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.

The 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 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 better 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 characterized 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 they may have carried from the periphery in the context of costimulation to naive, primary T cells (10, 11). The culmination of type I IFN induction is thus the initiation of an adaptive, antiviral, immune response.

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 activation by the TLR agonist polyinosinic-polycytidylic acid (polyI:C) (12). Given the role of DCs in the initiation of adaptive immunity (1416), 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, 1723). 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 particularly important to establish for the purposes of vaccine development, 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 IFNαR1 (IFNαR) (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 superfamily member OX40 ligand (OX40L). However, it remained unknown whether CD4+ T cells activated by polyI:C/CD40 were similarly dependent on signals through IFNαR. 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 pathway dependent on type I IFN (9). Furthermore, whereas a role for type I IFN in CD4+ T cell responses to polyI:C has been established (12), it was unknown whether the addition of a CD40 agonist would abolish the dependence of polyI:C responses on type I IFN.

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-αR–deficient (IFNαRKO) mice. This suggested that Ag-experienced CD4+ T cells were qualitatively different from naive CD4+ T cells and that the IFN dependency was unlikely due to direct IFN stimulation of the T cell. Indeed, the use of mixed bone marrow chimeras revealed that Rag1−/− bone marrow could rescue CD4+ T cell responses in otherwise IFNαRKO mice, demonstrating a role for IFNαR on innate, but not adaptive, cells. We further showed that polyI:C/CD40-stimulated IFNαRKO DCs had very low expression of OX40L, and restoration of OX40 signals using an agonistic Ab in IFNαRKO mice enhances CD4+, but not CD8+, T cell priming.

Six- to 8-wk-old female C57BL/6 mice were obtained through the National Cancer Institute or Harlan Laboratories. Mice deficient in the α1 receptor for type I IFN (B6.129PF2/ifnab) were obtained from Laurel Lenz at National Jewish Health and originally derived by Daniel Portnoy, University 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, National Jewish Health. Mice were housed at the Biological Resource Center at National Jewish Health. The Institutional Animal Care and Use Committee 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.

Unless indicated otherwise, mice were immunized i.p. with 500 μg whole chicken OVA protein (Sigma-Aldrich, St. Louis, MO), 100 μg Eα-derived 2W1S peptide (EAWGALANWAVDSA; custom synthesized by Pi Proteomics, Huntsville, AL) (27), 50 μg CD40 agonist Ab (clone FGK4.5; BioXCell, West Lebanon, NH), and 50 μg polyI:C (Amersham Biosciences/GE Healthcare, Piscataway, NJ). All vaccinations were prepared by mixing each component together in PBS and injected in 200 μl. PolyI:C was stored in frozen aliquots in PBS at −20°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 agonistic anti-OX40 (OX86) were obtained from BioXCell. OX40L blockade was facilitated by i.p. injection of 250 μg RM134L in PBS on days −1 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 μg 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 μl PBS containing 40 μg polyI:C, 40 μg FGK4.5, and 50 μg 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 International, Rockville, MD). All samples were resuspended in RPMI 1640 medium containing 2.5% heat-inactivated FCS, 2-ME, l-glutamine, nonessential amino acids, HEPES, sodium pyruvate, penicillin, and streptomycin. 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-IAb was 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-IAb was prepared as described (29). Kb reagents were purified as described (30) and loaded with OVA (SIINFEKL) Ag prior to staining. Cells were coincubated with tetramer for 1 h at 37°C prior to staining with surface Abs. Surface Abs were purchased from BioLegend (San Diego, CA) or eBioscience (San Diego, CA). For cytokine assays, cells were incubated for 3.5 h at 37°C with 6 μg/ml brefeldin A in complete media as above. Cells were restimulated with 10 μg/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 manufacturer’s instructions. Both DC and T cells were washed and stained in FACS buffer containing 10% 2.4G2 supernatant (B cell hybridoma blocking Fcγ 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), DX5, and CD3+ prior to gating on CD4+ and CD8+ events. DC were gated for forward scatter, side scatter, and pulse width and were CD19, CD3, and CD11c+.

Recipient mice were lethally irradiated with 900 rad in the morning and grafted via the tail vein in the afternoon with 4 × 106 total T cell-depleted donor bone marrow cells suspended in 200 μl 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 × 106 cells 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-sulfamethoxazole–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 immunization.

