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J. Exp. Med. Vol. 208 No. 10 2005-2016
Most tumors express antigens that can be rec-
ognized by T cells of the host immune system
(Huang et al., 1994; Boon and Old, 1997). De-
spite the expression of antigens, tumors grow
progressively and evade immunity. It has gen-
erally been assumed that immune evasion is a
result of a failure to initiate an antitumor adap-
tive immune response. However, recent results
have indicated that in many instances, sponta-
neous T cell responses against tumor antigens
can be detected in both human cancer patients
and in murine models, and that immune escape
in those cases appears to occur through domi-
nant inhibition by immunoregulatory pathways
(Vesely et al., 2011). For example, high fre-
quencies of CD8+ T cells specific for MelanA/
MART-1, MAGE-10, and NY-Eso-1 have
been detected in the blood of subsets of pa-
tients with metastatic melanoma (Pittet et al.,
1999; Valmori et al., 2001; Mortarini et al., 2003;
Peterson et al., 2003). Spontaneous antibody
responses against a range of tumor-associated anti-
gens have been previously described (Tan and
Zhang, 2008). Antibody responses in early
stage prostate cancer have been reported to be
detected before PSA becomes detectably ele-
vated (Wang et al., 2005). Moreover, we and
others have shown that some human melanoma
metastases contain activated CD8+ T cells, in-
cluding tumor-reactive cells (Anichini et al.,
1999; Harlin et al., 2009), suggesting that spon-
taneous immune responses can be generated all
the way through to the step of effector cell
migration into tumor sites. Expression of multiple
immune evasion mechanisms likely blunts im-
mune function at the effector phase and allows
tumor outgrowth in those instances (Rabinovich
et al., 2007).
The observation that a T cell response can
ever become spontaneously primed against a
growing tumor mass raises the question of how
this is possible given the tight regulation of
innate immune signals that dictate whether a
bridge to adaptive immunity can occur. Most
malignancies (including melanoma) lack an ob-
vious infectious etiology and therefore would
not contain abundant external ligands for Toll-
like receptors (TLRs). In this context, studies
Thomas F. Gajewski:
Abbreviations used: BMDC,
BM-derived DC; dsDNA, double-
stranded DNA; mDC, myeloid
DC; pDC, plasmacytoid DC;
TLR, Toll-like receptor.
M.B. Fuertes and A.K. Kacha contributed equally to
Host type I IFN signals are required
for antitumor CD8+ T cell responses
through CD8+ dendritic cells
Mercedes B. Fuertes,1 Aalok K. Kacha,1 Justin Kline,1 Seng-Ryong Woo,1
David M. Kranz,2 Kenneth M. Murphy,3 and Thomas F. Gajewski1
1Department of Pathology and Department of Medicine, Section of Hematology/Oncology, the University of Chicago,
Chicago, IL 60637
2University of Illinois, Champaign-Urbana, IL 61820
3Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110
Despite lack of tumor control in many models, spontaneous T cell priming occurs frequently
in response to a growing tumor. However, the innate immune mechanisms that promote
natural antitumor T cell responses are undefined. In human metastatic melanoma, there
was a correlation between a type I interferon (IFN) transcriptional profile and T cell mark-
ers in metastatic tumor tissue. In mice, IFN- was produced by CD11c+ cells after tumor
implantation, and tumor-induced T cell priming was defective in mice lacking IFN-/R or
Stat1. IFN signaling was required in the hematopoietic compartment at the level of host
antigen-presenting cells, and selectively for intratumoral accumulation of CD8+ dendritic
cells, which were demonstrated to be essential using Batf3/ mice. Thus, host type I IFNs are
critical for the innate immune recognition of a growing tumor through signaling on CD8+ DCs.
© 2011 Fuertes et al. This article is distributed under the terms of an Attribu-
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The Journal of Experimental Medicine
Type I IFNs and CD8+ DCs in antitumor immunity | Fuertes et al.
mediate sterile immunity against tumors arising from self tis-
sues. Reanalysis of the gene expression profiling data revealed
that several transcripts indicative of type I IFN signaling, such
as IRF1 and the IFN-induced protein of 30 kD were co-
expressed in those tumors that contained T cell transcripts (Fig. 1).
This correlation prompted mechanistic experiments to deter-
mine whether host type I IFN signals might be necessary for
spontaneous priming of CD8+ T cells against tumor antigens
when it does occur.
We investigated whether spontaneous priming of anti-
gen-specific CD8+ T cells could be detected in lymphoid or-
gans after subcutaneous implantation of murine cell lines
in immunocompetent mice. To provide a defined antigen,
tumor cells were transduced to express the model antigenic
peptide SIYRYYGL (SIY) that is presented by the class I
molecule Kb. The SIY antigen is advantageous because of the
set of tools we have assembled for monitoring details of the
host immune response, including peptide/MHC tetramers,
TCR transgenic T cells, and high-affinity TCR tetramers to
monitor processed antigen on APCs (Kline et al., 2008;
Zhang et al., 2008). B16 melanoma cells expressing the SIY
antigen (B16.SIY) induced a significant frequency of peptide-
specific IFN-–producing cells in the spleen 6 d after sub-
cutaneous tumor implantation in the flank (Fig. 2 A). This was
accompanied by an increase in the frequency of SIY-specific
CD8+ T cells detected by SIY/Kb tetramer staining (Fig. 2 B).
This T cell activation is completely dependent on cross-
presentation by host DCs, as it was ablated in CD11c-DTR
transgenic mice treated with diphtheria toxin (Fig. S1). Flow
cytometry confirmed that the majority of CD8+ and plas-
macytoid DCs (pDCs), and a fraction of myeloid DCs
(mDCs), were depleted with this approach (Fig. S1). It is in-
teresting to note that these tumors grow progressively over
time and are not ultimately rejected by the host, but this
failure is not caused by an absence of early T cell priming.
Rather, recent observations suggest that the immune response
wanes over time because of dominant-negative regulatory
mechanisms that eventually lead to tumor outgrowth (Kline
et al., 2008). Regardless of those late events, early T cell
from several groups have revealed that dying cells can release
endogenous adjuvants (Kono and Rock, 2008), providing
activation signals for DCs and other APCs that lead to up-
regulation of co-stimulatory molecules and consequently
yield productive T cell activation and differentiation (Kono
and Rock, 2008). Although these early results indicate that
tumor cells can, under certain conditions, liberate products
that can theoretically elicit innate immune signals, how these
or other signals may lead to the spontaneous activation of a
tumor-specific adaptive T cell response remains unclear.
