CN115040538A - Application of human CXCR5+ CD8+ T cell in preparing medicine for treating chronic hepatitis B - Google Patents
Application of human CXCR5+ CD8+ T cell in preparing medicine for treating chronic hepatitis B Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
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- G01N15/14—Electro-optical investigation, e.g. flow cytometers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
Abstract
The invention discloses application of human CXCR5+ CD8+ T cells in preparation of a medicine for treating chronic hepatitis B, application of a reagent for promoting proliferation of CXCR5+ CD8+ T cells in preparation of a medicine for treating chronic hepatitis B, and application of a reagent for promoting expression of CD8+ T cell surface functional molecule CXCR5 in preparation of a medicine for treating chronic hepatitis B. The invention clarifies an immune mechanism of the CXCR5+ CD8+ T cells playing an anti-virus role in the liver and spleen of a mouse through the establishment of a chronic HBV infection model of the mouse and a flow cytometry experiment. Although CXCR5+ CD8+ T cells are partially depleted, animal experiments have shown that their antiviral function is stronger than CXCR5-CD8+ T cells. The invention lays a foundation for researching and developing a new medicine and a new method for curing chronic hepatitis B.
Description
Technical Field
The invention relates to the technical field of biomedicine and immunology, in particular to application of human CXCR5+ CD8+ T cells in preparation of a medicament for treating chronic hepatitis B.
Background
It has been found that chronic lymphocyte-mediated choroidal clearance from meningitis virus (LCMV) which, upon infection of a mouse, results in the expression of a particular population of CXCR 5-positive CD 8-positive T lymphocytes, referred to as tfcs, in the lymphoid tissues of the mouse itself, which cells successfully migrate into B cells and into its lymphoid follicles, and that literature and experiments have shown that depletion and attenuation of the antiviral efficacy of CXCR5+ CD8+ T is significantly lower than that of its opposite negative subset. In some patients infected with HIV recently, researchers have also found that their specific CXCR5 positive CD8 positive Tfc cells, and that the level of its antiviral potency, as well as other immune related responses, is also high in the lymphoid follicles of their B cells, exhibiting higher antiviral potency than the same negative subset thereof. While Leong et al have also found virus-specific CXCR5 positive CD8 positive Tfc cells in chronic HIV-infected persons and in chronic LCMV-infected mouse models, experimental studies have shown that such a population of cells shares many features in common with other Tfc cells and still possesses some functions similar to memory T-lymphocyte packets, and that CXCR5 positive CD8 positive Tfc cells have a lower expression of cytotoxic molecules compared to the negative subset, and therefore their antiviral potency should be lower than that of CXCR5 negative CD8 positive cells, but because CXCR5 positive cells specifically express chemokines such as CXCR5, which makes them more readily localized in the B-cell lymphoid follicles, when the virus is infected, the specific CXCR5 positive CD8 positive Tfc cells are produced, and have a stronger antiviral potency against the virus infected in the B-lymphoid follicles and Tfc cells, can better eliminate the infected virus. Xiao et al found a population of CD8+ T cells in some tissues infected with chronic inflammatory nasal polyps that secreted IL-21 at significantly higher levels than other cells, and also expressed molecules such as TIM-3, PD-1, ICOS, CXCR5 and also secreted some effector factors such as IFN-r and TNF-a, and found that if CD8+ T cells were cultured with B cells for functional experimental studies, it was indeed more IgG antibody secreted by the cultured B cells.
Chronic hepatitis b virus is a kind of liver attacking mainly and causes liver cell damage through immune mediation. Due to individual differences, the differences in immune function among individuals vary greatly in the control of post-hepatitis B infection and the effectiveness of clinical treatments. CD8 + The immune function of T cells plays an important role in controlling chronic HBV infection in the whole HBV infection process, however, CD8+ T is easy to be exhausted, which is also an important reason for chronic hepatitis B. In recent years, different studies have shown that CD8+ T cells have many different subtypes, and the phenotype and function of these subgroups are very different. Different CD8 + The T cell subset should play a different antiviral role in chronic HBV infection. Thus, the study of CD8 is well-studied + The specific phenotype and function of different subpopulations of T cells may help to find new methods for curing chronic hepatitis b.
At present, the research on the effect of CXCR5+ CD8+ T cells after HBV infection is still relatively lacked, and whether CXCR5+ CD8+ T cells will function, have stronger antiviral ability than negative subset, and have the function of assisting humoral immunity during HBV infection, and these scientific problems still need to be further solved to develop new drugs and new methods for curing chronic hepatitis b.
