Subcellular Localization of Antigen in Keratinocytes Dictates Delivery of CD4+ T-cell Help for the CTL Response upon Therapeutic DNA Vaccination into the Skin

In a mouse model of therapeutic DNA vaccination, we studied how the subcellular localization of vaccine protein impacts antigen delivery to professional antigen-presenting cells and efficiency of CTL priming. Cytosolic, membrane-bound, nuclear, and secretory versions of ZsGreen fluorescent protein, conjugated to MHC class I and II ovalbumin (OVA) epitopes, were expressed in keratinocytes by DNA vaccination into the skin. ZsGreen-OVA versions reached B cells in the skin-draining lymph node (dLN) that proved irrelevant for CTL priming. ZsGreen-OVA versions were also actively transported to the dLN by dendritic cells (DC). In the dLN, vaccine proteins localized to classical (c)DCs of the migratory XCR1(+) and XCR- subtypes, and-to a lesser extent-to LN-resident cDCs. Secretory ZsGreen-OVA induced the best antitumor CTL response, even though its delivery to cDCs in the dLN was significantly less efficient than for other vaccine proteins. Secretory ZsGreen-OVA protein proved superior in CTL priming, because it led to in vivo engagement of antigen-loaded XCR1(+), but not XCR1(-), cDCs. Secretory ZsGreen-OVA also maximally solicited CD4(+) T-cell help. The suboptimal CTL response to the other ZsGreen-OVA versions was improved by engaging costimulatory receptor CD27, which mimics CD4(+) T-cell help. Thus, in therapeutic DNA vaccination into the skin, mere inclusion of helper epitopes does not ensure delivery of CD4(+) T-cell help for the CTL response. Targeting of the vaccine protein to the secretory route of keratinocytes is required to engage XCR1(+) cDC and CD4(+) T-cell help and thus to promote CTL priming. (C) 2018 AACR.