to alleviate GVHD.

[0005] ZNF683, also known as zinc finger protein 683, is a transcription factor that mediates the transcriptional program of various T lymphocytes in tissues, including memory T cells, NK cells, and NKT cells. It also plays an important role in the differentiation of thymic and peripheral T cells and NKT cells. Previous research by our team has found that in recipients who have developed immune tolerance after haploidentical hematopoietic stem cell transplantation, CD8 effector T cells (CD8 Teff) are significantly expanded and highly express the transcription factor ZNF683. Currently, research on ZNF683 in human primary T cells is limited to exploring its expression level. Previous studies have shown that ZNF683 is primarily expressed in circulating effector T cells; however, the regulatory role of ZNF683 in human primary T cells and the specific molecular mechanisms of this regulation have not been reported.

[0006] Therefore, in order to address the above shortcomings, this invention studies the regulatory role of ZNF683 in human primary T cells, and provides new applications for ZNF683 based on the research conclusions on the regulatory role. Summary of the Invention

[0007] This invention studies the regulatory effect of ZNF683 on human primary T cells and examines the feasibility of ZNF683 as a target for clinical post-transplant prognostic monitoring and treatment of GVHD.

[0008] In a first aspect, the present invention provides the use of ZNF683 in the preparation of reagents or kits for the diagnosis or prognostic monitoring of diseases related to T-cell immune tolerance regulation.

[0009] Furthermore, the T-cell immune tolerance regulation-related disease is a disease caused by allogeneic hematopoietic stem cell transplantation, preferably acute or chronic graft-versus-host disease (GVHD).

[0010] Furthermore, the reagent is a reagent for detecting the expression level of ZNF683, and the kit contains a reagent for detecting the expression level of ZNF683.

[0011] Furthermore, the reagents for detecting ZNF683 expression levels include reagents for detecting mRNA expression levels and / or protein expression levels.

[0012] Preferably, the reagents for detecting mRNA expression levels include those used in the following methods: PCR-based detection methods, Southern hybridization methods, Northern hybridization methods, dot hybridization methods, fluorescence in situ hybridization methods, DNA microarray methods, ASO methods, and high-throughput sequencing platform methods.

[0013] Preferably, the reagent for detecting the mRNA expression level of ZNF683 includes specific primers and / or probes.

[0014] The upstream primer of ZNF683:

[0015] 5'-CATATGTGGCAAGAGCTTTGG-3';

[0016] ZNF683 downstream primer:

[0017] 5'-GGCAAGTTGAGTGAAGCTCT-3';

[0018] 18S upstream primer: 5'-ACCGATTGGATGGTTTAGTGAG-3';

[0019] 18S downstream primer: 5'-CCTACGGAAACCTTGTTACGAC-3'.

[0020] Furthermore, the diagnosis or prognostic test is a prediction based on the test results of a patient's sample; preferably, the sample is tissue; preferably, the sample is tumor tissue.

[0021] In a second aspect, the present invention provides the use of ZNF683 in the preparation of medicaments for the treatment or prevention of diseases related to T-cell immune tolerance regulation.

[0022] Furthermore, the T-cell immune tolerance regulation-related disease is a disease caused by allogeneic hematopoietic stem cell transplantation, preferably an acute or chronic graft-versus-host disease.

[0023] Furthermore, the drug is a reagent for increasing the amount of ZNF683 gene or protein in the patient's body.

[0024] Furthermore, the reagent is an agonist of ZNF683, a lentiviral vector overexpressing ZNF683, or a peptide-mimicking inhibitor of the functional domain of ZNF683.

[0025] The beneficial effects achieved by this application are:

[0026] First, to explore the regulatory effect of ZNF683 on T cells, a lentiviral vector containing the coding region of the ZNF683 gene was constructed. ZNF683 was overexpressed in human primary T cells using lentivirus, and the proliferation, apoptosis, and cytokine secretion capacity of primary T cells with high ZNF683 expression were detected by flow cytometry.

[0027] Secondly, RNA-seq was used to explore the molecular mechanism by which ZNF683 inhibits T cell activation. The results showed that overexpression of ZNF683 in primary T cells reduced T cell proliferation, decreased apoptosis levels, and reduced secretion levels of cytokines such as IL-2, IL-4, and IL-10, as well as decreased expression of the cytotoxic molecule CD107a. RNA-seq results revealed that the RTEL gene, crucial for maintaining telomere stability, protection, and extension, was upregulated in CD8+ T cells overexpressing ZNF683. The Wnt signaling pathway, related to T cell activation, was downregulated in CD8+ T cells overexpressing ZNF683.

