Variation in the sequence of T cell epitopes between dengue virus (DENV) serotypes is believed to alter memory T cell responses during second heterologous infections contributing to pathology following DENV infection. We identified a highly conserved, novel, HLA-B57-restricted epitope on the DENV NS1 protein, NS_126-34. We predicted higher frequencies of NS_126-34-specific CD8⁺ T cells in PBMC from individuals undergoing secondary, rather than primary, DENV infection due to the expansion of memory CD8⁺T cells. We generated a tetramer against this epitope (B57-NS_126-34TET) and used it to assess the frequencies and phenotype of antigen-specific T cells in samples from a clinical cohort of children with acute DENV infection established in Bangkok, Thailand. High tetramer-positive T cell frequencies during acute infection were seen in only 1 of 9 subjects with secondary infection. B57-NS1_26-34-specific, other DENV epitope-specific CD8⁺ T cells, as well as total CD8⁺ T cells, expressed an activated phenotype (CD69⁺ and/or CD38⁺) during acute infection. In contrast, expression of CD71 was largely limited to DENV-specific CD8⁺ T cells. In vitro stimulation of CD8⁺ T cell lines, generated against three different DENV epitopes, indicated that CD71 expression was differentially sensitive to stimulation by homologous and heterologous variant peptides with substantial upregulation of CD71 detected to peptides which also elicited strong functional responses. CD71 may therefore represent a useful marker of antigenspecific T cell activation.

During the course of our analysis we found substantial binding of B57-NS1_26-34 TET to CD8^- cells. We demonstrated that the B57-NS1_26-34 TET bound KIR3DL1, an inhibitory receptor on natural killer (NK) cells. NK sensitive target cells presenting the NS1_26-34 peptide in the context of HLA-B57 were able to dampen functional responses of only KIR3DL1⁺ NK cells. Analysis of the activation of an NK enriched population in our Thai cohort revealed peak activation during the critical time phase in patients with severe dengue illness, dengue hemorrhagic fever, compared to people with mild illness.

Our data identified CD71 as biologically useful marker to study DENV-specific CD8⁺ T cell responses and highlighted the role of viral peptides in modulating NK cell activation through KIR-MHC class I interactions during DENV infection.