Disclosure of Invention
The invention utilizes high-pressure tail vein injection (HDI) to inject pAAV-HBV1.2 plasmid to establish HBV infected mouse model and detect CXCR5 + CD8 + Phenotypic and functional differences of T cells in peripheral blood, liver and spleen; finally, through a flow cytometry detection experiment of CXCR5+ CD8+ T cells, the antiviral effect of the CXCR5+ CD8+ T cells is determined:
1. a HBV chronic infection mouse model is established by injecting pAAV-HBV1.2 plasmid, and the frequency of CXCR5+ CD8+ T cells in spleen and liver of HBV chronic infection mice is higher than that of CXCR5-CD8+ T cells. Compared with CXCR5-CD8+ T cells, CXCR5+ CD8+ T cells highly express receptors such as TIM-3, PD-1, ICOS, CD40L and the like.
2. After separating the lymphocytes of the liver and the spleen, the spleen is discovered through the stimulation of HBV antigen peptideAnd CXCR5 of liver + CD8 + The T cell has obviously stronger capacity of secreting IFN-gamma, TNF-a and IL-21 than CXCR5-CD8 + T cells. While intrasplenic CXCR5 + CD8 + T cells have stronger IFN-r and TNF-a secretion ability than liver.
Based on the research result, the invention protects the following technical scheme:
application of human CXCR5+ CD8+ T cells in preparing medicine for treating chronic hepatitis B.
Application of a reagent for promoting proliferation of CXCR5+ CD8+ T cells in preparation of a medicament for treating chronic hepatitis B.
Application of a reagent for promoting expression of CD8+ T cell surface functional molecule CXCR5 in preparation of medicines for treating chronic hepatitis B.
The application of the agent for promoting the expression of TNF-alpha and IFN-gamma of CD8+ T cells in preparing the medicine for treating chronic hepatitis B.
A medicine for treating chronic hepatitis B contains human CXCR5+ CD8+ T cells as active component.
A medicament for the treatment of chronic hepatitis b comprising an agent which promotes the proliferation of CXCR5+ CD8+ T cells.
A medicament for the treatment of chronic hepatitis B comprising an agent which promotes the expression of CD8+ T cell surface function molecule CXCR 5.
A medicament for the treatment of chronic hepatitis B comprising an agent which promotes the expression of TNF- α and IFN- γ from CD8+ T cells.
The invention has the beneficial effects that: through the establishment of a chronic HBV infection model of a mouse, an immune mechanism of the CXCR5+ CD8+ T cell playing an anti-virus role in the liver and spleen of the mouse is clarified through a flow cytometry experiment. Although CXCR5+ CD8+ T cells are partially depleted, animal experiments have shown that their antiviral function is stronger than CXCR5-CD8+ T cells. The invention lays a foundation for researching and developing a new medicine and a new method for curing chronic hepatitis B.
Drawings
FIG. 1 shows the results of quantitative determination of HBV DNA and HBsAg in peripheral blood of mice at 7,14,28, and 56 days after transfection of pAAV-HBV1.2 plasmid.
FIG. 2 shows the frequency expression of HBV-specific CD8+ T cells and HBV-specific CXCR5+ CD8+ Tfc cells in spleen of mice at 7,14,28,56 days after transfection of pAAV-HBV1.2 plasmid.
FIG. 3 shows the inhibitory molecule expression on the surface of HBV specific CXCR5+ CD8+ Tfc cells and HBV specific CXCR5-CD8+ T cells in the spleen of transfected mice, the secretion of cytokines after HBc93-100 antigenic peptide stimulation, the expression of PD-1 and Tim-3, and the secretion of IFN-gamma and TNF-alpha after HBc93-100 antigenic peptide stimulation.
FIG. 4 shows the ICOS and CD40L expression on the surface of HBV-specific CXCR5+ CD8+ Tfc cells in the spleen of transfected mice, IL-21 secretion after HBc93-100 antigen peptide stimulation, ICOS and CD40L expression on the surface of cells, and IL-21 secretion after HBc93-100 antigen peptide stimulation on CXCR5+ CD8+ Tfc cells.
FIG. 5 is the frequency expression of HBV specific CD8+ T cells and HBV specific CXCR5+ CD8+ Tfc cells in the spleen of mice at 7,14,28,56 days after pAAV-HBV1.2 plasmid transfection.
FIG. 6 shows the inhibitory molecule expression on the surface of HBV specific CXCR5+ CD8+ Tfc cells and HBV specific CXCR5-CD8+ T cells in the liver of transfected mice, the secretion of cytokines after HBc93-100 antigen peptide stimulation, the expression of Tim-3 on the cell surface, and the secretion of TNF-alpha after HBc93-100 antigen peptide stimulation.
FIG. 7 is ICOS and CD40L expression on the surface of HBV-specific CXCR5+ CD8+ Tfc cells in spleen of chronic HBV transfected mice.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to be limiting.
The experimental procedures in the following examples are all conventional procedures unless otherwise specified; all chemical and biological reagents, unless otherwise specified, are conventional in the art.
