Method for establishing hbv-specific exhausted cd8+ t cell mouse model and application thereof

Abstract

The application discloses a mouse model of HBV-specific exhausted CD8+ T cells and application thereof. First, C57 mice of CD45.1 type are immunized by using HBsAg and HBcAg antigen peptides of HBV to obtain mice with HBV-specific immunity; C57 mice of CD45.2 type with liver-specific expression of HBV transgene are subjected to X-ray myeloablation; bone marrow of the CD45.1 type mice with HBV-specific immunity is collected, and the myeloablated CD45.2 type mice are transplanted through the orbital vein; after transplantation, flow cytometry is used to detect HBV-specific CD8+ T cells and exhaustion-related phenotypes, and establishment of the mouse model of HBV-specific exhausted T cells is confirmed. The mouse model has a high level of HBV-specific CD8+ T cells, and the exhaustion phenotype is significant, and can be applied to the formation mechanism of hepatitis B T cell exhaustion and research on related new targets and new strategies for diagnosis and treatment.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology inventions and relates to the establishment and application of a HBV-specific CD8+ T cell depleted mouse model. Specifically, HBV-specific immunity is obtained by immunizing CD45.1 mice with HBV antigen peptides. The bone marrow is then isolated and adoptively transplanted into myeloablated, irradiated CD45.2 HBV transgenic mice, thereby establishing a HBV-specific CD8+ T cell depleted mouse model. Background Art

[0002] Hepatitis B virus (HBV) infection is a significant issue affecting human health. Animal models are crucial for understanding the pathophysiology of HBV infection, the mechanisms of viral replication, the hepatocellular damage and fibrosis that occur during this process, and for evaluating drug efficacy.

[0003] The critical role of T cell responses in clearing HBV infection has been widely reported. Acute hepatitis B infection induces a robust, multifunctional CD8+ T cell response, while chronic hepatitis B infection causes HBV-specific CD8+ T cells to be affected in multiple ways, resulting in a decrease in number, suppressed function, and an inability to effectively clear the virus. Furthermore, as the infection progresses, exhausted HBV-specific CD8+ T cells are unable to effectively clear HBV, and repeated inflammation leads to tissue damage, which may eventually progress to cirrhosis and liver cancer. Therefore, research on the immune exhaustion caused by persistent HBV infection has become a hot topic in recent years.

[0004] Because HBV cannot naturally infect mice, even transgenic mice that express HBV-related components will not develop HBV-specific CD8+ T cells. Furthermore, while the HBV plasmid high-pressure hydrodynamic method can induce short-term expression of HBV components in mice and elicit HBV-specific CD8+ T cell responses, the acute infection does not result in an exhaustion phenotype. While humanized human-mouse liver chimeric mouse models can successfully construct a humanized immune system and allow HBV infection, the complex modeling process, difficulty sourcing human hepatocytes, and high costs limit the study of HBV-related immune exhaustion. While the AAV-HBV-1.3 virus can induce prolonged HBV expression in mice and induce HBV-specific CD8+ T cell exhaustion, its abundance is extremely low, hindering further investigation of the mechanisms of HBV-specific CD8+ T cell exhaustion and hindering the study of liver and systemic immunity during HBV infection. Therefore, establishing a convenient and cost-effective mouse model of HBV-specific CD8+ T cell exhaustion is crucial for studying HBV-related infection immunity. Summary of the Invention

[0005] The purpose of the present invention is to address the lack of mouse models for studying the mechanism of hepatitis B immune exhaustion. A method for establishing a HBV-specific exhausted CD8+ T cell mouse model is provided, which is achieved through the following technical solutions:

[0006] First, mice with HBV-specific immunity were established. HBV HBsAg (S190-197: VWLSVIWM) and HBcAg (Precore47-56: FLPSDFFPSV) antigen peptides were injected subcutaneously into the hind flank of CD45.1 C57 mice. The antigen peptide formula ratio was as follows: Peptide 50 μg + Poly I:C 50 μg + α-CD40 50 μg dissolved in 200 μl of PBS. A second immunization was performed 28-30 days after the second immunization. 28-30 days after the second immunization, peripheral blood mononuclear cells (PBMCs) were isolated from the mice and HBV-specific CD8+ T cells were detected using the flow cytometry panel shown in Table 1. IFN-γ was detected after stimulation with HBsAg (S190-197: VWLSVIWM) and HBcAg (Precore47-56: FLPSDFFPSV) antigen peptides. γ The results showed that CD45.1 mice with HBV-specific immunity could be obtained after two immunizations.

