Heat shock protein (HSP) acts as a vehicle to deliver its associated antigen (Ag) to antigen-presenting cells (APC), such as dendritic cells, and this then facilitates Ag cross-presentation to CD8+ T cells.[1-3] Various aspects of this pathway have been well characterized. For example, certain scavenger receptors such as CD91, LOX-1, and SREC-1 on APC are used for internalization of extracellular HSP for the cross-presentation pathway. This receptor-mediated endocytosis mechanism to internalize HSP-Ag complexes contributes to extremely efficient antigen cross-presentation, compared to Ag alone.
In addition to its role in the adaptive immune response, HSP has been implicated as a natural ligand for a variety of receptors involved in the activation of innate immunity. There are reports showing that Toll-like receptor (TLR)2 and TLR4 are receptors for Gp96 and HSP70 for activation of the NF-κB signaling pathway.[8, 9] CD40 and CCR5 also serve as receptors for HSP70. The interaction between HSP and these receptors is important in terms of providing a second signal required for full activation of T cells, together with the first signal provided by Ag recognition through the TCR. Despite a significant number of reports describing the importance of TLRs and the MyD88 adaptor molecule in HSP-mediated cross-priming, there are other conflicting reports. Very highly purified HSP did not activate APC, although it was able to stimulate cross-presentation, thus contamination with endotoxin-like molecules in HSP preparations used in many experiments is the most critical point to be carefully evaluated. Moreover, most results implicating TLRs have been obtained by in vitro experiments. Therefore, the potential involvement of TLRs in HSP-induced cross-priming needs to be revisited.
Given this controversy regarding TLR involvement, we decided to use a variety of TLR KO mice to define whether TLRs and their adaptor molecules MyD88 and IRAK4 are required for in vivo cross-priming and tumor rejection elicited by a mycobacterial HSP70–ovalbumin (OVA) fusion protein. We found that in vivo cross-priming, as evaluated by proliferation, γ-interferon (IFN-γ) production, and specific cytolytic activity of adoptively transferred OT-I CD8+ T cells, did not require TLR2, 3, 4, 7, or 9, or MyD88/IRAK4. Moreover, generation of OVA-specific CTLs from naïve mice immunized with mycobacterial HSP70-OVA again revealed MyD88 dispensability. In striking contrast, however, we found that MyD88 and IRAK4 are indispensable in mycobacterial HSP70-OVA-dependent rejection of a tumor expressing endogenous OVA. These results reveal an unexpected complexity in the requirement for the TLR-mediated signaling cascade: it is not essential for cross-priming by HSP70 but is required for tumor rejection.
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The possible contribution of endotoxin-like molecules to HSP-induced antitumor immunity has long been an enigma. The results presented here, at least in part, should help to resolve this issue. Mycobacterial HSP70-induced cross-priming as well as tumor rejection was not impaired in TLR2/4 double-KO mice, clearly indicating that these immune responses are not caused by a contaminating endotoxin, such as E. coli LPS. Moreover, TLR7/9 double-KO mice and TLR3 KO mice also showed no reduction in cross-priming or tumor rejection (Figs 5, 7). Strikingly, MyD88, which is a key adaptor molecule for most of the TLRs, was dispensable in proliferation and IFN-γ production by transferred OT-ICD8+ T cells and CTL generation in naïve mice (Figs 5, 6). These results strongly argue that the putative second signal provided by HSP-mediated immunity is not linked with the TLR/MyD88 signaling cascade.
Also implicated as a receptor for HSP70, CD40 facilitates endocytosis as well as activating APC. However, we observed no reduction of cross-priming ability or tumor rejection in CD40 KO mice (Figs 5-7). Involvement of other receptors, IL-18R and IL-1R, included upstream of MyD88, has not yet been tested, which is an important question to be answered in future experiments.
Recently, Pawaria and Binder have shown that CD91 serves as a signaling receptor for Gp96, HSP70, and calreticulin, promoting maturation of APC, secretion of cytokines, and priming of T-helper cells. Interestingly, interaction of Gp96 or calreticulin with CD91 triggers phosphorylation of membrane-proximal tyrosine 4508, whereas HSP70 binding results in phosphorylation of both tyrosine 4508 and a membrane-distal tyrosine 4474 of the CD91β chain to transduce signals. Although these results were obtained using RAW264.7 cells and peritoneal macrophages, our cross-priming results are consistent, because CD91 by itself, but not the TLRs, acts as a signaling receptor for HSP70.
Nonetheless, tumor rejection was absolutely dependent on MyD88/IRAK4, although TLR2, 3, 4, 7, and 9 were dispensable (Fig. 7). In this regard, it is possible that cross-priming by HSP70 is the minimum essential event but, by itself, is insufficient for tumor rejection. Keeping HSP70-primed T cells upfront as effector cells efficient enough to continuously kill cancer cells might require additional factors such as inflammatory signals that are likely provided by MyD88/IRAK4-dependent signaling. In this context, there is a report suggesting that ISCOMATRIX vaccine led to normal cross-presentation of DC isolated from draining lymph node in MyD88 KO mice, as determined by ex vivo proliferation of OT-ICD8+ T cells in the co-culture system. However, enodogenous T cell responses were significantly reduced in MyD88 KO, but not in TLR4 KO, mice, suggesting requirements of inflammatory signals provided by the MyD88-dependent cascade. In any case, the precise mechanisms underlying cross-priming and the resulting tumor rejection remain to be elucidated.
In summary, we have shown that TLRs, MyD88/IRAK4, and CD40 are not essential for HSP70-mediated cross-priming. We have also shown that MyD88/IRAK4, but not TLRs or CD40, are required for tumor rejection induced by the same reagents.