1 materials and methods
1.1 materials
1.1.1 Experimental animals
C57BL/6J (B6) mice, male, 6-8 weeks old, all weighed approximately 16-22 g. Provided by the animal testing center of Chongqing university of medicine, all mice were housed by the animal testing center of Chongqing university of medicine in an SPF-grade environment. The study was conducted with the consent of the ethical committee of the university of Chongqing medical science.
1.1.2 Primary antibodies
PE Class I Pentamers HBV core antigen 18-27 Proimmune F023-2B
PE Class I Pentamers HBV core antigen 93-100 Proimmune F1431-2B
PE-Cy7 Rat Anti-Mouse CD8a BD 552877
FITC hamster anti-mouse CD3e BD 553062
APC-Cy7 rat anti-mouse CD4 BD 552051
PerCP-Cy5.5 rat anti-mouse CXCR5 BD 560528
Alexa Fluor 647 rat anti-mouse CD278 BD 563469
APC hamster anti-mouse CD279 BD 562671
APC anti-mouse CD154(CD40L)eBioscience 17-1541-82
APC anti-mouse CD366(Tim-3)eBioscience 17-5871-82
PE anti-mouse IL-21 eBioscience 12-7211-82
PE rat anti-mouse IFN-γBD 554412
PE rat anti-mouse TNF BD 554419
1.1.2 Primary reagents
Phosphate Buffered Saline (PBS) was self-prepared in this laboratory
Fetal Bovine Serum (FBS) GIBCO
150Mm Ammonium chloride (NH4Cl) Shanghai Linkun Chemicals Co., Ltd
10Mm NaHCO3 Shanghai Linkuan Chemicals Co., Ltd
1Mm EDTA laboratory self-prepared
ddH2O self-contained in this laboratory
Plasmid pCDNA3.1-HBVl.3 this laboratory preservation
Plasmid pAAV-HBV1.2 this laboratory preservation
RPMI 1640 culture solution self-prepared in laboratory
Double antibody laboratory self-matching
HBc93-100 antigen peptide Biotechnology Ltd
Brefeldin A solution BD Co
Tianjin biochemistry pharmacy Co., Ltd of heparin sodium injection
Paraformaldehyde powder Shanghai Ika Biotechnology Ltd
Saponin Sigma Co
Sichuan Jinshan pharmaceutical Co., Ltd, 75% alcohol
1.1.5 Main reagent preparation
(1) FACS solution: 100ml of PBS buffer and 1ml of FBS were mixed and stored at 4 ℃.
(2) Erythrocyte lysate
150Mm Ammonium chloride(NH4Cl)0.16g、10Mm NaHCO3 1.7g、1Mm EDTA 0.4ml、ddH 2 O200 ml, mixing and storing at 4 ℃.
(3) Cell culture medium
Mixing RPMI 1640 culture solution 89ml, FBS 10ml and double antibody 1ml, and storing at 4 deg.C.
(4) 4% paraformaldehyde solution
4g of paraformaldehyde, 100ml of PBS solution and a few drops of NaOH solution are dissolved by magnetic stirring, the pH is adjusted to 7.4, and the mixture is stored at 4 ℃.
(5) 10% Saponin solution
Saponin 1g, PBS solution 9.9ml, FBS 0.1ml, mixing, 4 degrees C storage.
(6) 0.1% Saponin solution
1ml of 10% Saponin solution, 98ml of PBS solution and 1ml of FBS were mixed together.
(7) HBV core antigen 93-100(HBc 93-100) antigen peptide solution
1mg of HBc93-100 antigenic peptide and 1ml of PBS solution are mixed evenly and stored at the temperature of-20 ℃.
1.2 methods
1.2.1 establishment of acute and chronic HBV transfection mouse model by high-pressure water-powered method
(1) Establishment of chronic HBV transfection mouse model by high-pressure water power method
Mu.g of HBV full expression plasmid pAAV-HBV1.2 (reserved for viral hepatitis research institute at Chongqing medical university) having supergenomic DNA of HBV1.2 was dissolved in PBS solution corresponding to 8% of the mouse's physical mass and rapidly injected into male C57BL/6J (B6) mice through the tail vein within 5 to 8 seconds.
1.2.2 detection of viremia in HBV-transfected mice
(1) At days 7,14,28, and 56 after pAAV-HBV1.2 plasmid injection, the mouse whiskers were removed from the mouse, the mouse eyes were removed with forceps, and the peripheral blood in the eyes was taken out in a 1.5ml sterile EP tube and centrifuged in a centrifuge for 10min at 2500 rpm.
(2) After centrifugation, the upper serum was added into another 1.5ml sterile EP tube and sent to the liver disease laboratory in the southern Yangtze province of the second hospital affiliated to Chongqing medical university for high-accuracy quantification of HBsAg, HBeAg and HBV-DNA in the serum.
1.2.3 HBV transfection of mouse liver, lymphocyte isolation in spleen
Isolation of mouse spleen from lymphocytes in liver at days 7,14,28, and 56 after pAAV-HBV1.2 plasmid injection
1) The required materials and instruments are sterilized in a laboratory sterilizing device and then placed in a biosafety cabinet to be irradiated by ultraviolet rays for half an hour.
