Type I interferon is selectively required by dendritic cells for immune rejection of tumors

Dendritic cell responsiveness to type I interferon is required for the generation of antitumor T cell responses and tumor rejection.

The ability of the immune system to function as an extrinsic tumor suppressor and effectively eliminate, control, and/or sculpt developing tumors forms the basis of the cancer immunoedit ing hypothesis (Shankaran et al., 2001;Dunn et al., 2002Dunn et al., , 2004. There is strong experimental support for all three phases of cancer immuno editing, elimination, equilibrium, and escape, and many of the key cellular mediators and immune effector molecules involved in host protection from tumor development have been identified Smyth et al., 2006;Koebel et al., 2007;Schreiber et al., 2011;Vesely et al., 2011). The IFNs, both type I (IFN/) and type II (IFN), have emerged as critical compo nents of the cancer immunoediting process, and work is ongoing to define their respective roles in promoting antitumor immune responses.
Early studies supporting the existence of cancer immunoediting revealed an important function for IFN in suppressing tumor de velopment in models of both tumor transplan tation and primary tumor induction (Dighe et al., 1994;Kaplan et al., 1998;Shankaran et al., 2001;Street et al., 2001Street et al., , 2002. Specifi cally, IFN was found to induce effects on both tumor cells (Dighe et al., 1994;Kaplan et al., 1998;Shankaran et al., 2001;Dunn et al., 2005) and host cells (Mumberg et al., 1999;Qin and Blankenstein, 2000;Qin et al., 2003). Subsequently, an essential function for endog enous type I IFN in cancer immunoediting was established (Dunn et al., 2005;Swann et al., 2007). Genetargeted mice lacking the type I factor (CD8 + DCs and CD103 + DCs, hereafter referred to as CD8 + lineage DCs) was shown to play an important role in crosspresenting viral and tumor antigens, and mice lacking these cells fail to reject highly immunogenic unedited sarcomas (Hildner et al., 2008;Edelson et al., 2010). However, it remains unknown whether the crosspresenting activity of these cells re quires type I IFN to induce tumor immunity.
In the current study, we have investigated the host cell tar gets of endogenous type I IFN during the rejection of highly immunogenic, unedited tumors. We demonstrate that IFN/ acts early during the initiation of the immune response and IFN receptor developed more carcinogeninduced primary tumors than WT control mice (Dunn et al., 2005;Swann et al., 2007), and antibodymediated blockade of the IFN/ receptor in WT hosts abrogated rejection of immunogenic transplanted tumors (Dunn et al., 2005). The activity of endogenous type I IFN was mediated not by its direct effects on the tumor but by its actions on host cells, specifically on hematopoieticderived host cells (Dunn et al., 2005). Collec tively, these findings highlight a difference between the antitumor activities of the IFNs, wherein tumor cell respon siveness to IFN but not IFN/ and host cell responsive ness to both IFN and IFN/ are crucial for tumor rejection. However, the relevant host cell targets and anti tumor functions of IFN/ and IFN remain undefined because of the nearly ubiquitous expression of IFN/ and IFN receptors and the pleiotropic effects they induce.
Although initially defined by their antiviral activity, the type I IFNs are potent immunomodulators that shape host immunity through direct actions on innate and adaptive lymphocytes. The enhancement of NK cell cytotoxicity by IFN/ in the setting of viral infection was one of the earliest such effects to be recog nized (Biron et al., 1999). Type I IFN directly augments NK cell-mediated killing of virally infected or transformed cells and indirectly promotes the expansion and survival of NK cells through IL15 induction (Nguyen et al., 2002). Furthermore, in models of NK cell-dependent tumor rejection, host cell respon siveness to IFN/ was shown to be important for control of tumor growth and metastasis . Type I IFN can also act directly on T and B lymphocytes to modulate their activity and/or survival. Treatment with IFN/ in vitro pro longed the survival of activated T cells (Marrack et al., 1999) and augmented clonal expansion and effector differentiation of CD8 + T cells (Curtsinger et al., 2005) through cellintrinsic IFN/ receptor signaling. Similarly, type I IFN responsive ness in T cells was required in vivo for optimal clonal expansion of antigenspecific CD8 + and CD4 + T cells during viral infec tion (Kolumam et al., 2005;HavenarDaughton et al., 2006;Thompson et al., 2006) as well as for CD8 + T cell priming after immunization with antigen and IFN (Le Bon et al., 2006a). B cell differentiation, antibody production, and isotype class switching were also enhanced by type I IFN's effects either di rectly on B cells or indirectly via effects on T cells (Coro et al., 2006;Le Bon et al., 2006b) and DCs (Le Bon et al., 2001).
