Enumeration Of HBV Antigen-specific T Cells By Magnetic AAPC-microplate Method

Antigen-specific T lymphocytes (AST) play a critical role in anti-infection, anti-tumor and autoimmune diseases. Their number and function directly reflect the antigen-specific cellular immune function in patient. Thus, dynamic monitoring for ASTs has an important value in pathogenesis research, therapeutic regimen option, curative effect evaluation and vaccine developing. During recent 20 years, the detection techniques for AST have been developed rapidly from the traditional Cr51 release assay, limiting dilution method, intracellular cytokine staining to the current gold-standard pMHC multimer plus flow analysis. But few patients have been monitored for AST in clinics by now. It is mainly due to these disadvantages:commercial pMHC multimers are very expensive and flow cytometry is needed; the lack of commercial products for all patients with different HLA genotypes and for multiple antigenic epitopes from a pathogen; failure to high-throughput detection both for quantity and function in one assay.In our previous works, the artificial antigen-presenting cells (Kb/OVA beads) were prepared by immobilizing the commercial H-2Kb-Ig/OVA257-264 dimer onto 4.5?m magnetic beads; then OVA antigen-specific CD8+ T cells from OT-1 mouse spleen lymphocyte population were detected both for quantity and reactivity in 96-well plate. The methodology evaluation demonstrates the feasibility of this method is named as magnetic artificial antigen-presenting cells plus microplate technology (aAPC-microplate). To develop the detection kit based on aAPC-microplate with in-house pMHC multimers, in this paper, we fist improved the pMHC polymer technology, generated in-house H-2Kb/OVA single-chain trimer (SCT) and confirmed its conformation and ability binding to TCR; then, we used the in-house SCT molecules instead of commercial ones to prepare aAPC-beads, detect OVA antigen-specific CD8+T cell by aAPC-microplate followed by methodology evaluation; Finally, we used the aAPC-microplate to enumerate HBV antigen-specific CD8+T cells from PBMC of chronic HBV-infected patients. The main results are summarized below: 1. Overlap extension PCR and homologous recombination technology were used for the first time to construct H-2Kb/OVA SCT plasmid, high purity SCT protein was obtained after refolded and nickel column purification, then the biotinylated H-2Kb/OVA SCT protein was immobilized onto streptavidin-coated magnetic beads. H-2Kb conformation-specific mAb staining and flow analysis demonstrated the correct conformation of in-house H-2Kb/0VA SCT molecules; Finally, PE-H-2Kb/OVA SCT tetramer was generated to detect OVA antigen-specific CD8+T cells by flow cytometry, and compared with commercial H-2Kb-Ig/OVA dimer staining. Similar results (38.09% vs.35.32%) implied the binding ability of SCT molecules with TCR.2. H-2Kb/OVA SCT beads were used to sort and enumerate OVA antigen-specific CD8+ T cells in 96-well plate. The remaining cells in each well were co-incubated with ConA for 24h and were detected for IFN-y secretion by ELISPOT assay to evaluate their reactivity. Spleen cells from OT-1 mouse were seed into ELISPOT microplate at five different amounts with three replicate wells in each amount group. The percentage of bead-sorted cells in each amount group was represented by the mean value of three replicate wells. The final percentage of OVA antigen-specific T cells was adjusted by the linear regression equation depending on the data from five amount groups. The methodological accuracy:there is a very good amount-depending relation and linear relation between the seeded cells and aAPC-bead-sorted cells. R2 value is more than 0.99. The methodological specificity:the percentage of OVA-specific CD8+T cells was up to 92.35% and 86.39% for two independent experiments after aAPC-beads sorting in microplate. The methodological reproducibility or precision:the average intra-assay CV values for five amount groups in one assay was 6.44% and 4.01% for two independent experiments; Furthermore, the same cell sample was detected repeatedly in three independent experiments by aAPC-microplate method, the inter-assay CV value was 3.22%. The precision meets to the national standard for biological products permission. Correlation analysis with traditional pMHC multimers staining:No statistical difference was found between aAPC-microplate and traditional FACS assay for 5 OT-1 mice tests (paired t test, p=0.2397), the correlation coefficient (r value) between the two methods is 0.983, P<0.01, Y=2.229+0.815X. Reactivity evaluation:the percentages of IFN-y secreting cells in OVA-specific CD8+ T cell population after ConA stimulation were 43.83% and 41.87% for two independent tests. In addition, traditional ELISPOT assay was performed in parallel. After incubation with OVA antigenic peptides, the percentages of IFN-y secreting cells in spleen lymphocyte population were 4.44% and 4.01%(for two OT-1 mice), which much lower than that detected by both aAPC-microplate and traditional FACS assay.3. Five in-house SCT protein (HLA-A*0201/HBc18-27?A*0201/HBe183-191?A*0203/HBc18-27? A*0203/HBe183-191 and A*0206/HBc18-27) were used to prepare HLA-A2/HBV beads; Then HBV antigen-specific CD8+ T cells were enumerated from PBMCs of chronic HBV-infected patients. Preliminary results were summarized below:flow cytometry and PCR-SBT were used for HLA-A2 genotyping.5 HLA-A2-positive patients were collected from more than 20 patients. As detected by traditional HLA-A2/HBV dimer staining plus FACS assay, the frequency of HBc18-27 and HBe183-191epitopes-specific CD8+ T cells in PBMCs was 0.15%,0.08%,0.25%,0.08% and 0.28% respectively. Meanwhile, the frequency was 0.10%,0.11%,0.28%,0.10% and 0.23%, respectively, as detected by aAPC-microplate. No statistical difference was found between the two methods as analyzed by paired t test (p=0.9454). The correlation coefficient (r value) was 0.893, P<0.05,Y=0.028+0.811X.In this study, the H-2Kb/OVA single chain trimer protein in-house generated by new method was proved correct conformation and binding ability with TCR; Then H-2Kb/OVA SCT was used to establish aAPC-microplate method to evaluate the number and reactivity of OVA antigen-specific CD8+T cell, suggesting the feasibility of aAPC-microplate technology which used in-house self-critical reagent; Finally, aAPC-microplate was be used to detect HBV antigen-specific CD8+T cells from PBMCs of chronic HBV-infected patients, no statistical difference was found between aAPC-microplate and traditional HLA-A2/HBV FACS assay. Theses data will pave the way to the aAPC-microplate clinical kit.