Type I IFN-dependent T cell activation is mediated by IFN-dependent dendritic cell OX40 ligand expression and is independent of T cell IFNR expression.
ABSTRACT Type I IFNs are important for direct control of viral infection and generation of adaptive immune responses. Recently, direct stimulation of CD4(+) T cells via type I IFNR has been shown to be necessary for the formation of functional CD4(+) T cell responses. In contrast, we find that CD4(+) T cells do not require intrinsic type I IFN signals in response to combined TLR/anti-CD40 vaccination. Rather, the CD4 response is dependent on the expression of type I IFNR (IFNαR) on innate cells. Further, we find that dendritic cell (DC) expression of the TNF superfamily member OX40 ligand was dependent on type I IFN signaling in the DC, resulting in a reduced CD4(+) T cell response that could be substantially rescued by an agonistic Ab to the receptor OX40. Taken together, we show that the IFNαR dependence of the CD4(+) T cell response is accounted for exclusively by defects in DC activation.
- SourceAvailable from: Jukka Vakkila[Show abstract] [Hide abstract]
ABSTRACT: Abnormalities of dendritic cells (DCs) and STAT proteins have been reported in Crohn's disease (CD). Studies on JAK/STAT signaling in DCs are, however, lacking in CD. We applied a flowcytometric single-cell-based phosphoepitope assay to evaluate STAT1 and STAT3 pathways in DC subsets from CD patients. In addition, circulating DC counts were determined, together with the activation-related immunophenotype. We found that IL-6- and IFN-α-induced STAT3 phosphorylation and IFN-α-induced STAT1 phosphorylation were impaired in plasmacytoid DCs (pDCs) from CD patients (P = 0.005, P = 0.013, and P = 0.006, respectively). In myeloid DCs (mDCs), IFN-α-induced STAT1 and STAT3 phosphorylation were attenuated (P<0.001 and P = 0.048, respectively), but IL-10-induced STAT3 phosphorylation was enhanced (P = 0.026). IFN-γ-induced STAT1 signaling was intact in both DC subtypes. Elevated plasma IL-6 levels were detected in CD (P = 0.004), which strongly correlated with disease activity (ρ = 0.690, P<0.001) but not with IL-6-induced STAT3 phosphorylation. The numbers of pDCs and BDCA3+ mDCs were decreased, and CD40 expression on CD1c+ mDCs was increased in CD. When elucidating the effect of IL-6 signaling on pDC function, we observed that IL-6 treatment of healthy donor pDCs affected the maturation of and modified the T-cell priming by pDCs, favoring Th2 over Th1 type of response and the expression of IL-10 in T cells. Our results implicate DC signaling in human CD. Reduced IL-6 responsiveness in pDCs, together with the attenuated IFN-α-induced signaling in both DC subtypes, may contribute to the immunological dysregulation in CD patients.PLoS ONE 01/2013; 8(8):e70738. · 3.53 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: While the need for CD4 T cells in the generation of CD8 T cell memory has been well documented, the mechanism underlying their requirement remains unknown. Here, we detail an immunization method capable of generating CD8 memory T cells that are indifferent to CD4 T cell help. Using a subunit vaccination that combines polyIC and an agonistic CD40 antibody, we program protective CD4-independent CD8 T cell memory. When cells generated by combined polyIC/CD40 immunization are compared to cells produced following a CD4-dependent vaccination, Listeria monocytogenes, they display dramatic differences, both phenotypically and functionally. The memory cells generated in a CD4-deficient host by polyIC/CD40 immunization provide protection against secondary infectious challenge, whereas cells generated by LM immunization in the same environment do not. Interestingly, combined polyIC/CD40 immunization generates long-term memory cells with low Blimp-1 and elevated Eomes expression despite high expression of Blimp-1 during the primary response. The potency of combined polyIC/CD40 to elicit CD8+ T cell memory in the absence of CD4 T cells suggests that it could be considered as a vaccine adjuvant in clinical situations where CD4 responses/numbers are compromised.Immunologic Research 09/2012; · 3.53 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Antigen derived from viral infections with influenza and vesicular stomatitis virus can persist after resolution of infection. Here we show that antigen can similarly persist for weeks following viral challenge and vaccination. Antigen is captured by lymphatic endothelial cells (LECs) under conditions that induce LEC proliferation. Consistent with published data showing that viral antigen persistence impacts the function of circulating memory T cells, we find that vaccine-elicited antigen persistence, found on LECs, positively influences the degree of protective immunity provided by circulating memory CD8(+) T cells. The coupling of LEC proliferation and antigen capture identifies a mechanism by which the LECs store, or 'archive', antigens for extended periods of time after antigen challenge, thereby increasing IFNγ/IL-2 production and enhancing protection against infection. These findings therefore have the potential to have an impact on future vaccination strategies and our understanding of the role for persisting antigen in both vaccine and infectious settings.Nature Communications 06/2014; 5:3989. · 10.74 Impact Factor
of June 13, 2013.
This information is current as
of T Cell IFNR Expression
OX40 Ligand Expression and Is Independent
Mediated by IFN-Dependent Dendritic Cell
Type I IFN-Dependent T Cell Activation Is
McWilliams, Phillip J. Sanchez and Ross M. Kedl
Jonathan S. Kurche, Catherine Haluszczak, Jennifer A.
2012; 188:585-593; Prepublished online 7
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The Journal of Immunology
by guest on June 13, 2013
The Journal of Immunology
Type I IFN-Dependent T Cell Activation Is Mediated by
IFN-Dependent Dendritic Cell OX40 Ligand Expression and
Is Independent of T Cell IFNR Expression
Jonathan S. Kurche,1Catherine Haluszczak, Jennifer A. McWilliams, Phillip J. Sanchez,2
and Ross M. Kedl
Type I IFNs are important for direct control of viral infection and generation of adaptive immune responses. Recently, direct stim-
ulation of CD4+T cells via type I IFNR has been shown to be necessary for the formation of functional CD4+T cell responses. In
contrast, we find that CD4+T cells do not require intrinsic type I IFN signals in response to combined TLR/anti-CD40 vaccination.