[0028] Third, the study also found that T cells downregulate ZNF683 expression levels during activation. This indicates that ZNF683 is an immune checkpoint gene that maintains effector T cells in a relatively quiescent and long-lived state. T cells enter a quiescent state by upregulating ZNF683 expression levels and enter an activated state by downregulating ZNF683 levels. High expression of the ZNF683 gene can induce human T cells to enter a state of immune tolerance, and ZNF683 can serve as a key molecule for predicting and targeting the treatment of acute and chronic GVHD. Attached Figure Description

[0029] Figure 1 ZNF683 expression is upregulated in CD8+ T cells of haplotype transplant recipients; A: Comprehensive analysis of RNA-seq and ATAC-seq data. Differential genes from haplo-SCT recipients compared to their paired donors are shown in CD8 T cells.

[0030] Figure 2: Validation of ZNF683 overexpression in primary T cells in vitro. AB: ZNF683 was overexpressed in CD3+ T cells of healthy individuals via lentiviral infection. 48 hours after infection, A: qPCR was used to detect the overexpression efficiency of ZNF683. B: Flow cytometry typical plots of lentiviral infection efficiency in the non-infection group, the empty vector control group, and the ZNF683 overexpression group (ZNF683-OE).

[0031] Figure 3 Effect of ZNF683 overexpression on primary T cell proliferation. AE: ZNF683 was overexpressed in CD3+ T cells from healthy individuals via lentiviral infection, 48 hours after infection. AC: Cell cycle changes were detected by flow cytometry. DE: 48 hours after infection, cells were labeled with CellTrace™ Violet cell tracer for 3 days, and the proliferation of control T cells and ZNF683-overexpressing T cells was measured.

[0032] Figure 4 Effect of ZNF683 overexpression on apoptosis in primary T cells. AB: ZNF683 was overexpressed in CD3+ T cells of healthy individuals via lentiviral infection. T cell apoptosis was detected 48 hours after infection using Annexin V and 7-AAD co-staining. A: Typical flow cytometry plot. B: Statistical graph.

[0033] Figure 5 Effects of ZNF683 overexpression on cytokine secretion in primary T cells. AE: ZNF683 was overexpressed in CD3+ T cells of healthy individuals via lentiviral infection. 48 hours after infection, ZNF683-overexpressing T cells were sorted by flow cytometry. After 3 days of in vitro stimulation, the levels of IL-4, IL-10, IFN-γ, IL-2, and CD107a secreted by ZNF683-overexpressing GFP+ cells (A), CD4+ T cells (B), and CD8+ T cells (C) were detected by flow cytometry. DE; Statistical graph.

[0034] Figure 6 Transcriptional changes in primary CD8+ T cells with high ZNF683 expression

[0035] A: Heatmap showing differentially expressed genes between primary CD8+ T cells highly expressing ZNF683 and control CD8+ T cells. B: GO enrichment analysis of differentially expressed genes between primary CD8+ T cells highly expressing ZNF683 and control CD8+ T cells. C: CD8+ T cells from patients who underwent haploidentical hematopoietic stem cell transplantation more than one year ago were sorted, and ZNF683 expression levels were detected by qPCR after 3 days of in vitro stimulation with CD3 / 28beads. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] Example 1: Sample Source and Cell Culture

[0038] 1. Case data: Eight pairs of patients and healthy donors who underwent haploidentical and sibling-matched hematopoietic stem cell transplantation at the Institute of Hematology, Peking University People's Hospital were selected. The patients were aged ≤60 years. The hematopoietic stem cells were chimeric after transplantation. Immunosuppressants were discontinued. At the time of specimen collection, the patients had no chronic GVHD, no relapse, no poor engraftment, and no serious infection or other transplant-related complications.