2) The facial beard of the mouse is cut off, and the eyeball of the mouse is picked up to take the peripheral blood.
3) The mice are soaked in 75% alcohol for 10 seconds, taken out and placed in a biological safety cabinet to be fixed by a fixing device.
4) 1 plate was prepared, 5ml of RPMI 1640 medium was added thereto, and spleen was separated, and another plate was prepared, 5ml of PBS solution was added thereto, and liver was separated.
5) Spleen and liver were ground, and spleen was passed through 200 mesh membrane into 15ml centrifuge tube, and liver was passed through 200 mesh membrane into 50ml centrifuge tube.
6) Centrifuging: the rotation speed of the spleen is 2500 rpm for 10 min. The liver rotation speed is 2500 rpm, and the time is 5 min.
7) Discarding the supernatant, adding 1ml of erythrocyte lysate into the centrifuged liver and spleen cells, mixing well, and standing at room temperature for 5 min.
8) Centrifuging: the rotating speed is 2500 rpm, and the time is 5 min.
9) Discard the supernatant, add 1ml of FACS solution, blow and mix well.
10) Centrifuging at 2500 rpm for 5 min.
11) The supernatant was discarded, and 1ml of the prepared FACS solution was added to resuspend the cells for further use.
1.2.4 isolated mouse spleen lymphocyte cultures
1) All the materials and instruments to be used are put into a biological safety cabinet and are irradiated by ultraviolet rays for half an hour to achieve the aim of disinfection.
2) The splenic lymphocytes extracted in 1.2.3 were seeded in six well plates, and we seeded three wells for each mouse lymphocyte, so that we required 5 × 10 cells 5 One per well, the prepared cell culture medium was added to each well to make the solution 2ml, and each six well plate was marked with a marker.
3) To each well of the six well plates completed and prepared, 100. mu.l of the prepared HBc93-100 antigen peptide solution was added.
4) After fully mixing, the six-hole plate is covered and put into a laboratory with 5 percent CO 2 At 37 ℃ for 2 hours.
5) The six well plates were removed, the experiments were performed in biosafety cabinets that had been sterilized by UV and 2. mu.l of Brefeldin A solution was added to each well.
6) After fully mixing, the six-hole plate is covered, and 5 percent CO is put in 2 And then the culture is continued for 6 hours in an incubator at 37 ℃ for later use.
1.2.5 flow cytometry for detecting the frequency, phenotype and cytokine secretion of HBV specific CXCR5+ CD8+ Tfc cells and related cells in spleen of chronic HBV transfected mice
1) The cells in 1.2.4 were individually transferred into 15ml sterile centrifuge tubes.
2) Centrifuging at 2500 rpm for 5 min.
3) Discard the supernatant, add prepared 1ml FACS solution to the centrifuge tube, blow and mix well, transfer to 1.5ml sterile EP tube.
4) The spleen cells of the mice after centrifugation were divided into 7 tubes, and the number of spleen lymphocytes in each tube was controlled to about 200000, and the cells were numbered 1 to 7.
5) The divided lymphocytes were flicked by hand, 5. mu.l of 2.4G2(CD16/CD32, 10. mu.g/ml) was added to each tube, and then mixed by shaking or turbine and placed in a refrigerator at 4 ℃ for 10 min.
6) Adding the centrifuged PE-Class into sterile EP tubes of spleen numbers 1-4 according to the description
I Pentamers HBV core antigen 93-10010. mu.l, gently mixed, incubated at room temperature in the dark for 10 min.
7) 1ml of the prepared FACS solution was mixed into a sterile EP tube No. 1-4, and gently pipetted and mixed.
8) No. 1-4 sterile EP tubes were centrifuged at 2500 rpm for 5 min.
9) And pouring the supernatant out of the sterile EP tube No. 1-4, uniformly mixing spleen lymphocytes in all the EP tubes, accurately adding the fluorescent antibody into the sterile EP tube by using a pipette with reference to the amount marked in the specification of the surface-marked fluorescent antibody, placing the sterile EP tube into a refrigerator at 4 ℃ after light flicking, uniformly mixing the cells by light flicking one flick every 10min, dyeing for 30min, and keeping out of the sun.
The staining protocol was as follows:
fluorescent FITC APC-Cy7 PE-Cy7 PerCP-Cy5.5 APC
Sample tube 3 CD3 CD4 CD8 CXCR5 CD40L
Sample tube 4 CD3 CD4 CD8 CXCR5 Tim-3
Sample tube 6 CD3 CD4 CD8 CXCR5
Isotype control IgG1 IgG1 IgG2b IgG2b IgG1
11) To each EP tube, 1ml of the prepared FACS solution was added and flicked off with gentle blow.
12) Centrifuging at 2500 rpm for 5 min.