Spleen cells were harvested as described above using DNAse and collagenase and cultured unfractionated at 1 × 106 cells/ml in 24-well, flat-bottom plates. Cells were stimulated with 0.5 μg/ml anti-CD3ε (clone 2C11) in complete RPMI 1640 medium supplemented as above and with 10% FCS. In some cases, cells were incubated with 105 U/ml rIFN-α prepared as described (32). At indicated time points, cells were harvested by washing plates with FACS buffer and stained as above.

Spleen cells were quantified on a Vi-Cell cell viability analyzer (Beckman Coulter). Cytometry samples were acquired on a CyAn ADP (DakoCytomation) using Summit acquisition software. Samples were 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 between samples using the Student t test. In vivo experiments used in this manuscript were completed independently at least twice with a minimum three individuals per group. In vitro experiments were completed independently at least three times.

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 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 promotion of CD4+ T cell responses in our polyI:C/CD40 adjuvant system.

We began by immunizing wild-type (WT) B6 mice and IFNαRKO 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). Immunization 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 published results for CD8+ T cell responses (Fig. 1C) (26), absence of type I IFN signaling in IFNαRKO 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.

FIGURE 1.

CD4+ and CD8+ T cell responses to combined polyI:C/CD40 stimulus are IFN dependent. WT B6 or B6 IFNαRKO mice were immunized with Ag, polyI:C, and FGK4.5 anti-CD40 Ab as described in 1Materials 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 IFNαRKO 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, IFNαRKO mice demonstrate a defect in eliciting cytokine-producing cells following immunization. Cells from mice in B were stained with 2W1S-IAb tetramer as described in 1Materials 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.

FIGURE 1.

CD4+ and CD8+ T cell responses to combined polyI:C/CD40 stimulus are IFN dependent. WT B6 or B6 IFNαRKO mice were immunized with Ag, polyI:C, and FGK4.5 anti-CD40 Ab as described in 1Materials 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 IFNαRKO 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, IFNαRKO mice demonstrate a defect in eliciting cytokine-producing cells following immunization. Cells from mice in B were stained with 2W1S-IAb tetramer as described in 1Materials 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.

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FIGURE 2.

Primary CD4+ T cell responses are IFN and OX40L dependent, whereas secondary CD4+ T cell responses are IFN and OX40L independent. 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-γ+CD4+ T cells restimulated with 2W1S peptide over time relative to immunization on day 0 as in A. C, Fold expansion 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-γ+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 μg of RM134L i.p. on days −1 and 0 relative to priming, as described in 1Materials 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 μg of RM134L i.p. on days −1 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.

FIGURE 2.

Primary CD4+ T cell responses are IFN and OX40L dependent, whereas secondary CD4+ T cell responses are IFN and OX40L independent. 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-γ+CD4+ T cells restimulated with 2W1S peptide over time relative to immunization on day 0 as in A. C, Fold expansion 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-γ+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 μg of RM134L i.p. on days −1 and 0 relative to priming, as described in 1Materials 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 μg of RM134L i.p. on days −1 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.

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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 IFNαRKOs by using the blocking Ab RM134L. Mice were injected i.p. with 250 μg of blocking Abs on days −1 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 IFNαRKO mice were brought down even further in the presence of RM134L (Fig. 1D). Again, CD8+ T cell responses in IFNαRKO mice were unaffected by RM134L (data not shown). Thus, even the remnant CD4+ T cell response in the IFNαRKO mice was largely dependent on OX40/OX40L interactions. Collectively, the data demonstrate the central importance of both type I IFN and OX40 in the generation of CD4+ T cell immunity following combined polyI:C/CD40 immunization.

We were interested to know whether the defect in primary CD4+ T cell responses in IFNαRKO mice led to impaired secondary responses. To assess the role of type I IFN in CD4+ T cell memory, we immunized B6 or IFNαRKO mice with 2W1S Ag and polyI:C/CD40 as described above. Mice were followed by staining peripheral 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 (postboost).