[0007] Table 1 .

[0008] Myeloablation of CD45.2 transgenic mice expressing liver-specific HBV was then performed. Specifically, 5-week-old C57BL / 6J-Tg(Alb1HBV)44Bri / J mice (from Jackson Lab) expressing liver-specific HBV were prepared and myeloablated with an X-ray irradiator (Rs2000) at a dose of 4.5 Gy the day before bone marrow transplantation. The mice were then returned to the animal house for overnight housing.

[0009] Then bone marrow transplantation was performed to construct an HBV-specific exhausted CD8+ T cell mouse model. The specific plan is to first prepare crude fiber paper, 1.5ml centrifuge tubes, and 300μl centrifuge tubes (punch holes in the bottom with a needle) for the experiment, place them in a sterilization bag and sterilize them at 120 degrees Celsius for 30 minutes, then bake them in an oven for later use; prepare cell reselection solution (R2: 1640 culture medium + 1% calf serum + 1% antibiotics (streptomycin and penicillin); flow elution staining solution (PBS + 1% BSA). Then, on the day of transplantation, the CD45.1 mice immunized with HBV antigen peptides were killed, the femur and tibia of the mice were aseptically separated and the two ends were cut open with surgical scissors to expose the bone cavity. The exposed The femur and tibia in the bone cavity were placed in a 200μl centrifuge tube with a small hole in the bottom. The 200μl centrifuge tube was then placed in a 1.5ml centrifuge tube and centrifuged at 4000g for 40 seconds at 4°C. The femur and tibia in the 200μl centrifuge tube appeared white, with no red bone marrow in the bone cavity. However, a large number of red cell clumps were present at the bottom of the 1.5ml centrifuge tube. Red blood cells were removed using red blood cell lysis buffer, and the remaining cells were filtered through a 70μm filter. After cell count, 1M bone marrow cells were transplanted via orbital injection into irradiated myeloablated HBV liver-specific CD45.2 transgenic mice.

[0010] Finally, flow cytometry was used to evaluate the relevant immune indicators of the HBV-specific exhausted CD8+ T cell mouse model. The specific plan was to collect blood every week thereafter and observe the proliferation and differentiation of CD8+ T cells transplanted into the recipient mice by flow cytometry. The mice were killed on the 28th day and the liver-infiltrating T cells were isolated. They were cultured for 4-5 hours by in vitro stimulation with HBV peptides and detected using the flow cytometry combination shown in Table 2. The Ly108-Tim3+ positive ratio of IFN-γ positive cells was analyzed to evaluate the degree of exhaustion of HBV-specific CD8+ T cells. The flow cytometry analysis results showed that the proportion of CD8+;Ly108-;Tim3+ T cell subsets in the exhausted model mice was significantly increased compared with the control group, indicating that the model was successfully constructed ( Figure 3 ).

[0011] Table 2 .

[0012] Another object of the present invention is to provide the application of the mouse model in the study of the exhaustion mechanism of HBV-specific CD8+ T cells and the discovery of new targets for the diagnosis and treatment of severe chronic hepatitis B, including liver failure.

[0013] This study immunized CD45.1 mice with HBV antigen peptides to generate mice with HBV-specific immunity. The bone marrow from these mice was then isolated and adoptively transplanted into myeloablated, irradiated CD45.2 HBV transgenic mice, thereby creating a mouse model of HBV-specific CD8+ T cell depletion. This model provides a powerful tool for studying the mechanisms of HBV-specific CD8+ T cell depletion and for identifying new targets for the diagnosis and treatment of severe chronic hepatitis B.

