Human cytomegalovirus (HCMV) causes severe illness and poor outcome in immunocompromised hosts such as transplant recipients and HIV-infected patients. Cytotoxic CD8 T cells against HCMV antigens (pp65, IE1) presented by classical HLA class-I molecules are major cellular components of the protective anti-HCMV immunity. HLA-E-restricted CD8 T cells targeting HCMV UL40 leader peptides (Lp) have been recently reported as unconventional T-cell responses also observed in some hosts but they still need clinical and functional characterization. Our study aimed to provide a qualitative and quantitative ex vivo analysis of HLA-EUL40 CD8 Tcell responses, in a large cohort (n=144) of kidney transplant recipients and healthy volunteers, and to elucidate determining factors. HLA-EUL40 CD8 T-cells were detected in >30% of seropositive HCMV+ hosts and may represent >30% of total circulating CD8 αβT cells at a time point. We identified host-related genetic factors (HLA-A*02 and HLA-E genotype) and HCMV strain, determining the sequence of UL40Lp, as critical parameters for this response. HLA-EUL40 CD8 are effector memory T cells that appear early post-infection as monoclonal/oligoclonal populations and persist for life. Although specifically induced in response to HCMV infection, a key feature of these cells is their potential ability to be also responsive against self and allogeneic HLA resulting from sequence homology between HLA-ILp and UL40Lp. Thus we established that unconventional HLA-EUL40 CD8 T cells belong to the common immune arsenal against HCMV. Their functions remain to be defined toward infection as well as their potential side effect in contexts such as autoimmunity and transplantation.