We reasoned that poor primary responses would predict poor secondary responses in IFNαRKO mice. To our surprise, we found that secondary proliferative responses in IFNαRKO 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 IFNαRKO mice and B6 mice (Fig. 2C). Other authors 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 IFNαRKO mice relative to WT mice (Fig. 2B). However, in the spleen, the total number of cells producing IFN-γ were more similar between WT mice and IFNαRKOs (not shown), and, in all cases, the fold expansion of cytokine-producing cells from pre- to postboost was similar between WT and IFNαRKOs (Fig. 2D). Thus, our data indicate that secondary proliferation and cytokine production by CD4+ T cells are minimally dependent on type I IFN.

Curiously, we also found that WT and IFNαRKO mice responded 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 following rechallenge.

The fact that IFNαRKO CD4+ T cells could mount an effective secondary response suggested that the IFN dependency of the 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 IFNαR deficiency are T cell intrinsic or extrinsic, we generated mixed bone marrow chimeras in lethally irradiated IFNαRKO recipients to isolate defects in type I IFN signaling to bone marrow-derived cells (Fig. 3A). We used congenically marked CD45.1+ IFNαRKO bone marrow and mixed it 1:1 with WT B6 bone marrow (Fig. 3A). We reasoned that, if IFNαR were required on responding T cells, we would observe a disproportionate response of the WT bone marrow-derived T cells as compared with the congenic, IFNαRKO bone marrow-derived T cells. Instead, we found that T cells derived from IFNαRKO bone marrow competed very well with WT bone marrow, if anything, responding better than the WT T cells (Fig. 3B). This indicated that IFNαR 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 IFNαRKO hosts with RagKO bone marrow mixed 3:1 (to enhance engraftment of RagKO marrow) with IFNαRKO bone marrow. The resulting host has T cells that are exclusively IFNαR deficient, but both WT and IFNαR-deficient APCs. As anticipated, the CD4+ T cell responses were rescued in these RagKO × IFNαRKO chimeras (Fig. 3C). These data demonstrate that IFNαR is dispensable on responding CD4+ T cells and that cells derived from RagKO bone marrow are sufficient to restore IFNαR-dependent pathways in IFNαRKO mice.

FIGURE 3.

IFN dependency of CD4+ T cell responses is T cell extrinsic. A, Model for mixed bone marrow chimeras. Bone marrow was harvested from congenically marked, IFNαRKO (IFNαRKO.SJL), RagKO, CD40-deficient (CD40KO), or WT B6 mice, mixed as indicated, and transplanted via the tail vein into lethally irradiated IFNαRKO recipients. All mice were rested ≥12 wk prior to immunization with polyI:C/CD40 and Ag. B, Total of 2 × 106 T cell-depleted bone marrow cells derived from each IFNαRKO.SJL (CD45.1) and WT (CD45.2) mouse was mixed 1:1 (4 × 106 total cells) and injected into lethally irradiated CD4 IFNαRKO CD45.2 recipients. The ratio of IFNαRKO (CD45.1+) to WT (CD45.1) CD4+ T cells prior to immunization and the ratio of 2W1S-specific IFNαRKO (CD45.1+) to WT (CD45.1) following polyI:C/CD40 and 2W1S immunization is shown. Data represent consolidated samples from two independent experiments (n = 7). C, IFNαRKO bone marrow was harvested (IFNαRKO), mixed 1:3 with RagKO bone marrow (IFNαRKO+Rag), or 1:1 with WT bone marrow (IFNαRKO+B6) and transplanted into lethally irradiated IFNαRKO 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 IFNαRKO+B6 control (mean 1.3%). Data are representative of two independent experiments pooled with at least three mice per group. D, Lethally irradiated IFNαRKO mice were reconstituted with 4 × 106 T cell-depleted bone marrow cells from B6 mixed 1:1 with IFNαRKO, CD40KO mixed 1:1 with IFNαRKO, or IFNαRKO alone. Results represent two independent experiments containing three or more mice per group normalized to B6+IFNαRKO controls (mean 3.5 × 105 cells). Statistics were calculated using Student t test. *p < 0.05, **p < 0.01, ***p < 0.005.

FIGURE 3.

