| In the current market of medical diagnostic reagents, the kits detecting pathogen-specific genes, antigens and antibodies are very popular, thus face to strong competition. But no manufacturer focuses on the pathogen-specific T cells detection kit.Antigen-specific T lymphocytes (AST) are the core cells that mediate the adaptive immune response. They play a crucial role in controlling pathogen infections, monitoring tumor cells,and pathogenesis of autoimmune diseases, and so on. It is well known that cellular immune responses are more important than humoral immunity in antiviral and anti-tumor effectes, thus T cells are more important than B cells secreting antibody in infection or tumor cases. The enumeration and functional evaluation of diverse T cell populations in an individual’s T cell repertoire may provide a detailed picture of the physiological status, pathological course, and dynamic immune response to certain antigens associated with pathogens, allergens, cancer cells, self-proteins, allograft, as well as vaccines, and can be used to characterize individual-specific cellular immune response patterns that might help guide the design of immunological therapies.The detection techniques for AST have been developed rapidly from the traditional 51Cr release assay, limiting dilution assay (LDA), intracellular cytokine staining (ICS) to enzyme-linked immunospot assay (ELISPOT), pMHC multimer plus flow analysis, and pMHC protein array or chip. Although each technique has its advantages, but AST detection still sufferes from the obvious disadvantages of these motheds. Few patients have been monitored for AST in clinics in China by now. It is worthy to develop a convenient method for the concurrent emueration and functional evaluation of AST cells without the requirement of expensive instruments and HLA genotyping, which will facilitate the routine analysis of patient-specific immune response pattern to certain antigen in translational studies in the anticipation of precision medicine.Objective:In the present study, pMHC multimers, artificial antigen-presenting cells (aAPCs),magnetic-activated cell sorting (MACS), and ELISPOT techniques have been integrated into a micro well by coupling pMHC multimers onto cell-sized magnetic beads to characterize AST cell populations in a magnetic field in a 96-well microplate pre-immobilized with cytokine-capture antibodies. This method, termed artificial antigen-presenting cell microplate(aAPC-microplate), allows enumeration and local cytokine production of multiple AST cells in a single assay without using flow cytometry or fluorescence intensity scanning. First, we used the in-house H-2Kb/OVA257-264 single-chain trimer (SCT) molecules to prepare aAPC-beads, and detect the frequency and reactivity of OVA257-264 antigen-specific CD8+ T cell in the OT-1 trangenic mice by aAPC-microplate followed by methodological evaluation.Then,we generated HLA-A2/HBVpeptide mutilmers in a same manner, and fabricated aAPC-microplate to enumerate and functionally evaluate HBc18-27/HBs183-191antigen-specific CD8+ T cells in the peripheral blood mononuclear cells (PBMCs) from chronic hepatisis B patients, in a preliminary clinical validation for this new method.Methods:1) H-2Kb-Ig/OVA257-264 SCT fusion gene which contains OVA257-264, (GS4)3, β2m,(GS4)4,and H-2Kb (α1/α2/β3) fragments was synthesized using the overlap extension PCR, and inserted into plasmid pET28a by homologous recombination technology followed by expression in BL21 (DE3). Then,H-2Kb/OVA257-264 SCT molecues and tetramers were prepared and the structural conformation and binding ability to antiden-specific TCR were further confirmed. Finally, we used the in-house H-2Kb/OVA257-264 SCT aAPC-microplate to detect OVA257-264 antigen-specific CD8+ T cell from OT-1 transgenic mice, with the methodological evaluation for specificity, accuracy, sensitivity,reproducibility,and the comparison with commercial H-2Kb-Ig/OVA257-264 dimer staining plus flow cytometry by the two-tailed, paired Student’s test and Pearson correlation analysis. Meanwhile, aAPC-microplate method was also compared with traditional ELISPOT assay.2) Eighty-six healthy donors and 118 patients with type 2 diabetes mellitus (T2DM) were enrolled in HLA-A and B loci genotyping, respectively, using the gold-standard method,termed polymerase chain reaction