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. 2016 Mar 1;196(5):2205-2218.
doi: 10.4049/jimmunol.1502366. Epub 2016 Jan 25.

Extensive CD4 and CD8 T Cell Cross-Reactivity between Alphaherpesviruses

Affiliations

Extensive CD4 and CD8 T Cell Cross-Reactivity between Alphaherpesviruses

Lichen Jing et al. J Immunol. .

Abstract

The Alphaherpesvirinae subfamily includes HSV types 1 and 2 and the sequence-divergent pathogen varicella zoster virus (VZV). T cells, controlled by TCR and HLA molecules that tolerate limited epitope amino acid variation, might cross-react between these microbes. We show that memory PBMC expansion with either HSV or VZV enriches for CD4 T cell lines that recognize the other agent at the whole-virus, protein, and peptide levels, consistent with bidirectional cross-reactivity. HSV-specific CD4 T cells recovered from HSV-seronegative persons can be explained, in part, by such VZV cross-reactivity. HSV-1-reactive CD8 T cells also cross-react with VZV-infected cells, full-length VZV proteins, and VZV peptides, as well as kill VZV-infected dermal fibroblasts. Mono- and cross-reactive CD8 T cells use distinct TCRB CDR3 sequences. Cross-reactivity to VZV is reconstituted by cloning and expressing TCRA/TCRB receptors from T cells that are initially isolated using HSV reagents. Overall, we define 13 novel CD4 and CD8 HSV-VZV cross-reactive epitopes and strongly imply additional cross-reactive peptide sets. Viral proteins can harbor both CD4 and CD8 HSV/VZV cross-reactive epitopes. Quantitative estimates of HSV/VZV cross-reactivity for both CD4 and CD8 T cells vary from 10 to 50%. Based on these findings, we hypothesize that host herpesvirus immune history may influence the pathogenesis and clinical outcome of subsequent infections or vaccinations for related pathogens and that cross-reactive epitopes and TCRs may be useful for multi-alphaherpesvirus vaccine design and adoptive cellular therapy.

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Figures

Fig 1
Fig 1
CD4 T-cell cross-recognition of whole HSV-1. VZV-driven polyclonal CD4 T-cell responder lines were created from PBMC before (day 0) of 28 days after vOKA vaccination from persons of defined HSV serostatus (far left). The indicated antigens and PBMC as APC were used to detect cytokine responses by ICS. Gating is on live, CD3+ CD4+ cells. Numbers are percent gated live CD3+CD4+ responders with cytokine expression. At right, net responder cells expressing either IFN-γ or IL-2 or both for VZV compared to mock HET, or HSV-1 compared to mock Vero, are shown the proportion of HSV-1 responder cells compared to VZV responder cells. Day 0 in blue, day 28 in red.
Fig. 2
Fig. 2
A: Direct ex vivo PBMC cytokine responses (left) to VZV and mock viral antigens gated on live CD3+ CD4+ cells (not shown), and expression of CD137 18 hours after stimulation gated on live CD3+ cells. B: responses of sorted, expanded cells sorted for CD137 expression. Numbers in quadrants are percent of gated live CD3+CD4+ cells. C: Proliferative responses of bulk CD137high-origin UV-VZV-reactive CD4 T-cell line from subject 5 to whole virus and control antigens, and to each VZV protein. Top 8 VZV proteins indicated; O= ORF. Reactivity of same responders to each HSV-1/HSV-2 protein are also shown. The homologs of the top 8 VZV reactive proteins are color-coded. D: Proliferative responses of UV-HSV-1-reactive polylclonal CD4 T-cell lines from 18 participants to each HSV-1 and VZV protein. Proteins with homologs at top, followed by those found only in HSV-1 in the middle and only in VZV at bottom.
Fig. 3
Fig. 3
Cross-reactive CD4 T-cell antigens and epitopes. A: Proliferative responses by VZV-reactive polyclonal CD137high-origin CD4 T-cells to full-length or truncated VZV ORF68, HSV homologs, and negative control. B: Responses to 15-mer peptides at 1 μg/ml within reactive fragment 262-240. C, D: Dose-response for the same effectors to the indicated viral peptides for VZV-reactive polyclonal CD4 T-cells from a recent zoster vaccinee. All data from triplicate assays. E: Responses of CD4 T-cell clone from an HSV-seronegative person to whole viral and recombinant alphaherpes proteins. × axis labels are participant numbers (30-34) followed by clone number. F: Responses of CD4 T-cell clones to whole viral and peptide antigens (HSV-2 peptide details in Supplemental Table II).
Fig. 4
Fig. 4
Recognition of VZV-infected cells by polyclonal HSV-reactive CD8 T-cells derived from HSV-1 seropositive, HSV-2 seronegative healthy adults. A: CD137-selected effectors show brisk virus-specific-recognition of HSV-1-infected EBV-LCL with lesser activation when co-cultured with HLA-mismatched, HSV-1-infected EBV-LCL. B: The same effectors display virus- and self-restricted activation when co-cultured with VZV-infected autologous DFB. Cells in A and B are gated in live, CD8+ small lymphocytes and data are representative of triplicate assays. C: Polyclonal CD8 effectors purified with HLA A*0201 tetramers containing epitopes in the indicated HSV-1 proteins and specific for cross-reactive epitopes in VZV ORF18 or ORF34 show specific killing of DFB infected with VZV wild-type or vaccine strains in triplicate assays.
Fig. 5
Fig. 5
Presentation of full-length alphaherpes proteins to polyclonal CD8 T-cell lines selected from PBMC using cross-presentation of HSV-1 (top) or HSV-2 (bottom). Cos-7 cells were co-transfected in triplicate with the indicated HLA cDNA and viral genes. IFN-γ secretion was detected by ELISA.
Fig. 6
Fig. 6
Cross-reactive CD8 T-cell epitopes. A: Definition of minimal epitope in HSV-1 UL48 recognized by polyclonal HLA B*1502-restricted CD8 T-cells. EBV-LCL used as APC are matched to responder cells only at HLA B*1502. B: Comparison of shortest fully active HSV-1 UL48 and homolog VZV peptide for same responders and APC. C: Dual staining of same responder cell line with tetrameric complexes of HLA B*1502 and either the HSV-1 or VZV peptide. Numbers are percent of cells in square regions with overall gating on live CD3+CD8+ cells. D: Reactivity of polyclonal HSV-1-specific CD8 T-cell line to DMSO control or the indicated peptides at 1 μg/ml with autologous EBV-LCL as APC. Numbers are percent of gated live CD3+ cells. E: Reactivity of T-cell clones from subjects 21 and 24 to the same nonamer peptides. Clone TCC17 used for TCR sequencing and cloning indicated. F: Reactivity of lentivirally transduced CD8 PBMC expressing a CMV-specific control TCR or the TCR from TCC17, to HLA A*0201 (+) EBV-LCL as APC and 1 μg/ml viral peptide.

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