CN116731965B - Method for establishing immune response model and method for testing cell response - Google Patents
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Abstract
The invention belongs to the field of biology, and discloses a chicken TCR gamma delta + CD8α + The method for establishing the T cell immune response model comprises the following steps: step 1: taking spleen lymphocytes of chickens infected with avian influenza virus and inoculating the lymphocytes with the avian influenza virus to obtain antigen presenting cells; step 2: spleen lymphocytes and antigen presenting cells of chickens infected with avian influenza virus were mixed and cultured. The model established based on the method can better realize TCR gamma delta after the infection of the avian influenza virus of the H9N2 subtype + CD8α + T cell proliferation simulation, the model can reflect the virulence of the avian influenza virus of H9N2 subtype of different strains more accurately and TCRγdelta + CD8α + T cells are responsive to avian influenza virus of H9N2 subtypes of different strains. Meanwhile, the invention also discloses a chicken TCR gamma delta for avian influenza virus + CD8α + T cell immune response test method.
Description
Technical Field
The invention belongs to the field of biology, and particularly relates to a method for establishing an immune response model and a method for testing cell response.
Background
Avian Influenza (AI) is a highly contagious disease of infection of poultry and wild birds caused by influenza a virus (Avian influenza virus, AIV). Avian influenza virus is orthomyxoviridae, influenza a virus. The surface structural proteins Hemagglutinin (HA) can be classified according to their antigenicity. To date, avian influenza viruses have found 16 HA subtypes and 9 NA subtypes.
Avian influenza can be classified into highly pathogenic avian influenza (highly pathogenic avian influenza, HPAI) and less pathogenic avian influenza (low pathogenic avian influenza, LPAI) according to the pathogenicity. Although the H9N2 subtype AIV is a low pathogenic avian influenza virus, it can lead to reduced egg production in poultry, causes a tremendous economic loss to the poultry industry, and is considered to be one of the most potential avian influenza viruses to cause cross-species pandemics. Thus, enhanced control and research of H9N2 AIV is critical not only for poultry, but also for public health.
Neutralizing antibodies generated by inactivated vaccines to induce humoral immune responses have long been considered critical for host resistance to avian influenza virus invasion. However, under immune pressure, the HA protein of AIV is continuously mutated, so that the protective effect of an inactivated vaccine is obviously weakened, and the effect of cell immunity in anti-AIV immunity is more and more important. Chicken T cell immunity has been shown to play an important role in the protection against avian influenza virus infection, providing long lasting and cross strain protection and developing universal vaccines. However, there are relatively few studies of the T cell immune response induced after infection with AIV of the H9N2 subtype.
Chicken T cells can be divided into different subsets based on the expression of the T cell surface receptor (TCR) and the co-receptors CD4, CD 8. TCRs include 3 types: TCR1, TCR2, TCR3, wherein TCR1 is expressed predominantly on γδ T cells. There are studies showing that CD8 + Cytotoxic T lymphocytes (Cytotoxic T lymphocyte, CTL), typified by T cells, play an important role in the clearance of Infectious Bronchitis Virus (IBV), marek's Disease Virus (MDV) and AIV. Activated CTLs can kill infected cells directly to limit replication and transmission of invasive pathogens, while recruiting other immunityEpidemic cells secrete various cytokines and chemokines to clear the virus. Dai studies showed that CD8 after infection with H9N2 AIV + T cells are activated in PBL (peripheral blood lymphocytes) releasing cytotoxicity related genes (e.g., granzymeA, granzymek, PARP, IFN- γ, etc.), thus combating H9N2 AIV infection. In addition to traditional cd8+ T cells, there are also large numbers of TCR1 in chicken peripheral blood or peripheral organs + CD8 + T(TCRγδ + CD8 + T) cells, CD4 + CD8 + T cells also express CD8 receptor. However, the response of chicken tcrγδ+cd8+ T cells to avian influenza virus is not yet known.
Regarding the research on proliferation of T cells, the applicant has first proposed a mixed culture of memory cells and non-memory cells to increase the proliferation rate of T cells. The following are provided:
the applicant previously filed a patent ZL202110395246.3 discloses a method for promoting proliferation of duck T cells and application thereof, wherein the method for promoting proliferation of duck T cells comprises the following steps: the H5N1 HP AIV is inoculated into a duck in vivo and then separated from peripheral blood to obtain duck memory PBMC; H5N1 HP AIV in vitro infected duck memory PBMCs were mixed with uninfected memory PBMCs.
When the applicant tried to translate this method onto chicken and subjects of avian influenza of the H9N2 subtype, it was found that the above method failed to show our expected effect.