[0014] Compared with the existing technology, the present invention has the following beneficial effects: 1. The HBV-specific exhausted T cell mouse model established by this method has a higher level of HBV-specific T cells; 2. The HBV-specific T cell exhaustion phenotype in this model is significant; 3. This mouse model can be used to study the formation mechanism of T cell exhaustion in the context of chronic hepatitis B; 4. This mouse model can be used to study new targets and new drugs for HBV-specific CD8+ T cell exhaustion; 5. This mouse model is helpful for the study of the mechanism and new diagnosis and treatment technologies in the field of hepatitis B infection immunity. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 . Flowchart for the construction of HBV-specific CD8+ T cell-depleted mice.

[0016] Figure 2 . 57 weeks after mice were immunized twice with HBV peptides, peripheral blood was collected for flow cytometry detection of HBV-specific CD8+ T cells. Among the CD8+ T cells, 9.98% secreted IFN-γ after stimulation with HBsAg peptides, indicating successful immunization.

[0017] Figure 3 Figure 2. Flow cytometric analysis of the exhausted phenotype of CD8+ T cells infiltrating the liver of mice 28 days after bone marrow transplantation. Compared with CD8+ T cells transplanted from the bone marrow of mice not immunized with HBV peptides, the proportion of stem phenotype (TIM3-;Ly108+ cells) was significantly decreased, while the proportion of exhausted phenotype subpopulation (TIM3+;Ly108- cells) was increased.

[0018] Figure 4 Liver enzymes and the proportion of HBV-specific CD8+ T cells in mice depleted of HBV-specific CD8+ T cells in the ConA-induced mouse liver failure model. DETAILED DESCRIPTION

[0019] The present invention will be further described below in conjunction with the accompanying drawings and specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0020] Example 1 Preparation of HBV-specific immune mice:

[0021] according to Figure 1 The process shown first prepares HBV-specific immune mice:

[0022] 1. Commercially synthesized HBV S and C epitope peptides, which are epitope peptides specifically recognized by CD8+ T cells, have the sequences of HBsAg (S190-197: VWLSVIWM) and HBcAg (Precore47-56: FLPSDFFPSV), respectively. PBS is used to prepare 200μl of immune injection solution, which contains 50ug of peptide mixture, 50μg of Poly I:C, and 50μg of anti-mouse CD40 antibody.

[0023] 2. Subcutaneous injection immunization of mice: CD45.1 genotype C57 mice, about 5 weeks old, were injected with 200 μl of immune injection solution at a single point subcutaneously on the buttocks. A second booster immunization was given 28 days later. Orbital blood was collected on the 27th and 58th days for flow cytometry.

[0024] 3. Analysis of HBV-specific CD8+ T cell abundance: After lysing the red blood cells in the collected peripheral blood, PBMC were reselected in FACS buffer, centrifuged at 800g for 1 minute and the supernatant was discarded. The pellet was resuspended in flow cytometry antibody premix (Table 1), stained at 4°C for 20 minutes, then 200μl FACS buffer was added and centrifuged at 800g for 1 minute and the supernatant was discarded. Repeat this process once and then centrifuge. The pellet was resuspended in 400μl FACS buffer for flow cytometry detection. Figure 2 As shown, after HBV peptide stimulation, PBMCs of mice immunized with HBV antigen peptides could be detected to secrete IFN-γ CD8+ T cell subsets, indicating that HBV immunization was successful and specific CD8+ T cells targeting HBV peptides were obtained.

