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Involvement of CD4(+) helper T (Th) cells is crucial for CD8(+) cytotoxic T lymphocyte (CTL)-mediated immunity. However, CD4(+) Th's signals that govern CTL survival and functional memory are still not completely understood. In this study, we assessed the role of CD4(+) Th cells with acquired antigen-presenting machineries in determining CTL fates. We utilized an adoptive co-transfer into CD4(+) T cell-sufficient or -deficient mice of OTI CTLs and OTII Th cells or Th cells with various gene deficiencies pre-stimulated in vitro by ovalbumin (OVA)-pulsed dendritic cell (DCova). CTL survival was kinetically assessed in these mice using FITC-anti-CD8 and PE-H-2K(b)/OVA257-264 tetramer staining by flow cytometry. We show that by acting via endogenous CD40L and IL-2, and acquired peptide-MHC-I (pMHC-I) complex signaling, CD4(+) Th cells enhance survival of transferred effector CTLs and their differentiation into the functional memory CTLs capable of protecting against highly-metastasizing tumor challenge. Moreover, RT-PCR, flow cytometry and Western blot analysis demonstrate that increased survival of CD4(+) Th cell-helped CTLs is matched with enhanced Akt1/NF-κB activation, down-regulation of TRAIL, and altered expression profiles with up-regulation of prosurvival (Bcl-2) and down-regulation of proapoptotic (Bcl-10, Casp-3, Casp-4, Casp-7) molecules. Taken together, our results reveal a previously unexplored mechanistic role for CD4(+) Th cells in programming CTL survival and memory recall responses. This knowledge could also aid in the development of efficient adoptive CTL cancer therapy.

T cell-T cell Ag presentation is increasingly attracting attention. We previously showed that the in vitro OVA-pulsed dendritic cell (DC(OVA))-activated CD4(+) Th cells acquired OVA peptide/MHC (pMHC) class I and costimulatory molecules such as CD54 and CD80 from DC(OVA) and acted as CD4(+) Th-APC capable of stimulating OVA-specific CD8(+) CTL responses. In this study, we further applied the OVA-specific TCR-transgenic OT I and OT II mice with deficiency of various cytokines or costimulatory molecule genes useful for studying the molecular mechanisms underlying in Th-APC's stimulatory effect. We demonstrated that DC(OVA)-stimulated OT II CD4(+) Th-APC also acquired costimulatory molecules such as CD40, OX40L, and 4-1BBL and the functional pMHC II complexes by DC(OVA) activation. CD4(+) Th-APC with acquired pMHC II and I were capable of stimulating CD4(+) Th1 and central memory CD8(+)44(+)CD62L(high)IL-7R(+) T cell responses leading to antitumor immunity against OVA-expressing mouse B16 melanoma. Their stimulatory effect on CD8(+) CTL responses and antitumor immunity is mediated by IL-2 secretion, CD40L, and CD80 signaling and is specifically targeted to CD8(+) T cells in vivo via acquired pMHC I. In addition, CD4(+) Th-APC expressing OVA-specific TCR, FasL, and perforin were able to kill DC(OVA) and neighboring Th-APC expressing endogenous and acquired pMHC II. Taken together, we show that CD4(+) Th-APC can modulate immune responses by stimulating CD4(+) Th1 and central memory CD8(+) T cell responses and eliminating DC(OVA) and neighboring Th-APC. Therefore, our findings may have great impacts in not only the antitumor immunity, but also the regulatory T cell-dependent immune tolerance in vivo.