Type I IFN also directly enhances the function of DCs, which are central to the initiation of adaptive immune responses (Steinman and Banchereau, 2007). IFN/ induces DC matu ration, upregulates their costimulatory activity and enhances their capacity to present or crosspresent antigen (Luft et al., 1998;Gallucci et al., 1999;Montoya et al., 2002). For example, coinjection of IFN/ plus antigen (Gallucci et al., 1999;Le Bon et al., 2001 or injection of DCtargeted antigen in combination with the IFN/ inducer polyinosinic:polycyti dylic acid (polyI:C; Longhi et al., 2009) stimulated CD8 + T cell priming, humoral responses, and development of CD4 + Th1 responses in vivo. Recently, a subpopulation of DCs whose de velopment depends on expression of the BATF3 transcription mice or WT mice injected i.p. with either IFNAR1-specific MAR1-5A3 mAb or isotype control GIR-208 mAb 1 d prior were s.c. injected with 10 6 H31m1 tumor cells, and tumor size was measured over time. Data represent mean tumor diameter ± SEM of 12-16 mice per group from at least three independent experiments. (B-D) WT mice were injected with 10 6 H31m1 cells (at day 0) and treated beginning on the indicated day with MAR1-5A3 (B), IFN-specific H22 mAb (C), or a mixture of anti-CD4/anti-CD8/anti-IFN- mAbs GK1.5/YTS-169.4/H22 (D), and tumor growth was monitored. For each time point, groups of mice were treated in parallel with the respective isotypematched control mAb, and the data are presented as percent tumor growth over the control group. Results are from two to four experiments with 14-20 (ctrl/MAR1-5A3), 10-20 (ctrl/H22), or 6-11 (ctrl/cocktail) WT mice per group. The kinetics of tumor growth in individual mice is shown in Fig. S1. cells were rejected when transplanted into naive syngeneic WT mice either left untreated or pretreated with GIR208, the tumors grew progressively in WT mice pretreated with MAR1 5A3 (Fig. 1 A). Similarly, MAR15A3 treatment on day 4 or 6 (relative to tumor injection at day 0) blocked rejection in >50% of injected mice. In contrast, IFN/ receptor blockade at later time points did not inhibit rejection ( Fig. 1 B and Fig. S1). In parallel experiments, blockade of IFN via treatment with neu tralizing IFN-specific H22 mAb (Schreiber et al., 1985) re vealed a more prolonged requirement for the actions of IFN during H31m1 rejection (Fig. 1 C). Cohorts of mice were also treated with a mixture of mAbs that deplete CD4 + and CD8 + cells and neutralize IFN (GK1.5 [Dialynas et al., 1983], YTS169.4 [Cobbold et al., 1984], and H22, respectively) to broadly disrupt host immunity. In this group, progressively growing tumors were observed in a substantial proportion of mice treated as late as day 14 with the antiCD4/CD8/IFN mAb cocktail ( Fig. 1 D). Collectively, these data demon strate that the obligate functions of type I IFN are required only for initiating the immune response to tumors.

A tissue-restricted role for type I IFN during tumor rejection
To characterize the critical host cells responding to type I IFN during initiation of the antitumor response, we transplanted H31m1 tumor cells and cells from a second unedited MCA sarcoma, d38m2, into bone marrow chimeras with selective IFN/ sensitivity. These tumor cell lines were selected be cause we previously showed that their rejection required type I IFN responsiveness at the level of the host (Dunn et al., 2005). As reported previously, both cell lines were rejected when transplanted into immunocom petent WT mice but formed pro gressively growing tumors in either Rag2 / or Ifnar1 / mice (Fig. 2,A and B). We now show that both lines grew progressively in Ifnar1 / → Ifnar1 / bone marrow chimeras and that innate immune cells represent the essential responsive cells for the generation of protective antitumor immunity. Whereas type I IFN-unresponsive mice showed a defect in the priming of tumorspecific CTLs, reconstitution of IFN/ sensitivity in innate immune cells was sufficient to restore this deficit and resulted in tumor rejection. Within the innate immune com partment, we find no evidence of an essential role for NK cells or for type I IFN sensitivity in granulocytes or macrophages, but rather find that the actions of IFN/ on DCs are re quired for development of tumor immunity in vivo and play an important role in promoting the capacity of CD8 + lineage DCs to crosspresent antigen to CD8 + T cells. These results thus identify DCs and specifically CD8 + lineage DCs as key cellular targets of type I IFN in the development of protective adaptive immune responses to immunogenic tumors.

Early requirement for type I IFN during the antitumor response
We previously showed that blockade of type I IFN signaling by pretreatment of mice with the IFNAR1 (IFN/ receptor 1) specific MAR15A3 mAb (Sheehan et al., 2006) abrogated rejection of highly immunogenic sarcomas derived from 3methylcholanthrene (MCA)-treated Rag2 / mice (termed unedited tumors; Dunn et al., 2005). To dissect the temporal requirements for IFN/'s actions during the antitumor im mune response, we treated WT mice with either MAR15A3 or isotype control GIR208 mAb at different times after injec tion of unedited H31m1 MCA sarcoma cells. Whereas H31m1 was not caused by incomplete hematopoietic reconstitution, we confirmed normal cellularity and immune cell percent ages in the spleen, demonstrated normal functional immune reconstitution, and ruled out the presence of radioresistant tissueresident leukocytes within the tumor environment (Figs. S3-S5). These data not only establish an important role for IFN sensitivity in both hematopoietic and nonhemato poietic cells during tumor rejection but also reveal a differ ence between the broad cellular requirements for IFN as opposed to the tissuerestricted requirement for IFN/ during elimination of the same tumor. Ifnar1 / → Rag2 / chimeras (IFN/ sensitivity only in nonhematopoietic cells) but were rejected in WT → WT chimeras and WT → Ifnar1 / chimeras (IFN/ sensitivity only in hematopoietic cells). These results thus extend, to two additional tumors, our prior finding that type I IFN sensitivity within the hematopoietic compartment is both necessary and sufficient for tumor rejection (Dunn et al., 2005). Because the rejection of immunogenic sarcomas also re quires IFN sensitivity within the host (Fig. S2), we wanted to determine whether IFN/ and IFN were mediating their effects by acting on the same host cell compartment. We thus performed a similar set of experiments using chimeras with selective host cell IFN responsiveness. As expected, d38m2 tumor cells grew progressively in Rag2 / , Ifngr1 / , and Ifngr1 / → Ifngr1 / mice but were rejected in WT and WT → WT hosts (Fig. 2 C). Tumor growth was also observed in a significant fraction of Ifngr1 / → Rag2 / and WT → Ifngr1 / chimeras, though the defect in these mice (which selectively express the IFN receptor in either nonhemato poietic or hematopoietic cells, respectively) appeared less severe than that in globally insensitive Ifngr1 / → Ifngr1 / chimeras. To ensure that tumor growth in the chimeric mice Mixed bone marrow chimeras with selective IFNAR1 expression in innate or adaptive immune cells were generated by reconstitution of irradiated Ifnar1 / mice with mixtures of HSCs as described in Results. (A) Splenocytes were isolated from representative cohorts of control and mixed chimeric mice at least 12 wk after reconstitution, and IFNAR1 staining was analyzed by flow cytometry. Shown are the percentages of IFNAR1 + cells within the indicated immune cell subsets for 8-14 mice of each type. Horizontal bars represent the mean. (B-D) Control WT, Rag2 / , and Ifnar1 / mice and Ifnar1 / mixed chimeric mice were injected with 10 6 H31m1 (B), d38m2 (C), or F515 (D) tumor cells, and growth was monitored over time. Data are presented as mean tumor diameter ± SEM or the percentage or tumorpositive mice per group from two to three independent experiments with group sizes as indicated. WT mice treated with control or IFN-specific mAb were challenged with 10 6 F515 tumor cells, and growth was monitored (D, bottom). Mean tumor diameter ± SEM for 7-10 mice/group from two experiments is shown.
