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New in vivo evidence of CD4+T cells capable of directly stimulating effective CTL response. CD4+T cell help contributes critically to DC‐induced CD8+CTL immunity. However, precisely how these three cell populations interact and how CD4+T cell signals are delivered to CD8+T cells in vivo have been unclear. In this study, we developed a novel, two‐step approach, wherein CD4+T cells and antigen‐presenting DCs productively engaged one another in vivo in the absence of cognate CD8+T cells, after which, we selectively depleted the previously engaged CD4+T cells or DCs before allowing interactions of either population alone with naïve CD8+T cells. This protocol thus allows us to clearly document the importance of CD4+T‐licensed DCs and DC‐primed CD4+T cells in CTL immunity. Here, we provide direct in vivo evidence that primed CD4+T cells or licensed DCs can stimulate CTL response and memory, independent of DC‐CD4+T cell clusters. Our results suggest that primed CD4+T cells with acquired pMHC‐I from DCs represent crucial “immune intermediates” for rapid induction of CTL responses and for functional memory via CD40L signaling. Importantly, intravital, two‐photon microscopy elegantly provide unequivocal in vivo evidence for direct CD4‐CD8+T cell interactions via pMHC‐I engagement. This study corroborates the coexistence of direct and indirect mechanisms of T cell help for a CTL response in noninflammatory situations. These data suggest a new “dynamic model of three‐cell interactions” for CTL immunity derived from stimulation by dissociated, licensed DCs, primed CD4+T cells, and DC‐CD4+T cell clusters and may have significant implications for autoimmunity and vaccine design.

Conversion of tolerogenic DCs into immunogenic ones by time‐limited CpG signaling allows for stimulation of antitumor immunity via activation of CD4+Th1/Th17 and NK cell responses. Abstract TLR ligands have been reported to promote DC maturation and enhance CD8+CTL responses. We have demonstrated previously that CD4–8–DCs secreting TGF‐β stimulate CD4+Tr1 cell responses. Here, we have assessed whether TLR4 and TLR9 signaling through LPS and CpG stimulation can convert CD4−8−DC‐induced tolerance. We demonstrate that immature OVA‐pulsed CD4–8–DCs cultured in medium with LPS (2 μg/ml) and CpG (5 μg/ml) for 8 h became mature DCs (DCOVA) with no TGF‐β secretion. CpG‐treated, CD4–8–DCOVA‐secreting IL‐6/IL‐15 induced IFN‐γ/IL‐17‐secreting/T‐bet‐ and ROR‐γt‐expressing CD4+Th1/Th17, whereas LPS‐treated CD4–8–DCOVAstimulated IFN‐γ‐secreting/T‐bet‐expressing CD4+Th1 responses. The former also significantly stimulated more efficient OVA‐specific CD8+T cell responses and antitumor immunity against OVA‐expressing BL6‐10OVAtumor cells than the latter (P<0.05). CpG‐treated, CD4–8–DCOVA‐stimulated CD4+Th1/Th17 cell responses and antitumor immunity were found to be reduced by using neutralizing anti‐IL‐6, IL‐15, and NK1.1 antibodies in wild‐type C57BL/6 mice, IL‐15R−/−mice for immunization, or CD4–8–(IL‐6−/−) DCOVAfor immunization in C57BL/6 mice. Interestingly, in vitro‐generated CD4+Th17 cells significantly enhanced LPS‐treated, CD4–8–DCOVA‐induced in vivo antitumor immunity via increasing CD8+CTL responses (P<0.05), although they did not show any direct killing activity against tumor cells in vitro. In addition, prolonged 48 h CpG‐treated CD4–8–DCOVAdramatically diminished its cytokine secretion, stimulatory effect, and antitumor immunity. Taken together, our data demonstrate an effect of conversion of tolerogenic DCs into immunogenic ones capable of stimulating antitumor immunity via activating CD4+Th1/Th17 and NK cell responses by optimal CpG signaling, which may advance current understanding of the importance of TLR9 signaling in a DC‐based cancer vaccine.