with sequence-based genotyping (PCR-SBT). Then,recombinant plasmids of HLA-A (α1/α2/α3) for 13 alleles with a frequency of > 1% in Chinese population were constructed, respectively, while the recombinant genes of HBVpeotide-β2m for 8 antigenic peptides of HBV antigens were fused by traditional technology. The CTL epitopes derived from HBV antigens and restricted by HLA-A*01:01, A*03:01,A*30:01,A*31:01,A*32:01,A*33:03, or A*6801 were predicted by using the data bases of SYFEPITHI, BIMAS, SVMHC, IEDB, EpiJen, and NETMHC.3) Overlap extension PCR and homologous recombination technology were utilized to construct the SCT fusion genes and recombinant plasmids of HLA-A*0201/HBc18-27,HLA-A*0201/HBs183-191, HLA-A*0203/HBc18-27, HLA-A*0203/HBs183-191, and HLA-A*0201/HBc18-27. The resulting HLA-A2/HBc18-27/HBs183-191 complexes,tetramers,aAPC-beads,and aAPC-microplate were prepared. Concurrent enumeration and functional evaluation of HBc18-27/HBs183-191antigen-specific CD8+ T cells in PBMCs was carried out for the chronic hepatisis B patients and healthy donors. The results were further compared with that analyzed by traditional HLA-A2-Ig/HBc18-27/HBs183-191 dimer staining plus flow cytometry and ELISPOT assay.Results:1) H-2Kb/OVA257-264 SCT protein was successfully constructed and expressed. Magnetic beads pre-coupled with H-2Kb/OVA257-264 complexes could be strongly stained by the PE-labeled anti-mouse H-2Kb mAb (AF6-88.5), a conformation-specific mAb, as analyzed in flow cytometry, implying the correct conformation and appropriate orientation as well as accessibility of H-2Kb/OVA257-264 SCT molecules onto beads.Furthermore, PE-labeled H-2Kb/OVA257-264 tetramers were generated to detect OVA257-264 antigen-specific CD8+ T cells from four individual OT-1 mice by flow cytometry. No significant difference was found between the in-house H-2Kb/OVA257-264 tetramers staining and commercial H-2Kb-Ig/OVA257-264 dimers staining, suggesting the TCR-binding ability of in-house H-2Kb/OVA257-264 molecules similar to its commercial counterpart.2) The splenic lymphocytes from 7 OT-1 mice were detected by the aAPC-microplate method and conventional H-2Kb-Ig/OVA257-264 dimers staining plus flow cytometry for the frequency of OVA257-264-specific CD8+ T cells. No statistical difference was found between the aAPC-microplate method and the conventional flow cytometry for seven individual OT-1 mice tests (p=0.2464). The correlation coefficient (r value) between two methods was 0.946, P<0.01,Y=0.936X + 3.262, as analyzed by two-tailed Pearson. The result suggested that the methodological accuracy compared well with conventional pMHC multimer flow staining. Cells were collected from each well after aAPC-bead sorting and stained with H-2Kb-Ig/OVA257-264 dimer followed by flow cytometry. The purity of H-2Kb-Ig/OVA257-264 dimer+/CD8+ T cells was increased to 92.35%, 91.22% and 92.10% in tree independent assays. These data indicate a specificity of > 90% for the aAPC-microplate method. Seven OT-1 mice were detected independently by the aAPC-microplate method for the enumeration of OVA257-264-specific CD8+ T cells. The within-run coefficient variation (CV) of five cell density groups (3 wells per density) in each independent test was 6.44%, 4.01%, 6.31%, 9.08%, 6.63%, 4.24% and 3.11%,respectively,representing an acceptable well-to-well reproducibility. Moreover,a cell sample from the same individual OT-1 mouse was detected repeatedly in three independent experiments at different time points by the aAPC-microplate method. The proportion of OVA257-264-specific CD8+ T cells was 25.47%,25.59% and 24.49%. The within-run CV was 3.11%, 4.41% and 10.62%, and the between-run CV was 2.40%. The methodological precision meets to the national standard for biological products approvment of China. These data imply a reliable reproducibility and precision of the aAPC-microplate method. Comparison of the aAPC-microplate method with H-2Kb-Ig/OVA257-264 dimers staining assay was further carried out in the five cell samples with different concentrations of OVA257-264-specific CD8+ T cells (10%,1%,0.1%,0.05%,and 0.01%). No statistical difference was found between the two methods (p=0.3973).The correlation coefficient (r value) between two methods was 0.998, P<0.01, Y=0.943X+ 0.21, as analyzed by two-tailed Pearson. The detection limit of the aAPC-microplate