IFN dependency of CD4+ T cell responses is T cell extrinsic. A, Model for mixed bone marrow chimeras. Bone marrow was harvested from congenically marked, IFNαRKO (IFNαRKO.SJL), RagKO, CD40-deficient (CD40KO), or WT B6 mice, mixed as indicated, and transplanted via the tail vein into lethally irradiated IFNαRKO recipients. All mice were rested ≥12 wk prior to immunization with polyI:C/CD40 and Ag. B, Total of 2 × 106 T cell-depleted bone marrow cells derived from each IFNαRKO.SJL (CD45.1) and WT (CD45.2) mouse was mixed 1:1 (4 × 106 total cells) and injected into lethally irradiated CD4 IFNαRKO CD45.2 recipients. The ratio of IFNαRKO (CD45.1+) to WT (CD45.1) CD4+ T cells prior to immunization and the ratio of 2W1S-specific IFNαRKO (CD45.1+) to WT (CD45.1) following polyI:C/CD40 and 2W1S immunization is shown. Data represent consolidated samples from two independent experiments (n = 7). C, IFNαRKO bone marrow was harvested (IFNαRKO), mixed 1:3 with RagKO bone marrow (IFNαRKO+Rag), or 1:1 with WT bone marrow (IFNαRKO+B6) and transplanted into lethally irradiated IFNαRKO 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 IFNαRKO+B6 control (mean 1.3%). Data are representative of two independent experiments pooled with at least three mice per group. D, Lethally irradiated IFNαRKO mice were reconstituted with 4 × 106 T cell-depleted bone marrow cells from B6 mixed 1:1 with IFNαRKO, CD40KO mixed 1:1 with IFNαRKO, or IFNαRKO alone. Results represent two independent experiments containing three or more mice per group normalized to B6+IFNαRKO controls (mean 3.5 × 105 cells). Statistics were calculated using Student t test. *p < 0.05, **p < 0.01, ***p < 0.005.

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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 IFNαR expression was also sufficient on innate cells raised the question of whether CD40 and IFNαR were required on the same cells or whether 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 IFNαR were required on the same cells, we reconstituted lethally irradiated IFNαRKO mice with bone marrow derived from IFNαRKO.SJL mice alone, IFNαRKO.SJL and CD40KO bone marrow mixed 1:1, and IFNαRKO.SJL and B6 bone marrow mixed 1:1. We found that IFNαRKO.SJL and CD40KO bone marrow were not able to reconstitute responses in IFNαRKO recipients (Fig. 3D), suggesting that both CD40 and IFNαR 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 sufficient for the combined effects of both agonists.

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 IFNαR-dependent manner (8, 12). To understand how IFNαR deficiency might affect DC activation, we immunized B6 and IFNαRKO 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 IFNαRKO DCs (Fig. 4A) had relative defects in Ag uptake, MHC-II expression, CD69 expression, and CCR7 expression compared with WT mice (Fig. 4A). Importantly, DCs from IFNαRKO 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 IFNαRKO mice, but was impaired.

FIGURE 4.

DC OX40L expression is type I IFN dependent. A, Representative FACS histograms of popliteal CD11c+ cells harvested 24 h following footpad 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 IFNαRKO mice (thin black line), and contralateral 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 containing at least three mice per group. C and D, Representative FACS plots of ex vivo (left panels) and in vitro-cultured (right panels) splenocytes stimulated with 0.5 μg/ml anti-CD3 (2C11), 105 U/ml rIFN-α, or combined anti-CD3 and IFN-α for the indicated period. Shown are CD11c+ cells (C) and CD4+ T cells (D). Data are typical of four independent experiments.

FIGURE 4.

DC OX40L expression is type I IFN dependent. A, Representative FACS histograms of popliteal CD11c+ cells harvested 24 h following footpad 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 IFNαRKO mice (thin black line), and contralateral 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 containing at least three mice per group. C and D, Representative FACS plots of ex vivo (left panels) and in vitro-cultured (right panels) splenocytes stimulated with 0.5 μg/ml anti-CD3 (2C11), 105 U/ml rIFN-α, or combined anti-CD3 and IFN-α for the indicated period. Shown are CD11c+ cells (C) and CD4+ T cells (D). Data are typical of four independent experiments.

Close modal

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 IFNαRKOs relative to WT mice (Fig. 4B).