Type I IFNs have been studied extensively in the context
of viral infections (Stetson and Medzhitov, 2006b). During
various types of viral infection, type I IFNs induce the ex-
pression of an array of genes that act to prevent viral spread,
thus creating an antiviral state (Stark et al., 1998). But type
I IFNs also regulate antiviral immune effector responses and
play an important role in promoting the cross-presentation of
viral antigens to CD8+ T cells (Le Bon et al., 2003). Although
a role for type I IFNs has been described for immunosurveil-
lance against carcinogen-induced tumors and for rejection
of transplanted tumors (Dunn et al., 2005, 2006), the source
of type I IFNs and the mechanism of action of this cytokine
during the priming phase of an antitumor immune response
have not yet been elucidated. We have recently reported
that gene expression profiling of human melanoma metas-
tases revealed a subset of tumors that contained infiltrating
CD8+ T cells (Harlin et al., 2009). Reasoning that interro-
gation of those gene array data might provide an indication
regarding innate immune signals associated with the presence
of a T cell response, we herein report a correlation between
the presence of T cell–specific transcripts and a set of genes
known to be induced by type I IFNs. Using a series of
murine models, we show that shortly after tumor challenge
in vivo, type I IFN production was detected by DCs in tumor-
draining lymph nodes, and that host type I IFN signaling on
CD8+ DCs was required for spontaneous cross-priming of
tumor antigen–specific CD8+ T cells. Our results suggest a
model in which a growing tumor can elicit production of
type I IFNs from the host, which leads to accumulation of
CD8+ DCs that in turn promote CD8+ T cell activation
against tumor-derived antigens.
Tumors growing in immunocompetent hosts induce
T cell priming and IFN- production by CD11c+ cells
in tumor-draining lymph nodes
Gene expression profiling of human melanoma metastases
along with confirmatory assays revealed that a subset of
tumors showed evidence of spontaneous inflammation that
included the presence of infiltrating CD8+ T cells (Harlin et al.,
2009). Parallel studies have indicated that among tumor-
associated CD8+ T cells there are cells that recognize human
melanoma antigens as reflected by class I MHC/peptide tet-
ramer staining (Speiser et al., 2002; Harlin et al., 2006). This
evidence of spontaneous T cell priming against tumor anti-
gens raised the question of what innate immune signals might
Figure 1. Human melanoma metastases show a positive correla-
tion between T cell markers and IFN-induced transcripts. Tumor
samples were obtained by core biopsy or excisional biopsy or obtained
from material resected from patients as part of routine clinical manage-
ment. Total RNA was extracted from tumor samples (n = 52) and gene
levels were analyzed by Affymetrix. Arbitrary expression units according
to Affymetrix gene levels are shown. (IRF1, R2 = 0.648; p30, R2 = 0.658).
JEM Vol. 208, No. 10
As an alternative approach to determining whether DCs were
the dominant population producing IFN-, we depleted
DCs in vivo using CD11c-DTR transgenic mice treated
with diphtheria toxin. After tumor implantation, these mice
showed markedly reduced production of IFN- in the tumor-
draining lymph node compared with control mice (Fig. 2 E).
Together, these results indicate that DCs are the major source
of type I IFNs early after tumor challenge.
To determine whether this ability to induce a rapid host
immune response and type I IFN production was a general
phenomenon, an additional panel of C57BL/6-derived tumor
cells was transduced to express the SIY antigen. All the tumor
lines tested (EL4, MC57, and C1498) similarly induced a
spontaneous CD8+ T cell response as assessed by ELISPOT
(Fig. 2 F) and peptide/MHC tetramer analysis (not depicted),
and IFN- production in the tumor-draining lymph node
(Fig. 2 G). Together, these results indicated that it is not un-
common for implanted tumors to trigger type I IFN produc-
tion and a rapid CD8+ T cell response against tumor-associated
antigens in vivo.
Host IFN-/ signaling is critical for spontaneous
tumor-specific T cell priming
Having observed the early production of type I IFNs after
tumor inoculation, we sought to determine whether host
type I IFN signaling was necessary for spontaneous priming
of CD8+ T cells to tumor antigens in vivo. We therefore ex-
amined the effect of B16.SIY challenge on the CD8+ T cell
priming can be used in this model as a readout for determining
the host innate immune requirements for the initial recogni-
tion of tumor.
To determine whether implanted tumors might also in-
duce a type I IFN profile in mice, we inoculated B16 mela-
noma cells into recombination-activating gene 2-deficient
(Rag2/) mice, to eliminate a contribution of the adaptive
immune system. We compared gene expression of the tumors
recovered from the mice to the B16 cells grown in vitro, rea-
soning that the differentially expressed genes would be those
induced in the host by the presence of the tumor. Interest-
ingly, this gene expression profiling confirmed the up-
regulation of multiple transcripts reflective of innate immune
activation, including a panel of IFN-inducible genes (Table S1).
A repeat of this experiment in type I IFNR/ mice con-
firmed that induction of these transcripts required type I IFN
signaling on host cells (unpublished data). To assess directly
whether type I IFNs are produced early in response to a
growing tumor, we compared IFN- mRNA levels by
quantitative real-time PCR in lymph nodes from naive mice
and from draining lymph nodes of C57BL/6 mice challenged
with B16.SIY melanoma cells. We found that IFN- was
produced in tumor draining lymph nodes as early as 4 d after
tumor challenge (Fig. 2 C). We next sorted cells from tumor
draining lymph nodes on the basis of their expression of the
DC marker CD11c and analyzed IFN- mRNA levels,
which revealed that IFN- production after tumor challenge
was confined to the CD11c+ DC subpopulation (Fig. 2 D).
Figure 2. Tumors induce CD8+ T cell
priming, which is accompanied by IFN-
production by CD11c+ DCs in the tumor-
draining lymph nodes. (A and B) C57BL/6
mice were inoculated or not with 5 × 106 B16.
SIY tumor cells (s.c.), splenocytes were har-
vested 6 d later, and restimulated for 16 h in
the presence or absence of soluble SIY peptide
(A). The frequency of tumor-specific IFN-–
producing cells was assessed by ELISPOT.
***, P < 0.0001 versus No tumor. (B) Cells were
gated on CD8+CD4B220, and the frequency
of SIY-specific CD8+ T cells was assessed by
FACS using specific tetramers. **, P = 0.0063
versus No tumor. (C and D) C57BL/6 mice
were inoculated (s.c.) or not with 5 × 106 B16.