behavior of unedited MCA sarcoma cells (F515) that require lymphocytes and IFN but not host IFN/ responsiveness for their rejection. F515 tumor cells grew progressively when injected into Rag2 / mice and WT mice treated with IFNspecific H22 mAb but were rejected in WT mice, WT mice treated with isotype control PIP mAb, and Ifnar1 / hosts ( Fig. 3 D). Similar to Ifnar1 / mice, F515 cells were also re jected in Ifnar1 / mixed chimeras of each type, verifying functional reconstitution of the immune compartment. Third, to rule out a potential hyperactive immunological state in these reconstituted mice, we challenged Ifnar1 / mixed chimeras and control mice with MCA sarcoma cells derived from WT mice (1877). We have previously established that this tumor grows progressively when transplanted into naive WT mice (unpublished data). Similarly, these tumor cells grew progres sively in Ifnar1 / mixed chimeras of each type (Fig. 4 C).

Sensitivity to type I IFN in innate immune cells is required for the generation of tumor-specific CTL
To investigate the mechanism by which endogenous type I IFN promoted host antitumor responses, we looked specifi cally at the priming of tumorspecific T cells in WT and Ifnar1 / mice after tumor challenge. Splenocytes from WT hosts isolated 20 d after inoculation of H31m1 tumor cells showed robust cytolytic activity against H31m1 targets after in vitro restimulation (Fig. 5 A). In contrast, tumorspecific killing was largely absent from splenocytes derived from Ifnar1 / mice challenged with tumor cells. Similar results were observed using another highly immuno genic unedited MCA sarcoma (GAR4.GR1) or using IFN production as a readout (unpublished data). To ask whether type I IFN sensitivity in in nate immune cells was sufficient to generate tumor specific immune responses, we used the mixed bone marrow chimeras described previously (Fig. 3). These experiments showed that IFN/'s actions on the innate immune compartment were indeed both necessary and sufficient for development of adaptive chimeras). Control chimeras with responsiveness in both innate and adaptive compartments (Rag2 / + WT → Ifnar1 / ; innate + adaptive) or neither compartment (Ifnar1 / → Ifnar1 / ; "neither") were also generated. The phenotypes of mixed chimeras generated using this ap proach were confirmed by IFNAR1 staining of splenocyte subsets ( Fig. 3 A and Fig. S6). When challenged with H31m1 or d38m2 cells, Rag2 / and Ifnar1 / control mice and globally unresponsive "nei ther" chimeras developed progressively growing tumors. In contrast, WT controls and panhematopoietic responsive in nate + adaptive or WT → WT chimeras rejected the tumor challenge (Fig. 3, B and C), consistent with our previous re sults (Fig. 2). Importantly, H31m1 and d38m2 cells were re jected in mixed chimeras with IFN/ sensitivity only in innate immune cells (i.e., innate chimeras) but grew progres sively in chimeras with IFN/ sensitivity largely re stricted to the adaptive immune compartment (i.e., adaptive chimeras). These findings demonstrate that the essential anti tumor functions of type I IFN on host cells during tumor rejection are selectively directed toward cells of the innate immune compartment.
To confirm the functional hematopoietic reconstitution of Ifnar1 / mixed chimeras, we performed three experi ments. First, we confirmed the normal representation of vari ous immune cell subsets within the spleens of mixed chimeric mice (Fig. 4, A and B). Second, we assessed the in vivo growth genetically pure C57BL/6 Rag2 / mice and naive WT C57BL/6 mice as recipients because we could deplete NK cells in C57BL/6 mice with the NK1.1specific PK136 mAb (Koo and Peppard, 1984). Similar to results with unedited MCA sarcomas from 129/Sv mice, immunemediated rejec tion of two representative C57BL/6 strain unedited sarcomas (1969 and 7835) required IFN/ sensitivity at the level of the host (Fig. 6, A and B). When PK136treated WT mice were injected with unedited C57BL/6 tumor cells, they rejected these tumors with kinetics identical to control mice. We confirmed NK cell depletion by (a) flow cytometry, (b) the absence of ex vivo killing of YAC1 targets by spleno cytes from mAbtreated mice, and (c) the lack of in vivo control of RMAS tumor cell growth (Fig. 6, C-E). These data therefore indicate that NK1.1 + NK cells are not required for IFN/-dependent rejection of unedited MCA sarcomas.

Granulocytes and macrophages do not require type I IFN sensitivity for tumor rejection
To test whether type I IFN sensitivity is required by granulo cytes and macrophages for tumor rejection, we crossed C57BL/6 strain LysM-Cre + mice (Clausen et al., 1999) to C57BL/6 Ifnar1 f/f mice (Prinz et al., 2008; prepared by back crossing 129 strain Ifnar1 f/f mice >99% onto a C57BL/6 back ground using a speed congenic approach). The resulting LysM-Cre + Ifnar1 f/f mice displayed complete IFNAR1 deletion in peritoneal macrophages and PMNs and substantial deletion of IFNAR1 in monocytes (66%) and splenic macrophages (35%) but maintained undiminished IFNAR1 expression in DCs, NK cells, T cells, and B cells (Fig. 7, A and B). Perito neal macrophages from these mice were unresponsive to type I IFN and failed to phosphorylate STAT1 after IFN stimula tion (Fig. 7 C). However, LysM-Cre + Ifnar1 f/f mice still rejected highly immunogenic unedited B6 strain 1969 sarcoma cells similar to IFN/-responsive Ifnar1 f/f mice (Fig. 7 D). In contrast, these tumor cells formed progressively growing tumors in B6 strain Ifnar1 / control mice. Thus, protective tumor immunity does not require type I IFN sensitivity in granulocytes and at least some macrophage compartments.