Although the loss of DC OX40L expression was not absolute in the IFNαRKOs (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 dependent (Fig. 4B). These data suggested that the introduction rIFN-α 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-α, or anti-CD3 and rIFN-α. Intriguingly, anti-CD3 alone and anti-CD3 with IFN-α did not lead to appreciable expression of OX40L in these cultures (Fig. 4C). However, administration of rIFN-α alone was sufficient to massively upregulate OX40L expression on cultured DCs (Fig. 4C). Furthermore, DCs increased expression of CD70 in the presence of rIFN-α (Fig. 4C), regardless of the presence of anti-CD3.

It was interesting that IFN-α alone, but not IFN-α 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-α may have led to upregulation of OX40, which, in turn, might prevent detection of OX40L on the DCs, either by downmodulating 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 ± rIFN-α, but not on naive cells or on cells stimulated with IFN-α alone (Fig. 4D). This result is consistent with the finding that, in vivo, priming of CD4+ T cells does not require IFNαR on the cells themselves. Similarly, expression 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 regulation of the receptors on responding CD4+ T cells is likely to be a consequence of CD3-mediated TCR stimulus.

As OX40L signals were required for optimal CD4+ T cell priming, but defective in IFNαRKO mice, we hypothesized that restoration of signaling through OX40 might restore CD4+ T cell responses in IFNαRKOs. We therefore administered single doses of the OX40-agonistic Ab, OX86 (35, 36), to WT and IFNαRKO mice coincident 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 IFNαRKO 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 IFNαRKO mice can substantially enhance CD4+ T cell, but not CD8+ T cell primary responses.

FIGURE 5.

OX40 stimulation rescues primary CD4+ T cell responses in IFNαRKO mice. WT or IFNαRKO mice were immunized with Ag and polyI:C/CD40 and administered 250 μg 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-IAb tetramer (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 × 105 CD4+ T cells in A and 1.8 × 106 CD8+ 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.

FIGURE 5.

OX40 stimulation rescues primary CD4+ T cell responses in IFNαRKO mice. WT or IFNαRKO mice were immunized with Ag and polyI:C/CD40 and administered 250 μg 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-IAb tetramer (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 × 105 CD4+ T cells in A and 1.8 × 106 CD8+ 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.

Close modal

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. Several authors have written on the role of direct type I IFN signals for T cell immunity (12, 20, 3840) 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 IFNαRKO mice. Rather, we noted reduced responses across the board in IFNαRKOs 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 IFNαR on responding CD4+ T cells does not disfavor their participation in primary immune responses with competing WT cells. In fact, we show isolated IFNαRKO 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 differentiation (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 IFNαR 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 IFNαRKO mice dominates to limit expansion of CD4+ T cells dramatically compared 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 IFNαRKO 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 indirectly, through APCs, to rescue CD4+ T cell memory in IFNαRKO mice.

We have previously shown that rIFN-α 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-α would synergize with anti-CD40 to promote CD4+ T cell responses (data not shown). This implies that although IFNαR is indispensable for the effects of polyI:C/CD40 immunization, IFN-α 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 different priming conditions (26) and that these could be affected by the dose of recombinant IFNα 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-α. Alternatively, IFN-α is one of many types of type I IFN, all of which require the IFNαR to function (44), and any of these may differentially 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 paralog-dependent manner.

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 necessity 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 infection (discussed above) (21). Another possibility is that, during lymphotrophic viral infection such as lymphocytic choriomeningitis 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 IFNαRKO 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 secondary T cell expansion can occur after such a compromised primary burst size. One explanation is that larger clonal burst sizes lead to shorter t1/2 of 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 IFNαRKO mice regardless of costimulation. Thus, addition 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 proliferation yields extra effector cells, but not necessarily more memory. 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 upregulation on CD4+ T cells. We extrapolate that impaired TCR stimulus might impair CD4+ T cell competence for OX40 stimulus. This explains why IFNαRKO mice, in which MHC-II expression 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 IFNαR signaling can have very concrete molecular consequences for the ability of DCs to prime CD4+ T cells and that these defects may be partially reversed by agonist treatment of key pathways absent in IFNαRKOs.

Collectively, our data contribute to the growing 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 T cell immunity will need to take into consideration the fact that the 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 intervention 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.

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 Biodefense at National Jewish Health.

Abbreviations used in this article:

DC

dendritic cell

IFNαRKO

IFN-αR–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.