[0039] 2. Isolation and Culture of T Cells: Human bone marrow mononuclear cells (BMMCs) were isolated from the bone marrow of healthy donors using Ficoll density gradient centrifugation. CD3+ T cells were purified from BMMCs using CD3 MicroBeads (Miltenyi Biotec, 130-097-043), and the CD3+ T cells were resuspended in IMDM (Gibco, Invitrogen) + 10% 9500 BIT (Stemcell Technologies, 09500) (serum substitute to exclude the possible influence of TGF-β in serum on T cells) + IL-2 (100 U / ml) medium.

[0040] Example 2: Expression level of ZNF683 overexpression in lentivirus

[0041] The ZNF683 overexpression lentiviral vector was purchased from Sangon Biotech (Shanghai, China). CD3+ T cells were resuspended in IMDM medium containing 10% 9500 BIT and IL-2 (100 U / ml), and seeded in 24-well plates at 2×10⁻⁶ cells / well. 5 Cells / well were stimulated with CD3 / 28beads (Invitrogen, 11131D). After 24 hours, the culture medium in each well was concentrated to half its original volume. The appropriate volume of virus was added according to the viral titer to achieve an MOI of 30, and polybrene (Sigma, USA) was added to a final concentration of 6 μg / ml. The medium was changed after 24 hours of culture. Flow cytometry was used to detect infection efficiency 48 hours after virus addition, and the changes in T cell proliferation, apoptosis, and cytokine secretion after ZNF683 overexpression were analyzed.

[0042] ZNF683 is upregulated in CD8 T cells of haploidentical transplant recipients who have developed immune tolerance. Peripheral blood samples were collected from eight pairs of haploidentical hematopoietic stem cell transplant recipients with a survival of more than one year after discontinuing immunosuppressive drugs, and from their respective donors, provided that no infection or relapse occurred at the time of sample collection. We sorted CD8 T cells from the peripheral blood of both donors and recipients. + CD4 + and CD3 + CD8 + T cell subsets were sequenced at the transcriptome level (RNA-seq) and ATAC-seq (Assay for Transposase-Accessible Chromatin with high throughput sequencing). Our results indicate that in haplotype transplant recipients, ZNF683 is present not only in CD8... + T cells have more open chromatin accessibility, and ZNF683 is present in CD8. + T cell transcriptome expression was significantly upregulated. Figure 1 This represents a comprehensive analysis of RNA-seq and ATAC-seq data, revealing differentially expressed genes in CD8 T cells from the haplo-SCT receptor compared to its paired donor.

[0043] Validation of ZNF683 overexpression in primary T cells in vitro. ZNF683 was overexpressed in human primary T cells via lentiviral infection in vitro, and the overexpression level of ZNF683 after infection was verified by qPCR. Figure 2 A). Quantitative PCR results showed that the expression level of ZNF683 in the ZNF683 overexpression group (ZNF683-OE) was significantly higher than that in the control group (CT). The infection efficiency of the fluorescently labeled lentiviral vector was detected by flow cytometry. Figure 2 B).

[0044] Example 3. T cell cycle detection

[0045] Cells were resuspended in PBS at room temperature and incubated with flow cytometry antibodies in the dark for 20 minutes for surface staining with CD4-Percp-Cy5.5 (Biolegend, 357414) and CD8-APC-R700 (BD Horizon, 565165). T cells were fixed for 30 minutes using a fixation / perforation kit (BD Pharmingen, 562574), stained with Ki-67-APC antibody (Biolegend, 350514), and incubated at room temperature for 20 minutes. Cells were washed with PBS and analyzed using flow cytometry. DAPI was added before analysis. Monoclonal antibodies were purchased from BD and Biolegend.

[0046] ZNF683 inhibits T cell proliferation. ZNF683 may be a key molecule for inducing or maintaining CD8T+ cell immune tolerance after haploidentical hematopoietic stem cell transplantation. The eukaryotic cell cycle consists of four distinct phases: G1, S, G2, and M. On the one hand, T cell proliferation is crucial for generating an effective adaptive immune response, and a key factor in proliferation is cell entry into the cell cycle; on the other hand, non-proliferating (non-dividing) cells in eukaryotes typically enter a quiescent G0 state from G1 to remain quiescent for extended periods. To investigate the effect of ZNF683 on T cell cycle progression, we used Ki67 / DAPI staining followed by flow cytometry to divide the cell cycle into G0, G1, and S / G2 / M phases. The results showed that compared to T cells infected with an empty viral vector control, the proportion of primary T cells highly expressing ZNF683 in the G0 phase was significantly increased. Figure 3 A, Figure 3 C); Compared with the control group, the expression of Ki-67 in T cells overexpressing ZNF683 was decreased. Figure 3 A, Figure 3 B). Using CellTrace TM Violet labeling of primary T cells overexpressing ZNF683 and control primary T cells showed that the proliferation of T cells overexpressing ZNF683 was significantly reduced compared with that of control T cells. Figure 3 D, 3E).