CD8 + lineage DCs are important targets of type I IFN's actions
Having ruled out NK cells, PMNs, and certain macrophage subsets as the critical type I IFN responsive cellular popula tions, we turned our attention to DCs. We previously showed that the selective absence of CD8 + lineage DCs in 129 strain Batf3 / mice led to an impairment in tumorspecific CTL priming and an inability to reject 129 strain H31m1 or 1773 unedited sarcoma cells (Hildner et al., 2008). We sub sequently made similar observations using three other unedited 129 strain sarcoma cell lines (d38m2, d42m1, and GAR4. GR1) that require IFNAR1 in host cells for rejection (un published data). Given the effects of type I IFN in promoting DC maturation, we hypothesized that DCs, and specifically CD8 + lineage DCs, may be critical innate immune targets tumorspecific cytotoxicity (Fig. 5 B). In addition, treatment of splenocytes from innate chimeras with blocking CD4 or CD8specific antibodies confirmed the importance of CD8 + cells for in vitro cytotoxicity (Fig. 5 C). These results demon strate the selective importance of type I IFN on innate im mune cells to induce tumorspecific CTL priming.

NK cells are not required for type I IFN-dependent tumor rejection
Because NK cells have a hostprotective function in some tumor models and display enhanced cytotoxic activity in re sponse to type I IFN, we investigated the role of NK cells in the rejection of highly immunogenic sarcomas. We used comparable unedited MCA sarcoma cells generated from Figure 5. Impaired tumor-specific CTL priming in Ifnar1 / mice is restored by IFN-/-responsive innate immune cells. (A) Splenocytes from WT and Ifnar1 / mice were isolated 20 d after H31m1 tumor challenge (10 6 cells/mouse), co-cultured with IFN--treated, irradiated H31m1 cells, and 5 d later used as effectors in a cytotoxicity assay with 51 Cr-labeled H31m1 targets. Specific killing activity (in percentage ± SEM) at the indicated effector/target (E:T) ratios is shown for four to five mice per group assayed in duplicate from three independent experiments. (B) Splenocytes were harvested from the indicated chimeric mice 20 d after injection of 10 6 H31m1 tumor cells and were treated as in A. Data include representative results from three mice per group assayed in duplicate from two independent experiments. Splenocytes harvested from a naive mouse and treated similarly served as a negative control. (C) Effector cells from H31m1-challenged innate chimeras were co-cultured at the indicated effector/target ratios with 51 Cr-labeled H31m1 targets in the presence of 10 µg/ml control (PIP), anti-CD4 (GK1.5), or anti-CD8 (YTS-169.4) mAbs. Data show representative results from three mice per group assayed in duplicate from three independent experiments. Similar results were obtained when effector cells from H31m1-injected WT mice were used (not depicted). (B and C) Error bars represent SEM. expression corresponds to the selective expression of Cre recombinase in these cell types as indicated by expression of a bicistronic GFP gene that is contributed by the Itgax-Cre mouse (Fig. S8 B). Both CD8 + and CD4 + DCs from Itgax-Cre + Ifnar1 f/f mice exhibited significantly decreased respon siveness to type I IFN as detected by reduced accumulation of pSTAT1 (Fig. 8 C) and by impaired upregulation of CD86 upon stimulation with IFN (Fig. S8 C). In contrast, T cells and macrophages in Itgax-Cre + Ifnar1 f/f mice displayed type I IFN responsiveness that was comparable with cells from Ifnar1 f/f mice. The selective nature of IFNAR1 deletion and loss of function in DCs allowed us to examine whether these cells were obligate targets of type I IFN during devel opment of antitumor responses in vivo. Whereas unedited B6 strain 1969 sarcoma cells were rejected in WT or Ifnar1 f/f mice, they formed progressively growing tumors in Itgax-Cre + Ifnar1 f/f mice with growth kinetics indistinguishable from those in Ifnar1 / mice (Fig. 8 D). These results thus demon strate that type I IFN sensitivity is specifically required by DCs for development of hostprotective tumor immunity.

Adoptive transfer of type I IFN-responsive DCs into
Ifnar1 / mice promotes induction of antitumor responses. Third, we examined whether the adoptive transfer of CD11c + cells isolated from the spleens of naive WT or Ifnar1 / mice into Ifnar1 / recipients promoted tumor resistance in vivo. Whereas CD11c + cells from WT mice induced of type I IFN during tumor rejection. The following four sets of experiments were performed to test this hypothesis.

DC subsets develop normally in the absence of IFNAR1.
First, we assessed whether Ifnar1 / mice displayed a defi ciency in any DC populations. Analyses of splenic and LN cells revealed no difference between the numbers of each DC subset in WT and Ifnar1 / mice (Fig. S7). In addition, there was no defect in the ability of Ifnar1 / DCs to expand in vivo in response to flt3 (fmslike tyrosine kinase 3) ligandFc treatment (not depicted). Thus, the absence of type I IFN signaling did not affect the development of any DC subset.

IFN-/ signaling by DCs is required for rejection of tumors.