Rather, the CD4 response is dependent on the expression of type I IFNR (IFNaR) on innate cells. Further, we find that dendritic
cell (DC) expression of the TNF superfamily member OX40 ligand was dependent on type I IFN signaling in the DC, resulting in
a reduced CD4+T cell response that could be substantially rescued by an agonistic Ab to the receptor OX40. Taken together, we
show that the IFNaR dependence of the CD4+T cell response is accounted for exclusively by defects in DC activation.
Journal of Immunology, 2012, 188: 585–593.
immunity (1). The marked antiviral effects of type I IFN make it
clinically useful as an adjuvant to ribavirin as a treatment for
hepatitis C virus infection (1, 2). Furthermore, individuals lacking
key intermediates both upstream and downstream of type I IFN
signaling succumb to normally nonlethal viral infection (3, 4).
Thus, understanding type I IFN biology holds the promise of bet-
ter understanding human immunity.
In the periphery, type I IFN signaling induces infected cells and
bystanders to adopt an antiviral state (5) mediated by intracellular
factors such as protein kinase R, Mx1, and others and character-
ized by reduced protein synthesis, which limits viral replication
(1). Release of type I IFN in the context of infection (6), as well as
release of byproducts of infection such as dsRNA liberated from
lysed cells, leads to the activation of innate cells (7). Activation of
peripheral innate cells such as dendritic cells (DC) by type I IFN
leads to the trafficking of these cells to draining lymph nodes (8,
9). There, DC are autoenhanced by type I IFN (10) to present Ags
he type I IFN cytokine family is vitally important in
providing resistance to viral infections and plays a unique
role among cytokines by bridging innate and adaptive
they may have carried from the periphery in the context of co-
stimulation to naive, primary T cells (10, 11). The culmination of
type I IFN induction is thus the initiation of an adaptive, antiviral,
Although a picture of the role of type I IFN signaling for DC
at various points in the in vitro innate immune response has been
developed (10, 11), the function of type I IFN on DC activation
has only recently been explored in vivo (8, 9, 12). IFN-dependent
DC activation has been reported as required for vaccine adjuvant
efficacy in other model systems (13), and it was recently shown
that DC-intrinsic sensing of type I IFN was necessary for activa-
tion by the TLR agonist polyinosinic-polycytidylic acid (polyI:C)
(12). Given the role of DCs in the initiation of adaptive immunity
(14–16), it could be expected that defects in DC activation would
be a dominant hindrance to the generation of adaptive responses
in the absence of type I IFN signals. However, there is a body of
literature to suggest that type I IFNs can also have broad, direct
effects on responding adaptive immune cells, in particular CD4+
T cells (12, 17–23). Thus, it is presently unknown to what extent
failure of adaptive immune responses in the absence of type I IFN
are attributable to defects in priming DC and which are due to
defects in direct signals to T cells. These parameters are particu-
larly important to establish for the purposes of vaccine develop-
ment, as many of the adjuvants currently being explored have type
I IFN induction as a significant contributor to vaccine efficacy (24).
We have previously shown that combined agonists for TLRs and
CD40 (the anti-CD40 Ab FGK4.5) synergistically promote CD8+
T cell responses (25). Furthermore, CD8+responses to combined
polyI:C and anti-CD40 (polyI:C/CD40) were dependent on the
type I IFNR IFNaR1 (IFNaR) (25). Our experience with CD4+
T cells revealed that they could also be synergistically activated
by polyI:C/CD40 (26) and that the activation of CD4+T cells by
polyI:C/CD40 depended upon signals through the TNF super-
family member OX40 ligand (OX40L). However, it remained
unknown whether CD4+T cells activated by polyI:C/CD40 were
similarly dependent on signals through IFNaR. Unlike CD4+
T cells, CD8+T cells require DCs to take up exogenous Ag to be
processed and cross-presented on MHC class I (MHC-I), a path-
way dependent on type I IFN (9). Furthermore, whereas a role for
Integrated Department of Immunology, University of Colorado Denver and National
Jewish Health, Denver, CO 80206
1Current address: Department of Medicine, University of Colorado Denver, Aurora,
2Current address: Department of Biology, University of Iowa, Iowa City, IA.
Received for publication September 2, 2011. Accepted for publication November 9,
This work was supported by grants from the National Institutes of Health (AI06877
and AI066121) and the Department of Defense (W81XWH-07-1-0550). Department
of Defense support was associated with funding for the Center for Respiratory Bio-
defense at National Jewish Health.
Address correspondence and reprint requests to Dr. Ross M. Kedl, Associate Profes-
sor, Department of Immunology, University of Colorado Denver, National Jewish
Health, Goodman Building K825, Denver, CO 80206. E-mail address: ross.kedl@
Abbreviations used in this article: DC, dendritic cell; IFNaRKO, IFN-aR–deficient;
MHC-I, MHC class I; MHC-II, MHC class II; OX40L, OX40 ligand; polyI:C,
polyinosinic-polycytidylic acid; RagKO, Rag-recombinase deficient; WT, wild-type.
by guest on June 13, 2013
type I IFN in CD4+T cell responses to polyI:C has been estab-
lished (12), it was unknown whether the addition of a CD40 ag-
onist would abolish the dependence of polyI:C responses on type I
We set out to address the role of type I IFN in CD4+T cell
responses initiated by polyI:C/CD40 and confirmed that type I
IFN is necessary for CD4+T cell priming. To our surprise, we
found that secondary CD4+T cell responses were, unlike primary
responses, relatively intact in IFN-aR–deficient (IFNaRKO) mice.
This suggested that Ag-experienced CD4+T cells were qualita-
tively different from naive CD4+T cells and that the IFN de-
pendency was unlikely due to direct IFN stimulation of the T cell.
Indeed, the use of mixed bone marrow chimeras revealed that
Rag12/2bone marrow could rescue CD4+T cell responses in
otherwise IFNaRKO mice, demonstrating a role for IFNaR on
innate, but not adaptive, cells. We further showed that polyI:C/
CD40-stimulated IFNaRKO DCs had very low expression of
OX40L, and restoration of OX40 signals using an agonistic Ab in
IFNaRKO mice enhances CD4+, but not CD8+, T cell priming.
Materials and Methods
Six- to 8-wk-old female C57BL/6 mice were obtained through the National
Cancer Institute or Harlan Laboratories. Mice deficient in the a1 receptor
for type I IFN (B6.129PF2/ifnab) were obtained from Laurel Lenz at
National Jewish Health and originally derived by Daniel Portnoy, Uni-
versity of California, Berkeley. These mice were crossed to B6.SJL mice
(B6.SJL-PtprcaPep3b/BoyJ) obtained from The Jackson Laboratory.
