The role of pDCs in tumors has long been under debate. Clinical studies have shown negative correlation between the numbers of infiltrating pDCs and patient prognosis (33
). This was attributed to the finding that immature pDCs in tumors are weak inducers of T cell immunity or may even induce regulatory T cells (34
). In the present study, we demonstrate what we believe to be a novel function for pDCs as active and indispensable effectors of tumor killing after treatment with Imi. Moreover, we show that TLR7 and IFNAR1 signaling in pDCs is necessary for tumor killing upon Imi treatment.
We observed increased CCL2 levels in the dermis shortly after Imi stimulation, which was dependent on TLR7, MyD88, and IFNAR1 expression. Consistently, pDC recruitment to the dermis and tumors after Imi treatment was dramatically impaired, and the inflammatory response in the dermis was much weaker in these knockout mice. Mice deficient for CCL2 did not show increased numbers of pDCs in the skin after Imi application, thus providing strong evidence that CCL2 is necessary for pDC recruitment. Our results with BM-reconstituted mice demonstrate that Imi is still effective when TLR7 is missing in dermal cells of non-BM origin and that the tumoricidal effect of Imi strictly requires TLR7 expression on BM-derived cells. Since TLR7 signaling is required for CCL2 production, these results strongly suggest that a skin-resident, TLR7-expressing BM-derived cell is responsible for CCL2 production after Imi treatment. Dermal mast cells are strong candidates, as they express TLR7 and produce cytokines upon Imi treatment (24
). Recent studies have also shown that mast cells can produce CCL2 after anti-IgE stimulation (36
). We observed that mast cells produced significant amounts of CCL2 after Imi treatment, therefore strengthening our hypothesis that mast cells are responsible for CCL2 production and subsequent pDC recruitment after topical Imi application.
Although we found increased apoptosis and activation of the MAPK pathway in Imi-treated primary keratinocytes isolated from Tlr7–/–
mice, it is evident from our results that Imi-induced epidermal apoptosis does not significantly contribute to local inflammation and pDC infiltration. Imi can also directly induce apoptosis of melanoma cells. However, this alone is not sufficient for tumor resolution, since tumor regression is strictly dependent on the presence of pDCs. Imi loses its therapeutic effect in Tlr7–/–
, and Ifnar1–/–
mice. This is accompanied by reduction of CCL2 expression and tumor-infiltrating pDCs. TLR9-activated pDCs induce NK cell– and CD8+
T cell–mediated antitumor immunity by driving maturation of and cross-presentation by conventional DCs (22
). Although T cell and NK cell activity can be modulated by TLR7 agonists (37
), we show that the antitumor response induced by Imi remained effective, even in the absence of T and NK cells and IKDCs.
We provide definitive proof that pDCs are essential for Imi-induced tumor killing by depleting pDCs in tumor-bearing Bdca2
-DTR transgenic mice (9
). Lack of pDCs completely abolished the tumoricidal effect of Imi in these mice but did not influence other immune cell populations, emphasizing the central role for pDCs in the antitumor response induced by Imi. In vivo depletion of CD8α+
cells also abolished the tumoricidal effect of Imi, similar to that in pDC-depleted mice. Naive pDCs have been shown to express variable amounts of CD8α on their surface, and we show that Imi treatment induces TLR7-dependent upregulation of CD8α on pDCs in vivo and in vitro. Importantly, in vivo depletion of CD8α+
cells also ablated activated pDCs from tumors, providing an explanation for why Imi treatment was not active in mice lacking CD8α+
In response to TLR7, triggering pDCs can produce high amounts of type I IFN (4
), which can influence tumor growth by stimulating the adaptive immune system or by activating innate immune responses (9
). IFNAR1 knockout mice are impaired in their ability to induce adaptive immune responses in response to TLR7 ligation by the synthetic agonist polyUs21 (39
). Moreover, it has been shown that IFN-α/β can directly contribute to the clearance of tumor cells in a caspase-dependent manner (12
) or indirectly contribute by the upregulation of cytotoxic molecules (13
). We did not observe direct killing of tumor cells by IFN-α/β, as inhibition of IFNAR1 on tumor cells did not prevent killing induced by Imi-stimulated pDCs. Therefore, it is likely that IFN-α/β stimulates death receptor ligand expression in pDCs. Human pDCs have been implicated in exertion of cytolytic functions by upregulating TRAIL (17
) or influencing T cell responses by granzyme B secretion (40
). Furthermore, human pDCs have been shown to partially inhibit proliferation of a melanoma cell line in a type I IFN-dependent manner (41
A number of molecules responsible for tumor cell killing, like granzyme B, TRAIL, FasL, and TNF-α, were upregulated in Imi-stimulated pDCs, although to a variable degree. However, analysis of these factors was hampered by the fact that they may be stored and released within microvesicles or shed from the surface (42
). Expression of these lytic molecules was dependent on TLR7 expression and on IFNAR1 signaling in pDCs. Imi treatment did not lead to tumor clearance in Ifnar1–/–
mice, and Imi-induced cytotoxicity of Ifnar1–/–
pDCs toward melanoma cells was significantly reduced. Blocking of granzyme B led to a significant decrease of melanoma cell death by Imi-stimulated pDCs, providing strong evidence that the killing activity of pDCs is primarily mediated by granzyme B. TRAIL and FasL were both expressed only in a subpopulation of pDCs; however, their expression was only minimally overlapping (data not shown). In vitro blocking of TRAIL/DR5 significantly reduced tumor cell lysis, but it did not entirely inhibit killing, suggesting that TRAIL only partially contributes to the in vivo effect of Imi. Although pDCs do not produce perforin, which is thought to be essential for the delivery of granzyme B into the cytosol, it is still under debate whether pDCs are able to induce cell death via granzyme B alone. Evidence is growing that perforin-independent mechanisms also induce the uptake of granzyme B to induce killing of tumor cells (43
). It is therefore tempting to speculate that Imi could replace the requirement for perforin for granzyme B uptake by tumor cells.
Based on our results, we propose that topical application of Imi leads to skin inflammation via TLR7/MyD88-dependent and -independent mechanisms. Imi-stimulated keratinocytes show increased apoptosis, which is independent of TLR7/MyD88-signaling. Instead, Imi-induced CCL2 production by resident mast cells and subsequent pDC recruitment into skin and tumors is strictly dependent on TLR7/MyD88 and IFNAR1 expression. Once pDCs are recruited to the skin or tumors, Imi stimulation leads to TLR7-dependent upregulation of type I IFNs, which in turn act in an autocrine manner on pDCs and induce the secretion of cytolytic molecules like TRAIL and granzyme B via IFNAR1 signaling (Figure ). In mice lacking TLR7/MyD88 and IFNAR1, there is no induction of CCL2 and, as a consequence, no recruitment of pDCs and also no upregulation of cytolytic molecules, providing an explanation for the lack of therapeutic effect of Imi in these knockout mice. In conclusion, our data provide a very strong demonstration that pDCs are absolutely required for the antitumor response mediated by Imi. Therefore, strategies aiming at promoting the recruitment of pDCs to tumor sites and reprogramming them to become killer pDCs might prove to be effective stand-alone or adjuvant antitumor therapies.
Mechanism of Imi-mediated tumor cell killing by pDCs.