[0025] Example 2 Establishment and evaluation of HBV-specific exhausted CD8+ T cell mouse model:

[0026] according to Figure 1 The process shown here continues with the establishment of a HBV-specific CD8+ T cell-depleted mouse model and flow cytometry assessment of the T cell exhaustion phenotype:

[0027] 1. HBV-immunized mice were sacrificed after positive peripheral blood HBV-specific CD8+ T cells were detected by flow cytometry. The lower limbs of the mice were aseptically separated, the attached muscles were removed, and the femur and tibia were cut open at both ends to expose the bone marrow cavity. The cut femur and tibia were placed in a 300μl small centrifuge tube (with a pre-opened bottom), then placed in a 1.5ml centrifuge tube and centrifuged at 4000g for 40 seconds at 4 degrees Celsius. The red cell cluster at the bottom of the 1.5ml centrifuge tube was retained. The red blood cells were removed with red blood cell lysis buffer, and the remaining cells were centrifuged and resuspended in PBS. The final cell count was adjusted to a cell concentration of 7.5X10 6 / ml and place on ice until ready to use.

[0028] 2. Anesthetize the recipient mice with liver-specific expression of HBV using a mouse anesthesia system and transplant 1.5X10 6 After the mice regained consciousness at room temperature, they were placed back into the barrier for feeding.

[0029] 3. On day 28 after transplantation, peripheral blood was collected from the orbits. After erythrocyte lysis and removal of erythrocytes, peptide stimulation solution (0.2 μl of 1 mg / ml HBV peptide + 0.1 μl of 10 mg / ml DNA25 solution + 0.1 μl of brefeldin A + 0.1 μl of monesin) was added to the PBMCs and incubated in a 37°C cell incubator for 4-5 hours.

[0030] 4. After stimulation, the cells were centrifuged at 800 g for 1 minute, the supernatant was discarded, and the pellet was washed twice with FACS and then eluted with flow cytometry antibody premix ( Figure 2 ) and stain at 4°C for 20 minutes. Then, add 200 μl FACS buffer and centrifuge at 800 g for 1 minute. Discard the supernatant and repeat this process for 1 minute.

[0031] 5. After centrifugation, discard the supernatant, resuspend the pellet in 200 μl BD CytoPerm / CytoFix, and incubate at 4°C for 20 minutes.

[0032] 6. Wash twice with 200 μl BD Perm Wash Buffer.

[0033] 7. Prepare the antibody premix with 50 μl of BD Perm wash buffer (add 1 / 100 PE-labeled rabbit anti-mouse antibody), adjust the volume to 30 μl using a dispenser, pipette and aspirate repeatedly 5 times until there is no precipitation, and incubate at 4°C for 20 minutes.

[0034] 8. Wash twice with 200 μl BD Perm Wash Buffer.

[0035] 9. Wash the pellet again with FACS buffer.

[0036] 10. Finally, resuspend the pellet in 400 μl FACS buffer for flow cytometry detection.

[0037] like Figure 3 As shown, HBV-specific CD8+ T cells infiltrating the liver of mice exhibited an exhausted phenotype 28 days after bone marrow transplantation, with a significant increase in the proportion of Ly108-;TIM-3+ CD8+ T cell subsets. This indicates that a HBV-specific exhausted CD8+ T cell mouse model was successfully established.

[0038] Example 3 Application of HBV-specific CD8+ T cell depletion mouse model in the study of hepatitis B liver failure

[0039] The day before the experiment, HBV-specific CD8+ T cell depletion mice and control mice were given blood samples to test liver function indicators such as ALT and AST. After confirming that there was no difference between the two, 15 mg / kg of ConA was injected into the tail vein. Peripheral blood, spleen, and liver tissue samples were collected 6 hours after modeling to evaluate liver function. The results showed that HBV-specific CD8+ T cell depletion mice had more severe liver damage ( Figure 4 ), suggesting that the mice can simulate chronic hepatitis B liver failure.

