Our results identify type I IFNs as critical mediators in the spontaneous priming of an antitumor CD8+ T cell response. Our data show that IFN-β is produced shortly after tumor challenge and, through signaling at the level of CD8α+ DCs, promotes tumor antigen–specific T cell priming and tumor rejection. In vivo, endogenous type I IFNs induced the intratumoral accumulation of CD8α+ DCs, which were essential for spontaneous antitumor CD8+ T cell priming and which could explain the critical role of this cell lineage in spontaneous cross-priming of tumor antigen–specific CD8+ T cells.
Given the lack of external TLR ligands for most malignancies, the molecular mechanism by which a tumor can provide the right environment to stimulate immune activation remains poorly understood. Emerging evidence indicates that dying cells can release endogenous adjuvants capable of activating APCs (Kono and Rock, 2008
). Among these molecules are heat-shock proteins (Basu et al., 2000
), uric acid (Shi et al., 2003
), HMGB1 (high-mobility-group box 1; Scaffidi et al., 2002
), ATP (Haag et al., 2007
), and genomic double-stranded (ds) DNA (Ishii et al., 2001
). It has been recently demonstrated that after radiotherapy or chemotherapy, dying tumor cells can release HMGB1 that binds to TLR4 (Apetoh et al., 2007
) and ATP that activates the NALP3 inflammasome (Ghiringhelli et al., 2009
). Presumably there is some degree of spontaneous tumor cell death either upon implantation of transplantable tumor cell lines or, as tumor growth exceeds the available blood supply during attempted neoangiogenesis. However, the massive tumor cell death occurring with chemotherapy or radiation is unlikely to be present in our system of spontaneous T cell priming, so other mechanisms could be operational. Recently, it has been shown that dsDNA can be released from necrotic cells, reach the cytoplasm of APCs, and be recognized by a TLR-independent pathway leading to IRF3 activation and to type I IFN production (Stetson and Medzhitov, 2006a
). It is interesting to speculate that a TLR-independent cytosolic DNA recognition pathway might be involved in innate tumor recognition, IFN-β production, and spontaneous priming of tumor antigen–specific T cells.
Our data have demonstrated that, shortly after tumor challenge, IFN-β is produced by CD11c+
DCs in tumor-draining lymph nodes. However, the identity of the specific subset of DCs producing type I IFNs in response to tumor growth remains unclear. The fact that IFN-β production is still observed in Batf3−/−
mice strongly suggest that the CD8α+
DC subpopulation is not required for type I IFN production. Preliminary studies of depletion of pDCs using the anti-PDCA (Krug et al., 2004
) mAb have revealed that T cell priming and IFN-β production appear to be intact (unpublished data). Thus, it may be that conventional mDCs are capable of this function. Nonetheless, our data are consistent with a model in which at least two different DC subpopulations collaborate for the induction of spontaneous antitumor T cell priming. In this model, one DC subpopulation (likely either mDCs or pDCs) would sense the presence of the tumor and produce type I IFNs, which through signaling on CD8α+
DCs would promote effective cross-priming of CD8+
T cells. Consistent with this model, it has been recently shown using quantitative proteomics that the CD8α+
DC subpopulation selectively lacks the receptors and signaling molecules (such as DAI [Takaoka et al., 2007
] and Sting [Ishikawa et al., 2009
]) required for the detection of nucleotides in the cytoplasm (Luber et al., 2010
), so if this is indeed the pathway involved in type I IFN production to tumor, a non-CD8α+
DC subpopulation would need to be involved.
Although several studies have suggested that CD8α+
DC distribution is restricted to lymphoid organs (Randolph et al., 2008
) our results clearly indicate that CD8α+
DCs (defined as CD3−
cells) can infiltrate tumors. In agreement with our findings, it has been reported that CD8α+
DCs can infiltrate transplantable and spontaneous melanomas in B6 mice (Preynat-Seauve et al., 2006
) and sarcomas in BALB/c mice treated with Flt3L and GM-CSF (Berhanu et al., 2006
), and that such recruitment is associated with tumor rejection. Even though we found an augmented expression of XCR1 transcripts in tumors growing in wild-type hosts compared with type I IFN receptor–deficient mice, this difference could not be explained by a differential expression of its ligand, the chemokine XCL1, which was present in the tumor microenvironment in both hosts (unpublished data). The detailed mechanism by which type I IFNs induce the intratumoral accumulation of CD8α+
DCs will be a crucial area for future investigation.
It is noteworthy that, under conditions in which spontaneous priming of antitumor CD8+
T cells was not occurring, we detected expression of processed SIY peptide–Kb
complexes on the surface of several subsets of APCs. These results suggest that APC subtypes other than CD8α+
DCs are capable of processing antigen into the class I compartment. Similar results have been reported by others. Hans Schreiber’s laboratory has observed expression of processed tumor-derived antigen in tumor-infiltrating macrophages (Zhang et al., 2007
). In addition, in the TRAMP model, tumor-infiltrating DCs have been suggested to express processed antigen and behave in a tolerogenic rather than an activating fashion (Anderson et al., 2007
). Thus, although the CD8α+
DC subset is quantitatively superior at cross-presenting exogenous antigen into the class I compartment, it is likely that additional qualitative differences explain their ability to better initiate CD8+
T cell priming. Although it would have been ideal to characterize the DCs that had successfully processed antigen and then trafficked to the tumor-draining lymph node, we were unable to detect such cells with the TCR tetramer in the lymph node compartment, arguing that the presumably small number of cells is below the threshold of detection. It also should be pointed out that we studied DC subsets in the tumor microenvironment at relatively late time points, because in small tumors it simply wasn’t technically possible to detect them reliably. So we can only infer that a similar defect in CD8α+
DC accumulation is occurring at early times after tumor implantation. Nonetheless, our subsequent experiments solidified a requirement for CD8α+
DCs, and for type I IFN signaling on these cells, in order to attain spontaneous CD8+
T cell priming.
It is currently unknown what dictates why tumors in some patients are capable of inducing spontaneous tumor-antigen specific T cell priming whereas others are not. Single nucleotide polymorphisms (SNPs) in different genes involved in the type I IFN pathway have been reported, including IFNAR (Muldoon et al., 2001
) and Stat1 (Fortunato et al., 2008
), that could affect levels of expression of the mature proteins, leading to variation in the response to type I IFNs. Alternatively, activation of distinct combinations of oncogenic pathways in individual tumors could lead to expression of distinct sets of genes that facilitate innate immune recognition in vivo.
The involvement of type I IFNs in antitumor immune responses has been appreciated for a number of years. Although early clinical trials of systemic administration of type I IFNs showed encouraging results for the treatment of a broad range of tumors (Neidhart et al., 1984
; Motzer et al., 2002
), the mechanism by which exogenously administered type I IFNs induces antitumor activity has remained elusive. In addition, injection of mice with blocking antibody to IFN-α/β has been reported to enhance tumor growth, suggesting the importance of endogenous type I IFNs after tumor challenge and a role in inhibiting tumor growth in immunocompetent mice (Gresser et al., 1983
). Our findings now describe a link between spontaneous IFN-β production and signaling on CD8α+
DC which is essential for tumor antigen–specific CD8+
T cell priming. In addition, preliminary data have revealed potent rejection of B16 melanoma when transduced to express IFN-β (unpublished data). Collectively, our results have implications for human cancer therapy, providing a strong rationale for the intratumoral administration of type I IFNs, which are already FDA approved for other indications, in order to promote improved activation of tumor antigen–specific CD8+
T cells using the tumor itself as a source of antigen.