1
Sadler
A. J.
,
Williams
B. R.
.
2008
.
Interferon-inducible antiviral effectors.
Nat. Rev. Immunol.
8
:
559
568
.
2
Feld
J. J.
,
Lutchman
G. A.
,
Heller
T.
,
Hara
K.
,
Pfeiffer
J. K.
,
Leff
R. D.
,
Meek
C.
,
Rivera
M.
,
Ko
M.
,
Koh
C.
, et al
.
2010
.
Ribavirin improves early responses to peginterferon through improved interferon signaling.
Gastroenterology
139
:
154
162
.
e154
.
3
Jouanguy
E.
,
Zhang
S. Y.
,
Chapgier
A.
,
Sancho-Shimizu
V.
,
Puel
A.
,
Picard
C.
,
Boisson-Dupuis
S.
,
Abel
L.
,
Casanova
J. L.
.
2007
.
Human primary immunodeficiencies of type I interferons.
Biochimie
89
:
878
883
.
4
Müller
U.
,
Steinhoff
U.
,
Reis
L. F.
,
Hemmi
S.
,
Pavlovic
J.
,
Zinkernagel
R. M.
,
Aguet
M.
.
1994
.
Functional role of type I and type II interferons in antiviral defense.
Science
264
:
1918
1921
.
5
Isaacs
A.
,
Lindenmann
J.
.
1957
.
Virus interference. I. The interferon.
Proc. R. Soc. Lond. B Biol. Sci.
147
:
258
267
.
6
Gallucci
S.
,
Lolkema
M.
,
Matzinger
P.
.
1999
.
Natural adjuvants: endogenous activators of dendritic cells.
Nat. Med.
5
:
1249
1255
.
7
Schulz
O.
,
Diebold
S. S.
,
Chen
M.
,
Näslund
T. I.
,
Nolte
M. A.
,
Alexopoulou
L.
,
Azuma
Y. T.
,
Flavell
R. A.
,
Liljeström
P.
,
Reis e Sousa
C.
.
2005
.
Toll-like receptor 3 promotes cross-priming to virus-infected cells.
Nature
433
:
887
892
.
8
Shiow
L. R.
,
Rosen
D. B.
,
Brdicková
N.
,
Xu
Y.
,
An
J.
,
Lanier
L. L.
,
Cyster
J. G.
,
Matloubian
M.
.
2006
.
CD69 acts downstream of interferon-alpha/beta to inhibit S1P1 and lymphocyte egress from lymphoid organs.
Nature
440
:
540
544
.
9
Le Bon
A.
,
Etchart
N.
,
Rossmann
C.
,
Ashton
M.
,
Hou
S.
,
Gewert
D.
,
Borrow
P.
,
Tough
D. F.
.
2003
.
Cross-priming of CD8+ T cells stimulated by virus-induced type I interferon.
Nat. Immunol.
4
:
1009
1015
.
10
Montoya
M.
,
Schiavoni
G.
,
Mattei
F.
,
Gresser
I.
,
Belardelli
F.
,
Borrow
P.
,
Tough
D. F.
.
2002
.
Type I interferons produced by dendritic cells promote their phenotypic and functional activation.
Blood
99
:
3263
3271
.
11
Luft
T.
,
Pang
K. C.
,
Thomas
E.
,
Hertzog
P.
,
Hart
D. N.
,
Trapani
J.
,
Cebon
J.
.
1998
.
Type I IFNs enhance the terminal differentiation of dendritic cells.
J. Immunol.
161
:
1947
1953
.
12
Longhi
M. P.
,
Trumpfheller
C.
,
Idoyaga
J.
,
Caskey
M.
,
Matos
I.
,
Kluger
C.
,
Salazar
A. M.
,
Colonna
M.
,
Steinman
R. M.
.
2009
.
Dendritic cells require a systemic type I interferon response to mature and induce CD4+ Th1 immunity with poly IC as adjuvant.
J. Exp. Med.
206
:
1589
1602
.
13
Pilz
A.
,
Kratky
W.
,
Stockinger
S.
,
Simma
O.
,
Kalinke
U.
,
Lingnau
K.
,
von Gabain
A.
,
Stoiber
D.
,
Sexl
V.
,
Kolbe
T.
, et al
.
2009
.
Dendritic cells require STAT-1 phosphorylated at its transactivating domain for the induction of peptide-specific CTL.
J. Immunol.
183
:
2286
2293
.
14
Inaba
K.
,
Young
J. W.
,