Claims

1. A method for constructing an HBV-specific exhausted CD8+ T cell mouse model, characterized in that: This is achieved through the following scheme: (1) To establish mice with HBV-specific immunity, HBV HBsAg and HbcAg antigen peptides were subcutaneously injected into CD45.1 C57 mice. The second immunization was performed 28-30 days later. 28-30 days after the second immunization, peripheral blood mononuclear cells of the mice were isolated and HBV-specific CD8+ T cells were detected using flow cytometry. After stimulation with HBsAg and HbcAg antigen peptides, IFN- γ The release of HBV-specific CD45.1 mice was obtained after two immunizations. (2) Myeloablation of HBV liver-specifically expressed CD45.2 transgenic mice. Prepare HBV liver-specifically expressed mice of about 5 weeks old. The day before bone marrow transplantation, they are irradiated with an X-ray irradiator for myeloablation at a dose of 4.5 Gy and then kept overnight. (3) Bone marrow transplantation to construct HBV-specific exhausted CD8+ T cell mouse model: First, prepare experimental coarse fiber paper, 1.5ml centrifuge tubes, and 200μl centrifuge tubes with small holes at the bottom. Place them in a sterile bag and sterilize at 120 degrees Celsius for 30 minutes, then bake in an oven for use; prepare cell reselection solution and flow elution staining solution, and then aseptically separate the femur and tibia of CD45.1 mice immunized with HBV antigen peptides on the day of transplantation to expose the bone cavity. Then, place the exposed femur and tibia in a 200μl centrifuge tube with a small hole at the bottom. Then, place the 200μl centrifuge tube in a 1.5ml centrifuge tube and centrifuge at 4000g for 40 seconds at 4 degrees. At this time, the femur and tibia in the 200μl centrifuge tube appear white, there is no red bone marrow in the bone cavity, and there are a large number of red cell clumps at the bottom of the 1.5ml centrifuge tube. Remove the red blood cells with red blood cell lysis solution, and pass the remaining cells through a 70μM filter. After cell counting, transplant 1M bone marrow cells into irradiated myeloablated HBV liver-specific CD45.2 transgenic mice; (4) Flow cytometry was used to evaluate the relevant immune indicators of the HBV-specific exhausted CD8+ T cell mouse model. Blood samples were collected weekly to observe the proliferation and differentiation of CD8+ T cells transplanted into recipient mice by flow cytometry. On the 28th day, liver-infiltrating T cells were isolated and cultured for 4-5 hours after in vitro stimulation with HBV peptides. Flow cytometry was used to detect the Ly108-Tim3+ positive ratio of IFN-γ positive cells to evaluate the degree of exhaustion of HBV-specific CD8+ T cells. The flow cytometry analysis results showed that the proportion of CD8+;Ly108-;Tim3+ T cell subsets in the exhausted model mice was significantly increased compared with the control group, indicating that the model was successfully constructed.

2. The construction method according to claim 1, characterized in that The antigen peptide formula ratio described in step (1) is: Peptide 50 μg + Poly I:C 50 μg + α-CD40 50 μg dissolved in 200 μl PBS.

3. The construction method according to claim 1, characterized in that The flow cytometry detection combination 1 in step (1) is: APC-Cy7-NIR; FITC-CD45.1; Percp-cy5.5-CD44; APC-IFN- γ; BV605-CD8; AlexFlour700-B220.

4. The construction method according to claim 1, wherein The HBV liver-specific expression mice in step (2) were C57BL / 6J-Tg(Alb1HBV)44Bri / J, which were obtained from Jackson Lab.

5. The construction method according to claim 1, characterized in that The configuration of the cell reselection solution in step (3) is: 1640 culture medium + 1% calf serum + 1% antibiotic, wherein the antibiotic is streptomycin or penicillin; the flow elution staining solution is PBS + 1% BSA.

6. The construction method according to claim 1, characterized in that The flow cytometry detection combination 2 in step (4) is: APC-Cy7-NIR; FITC-CD45.1; Percp-cy5.5-CD44; PE-Ly108; BV421-Tim-3; BV605-CD8; AlexFlour700-B220.

7. Use of the mouse model constructed according to claim 1 in studying the exhaustion mechanism of HBV-specific CD8+ T cells.

8. The mouse model constructed according to claim 1 is used in the discovery of new targets for the diagnosis and treatment of severe chronic hepatitis B, including liver failure.

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