Therefore, the technical problem to be solved in the project is as follows: how to establish TCRγδ of chicken against H9N2 subtype of avian influenza virus infection + CD8α + T cell immune response model.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a chicken TCR gamma delta + CD8α + T cell immune response model establishment method, and model established based on the method can better realize TCRγdelta after H9N2 subtype avian influenza virus infection + CD8α + T cell proliferation simulation, the model can reflect the virulence of the avian influenza virus of H9N2 subtype of different strains more accurately and TCRγdelta + CD8α + T cell needleResponsiveness to avian influenza virus of H9N2 subtype of different strains.
A secondary object of the present invention is to provide a chicken TCRγδ for avian influenza virus + CD8α + T cell immune response test method.
In order to achieve the aim of the invention, the invention adopts the following technical scheme: chicken tcrγδ + CD8α + The method for establishing the T cell immune response model comprises the following steps:
step 1: taking spleen lymphocytes of chickens infected with avian influenza virus and inoculating the lymphocytes with the avian influenza virus to obtain antigen presenting cells;
step 2: spleen lymphocytes and antigen presenting cells of chickens infected with avian influenza virus were mixed and cultured.
In the above method for establishing a model, in the step 2, the ratio of the number of lymphocytes in spleen and antigen presenting cells in the chicken infected with avian influenza virus is: 5:1.
in the method for establishing a model, in the step 1, the avian influenza virus is inoculated in an inoculum size of moi=2 by 2×10 6 And lymphocytes.
In the above method for establishing a model, the method for preparing spleen lymphocytes of a chicken infected with avian influenza virus comprises the following sub-steps:
sub-step 11: killing chicken and taking spleen; the chicken is a chicken pre-infected with avian influenza virus; the chicken has been detoxified for 28 days, and no more toxin is discharged at this time;
sub-step 12: cutting spleen into small pieces, adding a proper amount of RP-10, blowing with a Pasteur pipette for multiple times, and filtering to obtain cell suspension;
sub-step 13: adding the cell suspension to the upper layer of a single nuclear lymphocyte separating medium of an equal volume of chicken viscera tissue, centrifuging, sucking the centrifuged intermediate layer cells, washing twice in a washing liquid, centrifuging, and re-suspending the cells by RP-10 to obtain the spleen lymphocytes of the chicken.
The chicken organ tissue single nuclear lymphocyte separating medium is a commercial kit; brands such as KIT, TBD2011CP and the like can be adopted; the operation is performed according to the instruction of the kit;
in the above method for establishing a model, the method for preparing antigen presenting cells specifically comprises: taking spleen lymphocytes of chickens infected with avian influenza virus, inoculating the lymphocytes with the avian influenza virus, incubating the virus for 0.5-2 h, replacing the virus with 1640 medium containing 0.25 mug/mLTPCK and 10% FBS, and incubating the virus at 39 ℃ for 4-6 h to obtain antigen presenting cells.
In the above method for establishing a model, the step 2 specifically includes: the spleen lymphocytes of chickens infected with avian influenza virus were used at 3×10 6 The density of individual cells/mL was plated in 48-well plates, and antigen presenting cells were added after 6h of culture.
In the method for establishing the model, the avian influenza is H9N2 subtype avian influenza.
Meanwhile, the invention also discloses a chicken TCR gamma delta aiming at the avian influenza virus + CD8α + A method for T cell immune response testing comprising the steps of:
step 100: modeling according to the method of any one of claims 1-7 as an experimental group; meanwhile, taking lymphocytes of spleen of the same batch of chickens infected with the avian influenza virus as a positive control group and a negative control group, wherein the lymphocytes are used for establishing a model; wherein ConA is added into the positive control group, and no reagent is added into the negative control group;
step 200: calculating TCRγδ of experimental group, positive control group and negative control group + CD8α + The number of T cells is changed and/or the chicken TCRgamma delta aiming at the avian influenza virus strain is obtained + CD8α + T cell immune response effects.
In the above test method, the positive control group was used in a 3X 10 ratio 6 Density of individual cells/mL spleen lymphocytes from chickens infected with avian influenza virus were plated in 48-well plates and 2.5. Mu.g/mL ConA was added.
In the above test method, the TCR gamma delta in the experimental group, the positive control group and the negative control group is detected by adopting a flow detection method + CD8α + Number of T cells.
Compared with the prior art, the invention has the following beneficial effects:
(1) Compared with the model based on Peripheral Blood (PBMC), the invention is based on a model which can be successfully established by spleen lymphocytes; the PBMC-based cannot be modeled.
(2) The invention compares lung lymphocytes with spleen lymphocytes, and only a model built by the spleen can accurately respond to TCR gamma delta + CD8α + The proliferation of T cells in response is differentiated.