CD40-deficient mice (CD40KO, CD40KO, B6.129P2-Cd40tm1Kik/J) were
also obtained from The Jackson Laboratory. Mice deficient for the Rag1
gene (B6.129S7-Rag1tm1Mom/J) were a gift of Dr. Phillippa Marrack, Na-
tional Jewish Health. Mice were housed at the Biological Resource Center
at National Jewish Health. The Institutional Animal Care and Use Com-
mittee at National Jewish Health approved all animal procedures. Mice
were maintained on Harlan Teklad 2919 chow (Harlan) and water ad
libitum for the breeding and duration of experiments.
Immunizations, depletions, and Ab blockade
Unless indicated otherwise, mice were immunized i.p. with 500 mg whole
chicken OVA protein (Sigma-Aldrich, St. Louis, MO), 100 mg Ea-derived
2W1S peptide (EAWGALANWAVDSA; custom synthesized by Pi Pro-
teomics, Huntsville, AL) (27), 50 mg CD40 agonist Ab (clone FGK4.5;
BioXCell, West Lebanon, NH), and 50 mg polyI:C (Amersham Biosci-
ences/GE Healthcare, Piscataway, NJ). All vaccinations were prepared
by mixing each component together in PBS and injected in 200 ml. PolyI:C
was stored in frozen aliquots in PBS at 220˚C and reconstituted prior
to injection by melting at 56˚C for 10 min and then allowing the solution
to cool to room temperature to limit concatamerization. All reagents were
found to contain minimal LPS content by Limulus amebocyte assay
(Lonza, Walkersville, MD). Blocking Ab for OX40L (RM134L) and ag-
onistic anti-OX40 (OX86) were obtained from BioXCell. OX40L blockade
was facilitated by i.p. injection of 250 mg RM134L in PBS on days 21 and
0 relative to immunization. On day 0, blocking Abs were mixed with
prepared vaccines, and both were delivered in a single injection. For OX40
agonist administration, 250 mg OX86 in PBS were mixed with prepared
vaccine on day 0 and given together as a single injection. For experiments
involving footpad immunization, mice were anesthetized with isofluorane
and injected with 50 ml PBS containing 40 mg polyI:C, 40 mg FGK4.5, and
50 mg OVA protein.
For T cell assays, mice were sacrificed 7 d post-primary immunization or
5 d post-boost immunization, peripheral blood was harvested from the
abdominal aorta, and spleens were harvested and minced with forceps in
HBSS containing 5 mM EDTA. Single-cell suspensions were made by
passing minced spleens through nylon mesh strainers. RBCs were lysed in
peripheral blood samples using ACK lysis solution (BioSource Interna-
tional, Rockville, MD). All samples were resuspended in RPMI 1640
medium containing 2.5% heat-inactivated FCS, 2-ME, L-glutamine, non-
essential amino acids, HEPES, sodium pyruvate, penicillin, and strepto-
mycin. Cells were stained with PE-labeled, tetramerized MHC molecules
bearing Ags of interest. Drosophila S2 cells transfected with 2W1S-IAb
monomers were a gift of Marc Jenkins at the University of Minnesota (28).
Secreted 2W1S-IAbwas harvested from supernatants as described (29) but
did not require additional biotinylation due to cotransfection of the BirA
enzyme (James Moon and Marc Jenkins). Tetramerized 3K-IAbwas pre-
pared as described (29). Kbreagents were purified as described (30) and
loaded with OVA (SIINFEKL) Ag prior to staining. Cells were coincu-
bated with tetramer for 1 h at 37˚C prior to staining with surface Abs.
Surface Abs were purchased from BioLegend (San Diego, CA) or eBio-
science (San Diego, CA). For cytokine assays, cells were incubated for 3.5
h at 37˚C with 6 mg/ml brefeldin A in complete media as above. Cells were
restimulated with 10 mg/ml 2W1S and SIINFEKL peptides, and following
the incubation period, cells were fixed, permeabilized, and stained for
intracellular cytokines as described (31). DC were isolated from spleens in
EHAA media (Invitrogen) containing DNAse (Worthington, Lakewood,
NJ) and Collagenase D (Roche Diagnostics, Indianapolis, IN) as described
(30). Crude preparations were made by passing digested spleens through
nylon mesh strainers and purified over Nycodenz (Nycoprep Universal;
Accurate Chemical & Scientific, Westbury, NY) according to the manu-
facturer’s instructions. Both DC and T cells were washed and stained in
FACS buffer containing 10% 2.4G2 supernatant (B cell hybridoma
blocking Fcg receptors). Cells were gated for forward scatter, side scatter,
and pulse width and, in the case of T cells, were MHC class II (MHC-II)2,
DX52, and CD3+prior to gating on CD4+and CD8+events. DC were
gated for forward scatter, side scatter, and pulse width and were CD192,
CD32, and CD11c+.
Mixed bone marrow chimeras
Recipient mice were lethally irradiated with 900 rad in the morning and
grafted via the tail vein in the afternoon with 4 3 106total T cell-depleted
donor bone marrow cells suspended in 200 ml PBS. Bone marrow was
depleted of T cells using magnetic removal of CD3+cells (Miltenyi Biotec,
Auburn, CA). Most mixed bone marrow chimeras were grafted at a ratio of
1:1, representing 2 3 106cells from each of two donors. However, Rag-
deficient bone marrow was enriched relative to competitor bone marrow
3:1 to enhance engraftment. Screening of recipients of B6.SJL and Rag-
recombinase deficient (RagKO) bone marrow revealed that the 3:1 ratio
was optimal for the generation of equal numbers of NK cells derived from
each donor. Chimeric mice were rested a minimum of 12 wk before being
immunized for experiments. Chimeric mice were fed tirmethoprim-sulfa-
methoxazole–containing chow (Harlan Teklad 6596; Harlan) for 6 wk
following reconstitution to reduce the risk of bacterial infection, but were
switched to standard chow (Harlan Teklad 2919; Harlan) well before im-
In vitro culture
Spleen cells were harvested as described above using DNAse and colla-
genase and cultured unfractionated at 1 3 106cells/ml in 24-well, flat-
bottom plates. Cells were stimulated with 0.5 mg/ml anti-CD3ε (clone
2C11) in complete RPMI 1640 medium supplemented as above and with
10% FCS. In some cases, cells were incubated with 105U/ml rIFN-a
prepared as described (32). At indicated time points, cells were harvested
by washing plates with FACS buffer and stained as above.
