All authors contributed to the article and approved the submitted version. Funding This study was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded from the Ministry of Health & Welfare, Republic of Korea (HI14C3417). Conflict of Interest H-JS and H-IC were employed by the company ViGenCell Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that may be construed like a potential conflict of interest. Acknowledgments We thank the Catholic Hematopoietic Stem Cell Standard bank, College of Medicine, The Catholic University or college of Korea, Seoul, Republic of Korea, for typing HLA. Supplementary Material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2020.602014/full#supplementary-material Click here for more data file.(622K, pdf). each HLA-DR, -DQ, and -DP locus. Interestingly, the frequencies of HLA-DR alleles and the positivity of specific allotypes showed an inverse correlation. One allotype within individuals is dominantly used in CD4+ T cell response in 49% of donors, and two allotypes showed PLCG2 that in 7% of donors, and any positive response was recognized in 44% of donors. Actually if one individual experienced several dominating alleles, CD4+ T cell reactions tended to become restricted by only one of them. Furthermore, CD8+ and CD4+ T cell reactions by HLA class I and class II were correlated. Our results demonstrate the CD4+ T cell preferentially make use of a few dominating HLA class II allotypes within individuals, similar GSK591 to CD8+ T cell response to CMV pp65. IFN- ELISPOT Assay The CMV pp65-specific CD4+ T cell reactions restricted by a single HLA class II allotype were measured by IFN ELISPOT assay as explained previously (34). Briefly, 100 ul of 5104 antigen-pulsed aAPCs and 5105 CD4+ T cells were incubated for 20h at 37C. The spot forming cells were counted using an AID ELISPOT Reader System (AID Diagnostika GmbH). The magnitude of HLA-restricted antigen-specific CD4+ T cell response was determined as [(response to aAPCs expressing HLA pulsed with peptide swimming pools) C (response to aAPCs expressing HLA)] C [(response to GSK591 aAPCs pulsed with peptide swimming pools) C (response to aAPCs)]. Statistical Analysis Statistical analyses were performed using GraphPad Prism 7 software. The results were from a single experiment on 45 donors. Statistical significance was determined by one-way ANOVA, Pearsons correlation analysis, Welchs 0.05 were considered significant. GraphPad Prism 7, NumPy (36), FlowJo v10 (BD) were used for generating numbers. Graphs are indicated as means standard deviation (SD) or standard error of the mean (SEM), and the sample sizes were offered in the numbers. Results Establishment of aAPC Panels Expressing Solitary HLA Class II Allele To measure solitary HLA allotype-restricted CD4+ T cell response, K562 cell collection expressing CD80, CD83, and CD137L was transduced with solitary HLA-DR, -DQ, or -DP allele ( Number GSK591 1A ). The aAPCs expressing 20 HLA-DR alleles, 16 HLA-DQ alleles, or 13 HLA-DP alleles were established to protect the common alleles, which are at frequencies above 1% in Koreans. It was confirmed that these aAPCs indicated 95% or more of each allele ( Supplementary Number S1 ). The antigen-specific CD4+ T cells were detected high in the concentrations of 6 nM to 60 nM of pp65 peptide pool, so the concentration of 60 nM was used in subsequent studies. ( Numbers 1BCD ). Open in a separate window Number 1 Optimization of artificial antigen-specific CD4+ T cells restricted by solitary HLA class II allotype. (A) K562 was transduced with CD80, CD83, CD137L, and DRA*01:01/DRB1*09:01 and purified by FACS to generate aAPC. (B) 1105 CD4+ T cells of HD21 were stimulated with 1104 aAPC loaded with cytomegalovirus (CMV) pp65 peptide pool at a concentration of 600 nM, 60 nM, 6 nM, 0.6 nM, or 0.06 nM, respectively. Data is definitely offered as mean SDs of triplicates. n.s., not significant; *P 0.05, ***P 0.001, ****P 0.0001 by one-way ANOVA. (C) Representative membrane manifestation of HLA-DR, -DQ, and -DP molecules on aAPCs indicated by HD09. (D) Representative IFN- ELISPOT assay of CD4+ T cells cocultured with aAPCs stably transduced with nothing (No HLA), or the HLA-DR, -DQ, and -DP alleles indicated by HD09 pulsed with nothing (-pp65) or CMV pp65 (+pp65). Table 1 shows the HLA class II genotypes of 45 healthy donors used in this study. Since the HLA class II is definitely polymorphic in the alpha chains and the beta chains, an individual can communicate heterodimers up to four. Consequently, based on haplotype analysis, probably the most probable combination of alpha chains and beta chains of HLA-DQ and HLA-DP was identified and applied to this study. Since HLA-DR offers very low polymorphism in the alpha chain, the most frequent allele, DRA*01:01, was.