SIY tumor cells, and inguinal lymph nodes
were recovered 4–6 d later. IFN- mRNA ex-
pression was assessed by real-time RT-PCR
analysis in total lymph nodes, and the results are expressed as 2Ct using GAPDH as endogenous control. **, P = 0.0046 versus No tumor (C) or in CD11c+
and CD11c cells sorted from lymph nodes. ***, P = 0.0008 versus CD11c (D). (E) Wild-type C57BL/6 mice (expressing the congenic marker CD45.1+) were
lethally irradiated and reconstituted with either wild-type (CD45.2+) or CD11c-DTR (CD45.2+) BM cells. Mice were allowed to reconstitute for 3 mo, and
then were injected i.p. with diphtheria toxin (100 ng in 100 µl of DPBS) once a day for 8 d, starting 2 d before s.c challenge with 5 × 106 B16.SIY tumor
cells in the left flank (n = 5). Inguinal lymph nodes were recovered 6 d later, and IFN- expression in total inguinal lymph nodes was measured by real-
time PCR. The results are expressed as 2Ct using 18s as endogenous control. (F and G) C57BL/6 mice were inoculated s.c. with the indicated tumor cell
lines (5 × 106; F), and splenocytes were harvested 6 d later and restimulated for 16 h in the presence or absence of soluble SIY peptide. Frequency of
tumor-specific IFN-–producing cells was assessed by ELISPOT. (G) Real-time RT-PCR analysis of IFN- mRNA expression in total inguinal lymph nodes.
The results are expressed as 2Ct using 18s as endogenous control. Data represent mean ± SEM (n = 5) and are representative of four independent ex-
periments (A–C) or two independent experiments (D–G).
Type I IFNs and CD8+ DCs in antitumor immunity | Fuertes et al.
reconstituted with wild-type BM cells rejected the tumor
normally, indicating that the expression of Stat1 in hemato-
poietic cells is sufficient for tumor rejection and that Stat1
expression is not required in non-BM-derived cells for tumor
response to the SIY tumor antigen in mice deficient for the
IFN-/R compared with WT mice. 17 d after tumor in-
oculation, splenocytes were assayed for the frequency of
IFN-–producing cells in response to soluble SIY peptide or
irradiated B16.SIY cells by ELISPOT. In comparison to the
vigorous response observed in the wild-type group, spleno-
cytes from IFN-/R/ mice displayed a dramatically re-
duced frequency of IFN-–producing effector cells after
tumor challenge (Fig. 3 A). To determine whether the effect of
type I IFNs on T cell priming was at the level of expansion
versus differentiation of CD8+ T cells, analysis using SIY/Kb
tetramers was performed. Whereas an expanded population of
tetramer-reactive CD8+ T cells was detected in the spleens of
wild-type mice, IFN-/R/, and IFN-/R//IFN-R/
mice failed to display an increased frequency of tumor
reactive CD8+ T cells (Fig. 3 B). The combined elimina-
tion of IFN-/R and IFN-R reduced T cell priming
completely to background levels, suggesting partial compen-
sation by IFN- in the absence of type I IFN signaling.
However, mice singly deficient in the IFN-R generated a
normal expanded frequency of SIY-specific CD8+ T cells
(Fig. 3 C), suggesting that type I IFNs are dominantly re-
quired and that IFN- is not necessary for this stage of an
antitumor T cell response.
As type I IFNs mediate signaling through Stat1, we
additionally analyzed spontaneous CD8+ T cell priming in
Stat1/ mice. Consistent with our previous results, Stat1/
mice also showed a markedly decreased priming of tumor
antigen–specific CD8+ T cells as assessed by ELISPOT and
SIY/Kb tetramer analysis (Fig. 3, D and E). Together, these
results indicate that host type I IFN signaling through Stat1 is
required for optimal expansion of tumor antigen–specific
CD8+ T cells after tumor challenge in vivo.
IFN signaling is required in the hematopoietic compartment
for spontaneous rejection of tumors in vivo
To determine whether the defect in natural CD8+ T cell prim-
ing in the absence of host IFN signaling would be associated
with failed tumor rejection, we used a model in which tumors
are spontaneously rejected. At the same time, it was of interest
to determine the cellular compartment in which IFN signaling
must occur. Toward this end, we turned to the immunogenic
variant of the P815 mastocytoma, P198, which initially grows
but is spontaneously rejected in syngeneic DBA/2 mice
(Fallarino et al., 1996). To determine if type I IFN signaling for
tumor rejection was required in the hematopoietic or nonhe-
matopoietic compartment, we generated radiation BM chime-
ras in which Stat1-sufficient versus Stat1/ DBA/2 mice were
irradiated and reconstituted with either wild-type or Stat1/
DBA/2 BM cells. After a period of at least 3 mo, to allow for
recovery of the immune system, mice received a tumor chal-
lenge with P198 cells on the flank and were monitored for
tumor progression. As expected, P198 cells were rejected by
wild-type mice that had been reconstituted with wild-type
BM, and tumors grew progressively in Stat1/ mice reconsti-
tuted with Stat1/ BM (Fig. 4 A). In contrast, Stat1/ mice
Figure 3. IFN-/, and not IFN- signaling, is critical for sponta-
neous CD8+ T cell priming to tumor-associated antigens.
(A and B) Wild-type, IFN-/R/, or IFN-/R//IFN-R/ mice were
inoculated s.c. with 106 B16.SIY tumor cells. Splenocytes were harvested
17 d later and restimulated for 16 h in the presence or absence of or sol-
uble SIY peptide (A). The frequency of tumor-specific IFN-–producing
cells was assessed by ELISPOT. ***, P < 0.0001 versus WT. (B) cells were
gated on CD8+CD4B220 and the frequency of SIY-specific CD8+ T cells
was assessed by FACS using specific tetramers. ***, P < 0.0009; **, P <
0.0027 versus WT. (C) Wild-type and IFN-R/ mice were inoculated s.c.
with 106 B16.SIY tumor cells, and splenocytes were harvested 17 d later
and restimulated for 16 h in the presence or absence of soluble SIY pep-
tide, and then the frequency of tumor-specific IFN-–producing cells was
assessed by ELISPOT. P = 0.268 versus WT. (D and E) Wild-type and
Stat1/ mice were inoculated s.c. with 106 B16.SIY tumor cells, and
splenocytes were harvested 17 d later and restimulated for 16 h in the
presence or absence of soluble SIY peptide (D). The frequency of tumor-
specific IFN-–producing cells was assessed by ELISPOT. ***, P < 0.0001
versus WT. (E) cells were gated on CD8+CD4B220, and the frequency
of SIY-specific CD8+ T cells was assessed by FACS using specific tetramers.