13) And (3) pouring off the supernatant, adding the prepared 300 mu l of FACS solution into a No. 1-4 sterile EP tube to resuspend the cells, transferring the cell into a flow tube, storing the cell on ice, keeping the cell away from light, immediately detecting the cell by using an up-flow cytometer, collecting at least 100000 lymphocytes in each tube, and analyzing the frequency and the phenotype expression of the target cells.
14) Add 200. mu.l of 4% paraformaldehyde solution to sterile EP tube No. 5-7, blow and mix well, protect from light for 30min at room temperature.
15) No. 5-7 sterile EP tubes were centrifuged at 2500 rpm for 5 min.
16) The supernatant was decanted, and 1ml of 0.1% Saponin solution was added to sterile EP tube nos. 5-7, and the mixture was flushed and mixed well, protected from light at room temperature for 10 min.
17) No. 5-7 sterile EP tubes were centrifuged at 2500 rpm for 5 min.
18) The supernatant was discarded, and 1ml of 0.1% Saponin solution was added to a sterile EP tube No. 5-7, and the mixture was pipetted and mixed.
19) No. 5-7 sterile EP tubes were centrifuged at 2500 rpm for 5 min.
20) Discarding the supernatant, keeping about 50 μ l of liquid as much as possible, mixing the cells, adding the fluorescent antibody into a sterile EP tube according to the dosage of the fluorescent dye required by the specification, slightly mixing the mixture, putting the mixture into a refrigerator at 4 ℃, slightly scattering and mixing the cells uniformly every 20min, and dyeing the mixture for 60min in a dark environment.
The staining protocol was as follows:
fluorescent PE
Sample tube 5IL-21
Sample tube 6 IFN-gamma
21) Add prepared 1ml FACS solution to sterile EP tube No. 5-7, blow gently to flick and mix well.
22) Centrifuging at 2500 rpm for 5 min.
23) And (3) pouring off the supernatant, adding the prepared 300 mu l FACS solution into a sterile EP tube No. 5-7 to resuspend the cells, storing on ice and keeping out of the light, transferring into a flow-type sample loading tube, immediately detecting by an up-flow cytometer, collecting at least 100000 lymphocytes in each tube, and analyzing the frequency of target cells and the secretion condition of cytokines.
1.2.8 statistical analysis
All data were analyzed using SPSS 20.0 statistical software, with data in median (range), percent or
And (4) showing. The chi-square test is used for comparison between two groups of classified variables, the Mann-Whitney U test is used for comparison between two groups of independent continuous variables, and the Wilcoxon paired signed rank test is used for detection of related continuous variables. For all statistical analyses, with P<A two-way test of 0.05 indicated that the difference was statistically significant.
2 results
2.1 establishment of HBV chronic transfection mouse model by high-pressure water-powered tail vein injection method
In order to test the transfection efficiency of the high-pressure water-powered HBV acute and chronic transfection models, the expression of HBV DNA and hepatitis B markers in mouse serum was tested at 7,14,28 and 56 days after the pAAV-HBV1.2 plasmid injection. As shown in FIG. 1, in the serum of pAAV-HBV 1.2-injected mice, the HBV DNA load was highest at day 7 after injection and then gradually decreased at days 14,28, and 56. As shown in FIG. 2, HBsAg level was highest on day 7 after the injection of pAAV-HBV1.2 and gradually decreased on days 14,28, and 56 after the injection.
2.2 frequency expression of HBV-specific CXCR5+ CD8+ Tfc cells in spleen of Chronic HBV transfected mouse model
To investigate whether CXCR5+ CD8+ Tfc cells were involved in the immune response after chronic HBV infection, we examined the frequency of HBV-specific CXCR5+ CD8+ Tfc cells in mouse spleen at 7,14,28,56 days after pAAV-HBV1.2 plasmid injection by flow cytometry. As a result, as shown in fig. 2, the expression of HBV-specific CD8+ T cells was detected at day 7 after pAAV-HBV1.2 injection, and was significantly reduced at day 14 after injection (p <0.01), followed by a reduction in expression at day 28 as compared to day 14, which was also reduced at day 56 as compared to day 28 after injection, but was still significantly lower than its expression at day 7 (p < 0.01). The frequency of HBV specific CXCR5+ CD8+ Tfc cells in spleen of pAAV-HBV1.2 transfected mice was similar to the change of HBV specific CD8+ T cell frequency, and the expression of the cells at 7 days after injection was statistically significant to the difference of the expression of the cells at 14,28 and 56 days after injection (p values are all < 0.01). The research results show that HBV-specific CXCR5+ CD8+ Tfc cells are involved in the anti-infection immune response of the body in chronic HBV infection.
