The viruses HIV-1, Epstein-Barr virus (EBV), cytomegalovirus (CMV) and hepatitis C virus (HCV) are characterized by the establishment of lifelong infection in the human host, where their replication is thought to be tightly controlled by virus-specific CD8+ T cells. Here we present detailed studies of the differentiation phenotype of these cells, which can be separated into three distinct subsets based on expression of the costimulatory receptors CD28 and CD27. Whereas CD8+ T cells specific for HIV, EBV and HCV exhibit similar characteristics during primary infection, there are significant enrichments at different stages of cellular differentiation in the chronic phase of persistent infection according to the viral specificity, which suggests that distinct memory T-cell populations are established in different virus infections. These findings challenge the current definitions of memory and effector subsets in humans, and suggest that ascribing effector and memory functions to subsets with different differentiation phenotypes is no longer appropriate.

The use of peptide–human histocompatibility leukocyte antigen (HLA) class I tetrameric complexes to identify antigen-specific CD8+ T cells has provided a major development in our understanding of their role in controlling viral infections. However, questions remain about the exact function of these cells, particularly in HIV infection. Virus-specific cytotoxic T lymphocytes exert much of their activity by secreting soluble factors such as cytokines and chemokines. We describe here a method that combines the use of tetramers and intracellular staining to examine the functional heterogeneity of antigen-specific CD8+ T cells ex vivo. After stimulation by specific peptide antigen, secretion of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, macrophage inflammatory protein (MIP)-1β, and perforin is analyzed by FACS® within the tetramer-positive population in peripheral blood. Using this method, we have assessed the functional phenotype of HIV-specific CD8+ T cells compared with cytomegalovirus (CMV)-specific CD8+ T cells in HIV chronic infection. We show that the majority of circulating CD8+ T cells specific for CMV and HIV antigens are functionally active with regards to the secretion of antiviral cytokines in response to antigen, although a subset of tetramer-staining cells was identified that secretes IFN-γ and MIP-1β but not TNF-α. However, a striking finding is that HIV-specific CD8+ T cells express significantly lower levels of perforin than CMV-specific CD8+ T cells. This lack of perforin is linked with persistent CD27 expression on HIV-specific cells, suggesting impaired maturation, and specific lysis ex vivo is lower for HIV-specific compared with CMV-specific cells from the same donor. Thus, HIV-specific CD8+ T cells are impaired in cytolytic activity.

Major histocompatibility complex (MHC)-E is a highly conserved, ubiquitously expressed, non-classical MHC-Ib molecule with limited polymorphism primarily involved in NK cell regulation. We found that vaccination of rhesus macaques (RM) with ΔRh157.5/.4 Rhesus Cytomegalovirus (RhCMV) vectors results in MHC-E-restricted presentation of highly varied peptide epitopes to CD8α/β+ T cells, approximately 4 distinct epitopes per 100 amino acids in all tested antigens. Computational structural analysis revealed that MHC-E provides heterogeneous chemical environments for diverse side chain interactions within a stable, open binding groove. Since MHC-E is up-regulated on cells infected with HIV/SIV and other persistent viruses to evade NK cell activity, MHC-E-restricted CD8+ T cell responses have the potential to exploit pathogen immune evasion adaptations, a capability that might endow these unconventional responses with superior efficacy.