Steinman
R. M.
.
1987
.
Direct activation of CD8+ cytotoxic T lymphocytes by dendritic cells.
J. Exp. Med.
166
:
182
194
.
15
Guéry
J. C.
,
Ria
F.
,
Adorini
L.
.
1996
.
Dendritic cells but not B cells present antigenic complexes to class II-restricted T cells after administration of protein in adjuvant.
J. Exp. Med.
183
:
751
757
.
16
Hildner
K.
,
Edelson
B. T.
,
Purtha
W. E.
,
Diamond
M.
,
Matsushita
H.
,
Kohyama
M.
,
Calderon
B.
,
Schraml
B. U.
,
Unanue
E. R.
,
Diamond
M. S.
, et al
.
2008
.
Batf3 deficiency reveals a critical role for CD8alpha+ dendritic cells in cytotoxic T cell immunity.
Science
322
:
1097
1100
.
17
Marrack
P.
,
Kappler
J.
,
Mitchell
T.
.
1999
.
Type I interferons keep activated T cells alive.
J. Exp. Med.
189
:
521
530
.
18
Ramos
H. J.
,
Davis
A. M.
,
George
T. C.
,
Farrar
J. D.
.
2007
.
IFN-alpha is not sufficient to drive Th1 development due to lack of stable T-bet expression.
J. Immunol.
179
:
3792
3803
.
19
Wenner
C. A.
,
Güler
M. L.
,
Macatonia
S. E.
,
O’Garra
A.
,
Murphy
K. M.
.
1996
.
Roles of IFN-gamma and IFN-alpha in IL-12-induced T helper cell-1 development.
J. Immunol.
156
:
1442
1447
.
20
Le Bon
A.
,
Durand
V.
,
Kamphuis
E.
,
Thompson
C.
,
Bulfone-Paus
S.
,
Rossmann
C.
,
Kalinke
U.
,
Tough
D. F.
.
2006
.
Direct stimulation of T cells by type I IFN enhances the CD8+ T cell response during cross-priming.
J. Immunol.
176
:
4682
4689
.
21
Havenar-Daughton
C.
,
Kolumam
G. A.
,
Murali-Krishna
K.
.
2006
.
Cutting Edge: The direct action of type I IFN on CD4 T cells is critical for sustaining clonal expansion in response to a viral but not a bacterial infection.
J. Immunol.
176
:
3315
3319
.
22
Gallagher
K. M.
,
Lauder
S.
,
Rees
I. W.
,
Gallimore
A. M.
,
Godkin
A. J.
.
2009
.
Type I interferon (IFN alpha) acts directly on human memory CD4+ T cells altering their response to antigen.
J. Immunol.
183
:
2915
2920
.
23
Davis
A. M.
,
Ramos
H. J.
,
Davis
L. S.
,
Farrar
J. D.
.
2008
.
Cutting edge: a T-bet-independent role for IFN-alpha/beta in regulating IL-2 secretion in human CD4+ central memory T cells.
J. Immunol.
181
:
8204
8208
.
24
Coffman
R. L.
,
Sher
A.
,
Seder
R. A.
.
2010
.
Vaccine adjuvants: putting innate immunity to work.
Immunity
33
:
492
503
.
25
Ahonen
C. L.
,
Doxsee
C. L.
,
McGurran
S. M.
,
Riter
T. R.
,
Wade
W. F.
,
Barth
R. J.
,
Vasilakos
J. P.
,
Noelle
R. J.
,
Kedl
R. M.
.
2004
.
Combined TLR and CD40 triggering induces potent CD8+ T cell expansion with variable dependence on type I IFN.
J. Exp. Med.
199
:
775
784
.
26
Kurche
J. S.
,
Burchill
M. A.
,
Sanchez
P. J.
,
Haluszczak
C.
,
Kedl
R. M.
.
2010
.
Comparison of OX40 ligand and CD70 in the promotion of CD4+ T cell responses.
J. Immunol.
185
:
2106
2115
.
27
Rees
W.
,
Bender
J.
,
Teague
T. K.
,
Kedl
R. M.
,
Crawford
F.
,
Marrack
P.
,
Kappler
J.
.
1999
.
