Research method for regulation mechanism of Tpex cells in chronic virus infection

Abstract

The invention discloses a method for researching a regulation mechanism of Tpex cells in chronic virus infection, and relates to the field of cellular immunology and gene editing. Comprising the following steps: constructing an in-vivo environment in which ARHGAP9 defective and wild CD8 + T cells coexist through a bone marrow chimeric model; comparing the stemness characteristics, differentiation trajectories and functional differences of the two groups of Tpex cells under the background of LCMV-CL13 infection; a Tpex cell differentiation path map is constructed by using a single cell sequencing technology, and a key node gene regulated and controlled by ARHGAP9 is identified. According to the invention, through a bone marrow chimeric model and single cell sequencing, a differentiation path of Tpex cells under ARHGAP9 deletion is drawn for the first time, and an in-vitro pathway inhibition experiment is combined to prove that an ARHGAP9-Rho axis is a key for regulation and control and is suitable for drug development and mechanism mining.

Description

Technical Field

[0001] This invention relates to the fields of cellular immunology and gene editing, and in particular to methods for studying the regulatory mechanisms of Tpex cells in chronic viral infections. Background Technology

[0002] In chronic viral infections (such as HIV and HCV) and the tumor microenvironment, the persistent presence of antigens leads to progressive exhaustion of CD8+ T cell function, manifested as loss of effector function, high expression of inhibitory receptors (such as PD-1 and Tim-3), and ultimately, an inability to effectively clear pathogens or cancer cells. Recent studies have revealed that the exhausted T cell pool is not a homogeneous population. A group of PD-1+ cells expressing the transcription factor TCF-1, namely exhausted T progenitor cells (Tpex), plays a crucial "stem cell-like" role in maintaining long-term immune responses. Tpex cells possess self-renewal capacity and pluripotent differentiation potential, serving as the source of terminally exhausted T cells (Tex) and the primary responder cells for immune checkpoint inhibitors such as PD-1 blockade. Therefore, a deep understanding of the fate determination mechanisms of Tpex cells, particularly the intrinsic regulatory factors maintaining their stemness and delaying their differentiation into a terminally exhausted state, is of great significance for developing novel immunotherapies to reverse T cell exhaustion.

[0003] However, our understanding of the regulatory network of Tpex cells remains incomplete. Existing research largely focuses on the dominant roles of transcription factors (such as TCF-1 and TOX) and epigenetic modifications. While these studies have laid an important foundation, their perspectives are relatively limited. A key scientific question that has long been neglected is: does the dynamic reorganization of the cytoskeleton and its associated signal transduction participate in regulating the fate of Tpex cells? The cytoskeleton is the structural basis for cells to sense microenvironment signals, establish polarity, and undergo activation and migration. Its dynamic changes are precisely regulated by the Rho GTPase family (such as RHOA, RAC1, and CDC42). In T cells, cytoskeleton reorganization is crucial for immune synapse formation, signal transduction, and functional performance, but its role in the functional transition process of T cell exhaustion, especially in determining the maintenance of stemness and differentiation of Tpex cells, remains almost entirely unknown.

[0004] ARHGAP9, a Rho GTPase activator protein (RhoGAP), negatively regulates cytoskeleton reorganization and dynamics by accelerating GTP hydrolysis and inactivating specific Rho GTPases (such as RAC1). Previous bioinformatics analyses have suggested a potential association between ARHGAP9 expression and T cell exhaustion; however, its specific functions under physiological and pathological conditions, particularly its regulatory role and molecular mechanisms on Tpex cells in chronic infection settings, remain unknown. Furthermore, current research lacks a systematic approach that integrates in vivo models (such as animal models that accurately simulate human chronic infection) with in vitro functional validation to elucidate the complete pathway by which cytoskeleton regulators like ARHGAP9 influence Tpex cell differentiation.

[0005] Therefore, the purpose of this invention is to fill the above-mentioned research gaps and provide a comprehensive research method that integrates bone marrow chimeric models, single-cell sequencing technology and in vitro pathway intervention, in order to reveal a new mechanism by which ARHGAP9 affects the maintenance of stemness and differentiation process of Tpex cells in chronic viral infection by regulating the Rho GTPase signaling pathway and cytoskeleton dynamics. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by proposing a method for studying the regulatory mechanism of Tpex cells in chronic viral infections.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: The research method for studying the regulatory mechanism of Tpex cells in chronic viral infection includes the following steps: S1: Construct an in vivo environment in which ARHGAP9-deficient and wild-type CD8+ T cells coexist using a bone marrow chimeric model; S2: Compare the stem cell characteristics, differentiation trajectory and functional differences of the two groups of Tpex cells in the context of LCMV-CL13 infection; S3: Construct a Tpex cell differentiation pathway map using single-cell sequencing technology to identify key node genes regulated by ARHGAP9.

