R848/TLR7-Mediated Stronger CD8+ T Immunity Is Dependent on DC-NK Cell Interactions

Abstract

Background: Toll-like receptor (TLR) 7 agonists are effective candidates for Th1 immune adjuvants, which compensate for the insufficient Th1 immune responses induced by traditional adjuvants. This effect is currently dependent on TLR7-mediated induction of dendritic cell (DC) maturation and increased IL-12 production. Methods: In vivo, we intraperitoneally injected TLR agonists with OVA, and LNs were collected for detection. In vitro, Activated DCs, natural killer (NK) cells, and CD8⁺ T cells were tested using flow cytometry for surface expression and enzyme-linked immunosorbent assay for cytokine production. NK cell migration was evaluated using transwell system. All experiments were performed in both C57BL/6 and BALB/C backgrounds. Results: Our findings revealed that the enhanced CD8⁺ T immunity characterized by CD8⁺ T accumulation, proliferation, and IFN-γ⁺CD8⁺ T induction induced by R848 was attributed to DC-dependent NK cell migration and DC-NK interactions. Our results demonstrated that R848 induced CD8⁺ T cell accumulation and IFN-γ⁺CD8⁺ T cells in lymph nodes (LNs) to a greater degree in vivo than TLR4 agonists (lipopolysaccharide) and TLR9 agonists (Class C CPG). R848-activated DCs enhanced CD8⁺ T cell proliferation and increased IFN-γ⁺CD8⁺ T cells with the assistance of NK cells. In contrast, depletion of NK cell decreased IFN-γ⁺CD8⁺ T cell production. Greater NK cell migration to LNs occurred in R848-immunized mice. A similar effect of R848 on NK cell migration was observed in an in vitro transwell study. When co-cultured, NK cells plus R848 could promote DCs maturation, and in turn, DCs in combination of R848 augmented NK cells activation. Further studies demonstrated that among several TLR agonists, R848 produced the largest amount of the chemokine CXCL9 from activated DCs, which is relevant to NK cell migration. CXCL9 blockade reduced the number of migrated NK cells, and the addition of CXCL9 increased the number of NK cells. Discussion: Taken together, R848-mediated stronger CD8⁺ T cell immunity does not depend on DC activation alone, rather that NK cells must also be considered. By increasing our immunological understanding of the effect of R848/TLR7, these findings provide a new perspective for applying R848 in future clinical studies.