Drawings
FIG. 1a shows the discharge of the cloaca after infection with H9N2 AIV according to the present invention;
FIG. 1b shows the oropharynx detoxification of H9N2 AIV infection according to the present invention
Statistical analysis using unpaired t-test, n=5,: p <0.05, significant differences; * *: p is less than 0.01, and the difference is extremely remarkable; * **: p <0.001, the difference is very significant.
FIG. 2a is a schematic view of a streaming portal policy;
FIG. 2b shows the ratio of TCRγδ+CD8α+T cells in the viscera of SPF chickens after challenge;
FIG. 2c shows the ratio of TCRγδ+CD8α+T cells in PBMC of SPF chickens after challenge;
statistical analysis using unpaired t-test,: p <0.05, significant differences; * *: p <0.01, the difference is very significant.
FIG. 3a shows the expression of cytotoxicity related genes in SPF chicken TCRγδ+CD8α+ T cells after challenge according to the present invention;
FIG. 3b shows the expression of immune-related genes in SPF chicken TCRγδ+CD8α+ T cells after challenge according to the present invention;
FIG. 3c shows helper T cell expression in post-challenge SPF chicken TCRγδ+CD8α+ T cells of the present invention;
statistical analysis was performed using the paired-t test with P <0.05, with significant differences; * P <0.01, the difference is very significant; FIG. 4 is a morphology of in vitro culture of chicken spleen lymphocytes;
FIG. 5 is a schematic representation of CFSE marker proliferation assay circle strategy;
FIG. 6a shows the results of the CFSE marker stream for H9N2 AIV stimulation;
wherein 1 is a sample with a sample name of 5DPI#1H9N2.fcs, a group name of single cells, and a cell count of 37219;2 is a sample with a sample name of 3DPI#1H9N2.fcs, a group name of single cells, and a cell count of 34995;
3 is a sample with a sample name of 2DPI#1H9N2.fcs, a group name of single cells, and a cell count of 33490;
4 is a sample named 2DPI#1control.fcs, a group named single cells, and a cell count of 14783;
FIG. 6b shows the results of the H9N2 AIV stimulation CFSE marker stream;
wherein, 1 is a sample with the sample name of 5DPI#2H9N2.Fcs, the group name of single cells and the cell count of 40226;2 is a sample with the sample name of 3DPI#2H9N2.Fcs, the group name of single cells, and the cell count of 30525;
3 is a sample with the sample name of 2DPI#2H9N2.fcs, the group name of single cells and the cell count of 30234;
4 is a sample named 2DPI#2control.fcs, a group named single cells, and a cell count of 24814;
FIG. 6c shows the results of the CFSE marker stream for H9N2 AIV stimulation;
wherein 1 is a sample with a sample name of 5DPI#3H9N2.fcs, a group name of single cells, and a cell count of 46099;2 is a sample with the sample name of 3DPI#3H9N2.fcs, the group name of single cells and the cell count of 48505;
3 is a sample with the sample name of 2DPI#3H9N2.fcs, the group name of single cells and the cell count of 40444;
4 is a sample named 2DPI#3control.fcs, a group named single cells, and a cell count of 24805;
FIG. 7 is a chart of the conA stimulation CFSE marker flow results;
wherein, 1 is a sample with the sample name of 5DPI#2ConA.fcs, the group name of single cells and the cell count of 43550;
2 is a sample with the sample name of 3DPI#2ConA.fcs, a group name of single cells, and a cell count of 46338;
3 is a sample with the sample name of 2DPI#2ConA.fcs, a group name of single cells, and a cell count of 34857;
4 is a sample named 2DPI#2control.fcs, a group named single cells, and a cell count of 24841;
FIG. 8 is a schematic representation of flow analysis of TCRγδ+CD8α+T cell loop gate strategy;
FIG. 9a is a graph showing the results of the change in the ratio of TCRγδ+CD8α+T cells after H9N2 AIV stimulation;
FIG. 9b is a graph showing the change in the number of TCRγδ+CD8α+T cells following H9N2 AIV stimulation;
FIG. 10a is a graph showing the results of three independent repeat ELISPot assays for IFN-gamma detection;
statistical analysis is carried out on experimental results by using unpaired-t test, wherein the P is less than 0.01, and the difference is extremely remarkable; * P <0.001, the difference is very significant.
FIG. 10b is a plot of the results of ELISPot assays performed on 3 samples of cells from the H9N2 AIV stimulated, positive control and unstimulated groups;
fig. 11 is an H9N2 stimulation picture of comparative example 1.
Fig. 12 is an unstimulated picture of comparative example 1.
FIG. 13 is a flow test CFSE of comparative example 1;
wherein, 1 is a sample with the sample name of 5DPI.H9N2.fcs, the group name of single cells and the cell count of 15486;
2 is a sample with the sample name of 3DPI.H9N2.fcs, a group name of single cells and a cell count of 21907;
3 is a sample with the sample name of 2DPI.H9N2.fcs, a group with the sample name of single cells and a cell count of 23911;
4 is a sample named 2DPI.2control.fcs, a group named single cells, and a cell count of 34915.