**, P = 0.0029 versus WT. Data represent mean ± SEM (n = 5), and are
representative of three independent experiments.
JEM Vol. 208, No. 10
compartment is necessary for spontaneous tumor rejection
in vivo and that Stat1 in non-BM-derived cells is not sufficient
for tumor rejection (Fig. 4 A).
IFN signaling is required in non-T cell BM-derived cells
for tumor antigen–specific T cell priming
Among the cells of the hematopoietic compartment, IFN
signaling could be playing a T cell–intrinsic role in the gen-
eration of an antitumor immune response and/or be impor-
tant in non–T cells, perhaps at the level of APCs. To address
whether T cell–intrinsic IFN signaling was required for the
acquisition of an effector phenotype, we compared wild-type
and Stat1/ T cells primed to become effector CTL in a
mixed lymphocyte culture in vitro. However, Stat1/ and
wild-type T cells were equally able to lyse allogeneic target
cells (Fig. 4 B), suggesting that T cell–intrinsic Stat1 is not
absolutely required for the development and execution of
lytic effector function by cytotoxic T lymphocytes. IFN-
production by Stat1/ CTL was also preserved (unpublished
data). To investigate more directly whether IFN signaling on
non–T cells was required for activation of tumor antigen–
specific CD8+ T cells, we CFSE labeled wild-type CD8+
T cells purified from 2C TCR Tg/Rag2/ mice (Sha et al.,
1988) specific for the SIY octameric peptide in the context of
Kb (Udaka et al., 1996) and adoptively transferred them into
wild-type or Stat1/ syngeneic mice. We then challenged
the mice with B16.SIY tumors. 7 d after tumor challenge
with B16.SIY, we analyzed CFSE dilution of SIY-specific
CD8+ T cells in the spleen. A large percentage of the 2C
CD8+ T cells transferred into wild-type mice displayed a
decreased intensity of CFSE fluorescence, consistent with
antigen-specific T cell proliferation and successful cross-
presentation of the SIY antigen. In contrast, CFSE dilution was
markedly reduced upon transfer into Stat1/ hosts (Fig. 4,
C and D). Finally, to determine whether T cell priming could
occur in the absence of T cell–intrinsic IFN signaling in vivo,
we vaccinated Stat1/ versus wild-type mice with wild-type
BM-derived DCs (BMDCs) loaded with SIY peptide. When
assayed 20 d later for T cell priming by IFN- ELISPOT,
comparable induction of SIY-specific T cells was observed in
both wild-type and Stat1/ hosts (Fig. 4 E). This result demon-
strates that T cell–intrinsic IFN signaling is not required for
T cell priming in vivo, and suggests that the defect in T cell
priming lies upstream, likely at the level of host APCs.
Analysis of DC subsets from mice deficient in IFN signaling
In a search for potential mechanisms responsible for the
lack of tumor antigen–specific CD8+ T cell priming in the
absence of host IFN signaling, we analyzed multiple pheno-
typic characteristics of DCs from wild-type versus Stat1/
or IFN-/R/ mice. We first compared frequencies and
absolute numbers of the different DC subpopulations (mDCs,
CD11C+B220CD8CD11b+; CD8+DCs, CD11C+
B220CD8+CD11b; and pDCs, CD11CintB220+PDCA+)
in the spleen and lymph nodes of unmanipulated or tumor-
bearing mice but found no difference between Stat1/ or
Figure 4. IFN signaling is required in non–T cell BMDCs for tumor-
specific T cell priming and for spontaneous rejection of immunogenic
tumors in vivo. (A) Wild-type and Stat1/ DBA/2 mice were lethally
irradiated and reconstituted with either wild-type or Stat1/ DBA/2 BM
cells, and 3 mo later they were challenged s.c. with 106 P198 cells in the left
flank (n = 5). Tumor size was measured at different time points. Results are
shown as mean tumor diameter ± SEM. Data are representative of two
independent experiments. (B) T cells from wild-type and Stat1/ (H-2b)
mice were stimulated with T cell–depleted irradiated splenocytes from
DBA/2 (H-2d) mice for 5 d in an allogeneic MLR. Cytotoxic activity was mea-
sured by standard 51Cr-release assay against P815 (H-2d, cognate targets)
and EL4 cells (H-2b, control targets). (C and D) CD8+ T cells were purified
from the spleens of 2C Tg/RAG2/ (Stat1-sufficient) mice, CFSE labeled,
and transferred by retroorbital injection to wild-type or Stat1/ mice. The
next day, those mice were inoculated s.c. with 106 B16.SIY cells in the flank,
and 7 d later splenocytes were harvested and CFSE dilution of 2C CD8+
T cells was assessed by FACS. (C) Percent of cells with decreased CFSE inten-
sity within the DAPICD8+1B2+ gate. Data show mean ± SEM of individual
mice in each group. Data are representative of two independent experi-
ments. (D) Representative dot plots of CFSE dilution. Numbers indicate per-
cent of cells in the indicated gate. (E) 106 SIY-pulsed wild-type BMDCs were
s.c. inoculated in the flank of wild-type or Stat1/ mice, and 20 d later
splenocytes were harvested and restimulated in vitro for 16 h in the pres-
ence of culture medium or soluble SIY peptide. The frequency of tumor-
specific IFN-–producing cells was assessed by ELISPOT. Data are
representative of two independent experiments.
immunity (Fig. 4 A). In addition, the failure of wild-type mice
reconstituted with Stat1/ BM to reject the P198 tumor
demonstrates that expression of Stat1 in the hematopoietic
Type I IFNs and CD8+ DCs in antitumor immunity | Fuertes et al.