[0008] Preferably, in step S1, the bone marrow chimeric model uses recipient mice irradiated with a dose of 9.5 Gy, transplanted with mixed bone marrow cells, and infected with LCMV-CL13 8 weeks after reconstruction, wherein the ratio of mixed bone marrow cells is: wild-type WT to ARHGAP9 knockout KO = 7:3.

[0009] Preferably, in step S2, stemness characteristics are assessed through TCF-1⁺CXCR5⁺ phenotype, self-renewal capacity (Ki67⁺ staining), and pluripotent differentiation potential (differentiation towards tTex and Tex).

[0010] Preferably, in step S2, the functional differences include cytokine secretion, degradation activity, and virus clearance ability; Functional cytokines include IFN-γ, TNF-α, and IL-2; Degradation activity was detected by measuring CD107a expression. Virus clearance capacity was assessed by tissue viral load qPCR assay.

[0011] Preferably, in step S3, the key node genes include RhoGTPase family members RHOA, RAC1, and CDC42, which are related to cytoskeleton reorganization and polarity establishment.

[0012] Preferably, it also includes using the CRISPR / dCas9 system to directionally regulate ARHGAP9 expression in Tpex cells and observe changes in differentiation markers.

[0013] An ARHGAP9-regulated Tpex cell differentiation model for studying the regulatory mechanism of Tpex cells in chronic viral infection, wherein the model includes the Tpex→tTex→Tex differentiation pathway, and ARHGAP9 deficiency delays this process.

[0014] A kit for assessing the immune status of chronic infection based on ARHGAP9 expression level includes ARHGAP9 detection primers, Tpex cell marker antibodies, and viral load detection reagents. The Tpex cell marker antibodies include anti-TCF-1 and CXCR5.

[0015] The beneficial effects of this invention are as follows: 1. This invention, through a bone marrow chimeric model and single-cell sequencing, for the first time mapped the differentiation pathway of Tpex cells lacking ARHGAP9. Combined with in vitro pathway inhibition experiments, it demonstrates that the ARHGAP9-Rho axis is a key regulator, applicable to drug development and mechanism discovery. Attached Figure Description

[0016] Figure 1 Flow cytometry and statistical graphs of inhibitory receptor expression in lymph nodes and spleen in the WT KO group of this invention; Figure 2 This is a statistical diagram of intracellular cytokines after stimulation with spleen and lymph node-specific peptides, as presented in this invention. Detailed Implementation

[0017] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0018] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," "linking," and "setting" should be interpreted broadly. For example, they can refer to a fixed connection or setting, a detachable connection or setting, or an integral connection or setting. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0019] Example 1: Establishment of a bone marrow chimerism model and analysis of Tpex cells Model building: Bone marrow cells from WT and ARHGAP9 KO mice were resuspended in PBS and mixed at a ratio of 7:3 (total 5 × 10⁻⁶ cells). 6 cell).

[0020] Receptor C57BL / 6J mice were irradiated with 9.5 Gy whole body and intravenously injected with mixed bone marrow cells within 24 hours.

[0021] After 8 weeks of feeding, the proportion of CD8+ T cells in peripheral blood was regularly monitored to confirm successful reconstitution.

[0022] Post-reconstruction infection with LCMV-CL13 (2×10) 6 PFU (iv), sample taken on day 28.

[0023] Tpex cell sorting and phenotypic analysis: After splenic lymphocytes were isolated, CD8+ T cells were enriched (using a biotinylated antibody combination (CD4, B220, Ly6G, NK1.1, CD11c, F4 / 80, Ter119), diluted 1:400, incubated with cell suspension for 30 minutes, and streptomycin-avidin magnetic beads (Beaver Company) were added for magnetic sorting of CD8+ T cells), and TCF-1⁺PD-1⁺Tim-3⁻ cells (Tpex) were sorted by flow cytometry.

[0024] Dryness detection: Ki67 nuclear staining showed that the proportion of Ki67⁺ cells in the KO group did not change significantly, but the proportion of TCF-1⁺CXCR5⁺ cells increased.

[0025] Inhibitory receptor detection: The proportion of Tim-3⁺PD-1⁺ cells was reduced in the KO group.

[0026] Functionality verification: Cytokine secretion: Splenic cells were stimulated with gp276 or gp33 peptide (1 μg / ml), and the expression of IFN-γ, TNF-α, and CD107a was detected. The results showed that the secretion capacity of the KO group was reduced.