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.
Material preparation
1.1 strain background
A/Chicken/HuNan/HN/2015 (H9N 2 subtype AIV) was isolated and stored by the zoonotic agent national local joint engineering laboratory.
1.2 chick embryo and laboratory animal
SPF chickens of 4 weeks of age and SPF chick embryos of 9 to 11 days of age were offered by the New Dahua farm eggs Inc., guangdong province.
1.3 major reagents
RPMI-1640 medium, FBS Australian fetal bovine serum, PBS pH7.4 basic1×, 10000UI diab, L-glutamine (200 mM), HEPES (100×), sodium pyruvate (100 mM), non-essential amino acids (100×), 0.25% Trypsin-EDTA pancreatin from GIBCO company, USA;
chicken peripheral blood lymphocyte separation liquid kit, chicken organ tissue single nuclear lymphocyte separation liquid kit and erythrocyte lysate are purchased from Tianjin's Chayo biological company;
chicken peripheral blood lymphocyte separation liquid kit, chicken organ tissue single nuclear lymphocyte separation liquid kit and erythrocyte lysate are purchased from Tianjin's Chayo biological company; 2-mercaptoethanol (55 mM), canavalin protein (ConA), TPCK-treated trypsin was purchased from Sigma, USA; chamQ SYRBR qPCR Master Mix from Nanjinouzan Biotechnology Co., ltd; TMB ELISPOT dedicated color development solution is purchased from the company of Daidae, china, and Chicken IFN-. Gamma.ELISPOTBIST ASIC kit is purchased from the company of Mabtech; the flow antibody Anti-chicken CD3 antibody, anti-chicken TCR gamma 6antibody, anti-chicken CD8 alpha Anti-body is available from southern Biotech company; CFSE-labeling kit was purchased from abcam corporation.
1.4 preparation of the Main reagent
(1) 10% complete medium (RP-10): 10% FBS+90%1640, and storing at 4deg.C after preparation;
(2) T cell medium: fully and uniformly mixing 10% FBS+1% nonessential amino acid, 1% glutamine, 1% sodium pyruvate, 1% diabody, beta-mercaptoethanol+90% RMPI1640, and sub-packaging at 4 ℃ for storage and use after preparation.
(3) Streaming Buffer:2% FBS+98% PBS, and after the preparation, the mixture is placed at 4 ℃ for standby.
(4) Cell cryopreservation solution: 10% DMSO+90% FBS, and placing at 4deg.C for use after preparation.
Early preparation of experiment
2.1 propagation of Virus
The H9N2 AIV prototoxin was diluted 1000-fold in sterile PBS after thawing.
After the SPF chick embryo of 9-11 days old is disinfected, the chick embryo is placed in a workbench, and 200 mu L of diluted virus liquid is inoculated into each chick embryo allantoic cavity. And (5) continuously culturing the sealed chick embryo, observing the death and alive of the chick embryo after 24 hours of inoculation, discarding the dead embryo, and continuously culturing the residual alive embryo until 72 hours to obtain the virus-free chick embryo. The allantoic fluid is sucked into a centrifuge tube by a liquid transfer device, the liquid is centrifuged for 10min at 2000rpm at 4 ℃, and the supernatant is taken out and split-packed after passing through a filter membrane with the diameter of 0.22 mu m, and is preserved at-80 ℃ for standby.
2.2 determination of hemagglutination titres
The potency determination is referred to the national standard latest edition (GB/T18936-2020).
1.3 EID 50 Is (are) determined by
Half-number infection of chick embryo (50% Embryo infective dose, EID) 50 ) The measurement of (2) is as follows: the amplified virus solution was thawed on ice and diluted 10-fold with PBS. Inoculating chick embryos with 10-4 to 10-9 dilutions of virus liquid according to the method of 2.1, and culturing, wherein each dilution is 5 chick embryos. After 72h, 25. Mu.L of allantoic fluid was collected from each chick embryo, the hemagglutination titers were determined by the 2.2 method, and the EID was calculated by the Spearman-Karber method 50 。
Establishment of 2.4H9N2 subtype AIV infection induced SPF chicken TCRγδ+CD8α+T cell immune response animal model
16 SPF chickens of 4 weeks of age were randomly divided into a challenge group and a control group, 8 per group. The virus challenge group adopts an eye-drop and nose-drop mode to inoculate H9N2 AIV, and the virus titer is 10 6 EID 50/200. Mu.L, control group was inoculated with 200. Mu.L of sterile PBS. Collecting corresponding samples after toxin attack, and carrying out the following detection:
2.4.1 detection of post-infection detoxification conditions
Collecting throat and cloaca swab of each chicken 3, 5, 7, 9, and 14 days after virus attack, placing into sterilized PBS containing diabody and 30% glycerol, and preserving at-80deg.C for use. And (5) measuring the EID50 and detecting the toxin expelling condition.
