SUMMARY CD8+ T cells play a critical role limiting peripheral virus replication, yet how they locate virus-infected cells within tissues is unknown. Here, we have examined the environmental signals that CD8+ T cells use to localize and eliminate virus-infected skin cells. Epicutaneous vaccinia virus (VV) infection, mimicking human smallpox vaccination, greatly increased expression of the CXCR3 chemokine receptor ligands CXCL9 and -10 in VV-infected skin. Despite normal T cell numbers in the skin, Cxcr3−/− mice exhibited dramatically impaired CD8+ T cell-dependent virus clearance. Intravital microscopy revealed that Cxcr3−/− T cells were markedly deficient in locating, engaging, and killing virus-infected cells. Further, transfer of wild-type CD8+ T cells restored viral clearance in Cxcr3−/−animals. These findings demonstrate a function for CXCR3 in enhancing the ability of tissue-localized CD8+ T cells to locate virus-infected cells and thereby exert anti-viral effector functions.

SUMMARY Despite extensive ex vivo investigation, the spatiotemporal organization of immune cells interacting with virus-infected cells in tissues remains uncertain. To address this, we used intravital multiphoton microscopy to visualize immune cell interactions with virus-infected cells following epicutaneous vaccinia virus (VV) infection of mice. VV infects keratinocytes in epidermal foci, and numerous migratory dermal inflammatory monocytes outlying the foci. We observed Ly6G+ innate immune cells infiltrating and controlling foci, while CD8+ T cells remained on the periphery killing infected monocytes. Most antigen-specific CD8+ T cells in the skin did not interact with virus-infected cells. Blocking the generation of reactive nitrogen species relocated CD8+ T cells into foci, modestly reducing viral titers. Depletion of Ly6G+ and CD8+ cells dramatically increased viral titers, consistent with their synergistic but spatially segregated viral clearance activities. These findings highlight previously unappreciated differences in the anatomic specialization of antiviral immune cell subsets.

Personalized cancer vaccines are a promising approach for inducing T cell immunity to tumor neoantigens. Using a self-assembling nanoparticle vaccine that links neoantigen peptides to a TLR7/8 agonist (SNP-7/8a), we show how the route and dose alter the magnitude and quality of neoantigen-specific CD8 + T cells. Intravenous vaccination (SNP-IV) induced a higher proportion of TCF1 + PD-1 + CD8 + T cells compared to subcutaneous immunization (SNP-SC). Single cell RNA-seq showed that SNP-IV induced stem-like genes ( Tcf7, Slamf6, Xcl1 ) whereas SNP-SC enriched for effector genes ( Gzmb, Klrg1, Cx3cr1) . Stem-like cells generated by SNP-IV proliferated and differentiated into effector cells upon checkpoint blockade leading to superior antitumor response compared to SNP-SC in a therapeutic model. The duration of antigen presentation by dendritic cells controlled the magnitude and quality of CD8 + T cells. These data demonstrate how to optimize antitumor immunity by modulating vaccine parameters for specific generation of effector or stem-like CD8 + T cells.