Presentation of an Immunodominant Immediate-Early CD8+ T Cell Epitope Resists Human Cytomegalovirus Immunoevasion.

Control of human cytomegalovirus (HCMV) depends on CD8+ T cell responses that are shaped by an individual's repertoire of MHC molecules. MHC class I presentation is modulated by a set of HCMV-encoded proteins. Here we show that HCMV immunoevasins differentially impair T cell recognition of epitopes from the same viral antigen, immediate-early 1 (IE-1), that are presented by different MHC class I allotypes. In the presence of immunoevasins, HLA-A- and HLA-B-restricted T cell clones were ineffective, but HLA-C*0702-restricted T cell clones recognized and killed infected cells. Resistance of HLA-C*0702 to viral immunoevasins US2 and US11 was mediated by the alpha3 domain and C-terminal region of the HLA heavy chain. In healthy donors, HLA-C*0702-restricted T cells dominated the T cell response to IE-1. The same HLA-C allotype specifically protected infected cells from attack by NK cells that expressed a corresponding HLA-C-specific KIR. Thus, allotype-specific viral immunoevasion allows HCMV to escape control by NK cells and HLA-A- and HLA-B-restricted T cells, while the virus becomes selectively vulnerable to an immunodominant population of HLA-C-restricted T cells. Our work identifies a T cell population that may be of particular efficiency in HCMV-specific immunotherapy

Characterization and clinical enrichment of HLA-C*07:02-restricted Cytomegalovirus-specific CD8+ T cells.

Human Cytomegalovirus (CMV) reactivation remains a major source of morbidity in patients after solid organ and hematopoietic stem cell transplantation (HSCT). Adoptive T cell therapy (ACT) with CMV-specific T cells is a promising therapeutic approach for HSCT recipients, but might be counteracted by CMV's immune evasion strategies. HLA-C*07:02 is less susceptible to viral immune evasion suggesting HLA-C*07:02-restricted viral epitopes as promising targets for ACT. For a better understanding of HLA-C*07:02-restricted CMV-specific T cells we used recently generated reversible HLA-C*07:02/IE-1 multimers (Streptamers) recognizing a CMV-derived Immediate-Early-1 (IE-1) epitope and analyzed phenotypic and functional T cell characteristics. Initially, we detected very high frequencies of HLA-C*07:02/IE-1 multimer+ T cells (median = 11.35%), as well as robust functional responses after stimulation with IE-1 peptide (IFNγ+; median = 5.02%) in healthy individuals. However, MHC-multimer+ and IFNγ-secreting T cell frequencies showed a relatively weak correlation (r2 = 0.77), which could be attributed to an unexpected contribution of CMV-epitope-independent KIR2DL2/3-binding of HLA-C*07:02/IE-1 multimers. Therefore, we developed a MHC-multimer double-staining approach against a cancer epitope-specific HLA-C*07:02 multimer to identify truly HLA-C*07:02/IE-1 epitope-specific T cells. The frequencies of these truly HLA-C*07:02/IE-1 multimer+ T cells were still high (median = 6.86%) and correlated now strongly (r2 = 0.96) with IFNγ-secretion. Interestingly, HLA-C*07:02/IE-1-restricted T cells contain substantial numbers with a central memory T cell phenotype, indicating high expansion potential e.g. for ACT. In line with that, we developed a clinical enrichment protocol avoiding epitope-independent KIR-binding to make HLA-C*07:02/IE-1-restricted T cells available for ACT. Initial depletion of KIR-expressing CD8+ T cells followed by HLA-C*07:02/IE-1 Streptamer positive selection using paramagnetic labeling techniques allowed to enrich successfully HLA-C*07:02/IE-1-restricted T cells. Such specifically enriched populations of functional HLA-C*07:02/IE-1-restricted T cells with significant central memory T cell content could become a potent source for ACT

Functional separation of epitope presentation and HCMV immunoevasion.

<p>WI-38 fibroblasts (negative for HLA-A*0201 and C*0702) were transfected with plasmids encoding HLA-A*0201, HLA-C*0702, or chimeric HLA class I heavy chains (HLA-A2/C7 or HLA-C7/A2) and subsequently infected with MVA-IE-1 or different HCMV derivatives. IFN-γ secretion was measured in ELISA after overnight incubation of 10 000 clonal T cells with 10 000 target cells. (A) Schematic representation of the native and chimeric HLA class I molecules that were tested. (B) HLA-transfected WI-38 cells were infected with MVA-IE-1, and presentation of IE-1 epitopes was detected by VLE- and CRV-specific T cell clones. Mean and SD of three replicates are shown for 3 T cell clones generated from 3 different donors for each specificity. (C) HLA-transfected WI-38 were infected with CMV-wt, CMV-Δall, CMV-US2 or CMV-US11, not infected (n.i.) or peptide-loaded (+pep). Three T cell clones generated from 3 different donors were used as effectors for each specificity. Data are shown as mean+SD of triplicate samples from one of two independent experiments.</p

Immunodominance of CRV/C*0702-specific T cells.