IFNs would induce chemokines by the DCs that would aid
in the recruitment of T cells to the lymph node for subse-
quent activation. Analysis of DCs stimulated with IFN/
revealed substantial production of CXCL9 and CXCL10,
which could potentially mediate recruitment of activated
CD8+ T cells via CXCR3 (unpublished data). However,
there was no defect in priming of SIY-specific CD8+ T cells
in response to B16.SIY tumors in CXCR3/ mice (unpub-
lished data), arguing for a lack of importance of this pathway
at the early stages of T cell priming in vivo. Finally, it was
reasoned that type I IFN signaling might be necessary for
DCs to be able to take up, process, and present tumor antigen
in the context of class I MHC molecules. This possibility was
addressed through the use of tetramers generated from a
high-affinity variant of the 2C TCR, which can bind to
SIY–Kb complexes with sufficient avidity to allow analysis
by flow cytometry. Analysis of the APC compartments was
performed in wild-type versus Stat1/ mice crossed to a
RAG2/ background, to eliminate the possibility that host
T cells might eliminate antigen-expressing APCs. In fact,
when tumor-derived CD11c+ or CD11b+ cells were ana-
lyzed from wild-type versus Stat1/ RAG2/ mice bear-
ing MC57.SIY tumors, comparable binding of the 2C TCR
tetramer was observed (Fig. 5, C and D). Absence of binding
to APCs derived from wild-type MC57 tumors confirmed
specificity of tetramer binding only when the SIY antigen
was present. These results suggest that the APC subsets that
accumulate in the tumor site process and present the SIY
tumor-derived antigen normally, even in the absence of host
type I IFN signaling.
In vivo, endogenous type I IFN signaling is required
for intratumoral accumulation of the CD8+ DC subset
Given the comparable properties of DC subsets in the lym-
phoid organs of wild-type and Stat1/ mice, and the intact
expression of SIY–Kb peptide–MHC complexes on the total
APC populations that are present within the tumor site, we
next addressed whether there was a defect at the level of
the specific DC subsets that accumulate within the tumor
microenvironment. We therefore challenged wild-type
and Stat1/ mice with B16.SIY cells and evaluated the fre-
quency and absolute number of the three major DC subpop-
ulations accumulating in the tumor at day 16. We found
that the mDC and pDC subpopulations accumulated in the
tumors of both groups of mice. However, the CD8+ DC
population, which accounted for up to 20% of the DCs infil-
trating the tumors in wild-type mice, was almost completely
absent in the tumors grown in Stat1/ mice (Fig. 6, A and B;
and Fig. S2). A similar defect in CD8+ DC accumulation
was observed in IFN-/R/ mice (Fig. 6 C). As an alter-
native quantitative approach to evaluate the presence of
CD8+ DCs, molecular markers were used. XCR1 is a
chemokine receptor exclusively expressed by CD8+ DCs
(Dorner et al., 2009) and Batf 3, a transcription factor prefer-
entially expressed by CD8+ DCs and absolutely required for
their development. Using quantitative RT-PCR to analyze
IFN-/R/ and wild-type mice (unpublished data). In
addition, the surface expression of CD80, CD86, CD40, and
class I and class II MHCs in CD11c+ tumor-draining lymph
node cells was comparable (Fig. 5 A and Table S2), and
LPS-induced IL-12 production was still detected in Stat1/
CD11c+ cells (not depicted). Moreover, adherent splenocytes
from wild-type and Stat1/ mice did not differ in their abil-
ity to stimulate 2C TCR Tg CD8+ T cells to produce IL-2
upon loading with the SIY peptide in vitro (Fig. 5 B).
It was conceivable that host IFN signaling was generally
important for the migration of DCs into the draining lymph
node compartment in vivo. To address this possibility, the
skin of wild-type and Stat1/ mice was painted with FITC
and the draining lymph nodes were analyzed for green fluo-
rescence on CD11c+ cells. However, comparable appearance
of FITC+ DCs was observed in both sets of mice (unpub-
lished data). We also considered the possibility that type I
Figure 5. Analysis of DCs from WT, Stat1/, and IFN-/R/
mice. (A) Wild-type and IFN-/R/ mice were inoculated s.c. with 106
B16.SIY cells. 6 d later, surface expression of CD80, CD86, CD40, and class I
and II MHC was assessed by FACS in tumor-draining lymph node cells
gated on CD11c+ cells. Filled histograms correspond to an isotype control (IC),
continuous line corresponds to wild-type, and dashed line corresponds
to IFN-/R/ mice. (B) Adherent splenocytes from wild-type and
Stat1/ mice were loaded with SIY peptide or left untreated and used
to stimulate 2C CD8+ T cells. IL-2 production was assessed by ELISA.
(C and D) Wild-type Rag1/ mice (top) or Rag1/Stat1/ mice (bottom)
were inoculated with 106 MC57 or MC57.SIY tumor cells. 14 d later SIY/Kb
expression was assessed by FACS using high-affinity 2C TCR tetramers
gated on the tumor-infiltrating CD11c+ population (C) and CD11b+ popu-
lation (D). Filled histograms correspond to staining with streptavidin-
phycoerythrin alone. Data are representative of two independent
experiments (n = 4).
JEM Vol. 208, No. 10
in Batf3/ and IFN-/R/ mice (Fig. S3, A and B). More-
over, parental B16 melanoma cells, without the SIY antigen,
also grew faster in Batf3/ mice compared with wild-type
(Fig. S3 C), indicating that this is a critical pathway for cross-
presentation of natural endogenous tumor antigens. To deter-
mine whether CD8+ DCs were themselves required for type I
IFN production in response to tumor implantation, induction of
IFN- mRNA was assessed in tumor-draining lymph nodes in
wild-type versus Batf3/ mice. However, IFN- induction
was comparable in both sets of mice (Fig. 7 C). Collectively,
these findings demonstrate that the CD8+ DC subpopulation
is critical for the spontaneous priming of tumor antigen–specific
CD8+ T cells in response to a growing tumor, downstream from
host type IFN production.
To determine whether the CD8+ DC lineage itself
must respond to type I IFNs, wild-type mice were irradiated
and reconstituted with wild-type, Batf3/ or IFN-/R/
BM cells, or a mix of wild-type and IFN-/R/ or
Batf3/ and IFN-/R/ in a 50/50 ratio. 3 mo later, those
chimeric mice were challenged with B16.SIY melanoma cells,
and splenocytes were assayed 6 d later for T cell priming by
SIY/Kb tetramer analysis and tumor size was measured. As
expected, mice reconstituted with Batf3/ or IFN-/R/
BM cells showed a dramatically reduced frequency of SIY-
specific CD8+ T cells as compared with mice reconstituted
with wild-type BM cells (Fig. 8 A). Mice reconstituted with a
mix of wild-type and IFN-/R/ cells showed restored
T cell priming to the level seen in wild-type mice. However,
mice reconstituted with a mix of Batf3/ and IFN-/R/
BM cells continued to show reduced priming of SIY-specific
T cells (Fig. 8 A). This reduced T cell priming was associated
with poor tumor growth control (Fig. 8 B). These results dem-
onstrate that type I IFNs must signal on the CD8+ DC
lineage for optimal priming of tumor antigen–specific CD8+
T cells after tumor challenge in vivo.
transcript abundance in tumors analyzed ex vivo, we found
that both transcripts were highly expressed in tumors grown
in wild-type mice, yet severely reduced in tumors grown in
IFN-/R/ mice (Fig. 6, D and E). Therefore, host IFN
signaling appeared to be required for intratumoral accumula-
tion of CD8+ DCs within the tumor microenvironment.