Second, we assessed tumor rejection in mice that displayed a selective deletion of IFNAR1 in DCs. We crossed the aforementioned C57BL/6 strain Ifnar1 f/f mice to a spe cific line of Itgax (CD11c)-Cre + mice generated on a pure C57BL/6 genetic background (Stranges et al., 2007). When compared with the same cell populations from control mice by flow cytometry, IFNAR1 was expressed in undiminished levels in B cells, T cells, NK cells, macrophages, granulo cytes, and plasmacytoid DCs (pDCs) from Itgax-Cre + Ifnar1 f/f mice (Fig. 8, A and B; and Fig. S8 A). In contrast, IFNAR1 expression was substantially reduced in CD8 + DCs, the highly related CD103 + DCs, and CD4 + DCs from Itgax-Cre + Ifnar1 f/f mice (Fig. 8, A and B). The reduction in IFNAR1 Data are presented as mean tumor diameter ± SEM of 4-13 (untreated) or 8 (treated) mice per group from at least two independent experiments. Error bars for Ifnar1 / mice reflect progressive growth of 1969 and 7835 tumors in 6/9 mice. (C) WT C57BL/6 mice were treated with either PBS or PK136 mAb, and splenocytes were harvested 2 d later and analyzed by flow cytometry using the NK cell markers DX5 and NKp46. Splenocytes were gated on CD3  cells, and the percentages of DX5 + NKp46 + cells are indicated. Similar results were found when harvested at day 6 (not depicted). (D) WT C57BL/6 mice were treated with PBS or PK136 followed by i.p. injection of 300 µg polyI:C 4 d later. After 24 h, splenocytes were harvested and used as effectors in a standard 4-h cytotoxicity assay with NK-sensitive YAC-1 targets. Specific lysis (in percentage ± SEM) at the indicated effector/ target (E:T) ratios is shown for four mice/group assayed in duplicate from two independent experiments. (E) WT C57BL/6 mice were treated with PBS, PK136, or a mixture of anti-CD4 (GK1.5) and anti-CD8 (YTS-169.4) mAbs and injected s.c. with 10 5 RMA-S cells, and tumor growth was monitored over time. Mean tumor diameter ± SEM for three mice/group is shown, and data are representative of two independent experiments. from WT mice were more effective than Ifnar1 / derived cells in inducing the proliferation of OTI T cells (Fig. 9 A), although this defect could be overcome at high doses of antigen. Crosspresentation by WT CD11c + cells was en hanced by treatment with exogenous IFN and inhibited by the addition of MAR15A3 mAb that blocked the type I IFN receptor on these cells (Fig. 9 B). When WT and Ifnar1 / DCs were further purified into CD8 + and CD4 + subsets, the CD8 + DC subset was shown to be the critical crosspresenting cell in this assay, and a more significant defi cit was observed in the capacity of Ifnar1 / CD8 + DCs to activate OTI T cells (Fig. 9 C). Importantly, the CD8 + DCs from Itgax-Cre + Ifnar1 f/f mice displayed an OVA antigen crosspresentation defect that was virtually identical to CD8 + DCs from Ifnar1 / mice (Fig. 10). Similar results were also obtained when MHC mismatched, IFN-insensitive CMS5IC tumor target cells that were transduced with an OVAexpressing retrovirus were used as a source of antigen (Fig. S10). These findings thus demonstrate that type I IFN acts directly on CD8 + DCs to en hance crosspresentation of antigen to naive CD8 + T cells.
tumorspecific CTL priming (Fig. S9 A), Ifnar1 / CD11c + cells did not. The transfer of WT CD11c + cells also delayed tumor growth but did not result in tumor rejection (Fig. S9 B).
In contrast, no effect on tumor growth was observed upon transfer of CD11c + cells derived from Ifnar1 / mice. This difference was statistically significant (P = 0.03). These results are consistent with our previous observation that transfer of purified DC populations into Batf3 / mice results in only partial reconstitution of the antitumor response, perhaps be cause of issues of DC trafficking (Hildner et al., 2008).
(D) Ifnar1 f/f , LysM-Cre + Ifnar1 f/f , and Ifnar1 / mice were injected s.c. with 10 6 1969 unedited sarcoma cells. Mean tumor diameter ± SEM from a representative experiment is shown, and the bar graph shows the percentage of tumor-positive mice per group from two independent experiments with indicated total group sizes.
IFN during tumor rejection are distinguishable from those of IFN both temporally and functionally, and they repre sent an important step toward mapping the critical molecular pathways involved in cancer immunoediting. Functionally active type I IFN receptors are expressed on nearly all nucleated cells, and previous studies documented effects of type I IFN on many immunologically relevant cell types (such as T cells, NK cells, and DCs) that theoretically should enhance the immune elimination of tumors . Thus, it was surprising to find an essential func tional requirement for type I IFN in only a single cellular compartment, namely DCs, during the development of pro tective tumorspecific immune responses in vivo. As further documented in vitro, type I IFN enhances the function of the CD8 + DC subset, which in a previous study was shown to play a critical role in the development of tumor and virus specific immune responses through its capacity to crosspresent antigen to CD8 + T cells (Hildner et al., 2008). These cells,

DISCUSSION
Previous work from our laboratory and others has shown that naturally occurring, hostprotective immune responses against many highly immunogenic tumors require the obligate par ticipation of endogenously produced type I IFN (Dunn et al., 2005;Swann et al., 2007). Although these earlier studies pointed to hematopoietic cells as the physiologically relevant targets of type I IFN action, they neither identified the spe cific cell populations affected nor defined the functions that they performed. The current study was undertaken to eluci date the role of endogenously produced type I IFN in driving hostprotective, antitumor responses. Herein we demonstrate that type I IFN exerts its activity early during the develop ment of the antitumor response, that its major physiological function is directed selectively toward a single host cell popu lation (i.e., DCs), and that, at least in part, it functions to en hance the capacity of CD8 + DCs to crosspresent antigen to CD8 + T cells. These data thus reveal that the actions of type I ). Cells were gated as follows: CD8 + DCs (CD8 + Dec205 + CD11c hi ), CD103 DCs (CD8  Dec205 + CD11c hi CD103 + ), CD4 + DCs (CD8  Dec205  CD11c hi CD4 + ), dermal DCs (CD8  CD11c hi CD103  ), pDCs (B220 + PDCA + CD11c int ), B cells (B220 + ), T cells (CD3 + ), NK cells (NK1.1 + ), macrophages (CD11b + F4/80 + ), and blood PMNs (CD11b + Gr1 + ). IFNAR1 expression was measured using the MAR1-5A3 mAb. Data represent three to five mice from at least three independent experiments. (**, P < 0.01). (C) Splenocytes from Itgax-Cre + Ifnar1 f/f mice were