<p>(A) Frequencies of CRV-specific T cells in 15 HLA-C*0702-positive blood donors, tested with the CRV nonameric peptide in ELISPOT assays. Black parts of bars indicate CRV-specific signal, grey parts indicate background (no peptide). (B) Specific T cells in PBMCs of HLA-C*0702 carriers were quantified by fluorescent staining with HLA-C*0702/CRV streptamer or HLA-B*0702/TPR pentamer and anti-CD8 antibody. Donors LT12 and SA03 are HCMV-seropositive, donor ASM is HCMV-seronegative. (C) Distribution of T-cell targets within the IE-1 sequence for 15 HLA-C*0702-positive donors, tested with overlapping peptides covering the entire IE-1 sequence of strain AD169. The 120 peptides were divided into 10 subpools, each comprising 12 successive 15-mer peptides with an overlap of 11 amino acids. The C-terminal amino acid position of each subpool is indicated. (D) Frequencies of CRV/C*0702-specific and VLE/A*0201-specific T cells in HLA-C*0702/A*0201-positive donors (n = 6). (E) Comparison of IE-1-specific T cell frequencies in C*0702-negative (n = 13) vs. C*0702-positive (n = 15) donors. (A, C–E) IFN-γ ELISPOT assays were performed with 200 000 peptide-loaded PBMCs in each well and with 2–4 replicates per condition.</p

HLA-mediated inhibition of NK cell recognition of HCMV infection.

<p>Experiments were performed with polyclonal NK cells (A, C, E, G) or NK cell clone #29 (B, D, F, G) from donor AJU. (A, B) Analysis of KIR expression by flow cytometry. (C, D) Killing by NK cells of the MHC class I-deficient cell lines K562, Daudi and L721.221, HLA-C*0602 or C*0702-transfected L721.221 cells, uninfected MRC-5 fibroblasts, or MRC-5 infected with CMV-wt at moi = 5, at an effector∶target ratio of 2. Data are shown as mean+SD of four replicates from one representative experiment out of four. (E, F) NK cell-mediated killing of uninfected (n.i.) and HCMV-infected fibroblasts over time after infection. Fibroblasts were or were not pretreated with IFN-γ before infection as indicated. (G) Blockade of NK cell mediated-killing by monoclonal antibodies specific for HLA-ABC or KIR2DL2/3 (both IgG2a) or a matched isotype control. Targets were pretreated with IFN-γ. Blockade of the non-KIR ligand HLA-A2 served as additional negative control. The HLA class I type of MRC-5 fibroblasts is HLA-A*0201, A*2902, B*0702, B*4402, C*0501, C*0702. HLA-C*0702 is the only ligand of KIR2DL3 expressed by MRC-5 cells. Killing was assessed at an effector∶target ratio of 2. Data are shown as mean+SD of triplicate samples from one out of two independent experiments.</p

Impact of individual HCMV immunoevasins on the recognition of IE-1 T cell epitopes.

<p>MRC-5 fibroblasts were infected with HCMV strain AD169 (wt), with AD169 derivative viruses that expressed only one of the four immunoevasins US2, US3, US6, or US11 as indicated, not infected (n.i.) or peptide-loaded (+pep), and their recognition by T cell clones specific for the IE-1 epitopes CRV/C*0702 and VLE/A*0201 was analyzed. Before infection, fibroblasts were precultured with IFN-γ for 72 hours, then infected at moi = 5 and cocultivated with T cells at 48 hours post infection (10 000 fibroblasts and 10 000 T cells per well). IFN-γ secretion was measured by ELISA. Data are shown as mean+SD of triplicate samples. Representative data are shown for one of 10 CRV-specific clones and one of 4 VLE-specific clones, assayed in two independent experiments.</p

Time course of pp65- and IE-1-specific CD8+ T cell recognition of HCMV-infected fibroblasts.

<p>pp65 and IE-1 T cell epitopes were analyzed for their HLA-A*0201 or -C*0702-restricted presentation to T cell clones at different time points post infection. MRC-5 fibroblasts (HLA-A*0201, C*0702) were not treated (A) or were treated (B) with IFN-γ for 72 hours prior to infection with HCMV AD169 at an moi of 5 or 10. At the indicated time points post infection, 10 000 fibroblasts were incubated with 10 000 T cells for 16–18 hours before measuring antigen-specific IFN-γ secretion by ELISA assay. Cells that were not infected (n.i.) or were peptide-loaded 48 hours post infection (+pep) served as controls. Data are shown as mean+SD of triplicate samples. One of three independent experiments with clones ALT#21 NLV, F61#38 VLE and AJJ#7 CRV is shown, representing experiments with a total of 3 NLV-, 6 VLE- and 12 CRV-specific T cell clones, from 3 different donors for each specificity.</p