2.3 functional discussion of HBV-specific CXCR5+ CD8+ Tfc cells in spleen of chronic HBV-transfected mouse model participating in cellular immune response
To investigate the degree of functional exhaustion of HBV-specific CXCR5+ CD8+ Tfc cells, flow cytometry is adopted to detect the expression of PD-1 and Tim-3 on the surfaces of HBV-specific CXCR5+ CD8+ Tfc cells, and simultaneously the functions of secreting IFN-gamma and TNF-alpha of CXCR5+ CD8+ Tfc cells after being stimulated by HBc93-100 antigen peptide are detected to observe the cytotoxicity effect of the cells. Results as shown in fig. 3, the study found that in chronic HBV transfected mice, both PD-1 and Tim-3 expressed on the cell surface of HBV-specific CXCR5+ CD8+ Tfc at each observation time point were significantly higher than their expression on the cell surface of HBV-specific CXCR5-CD8+ T cells (p <0.05 or p <0.01 or p < 0.001). However, in mice transfected with chronic HBV, the level of secretion of IFN-gamma and TNF-alpha by spleen CXCR5+ CD8+ Tfc cells at each observation time point is significantly higher than that of secretion of IFN-gamma and TNF-alpha by CXCR5-CD8+ T cells after HBc93-100 antigen peptide stimulation (p is both < 0.01).
The above research results indicate that, although the depletion degree of HBV-specific CXCR5+ CD8+ Tfc cells is higher than that of HBV-specific CXCR5-CD8+ T cells, the functions of secreting cytokines to exert cytotoxicity are stronger than that of HBV-specific CXCR5-CD8+ T cells.
2.4 functional discussion of HBV-specific CXCR5+ CD8+ Tfc cell-assisted humoral immune response in spleen of chronic HBV transfected mouse model
To investigate whether HBV-specific CXCR5+ CD8+ Tfc cells have the function of promoting humoral immune response like CXC5+ CD4+ Tfh cells in chronic HBV infection, we used flow cytometry to detect whether the surfaces of HBV-specific CXCR5+ CD8+ Tfc cells in the spleen of chronic HBV-infected mice express costimulatory molecules ICOS and CD40L, and simultaneously detect the secretion level of IL-21 of HBV-specific CXCR5+ CD8+ Tfc cells. Our studies found that ICOS and CD40L expression could be detected on the surface of HBV-specific CXCR5+ CD8+ Tfc cells, regardless of the chronic HBV transfected mouse model. In the chronic HBV-infected mice transfected with pAAV-HBV1.2 plasmid, the expression of HBV-specific CXCR5+ CD8+ Tfc cell surface ICOS was significantly higher at day 14 after injection than at days 7, 28,56 (p was all <0.01), and in addition, ICOS expression was also significantly higher at days 28,56 after injection than at day 7 after injection (p < 0.05). HBV-specific CXCR5+ CD8+ Tfc cells of chronic HBV-infected mice had higher levels of expression of CD40L at day 28 post-injection expression of HBV-specific CXCR5+ CD8+ Tfc cell surface CD40L at day 56 post-injection was significantly higher than its expression at day 7 (p values <0.01 and <0.05, respectively). As shown in FIG. 4, IL-21 secretion in CXCR5+ CD8+ Tfc cells was detected in the mouse model transfected with chronic HBV after HBc93-100 antigenic peptide stimulation. In chronic HBV transfected mice, the level of IL-21 secreted by CXCR5+ CD8+ Tfc cells after stimulation with HBc93-100 antigenic peptide did not differ significantly between time points.
The research results show that the HBV specific CXCR5+ CD8+ Tfc cell surface expresses ICOS and CD40L in acute or chronic HBV infection, and the HBV related antigen peptide stimulates the cell surface to secrete IL-21, which suggests that the cell surface may have the function of assisting humoral immune response.
2.5 frequent expression of HBV-specific CXCR5+ CD8+ Tfc cells in the liver of Chronic HBV transfected mouse model
To investigate whether CXCR5+ CD8+ Tfc cells were involved in immune responses after chronic HBV infection, we examined the frequency of HBV-specific CXCR5+ CD8+ Tfc cells in mouse livers at days 7,14,28,56 after pAAV-HBV1.2 plasmid injection by flow cytometry. The results are shown in fig. 5, where the expression of HBV-specific CD8+ T cells was detected at day 7 after pAAV-HBV1.2 injection, slightly decreased at day 14 after injection, followed by a significant decrease in expression at day 28 compared to day 14, which was also decreased at day 56 compared to day 28 after injection, but was still significantly lower than its expression at day 7 (p < 0.05). The frequency of HBV specific CXCR5+ CD8+ Tfc cells in the liver of pAAV-HBV1.2 transfected mice was similar to the change of the frequency of HBV specific CD8+ T cells, and the expression of the cells at 7 days after injection was statistically significant to the difference of the expression of the cells at 14,28 and 56 days after injection (p values are all < 0.01). The research results show that HBV-specific CXCR5+ CD8+ Tfc cells are involved in the anti-infection immune response of the body in chronic HBV infection.