Experimental and statistical analysis
Spleen cells were quantified on a Vi-Cell cell viability analyzer (Beckman
Coulter). Cytometry samples were acquired on a CyAn ADP (DakoCy-
tomation) usingSummitacquisitionsoftware.Sampleswere analyzed using
FlowJo software (Tree Star, Ashland, OR). In most cases, results from
FlowJo analysis were imported into Prism (GraphPad, La Jolla, CA), and
pairwise statistical analyses were made betweensamples usingthe Student t
test. In vivo experiments used in this manuscript were completed inde-
pendently at least twice with a minimum three individuals per group.
In vitro experiments were completed independently at least three times.
IFNaRKO mice have defective primary responses to combined
polyI:C and CD40 stimulus
We have previously found that combined adjuvants polyI:C and an
agonistic CD40 Ab (FGK4.5) synergistically promote CD4+T cell
responses (25, 26, 32), whereas responses to either single agonist
alone were as much as 10-fold lower than combined stimulus (26).
We also showed that polyI:C/CD40 stimulus promoted CD8+
T cell responses that were type I IFN dependent (25). Whereas
586IFN-DEPENDENT OX40L EXPRESSION DICTATES CD4 RESPONSE
by guest on June 13, 2013
CD4+T cell responses to immunizations containing polyI:C alone
as an adjuvant are known to be dependent on type I IFN (12), we
were interested in whether type I IFN was also required for pro-
motion of CD4+T cell responses in our polyI:C/CD40 adjuvant
We began by immunizing wild-type (WT) B6 mice and
IFNaRKO B6 mice i.p. with both a CD4+T cell Ag (the 2W1S Ag
EAWGALANWAVDSA) and a CD8+T cell Ag (whole OVA) in
the presence of polyI:C and CD40 agonists (polyI:C/CD40). Im-
munization with polyI:C/CD40 promoted robust CD4+T cell
proliferative responses in WT mice (Fig. 1A, 1B), as measured by
the percentages (Fig. 1A) and numbers (Fig. 1B) of 2W1S-IAb-
tetramer–specific cells in the spleen. Similar to previously pub-
lished results for CD8+T cell responses (Fig. 1C) (26), absence of
type I IFN signaling in IFNaRKO mice reduced CD4+T cell
proliferative responses to polyI:C/CD40 up to 10-fold in the
spleen (Fig. 1A, 1B) and peripheral blood (Fig. 2A). Thus, type I
IFN signaling is critical for the synergistic effects of polyI:C/
CD40 for both CD4+and CD8+T cells and is not unique to any
particular cell type.
We previously found that OX40L is required for optimal CD4+
T cell proliferation in WT mice (26). We verified the role of
OX40L in CD4+T cell responses in both WT and IFNaRKOs by
using the blocking Ab RM134L. Mice were injected i.p. with 250
mg of blocking Abs on days 21 and 0 relative to immunization
with 2W1S and OVA Ags and polyI:C/CD40. Consistent with
previous data (26), we found that CD4+T cell responses in WT
mice were reduced in the presence of RM134L blocking Ab (Fig.
1D), whereas CD8+T cell responses were unimpaired following
OX40L blockade (26, 33 and data not shown). Somewhat to our
surprise, the CD4+T cell response in unblocked IFNaRKO mice
were brought down even further in the presence of RM134L (Fig.
1D). Again, CD8+T cell responses in IFNaRKO mice were un-
affected by RM134L (data not shown). Thus, even the remnant
CD4+T cell response in the IFNaRKO mice was largely depen-
dent on OX40/OX40L interactions. Collectively, the data dem-
onstrate the central importance of both type I IFN and OX40 in the
generation of CD4+T cell immunity following combined polyI:C/
The defect in IFNaRKO mice is limited to primary but not
We were interested to know whether the defect in primary CD4+
T cell responses in IFNaRKO mice led to impaired secondary
responses. To assess the role of type I IFN in CD4+T cell memory,
we immunized B6 or IFNaRKO mice with 2W1S Ag and polyI:C/
CD40 as described above. Mice were followed by staining pe-
ripheral blood for the presence of Ag-specific CD4+T cells at
different time points. After allowing a recovery period of 40–70 d,
the peripheral blood was monitored for the numbers of Ag specific
memory cells as determined by tetramer staining (preboost). The
mice were the rechallenged with a boosting dose of 2W1S and
polyI:C/CD40, and secondary responses were measured 5 d later
We reasoned that poor primary responses would predict poor
secondary responses in IFNaRKO mice. To our surprise, we found
that secondary proliferative responses in IFNaRKO mice were
largely intact and almost equivalent to secondary responses in B6
mice (Fig. 2A). In particular, the fold expansion of 2W1S-specific
CD4+T cells from preboost levels to postboost levels was
equivalent in IFNaRKO mice and B6 mice (Fig. 2C). Other au-
thors have noted that defects in type I IFN signaling during
priming can lead to secondary responses with intact proliferation,
but defective cytokine production (34). In line with these previous
observations (34), we found that secondary cytokine responses
by CD4+T cells in peripheral blood were generally reduced in
IFNaRKO mice relative to WT mice (Fig. 2B). However, in the
spleen, the total number of cells producing IFN-g were more
similar between WT mice and IFNaRKOs (not shown), and, in all
cases, the fold expansion of cytokine-producing cells from pre- to
postboost was similar between WT and IFNaRKOs (Fig. 2D).