2.4.2 detection of the post-infection Tiger T cell subtype changes in tcrγδ+CD8α+
Peripheral blood lymphocytes were isolated, trypan blue stained and counted, cells were taken and stained with anti-chicken CD3, tcrγδ and CD8 α flow antibodies in flow tubes and incubated at 4 ℃ for 30min in the absence of light. After the incubation, PBS was added for washing, 400g was centrifuged for 5min, and the cells were resuspended by flow Buffer and checked on the machine.
2.4.3 detection of post-infection chicken TCRγδ+CD8α+T cell immune related Gene changes
Resuscitates 39 DPI challenged group PBMC and 39 DPI control group PBMC samples, each of which was taken at 6X 10 7 Flow staining was performed on individual cells, with reference to 2.4.2. Sorting TCR gamma delta by flow sorter + CD8α + T cells.
The TCRγδ obtained by the above-mentioned separation + CD8α + After centrifugation of T cells at 2000rpm for 10min, the supernatant was discarded, cellular RNA was extracted by TRIZOL method, and the concentration of RNA was detected by spectrophotometry.
RNA reverse transcription was performed using the TAKARA PrimeScriptTM RT Master Mix (Perfect Real Time) kit, and the system is shown in Table 1. The reaction procedure: the reaction was carried out at 37℃for 15min and at 85℃for inactivation.
TABLE 1 reverse transcription system
Fluorescent quantitative PCR amplification was performed on the reverse transcribed cDNA to detect changes in cellular immune related genes in PBMC. The target genes and primers are shown in Table 2, and the system is shown in Table 3. The reaction procedure: pre-denaturation at 95 ℃ for 30s; the cyclic reaction is carried out at 95 ℃,10s,60 ℃ and 30s for 40 cycles; dissolution profile analysis was conducted at 95 ℃,15s,60 ℃,60s,95 ℃ and 15s. The experimental results were statistically analyzed using GraphPadPrism8 software.
TABLE 2 cellular immune related gene qPCR primer
TABLE 3 fluorescent quantitative reaction System
Experimental results:
post infection SPF chicken detoxification conditions of H9N2 subtype AIV
The toxin expelling condition of the chicken throat swab and the cloaca swab after toxin expelling is shown in figures 1a and 1 b. In the H9N2 infected group, the virus persisted for approximately 9 days. Wherein the viral load of the throat swab peaks at 3DPI, decreases from 5DPI and disappears at 11 DPI. Cloaca swabs can detect detoxification from 3DPI to 7DPI, and no detoxification has been detected at 9 DPI. The control group was negative for detoxification tests (data not shown).
Changes in TCRγ6+CD8α+ T cells following infection with AIV of subtype H9N2
And (3) collecting peripheral blood of chickens for separating PBMC (peripheral blood cell) 3, 5, 7, 9 and 14 days after virus attack, killing and separating spleen and lung mononuclear lymphocytes 5 days after virus attack, and detecting the proportion of TCRgamma 6+CD8alpha+T cells in a flow mode. FIG. 2a is a flow staining loop door strategy, analysis of flow results using FlowJo software. As shown in fig. 2b, after infection of SPF chickens with H9N2 AIV, the tcrγ6+cd8α+ T cell fraction was significantly increased in pbmc at 5DPI, 7DPI and 9DPI (P<0.05，P<0.01，P<0.05). The proportion of tcrγ6+cd8α+ T cells in the H9N2 group in the spleen was significantly increased compared to the control group (P<0.05 However, there was no statistical difference between the challenge group and the control group in the lung. The results showed that at the time of virus detoxification decline (5 DPI), TCRγ6+CD8α+ T cells were significantly upregulated in both PBMC and spleen, suggesting that viral clearance might be comparable to TCRγ6 + CD8α + T cell immune responses are related.
Chicken tcrγ 6 after infection with h9n2 subtype AIV + CD8α + T cell immune related gene changes
To further verify TCRgamma 6 in SPF chickens + CD8α + T cells play a role in combating H9N2 AIV infection, further by flow sorting TCRgamma 6 in the offending and control PBMC + CD8α + T cells, RNA extraction and qRT-PCR detection of the change of the mRNA expression level of important immune genes, wherein the detection mainly comprises three parts: natural immune related genes, CTLs related genes and Th2 related genes.