untreated (gray) or stimulated for 15 min with 10 ng/ml IFN- v4 (black), and pSTAT1 accumulation in CD8 + and CD4 + DCs was measured by flow cytometry. Histograms show a representative experiment, and the bar graph summarizes results from four independent experiments (**, P < 0.01). (B and C) Error bars represent SEM. (D) C57BL/6 WT, Ifnar1 / , Ifnar1 f/f , and Itgax-Cre + Ifnar1 f/f mice were injected s.c. with 10 6 1969 unedited sarcoma cells. Mean tumor diameter ± SEM from a representative experiment is shown, and the bar graph shows a summary of the percentage of tumorpositive mice per group from three independent experiments with indicated groups sizes (P < 0.001 [WT vs Support for this conclusion comes directly from the find ing that bone marrow chimeric mice with selective recon stitution of type I IFN sensitivity in the innate immune compartment generated tumorspecific CTL and rejected immunogenic tumor cells, whereas the direct actions of type I IFN on T and B lymphocytes contributed little to the anti tumor response. It is important to stress that whereas the re sults of our analyses clearly show that T cells are not the essential type I IFN-sensitive cellular population, immune elimination of tumors nevertheless requires both CD4 + and CD8 + T cells. The lack of a requirement for type I IFN responsiveness in T lymphocytes contrasts with results from studies of CD8 + T cell priming and clonal expansion in the settings of viral infection or protein immunization which are dependent on the BATF3 transcription factor for their development, were originally identified as the CD8 + DCs that resided in lymphoid organs; yet subsequent work showed that they are closely related to another small DC subset residing in peripheral tissues that lack CD8 but express CD103 (Hildner et al., 2008;Ginhoux et al., 2009;Edelson et al., 2010). Although we find herein that optimal cross presenting activity of CD8 + DCs occurs only in response to type I IFN, our results do not exclude a requirement for type I IFN in regulating other DC populations such as CD4 + DCs. Thus, we conclude that the CD8 + DC subset represents one innate immune cell population that displays an obligate requirement for type I IFN to perform its function in the anti tumor response.  we focused our attention on DCs as likely innate immune targets of type I IFN's actions. Although type I IFN is a strong inducer of DC maturation (Luft et al., 1998;Gallucci et al., 1999;Montoya et al., 2002), the specific role of this cellular subset in the generation of protective antitumor responses has been difficult to establish. Some studies have indeed impli cated bone marrow-derived cells in the crosspresentation of tumorassociated antigen (Huang et al., 1994), whereas others have argued that direct priming may additionally be involved (Ochsenbein et al., 2001;Wolkers et al., 2001). Moreover, although the CD8 + DC subset is particularly adept at antigen crosspresentation, evidence also exists that other nonDC immune subsets as well as nonhematopoietic stromal cells might be capable of crosspresenting exogenous antigen in some circumstances (Ackerman and Cresswell, 2004;Heath et al., 2004;Spiotto et al., 2004).
The generation of mice lacking the transcription factor BATF3 provided a useful mechanism to study DC cross presentation in vivo because these mice have a cellintrinsic defect in the development of CD8 + DCs but normal repre sentation and function of the remaining DC subsets as well as other hematopoietic lineages (Hildner et al., 2008). Highly immunogenic MCA sarcoma cells, which are rejected in WT mice, formed progressively growing tumors in Batf3 / mice and displayed growth kinetics comparable with those in lym phocytedeficient Rag2 / hosts (Hildner et al., 2008), a re sult which we have corroborated in the current study. In addition, the defect in Batf3 / mice correlated with a lack of tumorspecific CTL priming (Hildner et al., 2008). These findings therefore demonstrated that crosspriming by CD8 + lineage DCs is critical for tumor rejection, although they do not address the nature of the innate immune signals necessary for activation, migration, and in vivo function of these cells. The importance of such stimuli is clear because cross presentation without activation can lead to tolerance rather than immunity (Steinman et al., 2003;Melief, 2008). A better understanding of this process could provide insight into the mechanisms that progressively growing tumors use to escape immune control.
We show in this study that type I IFN enhances the cross presentation of cellassociated antigen to naive CD8 + T cells via direct actions on CD8 + lineage DCs. When taken to gether with data demonstrating that (a) type I IFN promotes tumorspecific CTL priming, (b) type I IFN acts on innate immune cells to mediate its antitumor effects, (c) IFN/responsive CD11c + cells partially reconstitute in vivo CTL priming in Ifnar1 / mice, (d) CD8 + lineage DCs are re quired for CTL priming and tumor rejection, and (e) selective deletion of IFNAR1 in DCs abrogates tumor rejection, the collective evidence supports a hostprotective function involv ing direct actions of type I IFN on CD8 + lineage DCs.
The mechanism responsible for type I IFN's enhancement of CD8 + DC crosspriming remains to be determined. Type I IFN may induce multiple effects on the CD8 + lineage DCs, including the modulation of antigen capture or processing, peptide shuttling and MHC loading, MHC class I and/or (Kolumam et al., 2005;Le Bon et al., 2006a). Yet, it was noted in these studies that during infectioninduced clonal expansion, the relative importance of type I IFN's actions on CD8 + T cells depended on the specific microbial pathogen used (Thompson et al., 2006), with T cell expansion during lymphocytic choriomeningitis virus infection showing a profound dependence on type I IFN, but less prominent im pairments occurring when other viruses were used. In addi tion, another study reported no change in the generation of antigenspecific CTL in mice lacking the type I IFN receptor in the T cell compartment after immunization with peptide and IC31 (Pilz et al., 2009), an adjuvant based on Tolllike receptor 9 signaling. Given these data, it was suggested that distinct inflammatory environments might evoke expansion of CD8 + T cell subsets that differ in their dependence on type I IFN for survival and function and that such environ mental cues may include the levels of type I IFN and other signals that stem from innate cells (Stetson and Medzhitov, 2006;Thompson et al., 2006). Little is known about the magnitude and localization of type I IFN production (and that of other inflammatory cytokines) during immune re sponses to tumors, and further investigation is warranted.