[0027] Viral clearance: Liver, spleen, and lung tissues were collected, RNA was extracted, and viral load was detected. The titer in the KO group was significantly reduced.

[0028] Example 2: Single-cell sequencing and differentiation trajectory construction Cell preparation: Tpex cells (TCF-1⁺PD-1⁺) from mice infected on day 28 were sorted and washed with PBS.

[0029] Single-cell RNA sequencing: Library construction was performed using the 10x Genomics Chromium system, followed by Illumina sequencing. Data were processed using Cell Ranger, and pseudo-time trajectories were constructed using Monocle3 software.

[0030] Results analysis: In Tpex cells of the ARHGAP9 deletion group, the expression of cytoskeleton-related genes (such as RAC1 and CDC42) was downregulated. The differentiation pathway showed that Tpex cells of the KO group were more likely to maintain stemness and differentiate into tTex delayed.

[0031] Example 3: In vitro Rho GTPase pathway inhibition experiment Tpex cell culture: Tpex cells were sorted and cultured in RPMI-1640 complete medium (10% FBS).

[0032] Inhibitor treatment: Add Y-27632 (10 μM), NSC23766 (50 μM) or ML141 (5 μM), set up a DMSO control group, and incubate for 72 hours.

[0033] Detection indicators: TCF-1 and Tim-3 expression were detected by flow cytometry, and IFN-γ levels in the supernatant were detected by ELISA. Results: The proportion of TCF-1⁺ was increased in the inhibitor group, mimicking the ARH knockout phenotype.

[0034] Example 4: CRISPR / dCas9 Validation gRNA targeting the ARHGAP9 promoter was designed, and Tpex cells were transduced with lentivirus. Differentiation markers were detected 72 hours later. The results showed that ARHGAP9 knockdown delayed the exhaustion process.

[0035] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for investigating the regulatory mechanism of Tpex cells in chronic viral infection, characterized in that, Comprising the following steps: S1: Construct an in vivo environment where ARHGAP9-deficient and wild-type CD8+ T cells coexist by bone marrow chimeric model; S2: Compare the stemness characteristics, differentiation trajectories and functional differences of the two groups of Tpex cells under the background of LCMV-CL13 infection; S3: Construct a Tpex cell differentiation path map using single-cell sequencing technology, and identify key node genes regulated by ARHGAP9.

2. The method of claim 1, wherein the method is for investigating the regulatory mechanism of Tpex cells in chronic viral infection. In the S1 step, the bone marrow chimeric model uses a 9.5 Gy dose of radiation to the recipient mouse, transplants mixed bone marrow cells, and infects LCMV-CL13 after 8 weeks of reconstitution, wherein the proportion of mixed bone marrow cells is: wild type WT and ARHGAP9 knockout KO = 7:

3.

3. The method of claim 1, wherein the method is for investigating the regulatory mechanism of Tpex cells in chronic viral infection. In the S2 step, the stemness characteristics are evaluated by TCF-1⁺CXCR5⁺ phenotype, self-renewal ability and pluripotent differentiation potential.

4. The method of claim 1, wherein the method is for investigating the regulatory mechanism of Tpex cells in chronic viral infection. In the S2 step, the functional differences include cytokine secretion, degradation activity and virus clearance ability; The functional cytokines include IFN-γ, TNF-α and IL-2; The degradation activity is detected by detecting CD107a expression; The virus clearance ability is evaluated by tissue viral load qPCR detection.

5. The method of claim 1, wherein the method is for investigating the regulatory mechanism of Tpex cells in chronic viral infection. In the S3 step, the key node genes include RHOA, RAC1 and CDC42, which are members of the Rho GTPase family related to cytoskeleton reorganization and polarity establishment.

6. The method of claim 1, wherein the method is for investigating the regulatory mechanism of Tpex cells in chronic viral infection. It also includes using the CRISPR / dCas9 system to direct the regulation of ARHGAP9 expression in Tpex cells and observing changes in differentiation markers.

7. A model for the regulation of differentiation of Tpex cells by ARHGAP9 for use in the method of claim 1-6 for the study of the regulatory mechanisms of Tpex cells in chronic viral infections, characterized by, The model contains a differentiation path of Tpex→tTex→Tex, and the deletion of ARHGAP9 delays this process.

8. The application of the ARHGAP9-regulated Tpex cell differentiation model of claim 7 in screening antiviral drugs or immunoadjuvants.

9. A chronic infection immune status evaluation kit based on the expression level of ARHGAP9, characterized in that, It contains ARHGAP9 detection primers, Tpex cell marker antibodies and viral load detection reagents, and Tpex cell marker antibodies include anti-TCF-1 and CXCR5.

Images