[0047] Example 4. Detection of T cell apoptosis

[0048] Cells were resuspended in PBS at room temperature and incubated with flow cytometry antibodies in the dark for 20 minutes. Surface CD4-AF700 (Biolegend, 357418) and CD8-V500 (BD Horizon, 560774) were then added and incubated at room temperature for 20 minutes. After antibody incubation, cells were resuspended in 100 μl of 1× binding buffer, labeled with Annexin V-APC and 7-AAD-PerCP (BD Pharmingen, 550475) apoptosis kits, and incubated at room temperature in the dark for 15 minutes. Cells were washed with PBS and analyzed within 1 hour. Monoclonal antibodies were purchased from BD and Biolegend.

[0049] ZNF683 inhibits T cell apoptosis.

[0050] T cell apoptosis is crucial for T cell immune tolerance. On one hand, thymocytes with autoreactive TCRs are eliminated during development via apoptosis, and effector T cells proliferating during the immune response also undergo apoptosis (Activation-induced cell death, ACID). On the other hand, cell longevity is the basis for the continuous peripheral homeostasis and proliferation of T naive and memory T cells. To investigate the effect of ZNF683 on T cell apoptosis, we used flow cytometry to sort T cells overexpressing ZNF683 and assessed the effect of ZNF683 on T cell apoptosis using Annexin V and 7-AAD co-staining. The results showed that the proportion of early and late apoptosis in T cells with high ZNF683 expression was lower than that in the control group. Figure 4 A, Figure 4 B) These results indicate that ZNF683 is associated with the long lifespan of T cells.

[0051] Example 5: Detection of the expression of cell secretion factors and cytotoxic molecules

[0052] Four hours before cell collection, cells were resuspended in GolgiPlug (BD Pharmingen, 555029) at room temperature and incubated in PBS with flow cytometry antibodies in the dark for 20 minutes for surface staining with CD4-Percp-Cy5.5 (Biolegend, 357414) and CD8-APC-R700 (BD Horizon, 565165). T cells were fixed for 30 minutes using a fixation / perforation kit (BD Pharmingen, 562574), and then IFN-γ-BV510 (Biolegend, 502528) and CD107a-PE (BDPharmingen, 555801) antibodies were added 4 hours before cell collection. Cells were stained with IL-4-APC (Biolegend, 500714), IL-10-PE-CY7 (Biolegend, 501420), and IL-2-V450 (Biolegend, 560867) and incubated at room temperature for 20 minutes. Cells were washed with PBS and analyzed. Monoclonal antibodies were purchased from BD and Biolegend.

[0053] Example 6. CellTrace™ Violet Cell Proliferation Assay

[0054] Cells were resuspended in pre-warmed PBS at a concentration of 1×10⁶ / ml. 1 μL of CellTrace™ Violet (ThermoFisher, C34557) stock solution was added per 1 ml of cells, bringing the final concentration to 10 μM. The cells were incubated at 37°C for 30 min, and then 5 volumes of complete culture medium were added to terminate the staining. Cells were detected under 405 nm excitation light after 3 days of culture.

[0055] Example 7. Quantitative qPCR detection of ZNF683 mRNA expression

[0056] RNA was extracted from purified T cells from healthy donors using the RNeasy mini kit (QIAGEN, 74106). cDNA was synthesized using a cDNA reverse transcription kit (TaKaRa, RR047A). The expression of the target gene in cells was detected using the SYBR Green dye assay. The relative expression of the target gene mRNA was calculated using the ΔΔCT method with 18S as an internal control. Primer sequences were as follows: ZNF683 upstream primer: 5'-CATATGTGGCAAGAGCTTTGG-3'; ZNF683 downstream primer: 5'-GGCAAGTTGAGTGAAGCTCT-3'; 18S upstream primer: 5'-ACCGATTGGATGGTTTAGTGAG-3'; and 18S downstream primer: 5'-CCTACGGAAACCTTGTTACGAC-3'.