To further define the target cells within the innate im mune compartment affected by type I IFN, we bred Ifnar1 f/f mice to LysM-Cre mice, an accepted method of deleting floxed target genes in nonDC myeloid cells (Clausen et al., 1999). The resulting mice exhibited nearly complete deletion of IFNAR1 in peritoneal macrophages and PMNs and re duced levels of IFNAR1 in other myeloid populations in cluding monocytes and splenic macrophages. Nevertheless, targeting myeloid cell IFNAR1 to the levels observed did not compromise antitumor immunity. These findings exclude a prominent role for granulocyte type I IFN sensitivity in our tumor system contrasting with data in the B16 melanoma model, suggesting that direct effects of endogenous IFN on tumorinfiltrating neutrophils are responsible for its anti tumor functions by suppressing expression of proangiogenic factors (Jablonska et al., 2010). With respect to the contribu tions of monocyte/macrophage subsets, more work is needed to define whether specific populations contribute to tumor immunity in the MCA sarcoma model, whether they are the same populations targeted in the LysM-Cre mouse, and which functions, if any, are influenced by type I IFN. Others have nonetheless shown that LysM-Cre + Ifnar1 f/f mice exhibit a clear phenotype during experimental autoimmune encepha lomyelitis despite observing similar partial reductions of IFNAR1 in myeloid populations (Prinz et al., 2008). LysM-Cre + Ifnar1 f/f mice display undiminished IFNAR1 ex pression in DCs. Thus, LysM-Cre + Ifnar1 f/f mice also serve as a control to support the conclusion that IFNAR1 is required predominantly in DCs and that tumor immunity remains in tact when IFNAR1 is genetically deleted in nonDC innate immune compartments.
Given the findings that adaptive immune cells, granulo cytes, and macrophages function independently of type I IFN and that NK cells do not play an obligate role in our system,
Tumor transplantation. MCAinduced fibrosarcomas were derived from 129/Sv strain Rag2 / or WT mice and C57BL/6 strain Rag2 / mice as de scribed previously (Shankaran et al., 2001;Dunn et al., 2005;Koebel et al., 2007). The GAR4 tumor, derived from an MCAtreated 129/Sv Ifngr1 / Ifnar1 / mouse, as well as IFNGR1resconstituted GAR4.GR1 cells and IFNAR1reconstituted GAR4.AR1 cells have been described previously (Dunn et al., 2005). RMAS is an MHC class I-deficient variant of the C57BL/6 strain T lymphoma RMA (Kärre et al., 1986). Tumor cells were propagated in vitro and injected s.c. in a volume of 150 µl endotoxinfree PBS into the shaved flanks of recipient mice as described previously (Dunn et al., 2005). Injected cells were >90% viable as assessed by trypan blue exclusion. Tumor size was measured on the indicated days and is presented as the mean of two perpendicular diameters. When calculating percent tumor growth, mice with tumors >6 mm in diameter were considered positive.
Generation of bone marrow chimeras. Recipient mice were irradiated with a single dose of 9.5 Gy and reconstituted with donor HSCs isolated from embryonic day (E) 14.5 fetal livers or 5fluorouracil (5FU)-treated adult bone marrow as described previously (Christensen et al., 2004;Dunn et al., 2005). For harvest of fetal liver cells (FLCs), embryos were extracted at E14.5, livers were removed, washed in sterile endotoxinfree PBS, and homogenized through a cell strainer using a syringe plunger. 5FU-treated bone marrow was isolated 4-5 d after treatment of donor mice with 150 mg/kg 5FU by i.p. injection. Cells were injected i.v. at a dose of 5 × 10 6 (FLCs) or 10 6 (5FU-treated bone marrow) cells/mouse in 200 µl PBS. Total cell dose was determined by titration of FLCs (Fig. S3) or based on prior data (Dunn et al., 2005). For mixed chimeras, a 4:1 cell ratio was selected costimulatory molecule expression, cellular migration, sur vival, or the induction of secondary cytokines/chemokines. Although current understanding of the cell biology of cross presentation is incomplete, some data indicate that heightened or altered antigen processing, rather than better antigen cap ture, underlies the ability of the CD8 + DCs to efficiently crosspresent antigen (Dudziak et al., 2007;Melief, 2008). Inter estingly, a recent study suggested that steadystate produc tion of low levels of IFN promotes antigen presentation by DCs to both CD8 + and CD4 + T cells via upregulation of heat shock protein 70, which boosts formation of MHC-peptide complexes (Zietara et al., 2009). Another recent study dem onstrated that type I IFN contributes to crosspresentation by enhancing antigen retention and survival of CD8 + DCs (Lorenzi et al., 2011). Additional mechanisms must be in volved because baseline antigen presentation (in the presence of lowlevel IFN) induces crosstolerance in the absence of DC activation triggered by inflammatory signals such as en hanced type I IFN production (Melief, 2008). The presence of other inflammatory stimuli, which may collaborate with type I IFN to activate CD8 + DCs, is suggested by detection of residual lowlevel priming in the absence of type I IFN signaling and the somewhat more robust tumor growth in Batf3 / mice (lacking CD8 + DCs) compared with Ifnar1 / mice (containing normal numbers of type I IFNunresponsive CD8 + DCs). The involvement of other in flammatory stimuli and their influence on type I IFN's effects remain to be investigated.
