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This study investigated the frequency and peptide specificity of long-lasting HCMV-specific CD8 T cells in a cohort of 120 cytomegalovirus seropositive (HCMV+) healthy carriers with the aim of deciphering the relative contribution of unconventional HLA-E- versus conventional HLA-A2-specific CD8 T cells to long-term T cell memory expansion in HCMV immunity. The presence of HCMV-specific CD8 T cells was investigated by flow cytometry using five MHC/peptide tetramer complexes (HLA-A2/pp65, HLA-A2/IE1 and three different HLA-E/UL40). Here, we report that 50% of HCMV+ healthy individuals possess HCMV-specific CD8 T cells, representing ≥0.1% of total blood CD8 T cells years post-infection. Around a third (30.8%) of individuals possess HLA-A2-restricted (A2pp65 or A2IE1) and an equal proportion (27.5%) possess an HLA-E/UL40 CD8 T response. Concomitant HLA-E- and HLA-A2-reactive CD8 T cells were frequently found, and VMAPRTLIL peptide was the major target. The frequency of HLA-E/VMAPRTLIL among total blood CD8 T cells was significantly higher than the frequency of HLA-A2pp65 T cells (mean values: 5.9% versus 2.3%, p = 0.0354). HLA-EUL40 CD8 T cells display lower TCR avidity but similar levels of CD3 and CD8 coreceptors. In conclusion, HLA-E-restricted CD8 T cells against the VMAPRTLIL UL40 peptide constitute a predominant subset among long-lasting anti-HCMV CD8 T cells.

Infection with human cytomegalovirus (HCMV) is associated with substantial morbidity in immunosuppressed patients and after congenital infections. Therefore, development of a vaccine against HCMV is a main public health priority., Human cytomegalovirus (HCMV) may cause severe infections in lung transplant recipients (LTRs). In response to HCMV infections, a subset of NKG2C+ NK cells expands, which limits HCMV replication and is characterized by high expression of the activating NKG2C/CD94 and absence of the inhibitory NKG2A/CD94 receptor. Both receptors bind to HLA-E, which is stabilized by HCMV-encoded UL40 peptides. HLA-E and UL40 occur as different genetic variants. In this study, we investigated the interplay between the human NK cell response and the infecting HCMV-UL40 strain, and we assessed the impact of HCMV-UL40 and of donor- and recipient-encoded HLA-E*0101/0103 variants on HCMV replication after lung transplantation. We included 137 LTRs displaying either no or low- or high-level (>1,000 copies/ml plasma) viremia. HCMV-UL40 and HLA-E*0101/0103 variants were determined. UL40 diversity was investigated by next-generation sequencing. UL40 peptide-dependent NK cell cytotoxicity was assessed by flow cytometry. Donor-encoded HLA-E*0101/0103 was significantly associated with development of high-level viremia after transplantation (P = 0.007). The HCMV-UL40 variant VMAPRTLIL occurred significantly more frequently in highly viremic LTRs, and the variant VMTPRTLIL occurred significantly more frequently in low-viremic LTRs (P = 0.004). This difference was associated with a better inhibition of NKG2A+ NKG2C− NK cells by VMAPRTLIL (P

Human cytomegalovirus (HCMV) infection induces a persistent reconfiguration of the NK cell compartment in some individuals, promoting the development of a mature NK cell subset, hallmarked by high expression levels of the CD94/NKG2C activating receptor (NKG2Cbright), together with additional phenotypic and functional features. The mechanisms underlying the adaptive differentiation and expansion of NKG2C+ NK cells remain unknown. In the present work we addressed the relationship between adaptive NKG2C+ NK cells and down-regulation of FcεRγ (FcRγ) expression, a phenotypic trait also associated with HCMV infection. Our results showed that expansions of adaptive NKG2Cbright and detection of FcRγ– NK cells largely coincided in a subgroup of HCMV+ individuals, presumably representing events related with a common virus-host interaction pattern. In addition, the existence of adaptive FcRγ-defective NK cells lacking NKG2C and activating KIRs supported the existence of alternative activation pathways promoting their differentiation. Finally, we disclose an association between NKG2C copy number and the distribution pattern of adaptive NK cell subsets. Several observations indirectly support the involvement in this process of a cognate interaction of CD94/NKG2C with ligand(s) displayed by HCMV-infected cells. To approach this issue, we developed a sensitive reporter system stably expressing CD94/NKG2C and DAP12 in the human Jurkat leukemia T cell line, segregated from other NK cell receptors involved in theinteraction with HCMV-infected cells. Signalling was detected by transfection of a reporter plasmid encoding Luciferase under NFAT/AP1-dependent promoter. Although Jurkat-NKG2C+ cells were activated by anti-CD94/NKG2C mAbs and HLA-E+ 721.221 cells, no response was detected to fibroblasts infected with AD169 or Towne HCMV strains, regardless of their ability to preserve surface HLA-E expression. On the other hand, infection with clinical isolates or with the endotheliotropic TB40/E strain triggered Jurkat-NKG2C+ activation; yet, this response was not inhibited by blocking mAbs and was independent of CD94/NKG2C expression. These results are discussed in the framework of previous observations supporting the hypothetical existence of specific ligand(s) for CD94/NKG2C in HCMV-infected cells., La infección por el citomegalovirus humano (HCMV) promueve en algunos individuos el desarrollo persistente de una subpoblación de células Natural Killer (NK) maduras, que presentan una elevada expresión del receptor activador CD94/NKG2C (NKG2Cbright), junto a otras características fenotípicas y funcionales. Se desconocen los mecanismos que subyacen esta diferenciación y expansión adaptativa de las células NK. En este trabajo estudiamos la relación entre las células NK NKG2C+ y la perdida de expresión de FcR (FcR), otro rasgo fenotípico asociado a la infección por HCMV. Nuestros resultados indican que las expansiones de células adaptativas NKG2C+ y la detección de células NK FcR– coinciden en un subgrupo de individuos HCMV+, representando presumiblemente procesos relacionados con un patrón de interacción virus-huésped común. Además, la existencia de células NK FcR– que no expresan NKG2C ni KIRs activadores apuntan a la existencia de vías de diferenciación alternativas. Finalmente, hemos observado una asociación entre el número de copias del gen NKG2C y el patrón de distribución de las diferentes subpoblaciones de células NK adaptativas. Por otra parte, varias observaciones sugieren que la interacción del receptor CD94/NKG2C con un hipotético ligando en las células infectadas por HCMV contribuye al desarrollo de las células NK adaptativas. Para abordar la cuestión, desarrollamos un sistema sensor mediante la expresión estable de CD94/NKG2C y DAP12 en una línea leucémica humana T (Jurkat), que carece de otrosreceptores NK. La señalización se detecta transfectando un plásmido que codifica la luciferasa, con un promotor dependiente de NFAT/AP-1. Las células Jurkat-NKG2C+ se activan mediante la estimulación por anticuerpos monoclonales anti-CD94/NKG2C o células 721.221 HLA-E+, validando la sensibilidad del sistema. Sin embargo, no se detecta la respuesta a fibroblastos infectados por las cepas AD169 y Towne de HCMV, al margen de su expresión de HLA-E. Por el contrario, lad células infectadas por dos aislados clínicos de HCMV o por la cepa endoteliotrópica TB40/E activan a las células Jurkat-NKG2C+, si bien por un mecanismo independiente de CD94/NKG2C. Estos resultados se discuten en el marco de las observaciones que sustentan la hipotética existencia de ligando(s) específico(s) para CD94/NKG2C en las células infectadas por HCMV. xii