[0057] Example 8. RNA-seq experiment

[0058] RNA was extracted using the RNeasy Micro Kit (QIAGEN, 74004) and mRNA was purified using NEBNext Oligo d(T)25 beads (NEB, E7490). cDNA libraries were constructed using the NEBNext Ultra II RNA Library Prep Kit (NEB, E7770S). AMPure XP beads (Beckman Coulter, A63881) were used to purify cDNA libraries ranging from 300 bp to 500 bp. Paired-end sequencing of cDNA was performed on a NovaSeq 6000 (Illumina), yielding 28 to 35 million 150 bp paired-end reads per sample.

[0059] Statistical analysis: SPSS 20.0 was used for statistical analysis. Paired t-tests were used to assess the differences in apoptosis, cytokine secretion, and proliferation among the ZNF683-0E and CT groups. P < 0.05 was considered statistically significant.

[0060] Example 9: ZNF683 reduces T cytokine secretion levels

[0061] IL-2, primarily an important T cell growth factor, is not only a crucial immunostimulant but also plays a vital role in regulating T cell immune responses and maintaining peripheral tolerance. To investigate the effect of ZNF683 on T cell IL-2 secretion, flow cytometry was used to sort ZNF683-overexpressing T cells. After three days of in vitro culture stimulated with CD3 / 28 beads, the IL-2 secretion level of ZNF683-overexpressing T cells was assessed by flow cytometry. Our results showed a significant trend of decreased IL-2 secretion in GFP+ cells, CD4+ T cells, and CD8+ T cells that highly expressed ZNF683 compared to the control group. Figure 5 A, Figure 5 C Figure 5 D), the decreasing trend of IL-2 secretion in CD3+ and CD8+ T cells was statistically significant. Figure 5 E). Our results also showed that, compared with control T cells, GFP+ cells, CD4+ T cells, and CD8+ T cells expressing ZNF683 showed a trend of decreased secretion of IL-4 and IL-10. Figure 5 A, Figure 5 B), in which the decreasing trend of IL-4 in total T cells was statistically significant. Figure 5 D), and the decreasing trend of IL-10 in CD4+ T cells with high ZNF683 expression was statistically significant. Figure 5 D). CD107a is a marker of degranulation in NK and activated CD8+ T cells, and the indirect effect of IFN-γ usually enhances CD8+ T cell activity. Regarding the secretion of these two cytokines, our results showed that, compared to control T cells, GFP+ cells, CD4+ T cells, and CD8+ T cells in the ZNF683-high expression group exhibited decreased expression levels of INF-γ and CD107a, and INF-γ expression was also decreased. Specifically, in GFP+ cells and CD4+ T cells with high ZNF683 expression, the decrease in INF-γ expression was statistically significant. Figure 5 D), and the decreased expression level of CD107a was statistically significant in GFP+ cells and CD8+ T cells that highly expressed ZNF683. Figure 5 E).

[0062] Figure 5 Effects of ZNF683 overexpression on cytokine secretion in primary T cells

[0063] AE: ZNF683 was overexpressed in CD3+ T cells of healthy individuals via lentiviral infection. 48 hours after infection, ZNF683-overexpressing T cells were sorted by flow cytometry. After 3 days of in vitro stimulation, the levels of IL-4, IL-10, IFN-γ, IL-2, and CD107a secreted by ZNF683-overexpressing GFP+ cells (A), CD4+ T cells (B), and CD8+ T cells (C) were detected by flow cytometry. DE; Statistical graph.

[0064] Example 10. CD8+ T cells overexpressing ZNF683 activate telomere maintenance-related signaling pathways.