2.6 functional discussion of the involvement of HBV-specific CXCR5+ CD8+ Tfc cells in the cellular immune response in the liver of chronic HBV transfected mouse model
To investigate the degree of functional exhaustion of HBV-specific CXCR5+ CD8+ Tfc cells, flow cytometry was used to detect the expression of Tim-3 on the surface of HBV-specific CXCR5+ CD8+ Tfc cells, and the function of TNF-alpha secretion of CXCR5+ CD8+ Tfc cells stimulated by HBc93-100 antigen peptide was also detected to observe the cytotoxic effect of the cells. The study found that in the chronic HBV transfected mice, the expression Tim-3 on the surface of HBV specific CXCR5+ CD8+ Tfc cells at each observation time point is significantly higher than that on the surface of HBV specific CXCR5-CD8+ T cells, as shown in FIG. 6, however, in the chronic HBV transfected mice, after being stimulated by HBc93-100 antigen peptide, the level of TNF-alpha secretion of spleen CXCR5+ CD8+ Tfc cells at each observation time point is slightly higher than that of CXCR5-CD8+ T cells.
The above research results indicate that, although the depletion degree of HBV-specific CXCR5+ CD8+ Tfc cells is higher than that of HBV-specific CXCR5-CD8+ T cells, the functions of secreting cytokines to exert cytotoxicity are stronger than that of HBV-specific CXCR5-CD8+ T cells.
2.7 functional discussion of HBV-specific CXCR5+ CD8+ Tfc cell-assisted humoral immune response in chronic HBV-transfected mouse model liver
To investigate whether HBV-specific CXCR5+ CD8+ Tfc cells have the function of promoting humoral immune response like CXC5+ CD4+ Tfh cells in chronic HBV infection, we used flow cytometry to detect whether the surfaces of HBV-specific CXCR5+ CD8+ Tfc cells in the spleen of chronic HBV-infected mice express costimulatory molecules ICOS and CD 40L. Our studies found that both ICOS and CD40L expression could be detected on the surface of HBV-specific CXCR5+ CD8+ Tfc cells in a chronic HBV transfected mouse model, as shown in fig. 7, the expression of ICOS on the surface of HBV-specific CXCR5+ CD8+ Tfc cells at day 7 after injection was significantly higher than that on days 14,28, and 56 in a pAAV-HBV1.2 plasmid transfected chronic HBV infected mouse, the expression of CD40L on the surface of HBV-specific CXCR5+ CD8+ Tfc cells of a chronic HBV infected mouse at day 7 after injection, and the expression of CD40L on the surface of HBV-specific CXCR5+ CD8+ Tfc cells at day 56 after injection was slightly lower than that on day 7.
The results of the above studies indicate that in chronic HBV infection, both ICOS and CD40L are expressed on the surface of HBV-specific CXCR5+ CD8+ Tfc cells, suggesting that they may have the function of assisting humoral immune response.
3 analysis of
The natural history of HBV infection depends on the interaction between the virus, the host and the environment. The immune response plays an important role in controlling HBV infection. Acquired immunity is more critical in the pathological course of HBV infection. The virus-specific antibodies secreted by B cells can prevent HBV recurrence in humoral immunity by infecting hepatocytes and effectively eliminating HBV in vivo to prevent chronic HBV infection. The CD8+ T cells in the cellular immunity can not only directly kill HBV-infected liver cells, but also recover by secreting IFN-gamma/TNF-alpha. By reducing the HBV cccDNA content in HBV infected hepatocytes, persistent HBV infection can be inhibited. This study discusses some of the humoral and cellular immune responses following HBV infection. Research on specific immunoreaction after HBV infection shows that in the HBV infection process, HBV specific T lymphocytes are easy to apoptosis, the secretion and proliferation functions of cytokines are obviously reduced, and the functions and the number of the T lymphocytes are reduced, so that persistent hepatitis B virus replication is caused. Therefore, it is of particular interest to explore ways to improve the antiviral capacity of HBV-specific T lymphocytes or to find new, more effective subsets of HBV-specific T lymphocytes. Recent studies have shown that a low-depleted CD8+ T lymphocyte population expressing the chemokine CXCR5 can migrate to the lymphocyte follicles, where they act as antiviral agents. Meanwhile, a group of IL-21 and IFN secreting CD8+ T lymphocyte-gamma is found in nasal polyps, and the cells also express CXCR5 and can promote B cells to produce IgG.
