Comprehensive Analysis of CD4+ T Cell Responses to CMV pp65 Antigen Restricted by Single HLA-DR, -DQ, and -DP Allotype Within an Individual

Abstract

Within an individual, six different HLA class II heterodimers are expressed co-dominantly by two alleles of HLA-DR, -DQ, and -DP loci. However, it remained unclear which HLA allotypes were used in T cell responses to a given antigen. For the measurement of the CD4+ T cell responses restricted by a single HLA allotype, we established a panel of artificial antigen-presenting cells (aAPCs) expressing each single HLA allele of 20 HLA-DRB1, 16 HLA-DQ, and 13 HLA-DP alleles. CD4+ T cell responses to cytomegalovirus (CMV) pp65 restricted by single HLA class II allotype defined in 45 healthy donors. The average magnitude of CD4+ T cell responses by HLA-DR allotypes was higher than HLA-DQ and HLA-DP allotypes. CD4+ T cell responses by DRA*01:01/DRB1*04:06, DQA1*01:02/DQB1*06:02, DPA1*02:02/DPB1*05:01 were higher among the other alleles in 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 that in 7% of donors, and any positive response was detected in 44% of donors. Even if one individual had several dominant alleles, CD4+ T cell responses tended to be restricted by only one of them. Furthermore, CD8+ and CD4+ T cell responses by HLA class I and class II were correlated. Our results demonstrate that the CD4+ T cell preferentially use a few dominant HLA class II allotypes within individuals, similar to CD8+ T cell response to CMV pp65.

Comprehensive Analysis of CD4+ T Cell Response Cross-Reactive to SARS-CoV-2 Antigens at the Single Allele Level of HLA Class II

“…The magnitude of response by a locus was calculated by summing the responses of two alleles in each HLA-DR, -DQ, and -DP locus within an individual to understand which HLA class II locus was preferentially used in the response. Similar to the CMV pp65-specific CD4 + T cell response (26), the magnitude of response by the HLA-DR locus was significantly higher than that by the HLA-DQ locus (Figure 1A, one-way ANOVA, p = 0.0359). The magnitude of response of the HLA-DP locus was significantly higher than that of the HLA-DQ locus (Figure 1A, one-way ANOVA, p = 0.0002).…”

“…The membrane expression of HLA-DR was higher than that of HLA-DQ and -DP in peripheral blood mononuclear cells and aAPCs transduced with a single allele at the same MOI (26,30), suggesting that HLA-DR-restricted T cells are highly for selection against pathogens. Unlike the response to cytomegalovirus pp65, the SARS-CoV-2-specific T cell response restricted by HLA-DP was significantly higher than that by HLA-DQ, and the magnitude of the response was similar to that by HLA-DR (Figure 1).…”

“…Moreover, one allotype-restricted T cell responded mainly to one antigen among spike, membrane, and nucleocapsid proteins in 5/11 individuals (Figure 3A). The T cell response to pp65 of cytomegalovirus, which has infected most of the world's population, also showed the allele dominance within an individual (26,27). In CMV-specific T cell responses, diverse high-affinity clones were stimulated at early time points after infection; however, at late time points, a few low-affinity clones predominated (35,36).…”

“…As previously described ( 26 ), 5 × 10 6 293TN producer cells (System Biosciences) were transfected (Lipofectamine 2000; Invitrogen) with 1.3 pmol psPAX2 (RRID: Addgene_12260), 0.72 pmol pMD.2G (RRID: Addgene_12259), and 1.64 pmol single HLA class II allele-encoding pCDH, and the supernatant was harvested after 2 days. The K562-based aAPCs, which do not express HLA molecules, were transduced with lentiviruses encoding an alpha and a beta chain at a multiplicity of infection (MOI) of 20.…”

“…We further estimated the most likely haplotype for HLA-DQ and DP within individuals based on haplotype frequency. We established single HLA-DR, -DQ, and -DP allotype-expressing aAPCs of a haplotypic pair in 42 donors in addition to those previously established (Figure S1) (26). CD4 + T cells were stimulated with an allotype-matched aAPCs pulsed with a mixture of peptide pools of the spike, nucleocapsid, and membrane proteins.…”