Thus, our data indicate that secondary proliferation and cytokine
production by CD4+T cells are minimally dependent on type I
Curiously, we also found that WT and IFNaRKO mice re-
sponded equally well to secondary immunization, regardless of
whether OX40L was blocked during the primary (Fig. 2E) or
secondary (Fig. 2F) challenge. Blockade of OX40L during the
primary response, although reducing the primary expansion of Ag-
specific CD4+T cells, does not prevent the formation or response
of memory CD4+T cells (Fig. 2E). Further, blockade of OX40L
only after boosting vaccination has minimal impact on CD4+
T cell recall responses (Fig. 2F). Broadly, we conclude from these
data that OX40L plays an important role in determining the
magnitude of the primary CD4+T cell responses, but not the
generation of memory or the response of the memory cells fol-
The IFNaR dependency of primary CD4+T cell immune
responses is T cell extrinsic
The fact that IFNaRKO CD4+T cells could mount an effective
secondary response suggested that the IFN dependency of the
with Ag, polyI:C, and FGK4.5 anti-CD40 Ab as described in Materials and Methods. Seven days later, mice were sacrificed and spleens harvested and
stained for Ag-specific CD4+and CD8+T cells. A, Representative FACS plot depicting Ag-specific CD4+T cells stained with 2W1S-IAb tetramers and the
activation marker CD44 from WT (left panel) and IFNaRKO mice (right panel). B and C, Spleen cells from mice 7 d after immunization as in A were
quantified and stained with 2W1S-IAb (B) or OVA-Kb(C) tetramers. D, IFNaRKO mice demonstrate a defect in eliciting cytokine-producing cells fol-
lowing immunization. Cells from mice in B were stained with 2W1S-IAb tetramer as described in Materials and Methods. Shown are total numbers of
tetramer+CD4+T cells per spleen. Data are representative of at least two independent experiments with at least three mice per group. Error bars represent
SEM. Statistics were calculated using Student t test. **p , 0.01, ***p , 0.005.
CD4+and CD8+T cell responses to combined polyI:C/CD40 stimulus are IFN dependent. WT B6 or B6 IFNaRKO mice were immunized
The Journal of Immunology587
by guest on June 13, 2013
primary CD4+T cell response was not due to a requirement for
IFNR expression on T cells. To more specifically address whether
the effects of IFNaR deficiency are T cell intrinsic or extrinsic, we
generated mixed bone marrow chimeras in lethally irradiated
IFNaRKO recipients to isolate defects in type I IFN signaling
to bone marrow-derived cells (Fig. 3A). We used congenically
marked CD45.1+IFNaRKO bone marrow and mixed it 1:1 with
WT B6 bone marrow (Fig. 3A). We reasoned that, if IFNaR were
required on responding T cells, we would observe a dispropor-
tionate response of the WT bone marrow-derived T cells as
compared with the congenic, IFNaRKO bone marrow-derived
T cells. Instead, we found that T cells derived from IFNaRKO
bone marrow competed very well with WT bone marrow, if
anything, responding better than the WT T cells (Fig. 3B). This
indicated that IFNaR was dispensable on responding CD4+T cells
to promote immune responses. Similar to previous reports of
a CpG-based adjuvant system (13), our data suggested that
the dependency of the polyI:C/CD40-elicited response on type I
IFN must be due to a requirement for IFNR expression on the
APCs (12). We therefore reconstituted irradiated IFNaRKO hosts
with RagKO bone marrow mixed 3:1 (to enhance engraftment of
RagKO marrow) with IFNaRKO bone marrow. The resulting host
has T cells that are exclusively IFNaR deficient, but both WT and
IFNaR-deficient APCs. As anticipated, the CD4+T cell responses
were rescued in these RagKO 3 IFNaRKO chimeras (Fig. 3C).
These data demonstrate that IFNaR is dispensable on responding
CD4+T cells and that cells derived from RagKO bone marrow are
sufficient to restore IFNaR-dependent pathways in IFNaRKO
We have previously shown that CD40 expression on innate cells
was sufficient for the synergistic effects of agonistic anti-CD40
combined with polyI:C (26). Our finding that IFNaR expression
was also sufficient on innate cells raised the question of whether
CD40 and IFNaR were required on the same cells or whether
A, Plot of percent 2W1S-IAb tetramer-positive CD4+T cells in the peripheral blood of mice showing frequencies of Ag-specific CD4+T cells as determined
by 2W1S-IAb tetramer at different time points relative to immunization with 2W1S and polyI:C/CD40 (set to day 0 on x-axis) as described in Fig. 1. Mice
were restimulated with a second dose of 2W1S peptide and polyI:C/CD40 as indicated by the arrow (day 65) and harvested 5 d later. B, Plot showing the
frequency of 2W1S Ag-specific IFN-g+CD4+T cells restimulated with 2W1S peptide over time relative to immunization on day 0 as in A. C, Fold ex-
pansion of CD4+T cells calculated as the ratio of Ag-specific cells in peripheral blood pre- and postboost of a separate experiment performed as in A. D,
Fold expansion of 2W1S-specific IFN-g+CD4+T cells of a separate experiment performed as in B. E, Mice were immunized with 2W1S peptide, and polyI:
C/CD40 and OX40–OX40L interactions were blocked by injecting 250 mg of RM134L i.p. on days 21 and 0 relative to priming, as described in Materials
and Methods. Sixty-seven days later, mice were rechallenged with Ag and polyI:C/CD40, and after 5 d, spleens were harvested and stained with 2W1S-IAb
tetramer. Shown are percentages of Ag-specific CD4+T cells in peripheral blood over time. F, Mice were primed with Ag and polyI:C/CD40 and
reimmunized 70 d after priming with Ag and polyI:C/CD40. OX40L was blocked only during the secondary immunization by administering 250 mg of
RM134L i.p. on days 21 and 0 relative to rechallenge. Shown are numbers of Ag-specific splenic CD4+T cells 5 d following rechallenge. Data are
representative of at least two independent experiments containing at least three mice per group. Statistics were calculated using Student t test and are
pairwise comparisons to control unless otherwise indicated. **p , 0.01, ***p , 0.005.
Primary CD4+T cell responses are IFN and OX40L dependent, whereas secondary CD4+T cell responses are IFN and OX40L independent.
588IFN-DEPENDENT OX40L EXPRESSION DICTATES CD4 RESPONSE
by guest on June 13, 2013
CD40 stimulus and polyI:C stimulus could work in trans for
promotion of optimal CD4+T cell responses to polyI:C/CD40. To
determine whether CD40 and IFNaR were required on the same
cells, we reconstituted lethally irradiated IFNaRKO mice with
bone marrow derived from IFNaRKO.SJL mice alone, IFNaRKO.