CD8+ DCs have been shown to be the most important
DC population for cross-presentation of antigens to CD8+
T cells in the setting of viral infection (Belz et al., 2004). Recent
work has indicated that mice lacking the transcriptional regula-
tor, Batf3, have a specific deficiency in the development of the
CD8+ DC lineage (which includes a CD103+ DC subset;
Hildner et al., 2008). These mice also show defective cross-
priming of CD8+ T cells in response to viruses, and were defec-
tive in control of immunogenic tumors in vivo (Hildner et al.,
2008). We therefore investigated whether these DC subpopula-
tions were required at the level of priming of CD8+ T cells to
tumor-derived antigens. Wild-type and Batf3/ mice were
challenged with B16.SIY melanoma cells, and splenocytes were
assayed 6 d later for the frequency of IFN-–producing cells
by IFN- ELISPOT and SIY/Kb tetramer staining. Indeed,
Batf3/ mice showed a dramatically reduced frequency of
IFN-–producing effector cells as compared with wild-type
mice (Fig. 7, A and B). The poor T cell priming was comparable
to the level of deficiency observed in IFN-/R/ mice
(Fig. 3 A), as was the tumor growth rate, which was accelerated
Figure 6. Endogenous type I IFN signaling is required for intra-
tumoral accumulation of CD8+ DCs. (A and B) Wild-type and Stat1/
mice were inoculated s.c. with 106 B16.SIY cells, and 15 d later tumors
were harvested and frequency (A) and percentages (B) of CD8+ DCs,
mDCs, and pDCs infiltrating tumors were analyzed by FACS.
GFP+DAPI+CD3+ cells were gated out, and the different DCs subpopula-
tions were identified as follows: mDCs, CD11C+B220CD8CD11b+;
CD8+DCs, CD11C+B220CD8+CD11b; and pDCs, CD11CintB220+PDCA+.
Results are shown as mean ± SEM of 3 independent experiments (n = 4).
(C–E) Wild-type and IFN-/R/ mice were inoculated s.c. with 106 B16.
SIY cells, and 15 d later tumors were harvested and frequency of intra-
tumoral CD8+ DCs was assessed by FACS (C). (D and E) XCR1 mRNA ex-
pression (D) and Batf3 mRNA expression (E) were assessed by real-time
RT-PCR analysis on tumor homogenates. The results are expressed as
2Ct using 18s as endogenous control. Results are shown as mean ±
SEM of 2 independent experiments (n = 5).
Figure 7. CD8+ DCs are critical for antitumor CD8+ T cell prim-
ing. Wild-type and Batf3/ mice were inoculated s.c. with 106 B16.SIY
cells. 6 d later, splenocytes were harvested and restimulated for 16 h in
the presence of culture medium or soluble SIY peptide (A). The frequency
of tumor-specific IFN-–producing cells was assessed by ELISPOT. ***, P <
0.0001 versus WT. (B) the frequency of SIY-specific CD8+ T cells was
assessed by FACS using specific anti–Kb-SIY tetramers, cells were gated on
the CD8+CD4B220 population. ***, P < 0.001 versus WT. (C) IFN-
mRNA expression was assessed by real-time RT-PCR analysis in total
lymph nodes. The results are expressed as 2Ct using 18s as endogenous
control. Results are shown as mean ± SEM (n = 5) and are representative
of at least two experiments.
Type I IFNs and CD8+ DCs in antitumor immunity | Fuertes et al.
pathway leading to IRF3 activation and to type I IFN pro-
duction (Stetson and Medzhitov, 2006a). It is interesting to
speculate that a TLR-independent cytosolic DNA recogni-
tion pathway might be involved in innate tumor recognition,
IFN- production, and spontaneous priming of tumor antigen–
specific T cells.
Our data have demonstrated that, shortly after tumor
challenge, IFN- 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- production is
still observed in Batf3/ mice strongly suggest that the
CD8+ 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- 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 sponta-
neous antitumor T cell priming. In this model, one DC sub-
population (likely either mDCs or pDCs) would sense the
presence of the tumor and produce type I IFNs, which
through signaling on CD8+ DCs would promote effective
cross-priming of CD8+ T cells. Consistent with this model, it
has been recently shown using quantitative proteomics that
the CD8+ 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 in-
deed the pathway involved in type I IFN production to tumor,
a non-CD8+ DC subpopulation would need to be involved.
Although several studies have suggested that CD8+ DC
distribution is restricted to lymphoid organs (Randolph et al.,
2008) our results clearly indicate that CD8+ DCs (defined
as CD3CD11c+B220CD11bCD8+ cells) can infiltrate
tumors. In agreement with our findings, it has been reported
that CD8+ DCs can infiltrate transplantable and spontane-
ous 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 (un-
published data). The detailed mechanism by which type I
IFNs induce the intratumoral accumulation of CD8+ DCs
will be a crucial area for future investigation.
It is noteworthy that, under conditions in which sponta-
neous priming of antitumor CD8+ T cells was not occurring,
we detected expression of processed SIY peptide–Kb com-
plexes on the surface of several subsets of APCs. These results
suggest that APC subtypes other than CD8+ DCs are
capable of processing antigen into the class I compartment.
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- is produced shortly after tumor
challenge and, through signaling at the level of CD8+ DCs,
promotes tumor antigen–specific T cell priming and tumor
rejection. In vivo, endogenous type I IFNs induced the intra-
tumoral accumulation of CD8+ DCs, which were essential
for spontaneous antitumor CD8+ T cell priming and which
could explain the critical role of this cell lineage in spontane-
ous cross-priming of tumor antigen–specific CD8+ T cells.