Natural immunity gene part (FIG. 3 b), antiviral genes in the infected group compared with the control group
OASL (2 ',5' -Oligoadenylate synthetase-like) expression levels were significantly up-regulated (P < 0.05); th2 gene part (FIG. 3 a), MHC-II gene expression level significantly decreased (P < 0.01) compared to control group, CTLs gene part (FIG. 3 c), granzymeA, granzymek, IFN-gamma (Interferon gamma), performin gene expression level significantly increased (P < 0.05) compared to control group; the increased proportion of tcrγ6+cd8α+ T cells in PBMC and spleen in combination with 3 further demonstrates that H9N2 AIV infection successfully activates the immune response of tcrγ6+cd8α+ T cells in chickens and that tcrγ6+cd8α+ T cells exert a cytotoxic T cell effect in viral clearance.
In view of the above, H9N2 AIV infection induced SPF chicken TCRγ6+CD8α+T cell immune response model was successfully established.
Example 2
Establishment of 3.1H9N2 subtype AIV memory TCRgamma 6+CD8alpha+T cell in-vitro expansion culture method
3.2H9N2 subtype AIV stimulates expansion of chicken TCRγ6+CD8α+ T cells in vitro
(1) Spleen mononuclear lymphocyte preparation
Killing chicken 28 days after toxin attack, taking spleen, cutting spleen into small pieces by scissors, adding a proper amount of RP-10, lightly blowing for tens of times by using a Pasteur pipette, filtering by using a 40 mu m cell filter screen, adding the cell suspension obtained by filtering to 400g of the single nuclear lymphocyte separating liquid of the chicken viscera tissue of equal volume, and centrifuging for 15min. The intermediate layer cells were aspirated and washed twice in wash solution, centrifuged, and the cells resuspended in RP-10 and counted for 0.08% trypan blue.
(2) H9N2 subtype AIV infected antigen presenting cells
Virus was inoculated 2×10 at moi=2 6 After 1h incubation of the individual spleen mononuclear lymphocytes, the virus was replaced with 1640 medium containing 0.25. Mu.g/mLTPCK and 10% FBS and incubated at 39℃for 5h. Spleen mononuclear lymphocytes infected with H9N2 AIV were used as antigen presenting cells in the experiment (Antigen presenting cell, APC).
(3) APC-stimulated chicken spleen lymphocytes
Spleen mononuclear lymphocytes prepared by the above separation are divided into an H9N2 group, a positive control group and a negative control group. Each group of cells was plated at 3X 10 6 The density of individual cells/mL was plated in 48-well plates and after 6h incubation the experimental group was plated at 5: APC was added in 1 proportion, conA at 2.5. Mu.g/mL was added to the positive control, and the negative control was not treated at all. Culturing in 39 deg.c incubator, observing cell morphology change every day, trypan blue staining and counting, and recording cell number change rule.
(4) Flow cytometry detection of tcrγ6+cd8α+t cell ratio changes
After 3 days of culture, the cells of the experimental group and the control group were collected and counted, and the cells were flow stained according to the method of 2.4.2 to analyze the ratio change of tcrγ6+cd8α+t cells after H9N2 AIV stimulation.
3.3CFSE markers for detection of chicken T cell proliferation
(1) CFSE marked chicken spleen mononuclear lymphocyte
Before the experiment, sterilized PBS and RP-10 culture medium are placed in a water bath kettle at 37 ℃ for preheating. Washing the cells with PBS, centrifuging 400g for 5min, and precipitating the cells at 1X 10 7 The concentration of/mL was resuspended in PBS containing 0.5. Mu.M CFSE and incubated in a 37℃water bath in the dark for 10min. After completion of incubation, 400g was centrifuged for 5min, the supernatant was discarded, and the cells were washed by adding pre-warmed RP-10 medium. The supernatant was removed by centrifugation and resuspended in T cell medium.
(2) Spleen mononuclear lymphocytes after CFSE labeling are infected with H9N2 subtype AIV
Taking 2X 10 6 Individual CFSE-labeled chicken spleenNuclear lymphocytes were inoculated with H9N2 AIV at MOI=2 in biochemical reaction tubes, incubated for 1H and then replaced with 1640 medium containing 0.25. Mu.g/mLTPCK and 10% FBS, and incubated at 39℃for a further 5H.
(3) CFSE-APC stimulation of CFSE-spleen mononuclear lymphocytes
The chicken spleen mononuclear lymphocytes marked by CFSE are grouped and cultured according to a method of 3.1, the morphological change of the cells is observed every day, the cells are taken for flow detection, and the proliferation change of the cells is recorded.