SJL and CD40KO bone marrow mixed 1:1, and IFNaRKO.SJL
and B6 bone marrow mixed 1:1. We found that IFNaRKO.SJL
and CD40KO bone marrow were not able to reconstitute responses
in IFNaRKO recipients (Fig. 3D), suggesting that both CD40 and
IFNaR need to be expressed by the same APC in order for polyI:
C/CD40 to promote robust CD4+T cell immune responses. By
extension, trans signals elaborated by polyI:C/CD40 are not suf-
ficient for the combined effects of both agonists.
IFNaRKO DC demonstrate defective activation in response to
polyI:C and CD40 stimulus
The data thus far indicate that the dependency of the CD4+T cell
response elicited by combined polyI:C/CD40 immunization must
be due to the action of IFN on the APC. Other authors have also
shown that DCs can be regulated in an intrinsic IFNaR-dependent
manner (8, 12). To understand how IFNaR deficiency might affect
DC activation, we immunized B6 and IFNaRKO mice in one
footpad with fluorescent OVA Ag and polyI:C/CD40 stimulus and
isolated DCs from the ipsilateral and contralateral popliteal lymph
nodes 18–24 h later. FACS histograms revealed that IFNaRKO
DCs (Fig. 4A) had relative defects in Ag uptake, MHC-II ex-
pression, CD69 expression, and CCR7 expression compared with
WT mice (Fig. 4A). Importantly, DCs from IFNaRKO mice did
upregulate these molecules relative to cells from contralateral
nodes in WT mice (Fig. 4A, shaded area), suggesting that DC
activation was not completely absent in IFNaRKO mice, but was
Given the established importance of OX40/OX40L signaling
in mediating the CD4+T cell response to combined polyIC/CD40
immunization (Fig. 1) (26), we were interested to know whether
expression of OX40L on activated DCs was also influenced by the
presence or absence of IFN. We chose to focus on CD8+DCs, as
OX40L expression is more pronounced on this subset, and we
have found that CD8+DC OX40L expression correlates to CD4+
T cell responses (26). We found that both OX40L expression and
MHC-II expression on CD8+DCs were reduced at multiple time
points in IFNaRKOs relative to WT mice (Fig. 4B).
Although the loss of DC OX40L expression was not absolute in
the IFNaRKOs (as evidenced by the fact that OX40L blockade
can still reduce the residual T cell response in these hosts) (Fig.
1D), the majority of OX40L expression appeared to be IFN de-
pendent (Fig. 4B). These data suggested that the introduction
rIFN-a might be sufficient to promote DC OX40L expression. We
harvested naive splenocytes and stained them directly (Fig. 4C) or
cultured them in the presence of anti-CD3 (2C11), rIFN-a, or anti-
CD3 and rIFN-a. Intriguingly, anti-CD3 alone and anti-CD3 with
IFN-a did not lead to appreciable expression of OX40L in these
cultures (Fig. 4C). However, administration of rIFN-a alone was
sufficient to massively upregulate OX40L expression on cultured
DCs (Fig. 4C). Furthermore, DCs increased expression of CD70 in
the presence of rIFN-a (Fig. 4C), regardless of the presence of
It was interesting that IFN-a alone, but not IFN-a with anti-
CD3, led to upregulation of OX40L on cultured DCs (Fig. 4C).
The Ab used to detect OX40L expression, RM134L, is also useful
for blockade of OX40L–OX40 interactions (26). We hypothesized
that activation of responding T cells in the presence of anti-CD3
and rIFN-a may have led to upregulation of OX40, which, in turn,
might prevent detection of OX40L on the DCs, either by down-
modulating OX40L expression or directly interfering with Ab
binding to OX40L. We therefore assessed the expression of OX40
on responding CD4+T cells. We found that OX40 expression was
high in the presence of anti-CD3 alone 6 rIFN-a, but not on naive
cells or on cells stimulated with IFN-a alone (Fig. 4D). This result
is consistent with the finding that, in vivo, priming of CD4+T cells
does not require IFNaR on the cells themselves. Similarly, ex-
pression of another TNFR, CD27, was high on CD4+T cells under
all conditions, but appeared to increase in the presence of anti-
CD3 (Fig. 4D). This suggests that regulation of DC TNF ligand
expression could be a direct consequence of type I IFN, but reg-
ulation of the receptors on responding CD4+T cells is likely to be
a consequence of CD3-mediated TCR stimulus.
OX40 receptor stimulation rescues CD4+T cell responses in
As OX40L signals were required for optimal CD4+T cell priming,
but defective in IFNaRKO mice, we hypothesized that restoration
of signaling through OX40 might restore CD4+T cell responses in
IFNaRKOs. We therefore administered single doses of the OX40-
agonistic Ab, OX86 (35, 36), to WT and IFNaRKO mice coin-
sic. A, Model for mixed bone marrow chimeras. Bone marrow was har-
vested from congenically marked, IFNaRKO (IFNaRKO.SJL), RagKO,
CD40-deficient (CD40KO), or WT B6 mice, mixed as indicated, and trans-
planted via the tail vein into lethally irradiated IFNaRKO recipients.
All mice were rested $12 wk prior to immunization with polyI:C/CD40
and Ag. B, Total of 2 3 106T cell-depleted bone marrow cells derived
from each IFNaRKO.SJL (CD45.1) and WT (CD45.2) mouse was mixed
1:1 (4 3 106total cells) and injected into lethally irradiated CD4
IFNaRKO CD45.2 recipients. The ratio of IFNaRKO (CD45.1+) to WT
(CD45.12) CD4+T cells prior to immunization and the ratio of 2W1S-
specific IFNaRKO (CD45.1+) to WT (CD45.12) following polyI:C/CD40
and 2W1S immunization is shown. Data represent consolidated sam-
ples from two independent experiments (n = 7). C, IFNaRKO bone mar-
row was harvested (IFNaRKO), mixed 1:3 with RagKO bone marrow
(IFNaRKO+Rag), or 1:1 with WT bone marrow (IFNaRKO+B6) and
transplanted into lethally irradiated IFNaRKO recipients. Peripheral blood
from chimeric mice was stained with 2W1S-IAb tetramers 7 d following
immunization with 2W1S and polyI:C/CD40. Results were normalized to
the percent of tetramer+CD4+T cells in the IFNaRKO+B6 control (mean
1.3%). Data are representative of two independent experiments pooled
with at least three mice per group. D, Lethally irradiated IFNaRKO mice
were reconstituted with 4 3 106T cell-depleted bone marrow cells from
B6 mixed 1:1 with IFNaRKO, CD40KO mixed 1:1 with IFNaRKO, or
IFNaRKO alone. Results represent two independent experiments con-
taining three or more mice per group normalized to B6+IFNaRKO con-
trols (mean 3.5 3 105cells). Statistics were calculated using Student t test.