Given the lack of external TLR ligands for most malig-
nancies, the molecular mechanism by which a tumor can
provide the right environment to stimulate immune activa-
tion remains poorly understood. Emerging evidence indicates
that dying cells can release endogenous adjuvants capable of
activating APCs (Kono and Rock, 2008). Among these mol-
ecules 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 re-
cently 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 in-
flammasome (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
Figure 8. Type I IFN signaling must occur on the CD8+ DC lineage
for antitumor CD8+ T cell priming to occur. (A) Wild-type mice were
lethally irradiated and reconstituted with wild-type, Batf3/, or IFN-
/R/ BM cells, or a mix of wild-type and IFN-/R/ or Batf3/
and IFN-/R/ in a 50/50 proportion. Mice were allowed to reconsti-
tute for 3 mo, and were then inoculated s.c. with 106 B16.SIY cells.
(A) splenocytes were harvested 6 d later, and the frequency of SIY-specific
CD8+ T cells was assessed by FACS using specific tetramers. Cells were
gated on CD8+CD4B220. (B) Tumor size was measured at the end of
the experiment. *, P < 0.05 versus IFN-/R/ + WT. Results are shown
as mean ± SEM of 2 independent experiments (n = 4 each).
JEM Vol. 208, No. 10
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.
MATERIALS AND METHODS
Human samples and gene array analysis. Biopsy processing and gene
array analysis were described previously (Harlin et al., 2009). Data were
interrogated for expression of IFN-regulated genes and referenced to TCR
transcripts in individual tumors.
Mice. C57BL/6 mice, 129 mice, and Stat1/ mice were purchased from
Taconic. For the indicated experiments, Stat1/ mice were backcrossed for
six generations onto DBA/2 mice (Jackson ImmunoResearch Laboratories).
IFN-R/ mice were obtained from The Jackson Laboratory. IFN-
R/ mice and IFN-/R/IFN-R/ mice were purchased from
B&K Universal. Batf3/ mice (Hildner et al., 2008) were obtained from
K. Murphy (Washington University School of Medicine, St. Louis, MO).
Experiments in these strains were done either on the 129 or the C57BL/6
background (at least 5 generations) with similar results. 2C/RAG2/ mice
(Sha et al., 1988) were obtained from D. Loh (Washington University
School of Medicine, St. Louis, MO). CD11c-DTR mice (Jung et al., 2002)
were provided by D. Littman (New York University School of Medicine,
New York, NY). All mice were used between 6 and 10 wk of age and were
maintained in specific pathogen–free conditions in a barrier facility at the
University of Chicago (Chicago, Illinois). All animal experiments were per-
formed in accordance with protocol approved by the University of Chicago
Institutional Animal Care and Use Committee.
Tumor cell lines. The mutagenized DBA/2-derived mastocytoma cell line
P198 and the C57BL/6 derived melanoma cell lines B16.F10 and B16.F10.
SIY (henceforth referred to as B16.SIY), thymoma cell lines EL4 and EL4.
SIY, fibrosarcoma cell lines MC-57 and MC-57.SIY, and the leukemia cell
line C1498.SIY were used for experiments. All cells were maintained at
37°C with 7.5% CO2 in DME supplemented with 10% heat-inactivated
FCS, MOPS, 2-mercaptoethanol, penicillin, streptomycin, l-arginine,
l-glutamine, folic acid, and l-asparagine.
In vivo tumor experiments. Cultured tumor cells were washed three
times with Dulbecco’s PBS (DPBS), and 106 living cells were injected s.c. in
100 µl DPBS on the flank. For tumor growth experiments, the longest and
shortest diameters were measured twice per week using calipers, and a mean
and SD were calculated. For ELISPOT, tetramer staining, and CFSE dilu-
tion splenocytes were analyzed at the indicated time points after tumor chal-
lenge. For RT-PCR analysis of IFN-, tumor-draining inguinal lymph
nodes were collected and analyzed 4 to 6 d after tumor challenge. For analy-
sis of tumor-infiltrating DC subpopulations, tumors were recovered 15 d
after tumor challenge and were disrupted in complete DME medium con-
taining 1 mg/ml collagenase IV (Sigma-Aldrich) for 30 min at 37°C. Data
from groups of three to seven mice were analyzed.
IFN- ELISPOT. The enzyme-linked Immunospot assay (ELISPOT) was
conducted with the BD mouse IFN- kit according to the manufacturer’s
protocol. Splenocytes were plated at 106 cells/well and stimulated overnight
with irradiated (10,000 rad) B16.SIY cells (5 × 104/well), SIY peptide
(80 nM), or PMA (50 ng/ml) and ionomycin (0.5 µM). In experiments ana-
lyzing priming by BMDCs, the restimulation was performed in FCS-free
culture medium and plates were not blocked. IFN- spots were detected
using biotinylated antibody and avidin-peroxidase and developed using AEC
substrate (Sigma-Aldrich). Plates were read in an Immunospot Series 3 Ana-
lyzer and analyzed with ImmunoSpot software (Cellular Technology Ltd).
Flow cytometry. Cells were incubated for 15 min at 4°C with anti-CD16
monoclonal antibody (2.4G2) to block potential nonspecific binding and
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 CD8+
DC subset is quantitatively superior at cross-presenting exog-
enous 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 tet-
ramer in the lymph node compartment, arguing that the pre-
sumably 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 CD8+ DC accumulation is occurring at
early times after tumor implantation. Nonetheless, our subse-
quent experiments solidified a requirement for CD8+ 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 onco-
genic pathways in individual tumors could lead to expression
of distinct sets of genes that facilitate innate immune recogni-
tion in vivo.
The involvement of type I IFNs in antitumor immune
responses has been appreciated for a number of years. Al-
though 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-/ has been reported to enhance tumor growth, sug-
gesting 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- production
and signaling on CD8+ 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- (unpublished data). Collectively,
our results have implications for human cancer therapy, pro-
viding a strong rationale for the intratumoral administration
Type I IFNs and CD8+ DCs in antitumor immunity | Fuertes et al.
BMDC immunization. BMDCs were generated according to a modified
version of a published protocol (Inaba et al., 1992). BM cells from the tibiae
and femora were ACK-lysed and incubated for 10 d in complete DME me-
dium with 20 ng/ml rmGM-CSF (R&D Systems) with the addition of
200 ng/ml LPS (Sigma-Aldrich) for the last 24 h. On day 10, cells were ex-
posed to 10 µM SIY peptide for 1 h at 37°C and washed 3 times with DPBS.
Mice were injected s.c. in the flank with 106 BMDCs in 100 µl DPBS.