Exogenous administration of recombinant IFN has shown efficacy in the treatment of human cancer patients . However, despite many years of clin ical use, surprisingly little is known regarding its mechanism of action in this setting and the reason IFN treatment is effective in only a subset of patients. A host immunostimu latory mechanism is likely given the correlation between favorable responses to systemic IFN and the appearance of autoimmune sequelae in metastatic melanoma patients (Gogas et al., 2006). Animal studies have also confirmed that type I IFN activity on host cells, rather than actions on the tumor, mediate the protective effect of IFN/ administration . Whereas current treatments generally involve systemic injection of highdose IFN, it is possible that more targeted therapy based on a better understanding of the relevant underlying mechanism of action of type I IFN will enhance therapeutic efficacy while reducing undesirable side effects.
In summary, the findings made herein reveal that DCs represent the major targets of type I IFN actions during the induction of spontaneous tumorspecific CD8 + T cell re sponses and that these responses result, at least in part, from an enhanced capacity of CD8 + DCs to crosspresent anti gen to CD8 + T cells. These findings provide a strong ratio nale for future studies aimed at elucidating the precise DC functions that are regulated by type I IFN that ultimately promote development of naturally occurring or therapeutic immune responses to cancer. Thy1.2, DX5, and CD8 negative selection, followed by CD4 positive se lection. In all cases, purity of the population of interest was >97%. Spleno cytes from K b/ D b/  2 m / mice were prepared in serumfree medium, loaded with 10 mg/ml ovalbumin (EMD) by osmotic shock, and irradiated (13.5 Gy) as described previously (Hildner et al., 2008). OTI T cells were purified from OTI/Rag1 / mice by CD11c and DX5 negative selection followed by positive selection with CD8 microbeads (purity >99%). T cells were fluorescently labeled by incubation with 1 µM CFSE (SigmaAldrich) for 9 min at 25°C at a density of 2 × 10 7 cells/ml. For the assay, 5 × 10 4 puri fied DCs were incubated with 5 × 10 4 CFSElabeled OTI T cells in the presence of varying numbers of irradiated, ovalbuminloaded K b/ D b/  2 m / splenocytes. In some assays, the irradiated target cells were mis matched (BALB/c) tumor cells expressing a truncated version of the IFN receptor to render them IFN insensitive and in which ovalbumin was retrovirally enforced (CMS5IC). Ovalbumin expression was confirmed by coexpression of GFP by flow cytometry and by Western blot using a mouse antiovalbumin mAb (Santa Cruz Biotechnology, Inc.). After 3 d, cells were stained with antiCD8APC and CFSE, or cell proliferation dye (eBioscience) dilution was measured by flow cytometry. For IFN treat ment, recombinant mouse IFN5 (a gift from D. Fremont, Washington University in St. Louis) was added at 1,000 U/ml, whereas IFN/ recep tor blockade was achieved by incubation with 5 µg/ml IFNAR1specific MAR15A3 mAb.
Online supplemental material. Fig. S1 shows the kinetics of tumor growth in mice treated with blocking IFNAR1specific mAb. Fig. S2 demonstrates the importance of host IFN sensitivity for rejection of unedited sarcomas. Fig. S3 presents a titration of FLCs for generation of bone marrow chimeras. Figs. S4 and S5 show the normal functional im mune reconstitution of Ifngr1 / bone marrow chimeras (Fig. S4) and the absence of radioresistant, tissueresident leukocytes in the tumors of these mice (Fig. S5). Fig. S6 shows a determination of the HSC mixing ratio used to generate mixed bone marrow chimeras. Fig. S7 shows an analysis of DC subsets in Ifnar1 / mice. Fig. S8 shows further characterization of the Itgax-Cre + Ifnar1 f/f mice. Fig. S9 shows adoptive transfer experiments of WT and Ifnar1 / CD11c + cells into Ifnar1 / recipient mice. Fig. S10 shows decreased crosspresentation by CD8 + DCs from Itgax-Cre + Ifnar1 f/f mice compared with Ifnar1 f/f mice using retrovirally transduced tumor cells as a source of antigen. Online supplemental material is available at http://www .jem.org/cgi/content/full/jem.20101158/DC1. based on testing of different mixing ratios (Fig. S6). Animals were main tained on trimethoprimsulfamethoxazole (HiTech Pharmacal) antibiotic water prepared as described previously (Dunn et al., 2005) for 4 wk after irradiation, and tumor transplantation of chimeric mice was performed at least 12 wk after reconstitution. Hematopoietic reconstitution of all animals was verified by FACS staining of splenocytes at the completion of tumor trans plantation experiments. Similar experimental results were obtained with mice reconstituted using FLCs or 5FU-treated bone marrow as donor cells.
Tumor-specific CTL killing assay. Spleens were harvested from mice 20 d after tumor implantation, and single cell suspensions were prepared by homogenization using frosted glass slides. 4 × 10 7 splenocytes were cultured with 2 × 10 6 IFN-treated, irradiated (100 Gy) tumor cells. 5 d later, the cells were harvested and used as CTL effector cells in a standard 4h 51 Crrelease cytotoxicity assay that used tumor cell targets seeded at 10,000 cells/well and pretreated with 100 U/ml IFN for 48 h. For blocking assays, 10 µg/ml antiCD8 (YTS169.4), antiCD4 (GK1.5), or control mAb (PIP) was added to the cell culture of effector and target cells. Percent spe cific killing was defined as (experimental condition cpm  spontaneous cpm)/(maximal (detergent) cpm  spontaneous cpm) × 100.
NK cell cytotoxicity assay. Splenocytes were isolated from mice treated with 300 µg polyI:C (SigmaAldrich) by i.p. injection 24 h prior and were used as effector cells with 5,000 51 Crlabeled YAC1 tumor targets. Percent specific killing was assessed after 4h coincubation. Each sample was assayed in duplicate, and experiments were performed at least twice.