*p , 0.05, **p , 0.01, ***p , 0.005.
IFN dependency of CD4+T cell responses is T cell extrin-
The Journal of Immunology 589
by guest on June 13, 2013
cident with immunization by Ag and polyI:C/CD40. We found
that agonistic OX40 Ab largely enhanced CD4+T cell responses
(Fig. 5A), but not CD8+T cell responses (Fig. 5B), in WT and
IFNaRKO mice. These data confirmed a unique role for OX40 in
CD4+T cell, but not CD8+T cell, priming, which is reflected by
the literature (37). In all cases, cytokine production by CD4+and
CD8+T cells reflected the tetramer responses shown in this study
(not shown). Thus, augmentation of OX40–OX40L signals in
IFNaRKO mice can substantially enhance CD4+T cell, but not
CD8+T cell primary responses.
Collectively, our data contribute to the growing literature of the
influence of type I IFN on adaptive immunity by adding a critical
role for type I IFN in DC OX40L expression and subsequent
stimulation CD4+T cells through OX40. In addition, our data
challenge the paradigm that direct stimulation of T cells by IFN is
required for their capacity to respond to primary challenge. Sev-
eral authors have written on the role of direct type I IFN signals
for T cell immunity (12, 20, 38–40) and in particular the role
of type I IFN in Th1-type immune responses is controversial
(39). However, we could find no evidence that the Th1 phenotype
of responding CD4+T cells was different between WT and
IFNaRKO mice. Rather, we noted reduced responses across the
board in IFNaRKOs relative to B6 controls in both proliferation
and cytokine production, suggesting a defect in CD4+T cell
proliferation, but not necessarily differentiation. Furthermore,
using a mixed bone marrow chimeric system, we were able to
show that absence of IFNaR on responding CD4+T cells does
not disfavor their participation in primary immune responses with
dependent. A, Representative FACS histograms of
popliteal CD11c+cells harvested 24 h following foot-
pad immunization with polyI:C/CD40. Shown from
left to right are fluorescent Ag (OVA) uptake, MHC-II
expression, CD69 expression, and CCR7 expression.
Shown are (relative to injection site) ipselateral nodes
from WT mice (thick black line), ipselateral nodes
from IFNaRKO mice (thin black line), and contralat-
eral nodes from WT mice (gray shading). Data are
typical of three independent experiments containing
three or more mice per group. B, Representative FACS
plots of splenic CD8+CD11c+cells harvested at the
given time points following i.p. immunization with
polyI:C/CD40 and stained for OX40L and MHC-II.
Data are typical of two independent experiments con-
taining at least three mice per group. C and D, Rep-
resentative FACS plots of ex vivo (left panels) and
in vitro-cultured (right panels) splenocytes stimulated
with 0.5 mg/ml anti-CD3 (2C11), 105 U/ml rIFN-a, or
combined anti-CD3 and IFN-a for the indicated pe-
riod. Shown are CD11c+cells (C) and CD4+T cells
(D). Data are typical of four independent experiments.
DC OX40L expression is type I IFN
CD40 and administered 250 mg of an agonistic Ab for OX40 (OX86) i.p. on day 0 relative to primary immunization. Seven days following immunization,
spleens were harvested and stained with 2W1S-IAbtetramer (A) or OVA-Kb (B). Data represent combined results from three independent experiments
containing three or more mice per group normalized to control (WT), with mean responses of 7.2 3 105CD4+T cells in A and 1.8 3 106CD8+T cells in B.
Statistics represent pairwise comparisons to WT controls unless otherwise indicated. Error bars represent SEM. The p values were calculated using
a Student t test. *p , 0.05, **p , 0.01, ***p , 0.005.
OX40 stimulation rescues primary CD4+T cell responses in IFNaRKO mice. WTor IFNaRKO mice were immunized with Ag and polyI:C/
590IFN-DEPENDENT OX40L EXPRESSION DICTATES CD4 RESPONSE
by guest on June 13, 2013
competing WT cells. In fact, we show isolated IFNaRKO CD4+
T cells in a system without any cells able to sense type I IFN,
save for bone-marrow-derived, Rag-independent cells, have the
capacity to mount effective primary immune responses to IFN-
dependent stimuli. Granted, the outcomes we measure, such as
CD4+T cell proliferation, are different from the outcomes others
have measured, such as Ab production (38) or CD8+T cell dif-
ferentiation (20). Nevertheless, our data support a minimal role for
type I IFN signaling to CD4+T cells during primary responses.
