Genome-Wide Approach to the CD4 T-Cell Response to Human Herpesvirus 6B

Abstract

Human herpesvirus 6 (HHV-6) and cytomegalovirus (CMV) are populationprevalent betaherpesviruses with intermittent lytic replication that can be pathogenic in immunocompromised hosts. Elucidation of the adaptive immune response is valuable for understanding pathogenesis and designing novel treatments. Knowledge of T-cell antigens has reached the genome-wide level for CMV and other human herpesviruses, but study of HHV-6 is at an earlier stage. Using rare-cell enrichment combined with an HLA-agnostic, proteome-wide approach, we queried HHV-6B-specific CD4 T cells from 18 healthy donors with each known HHV-6B protein. We detected a low abundance of HHV-6-specific CD4 T cells in blood; however, the within-person CD4 T-cell response is quite broad: the median number of open reading frame (ORF) products recognized was nine per person. Overall, the data expand the number of documented HHV-6B CD4 T-cell antigens from approximately 11 to 60. Epitopes in the proteins encoded by U14, U90, and U95 were mapped with synthetic peptides, and HLA restriction was defined for some responses. Intriguingly, CD4 T-cell antigens newly described in this report are among the most population prevalent, including U73, U72, U95, and U30. Our results indicate that selection of HHV-6B ORFs for immunotherapy should consider this expanded panel of HHV-6B antigens. RESULTS Peripheral blood of healthy donors contains HHV-6B-specific CD4 T cells that can be enriched in vitro.The initial cohort of 53 healthy donors was 34% male, and the median age at the time of blood draw was 40 years (range, 21 to 68 years). To ascertain the frequency of HHV-6B-specific CD4 T cells in peripheral blood, peripheral blood mononuclear cells (PBMCs) were assayed by an interleukin-2 (IL-2)/interferon gamma (IFN-␥) intracellular cytokine cytometry (ICC) assay (Fig. 1A). Donors had virus-specific cell populations that were of low abundance but clearly discernible in most subjects. Responses were typically less than 0.1% of total CD4 T cells, with an overall median of 0.048% (Fig. 1B).Given the low abundance of HHV-6-reactive CD4 T cells in PBMCs, we enriched these cells from 42 donors prior to interrogation of the HHV-6B protein library. These donors were chosen based on PBMC availability and a detectable response to HHV-6B ex vivo.

Proteome-Wide Zika Virus CD4 T Cell Epitope and HLA Restriction Determination

“…In addition to these practical and medical considerations, the magnitude of the pathogen-specific T cell response, especially at memory time points, can be very low, providing an additional challenge to direct ex vivo approaches. For example, the integrated abundance of HHV-6B-reactive CD4 T cells in PBMC is on the order of 0.05% (19). Using cells expressing multiple AIM molecules (CD137, CD69, and CD154), we were able to reliably enrich virusreactive cells to levels of up to 50% (19).…”

“…For example, the integrated abundance of HHV-6B-reactive CD4 T cells in PBMC is on the order of 0.05% (19). Using cells expressing multiple AIM molecules (CD137, CD69, and CD154), we were able to reliably enrich virusreactive cells to levels of up to 50% (19). This ZIKV project featured both low immune responses and very limited blood volumes.…”

“…We modified AIM-based sorting (6,8,(17)(18)(19) to enrich ZIKVreactive cells. Thawed PBMC were cultured at 2-4 3 10 6 cells per well in 24-well plates in 2 ml/well T cell medium (TCM; RPMI 1640 with 25 mM HEPES, 1% penicillin-streptomycin, 2 mM L-glutamine, 5% FCS [Thermo Fisher Scientific], and 5% human serum [Valley Biomedical, Winchester, VA]).…”

“…The threshold for CD137 positivity was set using PBMC from separate persons seropositive for HSV-1 and stimulation with UV-treated HSV-1 Ag, as described previously (6). CD137 high cells were expanded polyclonally once with PHA as mitogen and further expanded with anti-CD3 mAb as mitogen (19).…”

“…For some donors, the proportion of CD4 T cells expressing CD137 was ,0.01%, and there was no mathematical difference from mock stimulation, but cells were collected regardless. Previously, we used PHA as a functional viability control, leading to CD137 expression by 5-50% of T cells (8,19). We omitted this to use as many cells as possible for ZIKV stimulation.…”