Enumeration And Functional Evaluation Of Antigen-specific T Cells By Magnetic AAPC-microplate Method

Antigen-specific T lymphocytes (AST) are the critical cells that mediate the adaptive immune response. They play an important role in anti-infection, anti-tumor, hypersensitivity, autoimmune diseases as well as in transplant rejection. Enumeration and functional evaluation of AST can directly reflect the specific cellular immune function in the body. However, compared with the antibody detection, the methods for analyzing AST are more difficult and complicated. By now, pMHC multimer technique is a gold standard for evaluating antigen-specific T cells. But the application is limited due to the rare commercial products. In view of the techniques which apply for analysis of AST, whether traditional ELISPOT or pMHC tetramers or pMHC chip, have their strengths and weaknesses. Each method alone cannot simultaneously analyze the enumeration and function. Thus it is worth exploring a simple and convenient method for the analysis of AST enumeration as well as reactivity simultaneously.In this report, we established a new and simple method named as aAPC-microplate technique with which to enumerate and functionalize AST in one-step. Cell-free artificial antigen-presenting cells (aAPC-beads) were made by covalently coupling commercial H-2Kb-Ig/OVA257-264 dimer onto cell-sized magnetic beads, followed by incubation with OT-1 mice spleen cells in a ratio of 1:1 in 96-well ELISPOT plate to sort and enumerate OVA antigen-specific T cells in magnetic aAPC-microplate, and further to evaluate the secretion of IFN-y by ELISPOT after the stimulation with PHA, OVA peptide plus anti-CD28 or aAPC-beads co-coupled with anti-CD28 and H-2Kb-Ig/OVA dimer. Further, because the commercial H-2Kb-Ig/dimer is very expensive, and in future we have to demonstrate the methodology with homemade polymer, so we need re-composite fusion gene with OVA257-264 antigenic peptides, β2m, extracellular domain of H-2Kb α chain, Avi tag and His6 tag. Then the recombinant H-2Kb-Ig/OVA SCT complex was expressed, extracted and purified. The preliminary results are summarized as followed:1. Enumeration of OVA antigen-specific T cells by the optimized aAPC-microplate procedure with CD8+ T cells sorting:spleen cells from transgenic 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 bead-sorted cells and the cells before sorting. R2 value is more than 0.99. The percentage of OVA antigen-specific T cells in the populations of five mice spleen lymphocytes was 11.27%,9.40%,12.62%,14.77% and 21.85%, respectively, as detected by aAPC-microplate method. In parallel, the percentage was 13.69%,10.13%,16.88%,16.77% and 21.45%, respectively, as detected by the traditional H-2Kb-Ig/OVA dimer staining and flow cytometry. No significant difference was found between the two groups (paired t test). The correlation coefficient (r value) between the two methods is 0.9280. However, (he percentage was increased significantly when analyzed by PE-anti-mouse TCR Vα2 and flow cytometry (15.94%,18.23%,32.08%,31.29% and 42.88%). The methodological specificity: When sorted by Kb/TRP2-beads which loading with irrelative antigenic peptide, the percentage was decreased markedly to around 0.45%. Furthermore, cells and beads were collected after magnetic sorting in aAPC-microplate, followed by H-2Kb-Ig/OVA dimer staining and flow cytometry. The data showed that the percentage of positive-staining cells was up to 84.48% and 89.80%(two independent experiments) after Kb/OVA-beads sorting while the percentage was 13.69% and 10.13% before sorting. The methodological repeatability:In an independent experiment, the average of CV values was 4.4% among five cell seeding amount groups for the same cell sample detection. Furthermore, the same cell sample was detected repeatedly in three independent experiments by aAPC-microplate method. The proportion of OVA antigen-specific T cells was 9.40%,9.77% and 9.47%. R2 value was 0.9959,0.9970 and 0.9960 respectively. CV value was 13.5%. These data implied that the optimized aAPC-microplate method revealed high specificity, accuracy, repeatability and high correlation coefficient with traditional methods.2. Enumeration and functional evaluation for OVA antigen-specific T cells in one-step: Spleen cells from two OT-1 mice were detected by aAPC-microplate method, the percentage of OVA antigen-specific T cells was 12.62%and 21.85%, which was similar with the percentages (16.88% and 21.45%) detected by H-2Kb-Ig/OVA dimer staining and flow cytometry. After magnetic sorting in aAPC-microplate, the cells were co-cultured for 24h with different forms and nature of the stimulus-es, followed by IFN-y detection with modified ELISPOT method. The percentage of IFN-y-secreted cells in the beads-sorted cells was 64.01%,47.30% and 41.62% under stimulus-es of PHA, OVA plus anti-CD28 and aAPC-beads, respectively. For another OT-1 mouse, the percentage of IFN-y-secreted cells was 64%,55.3% and 48.8%. These data demonstrated the feasibility of aAPC-microplate method to evaluate functionally AST.3. Here, the dominant T cell epitope of OVA antigen was connected with mouse β2m and a chain of H-2Kb molecules through flexible linker joint. The recombinant gene was constructed by SOE-PCR technique and cloned into recombinant pET28a plasmid, followed by protein expression in BL21 after the gene sequencing. Then the H-2Kb/OVA single chain protein (SCT) was extracted and purified, and further refolded by dilution renaturation method. Then the H-2Kb/OVA SCT complex was coated onto cell-size magnetic beads followed by PE-anti-H-2Kb mAb staining and flow cytometry. Finally, the PE-conjugated H-2Kb/OVA SCT tetramer was generated to detect the OVA antigen-specific T cells from OT-1 mice spleen cells:Data showed that the in-home generated H-2Kb/OV A SCT molecule has correct conformation and can effectively bind to specific TCR as compared with commercial H-2Kb/OVA dimer staining. These works will help us to re-characterize the aAPC-microplate method using the in-home generated pMHC multimers.