[0065] Having established the regulatory role of ZNF683 expression in inducing quiescent and long-lived T cells, we investigated the specific molecular mechanism by which ZNF683 regulates T cells. We performed RNA-seq on CD8+ T cells with high ZNF683 expression and control CD8+ T cells. Our results showed that ZNF683 expression was upregulated in the high-ZNF683 group, and compared to control CD8+ T cells, RTEL1, crucial for maintaining telomere stability, protection, and elongation, was upregulated in CD8+ T cells with high ZNF683 expression. Figure 6 A) Genes involved in t-loop formation and telomeric loop disintegration, which are involved in telomere maintenance, are enriched in CD8+ T cells overexpressing ZNF683. Figure 6 B). Furthermore, compared to control CD8+ T cells, Wnt signaling pathway-related genes, such as PORCN, were downregulated in CD8 T cells overexpressing ZNF683. Figure 6 (AB). Most interestingly, we found that ZNF683 expression levels decreased in CD8+ T cells from patients who underwent haploidentical hematopoietic stem cell transplantation more than one year prior, after in vitro stimulation with CD3 / 28 beads. These results indicate that high expression of ZNF683 inactivates CD8+ T cells; simultaneously, ZNF683 maintains telomere replication in CD8+ T cells and inhibits CD8+ T cell apoptosis, potentially making it an important regulatory gene for inhibiting CD8+ T cell senescence. Furthermore, ZNF683 expression was downregulated during in vitro activation of CD8+ T cells, suggesting that ZNF683 may be an immune checkpoint for maintaining CD8+ T cells in a resting state.

[0066] Example 11: Treatment of GvHD model mice

[0067] A radiation-induced hematopoietic injury model was established by subjecting BALB / c mice (H-2d) to whole-body lethal dose 8 Gy irradiation for myeloablation. T-cell-depleted bone marrow cells (TCellDepletion-BM) from C57BL / 6 mice were then reinfused with spleen-derived T cells (SpleenT).

[0068] The control group (20 animals) received a tail vein injection of 5×10 6 TCD-BM+3×10 6 SpleenT;

[0069] The treatment group (20 animals) received a tail vein injection of 5×10 6 TCD-BM+3×10 6 SpleenT-ZNF683-OE utilizes lentiviruses to overexpress the ZNF683 gene in spleen-derived T cells.

[0070] Observe and record the survival time of the two experimental groups after receiving allogeneic HSC transplantation.

[0071] The experimental results showed that after injecting allogeneic hematopoietic stem cells into the control group of hematopoietic-damaged mice, all mice developed severe GVHD, manifested as more than one-third of their body weight loss and death due to severe GVHD. The survival rate of the control group decreased to 50% within 8 days, and all mice died from severe GVHD within 14 days.

[0072] In the treatment group, mice that received the ZNF683 lentiviral vector showed a significant reduction in GVHD symptoms, with their survival rate increasing to 80% within 8 days and more than 50% surviving within 14 days.

[0073] This embodiment demonstrates that overexpression of ZNF683 can treat or prevent graft-versus-host disease (GVHD), thereby improving the survival rate of GVHD model mice.

[0074] In summary, in vitro experiments showed that high expression of ZNF683 inhibits T cell proliferation and cytotoxicity, and also suppresses T cell apoptosis. Further omics data analysis revealed that in CD8 T cells with high ZNF683 expression, the expression of RTEL genes, crucial for maintaining telomere stability, protection, and elongation, was upregulated. This indicates that ZNF683 is associated with the persistent presence of specific T cells after hCMV infection and T cells after adoptive immunotherapy. Simultaneously, experiments showed that high expression of ZNF683 downregulated genes related to the Wnt signaling pathway in T cell activation. These in vitro experiments demonstrate that ZNF683 induces a quiescent and long-lived state in T cells. Additionally, it was found that CD8 T cells in haplo-SCT recipients… + T cells and CD8 from healthy donors + ZNF683 expression decreased when T cells were stimulated in vitro. These results indicate that ZNF683 is an immune checkpoint for CD8 effector T cells in a long-lived, quiescent state.

[0075] Therefore, CD8+ in Haplo-SCT recipients who develop immune tolerance + High expression of ZNF683 on T cells indicates that ZNF683 is a key regulatory molecule for maintaining immune tolerance in haploidentical hematopoietic stem cell transplantation recipients. In summary, this invention represents a potential key molecule for monitoring, predicting, and targeting the treatment of acute and chronic GVHD following haploidentical hematopoietic stem cell transplantation.

[0076] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a lentiviral vector overexpressing ZNF683 in the preparation of a drug for treating T-cell immune tolerance regulation-related diseases, wherein the T-cell immune tolerance regulation-related diseases are acute or chronic graft-versus-host diseases.

2. The use according to claim 1, characterized in that, The drug is a reagent used to increase the amount of the ZNF683 gene in a patient's body.

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