method was 0.01% - 0.05%, which meets the requirement for clinical tests and is comparable to that obtained by standard state-of-the-art flow cytometry.3) Three OT-1 mice were detected independently by both the aAPC-microplate method and traditional ELISPOT assay for the functional evaluation of OVA257-264-specific CD8+ T cells. The percentage of IFN-y-secreting cells in the OVA257-264-specific CD8+ T cell population was 43.83%, 41.87%, and 48.99% after stimulation with ConA. As detected by traditional ELISPOT assay, the percentage of IFN-y-secreting cells in the population of lymphocytes isolated from OT-1 mouse spleen was 4.44%, 4.01% and 4.98%,respectively, after stimulation with OVA257-264 antigenic peptide. Due to the distinct methodological principles and presentation of results, we cannot directly compare the results obtained by the aAPC-microplate method and traditional ELISPOT assay.4) We have collected 13 HLA-A alleles and 32 HLA-B alleles with a frequency of > 1% for each allele in Chinese population; constructed and experessed 13 recombinant plasmids of the HLA-A alleles (al/a2/a3) and 8 fusion genes of peptide-(32m with different antigenic peptides of HBV antigens; and obtained several high-affinity antigenic epitopes derived from HBsAg (P03138),HBcAg (P03146),HBpol (P03156),or HBx (P03165),for each of HLA-A*01:01, A*03:01, A*30:01, A*31:01, A*32:01, A*33:03, and A*6801,by prediction based on the data bases.5) HLA-A*0201/HBc18-27, HLA-A*0201/HBs183-191, HLA-A*0203/HBc18-27 HLA-A*0203/HBs183-191, and HLA-A*0206/HBc18-27 SCT proteins were successfully constructed, experessed and refloded, and were coupled onto magnetic beads, respectively,to prepare HLA-A2/HBc18-27/HBs183-191 beads. Each type of HLA-A2/HBc18-27/HBs183-191 aAPC-beads was mixed together at a ratio of 1:1 to fabricate the HLA-A2/HBc18-27/HBs183-191 SCT aAPC-microplate.6) The percentages of HBc18-27/HBs183-191-specific CD8+ T cells in the PBMCs from 10 HLA-A2-positive chronic hepatisis B patients, 15 HLA-A2-negative chronic gepatisis B patients, and 15 HLA-A2-positive healthy donors were detected by the aAPC-microplatemethod and traditional flow cytometry. No statistical difference was found between the two methods for each subject group, with the p values of 0.0821, 0.0529, and 0.0594,respectively. The correlation coefficient (r value) between two methods was 0.980,P<0.01, Y=0.984X-0.05, as analyzed by Pearson test. Notably, HLA-A2-positive patients with chronic hepatisis B displayed obviously higher frequencies of HBc18-27/HBs183-191-specific CD8+ T cells in PMBCs than HLA-A2-negative patients or HLA-A2-positive healthy donors, who only presented background level of the AST cells in PBMCs (0.1957%±0.0672% vs. 0.0176% ± 0.0068%,0.1957% ± 0.0672% vs.0.0249% ±0.0077%).7) Ten HLA-A2-positve chronic hepatisis B patients were detected independently by the aAPC-microplate for the functional elvaluation of HBc18-27/HBs183-191-specific CD8+ T cells after aAPC-bead sorting. The percentage of IFN-y-secreting cells in the sorted HBc18-27/HBs183-i9i-specific CD8+ T cell population was 22.79% ± 3.40% after stimulation with PHA. However, the percentage of IFN-y-secreting cells in the T cell population was 36.35% ± 3.58% after stimulation with PHA. These data indicate that Chronic Hepatisis B patients displayed obviously higher frequencies of HBV-specific CD8+ T cells in PBMCs than healthy donors, but the reactivity of HBV-specific CD8+ T cells was lower than the unrelated T cells. This may be one of the reasons leading to chronic development of HBV infection.Conclusion:The aAPC-microplate fabricated using in-house pMHC multimer was successfully applied to enumerate and functionally evaluate OVA257-264-specific CD8+ T cells in a single assay. The high accuracy,specificity,reproducibility, and sensitivity of the aAPC microplate process compares well with conventional methods. The detection limit was down to 0.01% -0.05%. Concurrent enumeration and functional evaluation of HBc18-27/HBs183-191-specific CD8+ T cells in the PBMCs of chronic hepatisis B patients were carried out successfully by aAPC-microplate prepared using the in-house HLA,A2/4Bc18-27/HBs183-191 SCT multimers.No statistical difference was found between the aAPC-microplate method and traditional flow cytometry with a high goodness of fit. |
PDF Full Text Request