The research adopts a chronic HBV transfection mouse model injected by high-pressure tail vein, and adopts a flow cytometer to analyze the frequency of HBV specific CXCR5+ CD8+ TFC cells in the spleen of the mouse and the expression of surface molecules and cytokines. Our studies found that in the plasmid-transfected chronic HBV-infected mouse model, in the HBV-specific CXCR5+ CD8+ TFC cells in the mouse spleen, the cell frequency of HBV-specific CD8+ T cells decreased continuously with the development of viremia, which peaked at 7 days and had a significant decline after 14 days, while the rule of cell frequency of specific CXCR5+ CD8+ T cells was essentially the same as that of HBV-specific CD8+ T cells, indicating that both cells are involved and play an important role in the clearance of viremia. In the liver model of plasmid-transfected chronic HBV mice, the cell frequency of HBV-specific CD8+ T cells also decreased with the development of viremia, which peaked at 7 days and had a more significant decline after 14 days, while the rule of cell frequency of specific CXCR5+ CD8+ T cells was basically the same as that of HBV-specific CD8+ T cells, indicating that both cells are involved and play an important role in the clarification of viremia. Therefore, CXCR5+ CD8+ T cells in the spleen of the liver play an important antiviral role.
In the study, we used flow cytometry to detect the expression of PD-1 and Tim-3 on the surface of HBV specific CXCR5+ CD8+ Tfc cells in the spleen of chronic HBV transfected mice, and as a result, the expression of two inhibitory molecules, PD-1 and TIM-3 expressed on the surface of CXCR5+ CD8+ T cells in the spleen is higher than that of CXCR5-CD8+ T cells, which indicates that the degree of exhaustion of CXCR5+ CD8+ T cells is higher in the process of HBV clearance, and is greatly different from the idea that we begin. Then, the antiviral ability of CXCR5+ CD8+ T cells is further researched, and the secretion levels of IFN-gamma and TNF-alpha of spleen CXCR5+ CD8+ Tfc cells after being stimulated by HBc93-100 antigen peptide are detected. As a result, the secretion levels of IFN-r and TNF-a secreted by CXCR5+ CD8+ T cells in spleen at four different time points are obviously higher than that of CXCR5-CD8+ T cells, so that the antiviral capacity of the CXCR5+ CD8+ T cells is considered to be obviously higher than that of the CXCR5-CD8+ T cells. Since PD-1 and Tim-3 are thought to be markers of T cell failure and both molecules are thought to be associated with decreased cytokine secretion by cells, additional studies have shown that they may be associated with enhancement of effector and memory T cells. IFN-r and TNF-a are generally considered to have antiviral efficacy. We can therefore see in the spleen model that CXCR5+ CD8+ T cells, although depleted to a greater extent, do have greater antiviral efficacy. In a liver model, the expression of TIM3 and the expression of TNF-a are detected, and research results show that the expression of TIM-3 of CXCR5+ CD8+ T cells is slightly higher than that of CXCR5-CD8+ T cells at each time point, while the expression of CXCR5+ CD8+ T cells is higher than that of CXCR5-CD8+ T cells at each time point, so that the consumption degree of CXCR5+ CD8+ T cells in the liver model is considered to be slightly higher than that of a negative group, but the antiviral efficacy is higher than that of the negative group.
CXCR5+ CD4+ Tfh cells are coupled to B cells in the lymphoid follicles by a variety of surface molecules. For example, ICOS expressed by Tfh cells interacts with ICOSL expressed by B cells, promoting the production of Tfh cells and the formation of germinal centers. Meanwhile, the mutual combination of the Tfh cell surface expressed CD40L and the B cell surface expressed CD40 can promote the maturation of antibody affinity. In addition, Tfh cells secrete the cytokine IL-21, which binds to the B cell surface receptor IL-21R, promoting the activation, proliferation, differentiation and plasma cell production of B cells. Tfh cells are characterized by sustained high expression of the chemokine receptor CXCR5, which functions to assist the humoral immune response by interacting with B cells by expressing CXCR5 to localize to the lymphoid follicles.
Claims (8)
1. Application of human CXCR5+ CD8+ T cells in preparing medicines for treating chronic hepatitis B is disclosed.
2. Application of a reagent for promoting proliferation of CXCR5+ CD8+ T cells in preparation of a medicament for treating chronic hepatitis B.
3. Application of a reagent for promoting expression of CD8+ T cell surface functional molecule CXCR5 in preparation of a medicament for treating chronic hepatitis B.
4. The application of a reagent for promoting CD8+ T cells to secrete TNF-alpha and IFN-gamma in preparing a medicament for treating chronic hepatitis B.
5. A medicine for treating chronic hepatitis B is characterized in that: the active ingredient comprises human CXCR5+ CD8+ T cells.
6. A medicine for treating chronic hepatitis B is characterized in that: comprising an agent that promotes proliferation of CXCR5+ CD8+ T cells.
7. The medicine for treating chronic hepatitis B is characterized in that: comprising an agent that promotes the expression of the CD8+ T cell surface functional molecule CXCR 5.
8. A medicine for treating chronic hepatitis B is characterized in that: comprising an agent that promotes the secretion of TNF-alpha and IFN-gamma by CD8+ T cells.
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2022
- 2022-05-23 CN CN202210561452.1A patent/CN115040538A/en active Pending
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