We note also that, in our dual-adjuvant system, it is unlikely that
CD40 stimulus is rescuing IFN-dependent responses. We have
shown that CD40 is dispensable on responding CD4+T cells in the
setting of polyI:C/CD40 stimulus (26), and we show in this study
that IFNaR and CD40 must be on the same cells to promote
synergistic immune responses. This is consistent with our previous
data as well, in which we have shown that CD40 is sufficient on
innate cells for polyI:C/CD40-induced priming (26). Although
CD40 agonist by itself is a sufficient adjuvant to initiate adaptive
immune responses (41) and is not known to be dependent on type I
IFNs, it is interesting that the phenotype of IFNaRKO mice
dominates to limit expansion of CD4+T cells dramatically com-
pared with WT mice. It is possible, however, that CD40 agonism
may reverse some of the defects in CD4+T cell responses that
other authors have observed in IFNaRKO mice (17, 21). If true,
taken with our previous data that CD40 is necessary on innate
cells (26), our data would suggest that CD40 agonism acts indi-
rectly, through APCs, to rescue CD4+T cell memory in IFNaRKO
We have previously shown that rIFN-a will directly synergize
with agonistic anti-CD40 Ab to promote CD8+T cell responses
when combined together (32). However, we have not been able to
show that rIFN-a would synergize with anti-CD40 to promote
CD4+T cell responses (data not shown). This implies that al-
though IFNaR is indispensable for the effects of polyI:C/CD40
immunization, IFN-a itself is not sufficient to synergize with
CD40 agonists for CD4+T cells. We account for this by noting
that CD4+and CD8+T cells in this setting require slightly dif-
ferent priming conditions (26) and that these could be affected by
the dose of recombinant IFNa used. It is possible that CD4+and
CD8+T cell priming is initiated by different APC subsets (42, 43),
and these might have different sensitivities to rIFN-a. Alterna-
tively, IFN-a is one of many types of type I IFN, all of which
require the IFNaR to function (44), and any of these may dif-
ferentially impact Ag presentation to CD4+and CD8+T cells,
either by having variable effects on different APC subsets or by
directly impacting presentation through MHC-II or MHC-I in an
A larger question raised by these data concerns our findings that
both IFN and OX40 are dispensable for secondary CD4+T cell
responses but required for primary responses. This disagrees with
previous observations (17, 21, 23) and is particularly interesting
given the finding that, in the case of lymphocytic choriomeningitis
viral infection, survival of CD4+T cells is dependent on cell-
intrinsic type I IFN signaling (21). In contrast, our bone marrow
chimera suggests that intrinsic roles for type I IFN are dispensable
and that the primary role for IFN in CD4+T cell responses is
T cell extrinsic. Consistent with this, cell-intrinsic type I IFN
signaling was not required for CD4+T cell responses to bacterial
infection (21), whereas studies conflict with regard to the neces-
sity for direct IFN signaling to T cells in response to vaccinia virus
challenge (40, 45). There are a number of non-mutually exclusive
ways to reconcile our data with these findings. One is that our
dual-agonist immunization system exploits a pathway that permits
CD4+T cell survival similar to the result found for bacterial in-
fection (discussed above) (21). Another possibility is that, during
lymphotrophic viral infection such as lymphocytic choriome-
ningitis virus, the inability to respond to IFN signals leads to
killing of CD4+T cells by direct infection. Third, as has been
previously suggested (40), the inflammatory environment present
during the initial stimulation of the T cells may dictate the degree
of dependence or independence of direct IFN signaling into the
T cells. A final possibility is that aberrant IFN responses by CD4+
T cells in infected tissue leads to lysis by NK cells or CTL.
Our data show that the causes of poor primary responses that are
attributable to IFNaRKO mice are not as relevant for secondary
responses as they are for primary responses. To some extent, this
is not surprising, as primary and memory CD4+T cells are
qualitatively different from each other (46, 47), and our work adds
to this body of literature. However, our data showing memory
responses either in the absence of type I IFN signaling or during
OX40L blockade begs the question of how WT levels of sec-
ondary T cell expansion can occur after such a compromised
primary burst size. One explanation is that larger clonal burst sizes
lead to shorter t1/2of daughter progeny, which is an extrapolation
of recent data showing that population size of CD4+T cells is
a determinant of survival (48). An alternative, but not mutually
exclusive, explanation for these data is that a population of T cells
with high avidity for Ag, which is thought to be important for
CD4+T cell memory formation (47), is able to become activated
in WT and IFNaRKO mice regardless of costimulation. Thus, ad-
dition of extra costimulation and Ag presentation in WT mice in
this model might provide activation and recruitment of more lower
avidity cells, increasing the primary burst size but not the memory
pool. Similarly, it is possible that memory formation is facilitated
by a minimal threshold of activation beyond which extra prolif-
eration yields extra effector cells, but not necessarily more mem-
ory. Thus, type I IFN and OX40L costimulation may promote
effector cell generation beyond what is required for memory
formation, and the blockade or loss of these signaling pathways
reduces clonal burst size by reducing effectors. All possibilities
outlined above are validated for OX40 by recent data (49, 50).
Although we observed what appears to be a causal association
among CD4+T cell priming, IFN, and DC OX40L expression,
there are likely other defects in APC function that contribute to the
IFN dependency of the T cell response. DC activation (10, 12, 13),
trafficking (8), and Ag presentation (9) are all impaired in the
absence of type I IFN. We have confirmed many of these defects
in our system by observing reduced Ag uptake, expression of
MHC-II, CD69, CCR7, CD80, and CD86 (Fig. 4 and data not
shown), all of which contribute to poor CD4+T cell priming in the
absence of IFN. In particular, we show that stimulation by anti-
CD3 Abs through the TCR is important in vitro for OX40 up-
regulation on CD4+T cells. We extrapolate that impaired TCR
stimulus might impair CD4+T cell competence for OX40 stim-
ulus. This explains why IFNaRKO mice, in which MHC-II ex-
pression is impaired, are not completely rescued by OX86 to the
level of WT mice treated with OX86. Nevertheless, the connection
we make to OX40L demonstrates that defects in IFNaR signaling
can have very concrete molecular consequences for the ability of
DCs to prime CD4+T cells and that these defects may be par-
tially reversed by agonist treatment of key pathways absent in
Collectively,our data contribute to thegrowing body of evidence
that the induction of type I IFN in a vaccine setting can have a
powerful influence on the generation of cellular responses largely
through an influence on APC function. These data imply that the
application of IFN or IFN-inducing modalities to the generation of
The Journal of Immunology 591
by guest on June 13, 2013
primary function of IFN would be early in the generation of the
response and that continued application of IFN beyond the window
of necessary APC function will likely only be self-limiting. Thus,
although the primary clinical regimen of chronic IFN dosing is
appropriate to capitalize on the direct antiviral and cytostatic
properties of IFN signaling, the use of IFN as a therapeutic in-
tervention to augment the adaptive response will likely need to
consider a shorter course of IFN treatment. Our data indicate that
OX40–OX40L interactions will be a critical component by which
type I IFN elicits CD4+T cell responses with hopeful protective
and/or therapeutic potential.
We thank Hideo Yagita, Juntendo University School of Medicine, Tokyo,
Japan, for providing the OX40L and CD70 Abs.
R.M.K. is a founder of ImmuRx Inc., a vaccine company for which in-
tellectual property is based on the combined TLR agonist/anti-CD40 im-
munization platform. R.M.K., C.H., and P.J.S. are inventors on patent
applications filed by the University of Colorado and licensed by ImmuRx
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