3.4ELISPot assay to detect T cell effector responses
The chicken spleen mononuclear lymphocyte proliferation is stimulated in vitro according to the method 3.1, and the condition of IFN-gamma generation after the activation of the chicken spleen mononuclear lymphocyte is detected by an ELISPot test, which comprises the following specific steps:
(1) Activated PVDF96 well plates: adding 15 mu L of 35% ethanol into each hole, reacting for 1min at most, and washing with sterile water for 5 times;
(2) Coating an antibody: diluting anti-chicken IFN-gamma to 15 mug/mL, adding 100 mug/hole, and acting at 4 ℃ overnight;
(3) Closing: pouring the coating liquid, washing for 5 times by using DPBS, and then buckling a dry plate on sterilized absorbent paper. Adding RMPI1640 culture medium containing 10% FBS into PVDF 96-well plates at a ratio of 200 μl/well, and blocking at room temperature for 30min;
(4) Stimulation: the blocking solution was removed and 100. Mu.L of lymphocyte suspension at a cell concentration of 1X 107cells/mL was added to each well of H9N2 AIV-stimulated, positive and negative control cells. After all samples are added, putting the PVDF96 well plate into a CO2 incubator, and reacting at 37 ℃ for 24-48 hours;
(5) Secondary antibody incubation: after the culture is finished, the cells are thrown off, the cells are washed by DPBS for 5 times, 100 mu L of biotin-labeled secondary antibody with the concentration of 1 mu g/mL is added into each hole, and the cells are incubated for 2 hours at room temperature;
(6) HRP incubation: the secondary antibody liquid is thrown away, the secondary antibody liquid is washed by DPBS for 5 times, and then 100 mu L of streptavidin marked HRP is added into the hole for incubation for 1h at room temperature;
(7) Color development: throwing away HRP incubation liquid, washing with DPBS for 5 times, adding 100 mu LTMB chromogenic substrate into each hole, reacting at room temperature or 37 ℃ for 15-30min, throwing away chromogenic liquid when obvious spots appear in the positive control group, stopping chromogenic with pure water, and air-drying the plate.
Experimental results:
in vitro culture of h9n2 subtype AIV memory tcrγ6+cd8α+ T cells
h9N2 subtype AIV in vitro stimulated TCRγ6+CD8α+ T cell proliferation culture
(1) In vitro stimulation of cell morphology changes after proliferation of tcrγ6+cd8α+ T cells by AIV of H9N2 subtype
The cell morphology was observed microscopically at various times after proliferation culture of h9n2aiv stimulated chicken tcrγ6+cd8α+ T cells. Compared with the cells of the control group, the cells of the H9N2 AIV-stimulated group became larger and round, and an aggregation growth phenomenon occurred (FIG. 4), and the number of cell death increased with the increase of time.
(2) CFSE markers to detect T cell proliferation
CFSE labeling and flow detection of chicken spleen lymphocytes were performed according to the method in 3.3, and flow results were analyzed with FlowJo software according to the flow gate strategy shown in fig. 5.
The flow results are shown in FIGS. 6a-6c and 7, and the proliferation peaks of ConA and H9N2 AIV stimulated cells appear on the 3 rd day after culture, which indicates that the proliferation of virus memory T cells can be promoted after the H9N2 AIV stimulates chicken spleen lymphocytes.
(3) Flow detection of TCRγ6+CD8α+ T cell ratio changes after H9N2 subtype AIV stimulated proliferation
After 3 days of culture, cells of the H9N2 subtype AIV-stimulated and control groups were stained for TCRγ6+CD8α+T cells, e.g., 2.4.2. Flow staining loop gate strategy as in fig. 8, flow results were analyzed with FlowJo software and statistically analyzed. As a result, as shown in fig. 9a, H9N2 AIV stimulated proliferation of memory tcrγ6+cd8α+ T cells in chicken spleen lymphocytes, and the ratio of tcrγ6+cd8α+ T cells to cell number was significantly higher than that of the negative control group (P < 0.05) after H9N2 AIV stimulation.
3.5ELISPot assay to detect T cell effector response
To further demonstrate the effector response of H9N2 AIV after stimulation of chicken T cell proliferation, 3 samples of cells from the H9N2 AIV stimulated, positive control and unstimulated groups were collected and subjected to ELISpot assays.
The results are shown in FIG. 10b, on the premise that the positive control group is established, the H9N2 AIV stimulated group and the positive control group can obviously generate more spots (P <0.01 and P < 0.001) compared with the negative control group, which indicates that chicken T cells stimulated and proliferated and activated in vitro can secrete IFN-gamma to exert effect, and the spot results of three independent repeated experiments are shown in FIG. 10 a.
Comparative example 1
At 28 days post challenge, peripheral blood was collected and PBMCs were isolated using peripheral blood lymphocyte separation fluid, and Antigen Presenting Cells (APC) were incubated and CFSE stained according to the method in 3.2 (2).
