Aim: S. aureus bloodstream infections (SAB) are a frequent complication for hospitalized patients. Prior studies associated SAB risk with single nucleotide variants (SNV) within the DRA1/DRB1 HLA class II region (DRB1*04:01). To further investigate, we conducted a regional genome association study in 3,194 inpatients (1,616 cases, 1,578 controls) and CD4 T cell activation studies testing different HLA-DRB1 alleles.
Methods: Illumina GSA array data and TopMed Imputation Server imputed 38,077 HLA SNV (chr6: 29,000,111-33,999,677). Michigan Imputation Server imputed HLA class II haplotypes. 3,236 SNVs (or haplotypes) were examined via Logistic regression constructed in GENESIS and 10 principal components (generated by PCAir) controlled for population structure. CD4 cells were isolated from 35 controls and cocultured with homozygous, DRB1-matched B cell antigen presenting cells (APCs). APCs were pulsed overnight with increasing concentrations of inactivated S. aureus (multiplicity of infection=MOI: 0.5, 5, 50) or no antigen (MOI=0). Fig1A: CD4 T cell activation (CD69+ CD25+) was determined via flow cytometry (BD Fortessa, FlowJo) using CD4-PerCP-Cy5.5, CD69-PE, and CD25-APC antibodies. Data was analyzed and means calculated in Prism.
Results: No HLA-DRB1 allele (one or two field) was significantly associated with SAB, but DRB1*03:01 or DRB1*04:01 genotypes were increased in SAB cases vs controls (OR=1.1, 0.96-1.27; p=.17 and OR=1.12, 0.95-1.32; p=.17, respectively). Considering coding sequences, 3 of 167 amino acid changes in pocket 4 (P4) of DRB1 were associated with SAB. 71K was positively associated with SAB (OR=1.12, 1.01-1.24; p=.03) and 67I and 71E were negatively associated (OR=0.91, 0.83-0.99; p=.02 and OR=0.9, 0.77-0.98; p=.03, respectively). Notably, 71K is present on both DRB1*03:01 and DRB1*04:01 alleles. CD4 T cell activation (Fig 1B) was highest when S. aureus peptides were presented by DRB1*07:01 APCs, containing protective 67I and 71R in P4, compared to DRB1*03:01 and DRB1*04:01 APCs containing risk 71K in P4. However, lowest CD4 T cell activation was observed for DRB1*15:01 APCs that share the protective 67I with DRB1*07:01 but differ at 71A.
Conclusion: We provide genetic and in vitro evidence that HLA-DRB1 P4 variation influences S. aureus peptide presentation and activation of CD4 T cells, supporting that S. aureus blood infection is in part influenced by HLA variation.