Adherent splenocyte stimulation of CD8+ T cells. Splenocytes were
ACK-lysed, irradiated (5,000 rad), and plated at 106 cells/well. After 2 h of
incubation, nonadherent cells were removed by 2 washes with DPBS. To
stimulate CD8+ T cells, SIY peptide (10 µM) or culture medium was added
to the adherent splenocytes, followed by the addition of 5 × 104 naive 2C/
RAG2/ CD8+ T cells/well. Supernatants were collected after 18 h and
IL-2 production was determined by ELISA.
Statistical methods. Differences between datasets were analyzed with the
two-sided Student’s t test, and correlation was analyzed with Pearson test and
Prism software (GraphPad).
Online supplemental material. Fig. S1 shows that in CD11c-DTR trans-
genic mice treated with diphtheria toxin, the majority of CD8+ DCs and
pDCs, and a fraction of mDCs, were depleted and that these cell populations
are critical for spontaneous CD8+ T cell priming to tumor-associated anti-
gens. Fig. S2 shows that CD8+ DCs fail to accumulate in the tumors grown
in Stat1/ mice. Fig. S3 shows that parental and SIY-expressing B16 mela-
nomas grow faster in Batf3/ and IFN-/R/ mice compared with
wild-type. Table S1 shows selected immune-related genes up-regulated with
B16 tumors, including a panel of IFN-inducible genes. Table S2 shows that
there is no difference in surface expression levels of CD40, CD80, and
CD86 in CD11c+ tumor-draining lymph node cells from WT and IFN-/
R/ mice. Online supplemental material is available at http://www.jem
We thank Hans Schreiber and Bin Zhang (University of Chicago) for helping with
the TCR tetramer staining experiments; and Long Zhang and Michelle Gao for
helpful technical assistance.
This work was funded by a Burroughs Wellcome Fund Translational Research
Award and P01 CA97296 from the National Cancer Institute. M.B. Fuertes was
supported by the University of Chicago Committee on Cancer Biology Fellowship
Program. A.K. Kacha was supported by the Medical Science Training Program
The authors have no conflicting financial interests.
Submitted: 10 June 2011
Accepted: 17 August 2011
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with the fluorochrome-coupled antibodies against the following molecules
for 30 min at 4°C: CD3, CD4, CD8, CD11b, CD11c, CD40, CD45R
(B220), CD80, CD86, H-2Kb, and I-Ab/I-Eb (BD) and PDCA (Miltenyi
Biotec). For tetramer staining, cells were labeled with PE-MHC class I tet-
ramers (Beckman Coulter or Proimmune) consisting of murine H-2Kb com-
plexed to either SIYRYYGL (SIY) peptide or SIINFEKL (OVA) peptide,
anti–CD8-APC, anti–B220-PerCP-Cy5.5, and anti–CD4-PerCP-Cy5.5.
For CFSE dilution analysis, splenocytes were labeled with DAPI, anti–CD8-
APC, and 1B2-PE (anti–2C-TCR; Kranz et al., 1984). For TCR tetramer
staining, cells were stained with anti–CD11c-APC, anti–CD11b-PerCp.
Cy5.5, and biotin-2C-m67 TCR tetramer (Holler et al., 2003), followed by
streptavidin-PE. FACS analysis was performed using FACSCanto or LSR II
cytometers with FACSDiva software (BD). Data analysis was conducted
with FlowJo software (Tree Star). For DC sorting experiments, pooled
tumor-draining lymph node cells were stained with anti–CD11c-APC and
sorted in a FACSAria cell sorter (BD).
Quantitative real-time RT-PCR. Total RNA was purified using the
RNeasy mini kit (QIAGEN) and analyzed in a 7300 Real Time PCR System
(Applied Biosystems) using primer and probe sets from TaqMan Gene Ex-
pression Assays (Applied Biosystems) and TaqMan-based quantification. The
results are expressed as 2Ct using GAPDH or 18s as endogenous control.
BM chimeras. Mice were maintained on trimethoprim and sulfamethoxa-
zole for at least 3 d before the start of the experiment. Groups of 3–5 mice
were lethally irradiated (900 rad) and maintained on antibiotics. The follow-
ing day, pooled tibial and femoral BM cells from donor mice were ACK-
lysed and 10 × 106 cells were injected retroorbitally into recipient mice.
Mice were allowed to reconstitute for at least 8 wk before tumor challenge.
Allogeneic MLR. MLR stimulation to generate effector cells for cytokine
and CTL analysis was adopted from a previously published protocol (Gajewski
et al., 1995). Total T cells were purified from spleens by negative selection
with antibodies and magnetic beads from StemCell Technologies according
to the manufacturer’s protocol. These responder cells were plated at 106/
well containing stimulator cells consisting of allogeneic T cell–depleted irra-
diated (5,000 rad) splenocytes at 5 × 106/well. After 5 d, cells were used in
chromium release assays and ELISA.
Chromium release assay. Chromium release assays were performed as
previously described (Kacha et al., 2000). Briefly, 51Cr-labeled targets
(2 × 103) were plated with effectors cells at the indicated E/T ratios from
100:1 to 3.7:1. After 4 h of incubation at 37°C, 50 µl of supernatant was
transferred to a LumaPlate-96 (PerkinElmer) and allowed to dry overnight.
Plates were then counted using a TopCount-NXT plate reader (PerkinElmer).
Percent specific lysis was calculated using standard methods.
Cytokine ELISA. For cytokine analysis, tissue culture–treated 96-well flat
bottom plates were coated with either DPBS alone, 2C11 (1 µg/ml; anti-
CD3), or 2C11 and PV-1 (2 µg/ml; anti-CD28) in DPBS overnight at room
temperature and washed with culture medium. Effector cells were incubated
on the antibody-coated plates overnight, and supernatants were collected for
measurement of IFN- concentration by ELISA. Mouse IL-2 and IFN- anti-
body sets were obtained from BD. Cytokine concentrations were deter-
mined with the Softmax PRO data analysis program (Molecular Devices).
2C CD8+ T cells purification, CFSE staining, and adoptive transfer.
CD8+ T cells were purified from spleens of 2C/RAG2/ mice by negative
selection with antibodies and magnetic beads from StemCell Technologies
according to the manufacturer’s protocol. T cells were stained with 2.5 mM
CFSE at room temperature for 6 min and thoroughly washed with an excess
volume of cold FCS. 107 CFSE-labeled T cells in 100 µl of DPBS were
transferred by retroorbital injection into venous plexus of anesthetized mice.
Mice were challenged with 106 B16.SIY cells, and 7 d later splenocytes from
recipient mice were analyzed by flow cytometry.
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