Co-incubation of CFSE-APC and CFSE-PBMC according to the method of 3.2 (3), in vitro stimulated culture of H9N 2-specific TCRγδ + CD8 + T cells, but cells do not appear in aggregated form, while flow detection of CFSE does not appear as a proliferation peak. As shown in fig. 11-13:
figures 11 and 12 can be seen to: neither the control nor the experimental group showed a clumping of the cell morphology and a cell death morphology.
The flow label results of fig. 13 can be seen: CFSE-labeled cells were not observed to proliferate by flow-through detection, and the number of dead cells decreased with increasing days of culture after stimulation.
Results overview:
1. from the analysis of example 2 and comparative example 1, it was found that although PBMC and spleen mononuclear lymphocytes can both exhibit a significant increase in the proportion of tcrγ6+cd8α+t cells, PBMC could not be modeled stably;
the possible reasons for this are: the PBMC survival rate after the same condition stimulation is lower, and the PBMC will die in the same post-culture and non-stimulated groups.
2. In the experimental process, the establishment of the in-vitro culture model is mainly based on experimental materials obtained in early animal virus attack experiments, and because the virus attacked by early animal virus attack is H9N2 subtype low-pathogenicity avian influenza virus, the whole process of the invention is based on the experimental materials, and the later culture is the memory T cells with H9N2 specificity.
The invention adopts other highly pathogenic avian influenza, and the chickens die after the toxicity attack, so that experimental materials can not be obtained.
This also illustrates the specificity and severity of the selection of viral objects of the present invention.
3. In the early experiments, the optimal ratio of lymphocytes to antigen presenting cells was selected, and neither too high nor too low (e.g., 6:1 and 4:1) stimulated T cell proliferation, demonstrating the specificity of the invention in selecting the ratio of lymphocytes to antigen presenting cells.
4. Early experiments screened the best vaccination dose, either too high or too low (e.g., moi=1 or moi=3) to stimulate T cell proliferation in the case of challenged chickens. The invention has specificity when selecting the toxin attacking dosage.
The applicant states that the process of the invention is illustrated by the above examples, but the invention is not limited to the above process steps, which do not mean that the invention must be carried out in dependence on the above process steps. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of selected raw materials, addition of auxiliary components, selection of specific modes, etc. fall within the scope of the present invention and the scope of disclosure.
Claims (3)
1. Chicken tcrγδ + CD8α + The method for establishing the T cell immune response model is characterized by comprising the following steps of:
step 1: taking lymphocytes of spleen of a chicken infected with H9N2 subtype avian influenza virus and inoculating the lymphocytes with the H9N2 subtype avian influenza virus to obtain antigen presenting cells;
step 2: mixing and culturing spleen lymphocytes and antigen presenting cells of chicken infected with H9N2 subtype avian influenza virus;
in the step 2, the ratio of the number of lymphocytes in the spleen of the chicken infected with the avian influenza virus to the number of antigen presenting cells is: 5:1, a step of;
in the step 1, the H9N2 subtype avian influenza virus is inoculated with the inoculum size of MOI=22×10 6 A lymphocyte;
lymphocytes of the spleen of the chicken are derived from 4-week-old SPF chicken;
the preparation method of the antigen presenting cell specifically comprises the following steps: taking spleen lymphocytes of chickens infected with avian influenza virus, inoculating the lymphocytes with the avian influenza virus, incubating the virus for 0.5-2 h, replacing the virus with 1640 medium containing 0.25 mug/mLTPCK-treated trypsin and 10% FBS, and incubating the virus at 39 ℃ for 4-6 h to obtain the antigen presenting cells.
2. The method for establishing a model according to claim 1, wherein the method for preparing lymphocytes of the spleen of a chicken infected with avian influenza virus comprises the following sub-steps:
sub-step 11: killing chicken and taking spleen; the chicken is infected with avian influenza virus;
sub-step 12: cutting spleen into small pieces, adding a proper amount of RP-10, blowing with a Pasteur pipette for multiple times, and filtering to obtain cell suspension;
sub-step 13: adding the cell suspension to the upper layer of a single nuclear lymphocyte separating medium of an equal volume of chicken viscera tissue, centrifuging, sucking the centrifuged intermediate layer cells, washing twice in a washing liquid, centrifuging, and re-suspending the cells by RP-10 to obtain the spleen lymphocytes of the chicken.
3. The method for building a model according to claim 1, wherein the step 2 specifically comprises: the spleen lymphocytes of chickens infected with avian influenza virus were used at 3×10 6 The density of individual cells/mL was plated in 48-well plates, and antigen presenting cells were added after 6h of culture.
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| CN113105531A (en) * | 2021-03-24 | 2021-07-13 | 中国农业大学 | T cell epitope polypeptide of avian infectious bronchitis virus N protein and application thereof |
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