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

Fabian Schlott,Dirk H Busch,Dominik Steubl,Stefanie Ameres,Uwe Heemann,Michael Neuenhahn,Volker Hösel,Andreas Moosmann,Stefan Dreher

doi:10.1371/journal.pone.0193554

Abstract

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.

Highlights

Human Cytomegalovirus (CMV), a β-herpesvirus, causes lifelong latent infections in humans, reaching a seroprevalence of 50–90% [1, 2]
Due to a known linkage disequilibrium, HLA-CÃ07:02 is nearly always accompanied in Caucasians by HLA-BÃ07:02 [24], which is known to present an immunodominant epitope of the CMV tegument protein pp65 [27]
In order to confirm the specificity of the MHC multimer staining, we performed within the same donor intracellular cytokine staining (ICS) after peripheral blood mononuclear cells (PBMC) stimulation with the corresponding immediate early-1 (IE-1) and pp65 peptides

Summary

Introduction

Human Cytomegalovirus (CMV), a β-herpesvirus, causes lifelong latent infections in humans, reaching a seroprevalence of 50–90% [1, 2]. A possible evolutionarily beneficial inheritance from the Neandertalian genome [23] and its high allelic frequency of approximately 15% within the Caucasian population [24] hint both to a putative selection advantage of HLA-CÃ07:02 [25]. This makes the HLA-CÃ07:02-presented CMV epitope IE-1309−317 an interesting new target for vaccination, adoptive T cell therapy and immune monitoring. Depletion of T cell subsets mediating epitope-independent KIR-binding allowed to develop a novel enrichment protocol for robust and highly efficient enrichment of HLA-CÃ07:02-restricted CMV IE-1-specific T cell populations

Methods

Results

Conclusion