An inverse relationship between T cell receptor affinity and antigen dose during CD4(+) T cell responses in vivo and in vitro.
Proc. Natl. Acad. Sci. USA
96
:
9781
9786
.
28
Moon
J. J.
,
Chu
H. H.
,
Pepper
M.
,
McSorley
S. J.
,
Jameson
S. C.
,
Kedl
R. M.
,
Jenkins
M. K.
.
2007
.
Naive CD4(+) T cell frequency varies for different epitopes and predicts repertoire diversity and response magnitude.
Immunity
27
:
203
213
.
29
Crawford
F.
,
Kozono
H.
,
White
J.
,
Marrack
P.
,
Kappler
J.
.
1998
.
Detection of antigen-specific T cells with multivalent soluble class II MHC covalent peptide complexes.
Immunity
8
:
675
682
.
30
Kedl
R. M.
,
Rees
W. A.
,
Hildeman
D. A.
,
Schaefer
B.
,
Mitchell
T.
,
Kappler
J.
,
Marrack
P.
.
2000
.
T cells compete for access to antigen-bearing antigen-presenting cells.
J. Exp. Med.
192
:
1105
1113
.
31
Haluszczak
C.
,
Akue
A. D.
,
Hamilton
S. E.
,
Johnson
L. D.
,
Pujanauski
L.
,
Teodorovic
L.
,
Jameson
S. C.
,
Kedl
R. M.
.
2009
.
The antigen-specific CD8+ T cell repertoire in unimmunized mice includes memory phenotype cells bearing markers of homeostatic expansion.
J. Exp. Med.
206:
435
448
.
32
McWilliams
J. A.
,
Sanchez
P. J.
,
Haluszczak
C.
,
Gapin
L.
,
Kedl
R. M.
.
2010
.
Multiple innate signaling pathways cooperate with CD40 to induce potent, CD70-dependent cellular immunity.
Vaccine
28
:
1468
1476
.
33
Sanchez
P. J.
,
McWilliams
J. A.
,
Haluszczak
C.
,
Yagita
H.
,
Kedl
R. M.
.
2007
.
Combined TLR/CD40 stimulation mediates potent cellular immunity by regulating dendritic cell expression of CD70 in vivo.
J. Immunol.
178
:
1564
1572
.
34
Curtsinger
J. M.
,
Lins
D. C.
,
Mescher
M. F.
.
2003
.
Signal 3 determines tolerance versus full activation of naive CD8 T cells: dissociating proliferation and development of effector function.
J. Exp. Med.
197
:
1141
1151
.
35
al-Shamkhani
A.
,
Birkeland
M. L.
,
Puklavec
M.
,
Brown
M. H.
,
James
W.
,
Barclay
A. N.
.
1996
.
OX40 is differentially expressed on activated rat and mouse T cells and is the sole receptor for the OX40 ligand.
Eur. J. Immunol.
26
:
1695
1699
.
36
Maxwell
J. R.
,
Weinberg
A.
,
Prell
R. A.
,
Vella
A. T.
.
2000
.
Danger and OX40 receptor signaling synergize to enhance memory T cell survival by inhibiting peripheral deletion.
J. Immunol.
164
:
107
112
.
37
Kopf
M.
,
Ruedl
C.
,
Schmitz
N.
,
Gallimore
A.
,
Lefrang
K.
,
Ecabert
B.
,
Odermatt
B.
,
Bachmann
M. F.
.
1999
.
OX40-deficient mice are defective in Th cell proliferation but are competent in generating B cell and CTL Responses after virus infection.
Immunity
11
:
699
708
.
38
Le Bon
A.
,
Thompson
C.
,
Kamphuis
E.
,
Durand
V.
,
Rossmann
C.
,
Kalinke
U.
,
Tough
D. F.
.
2006
.
Cutting edge: enhancement of antibody responses through direct stimulation of B and T cells by type I IFN.
J. Immunol.
176
:
2074
2078
.
39
Berenson
L. S.
,
Gavrieli
M.
,
Farrar
J. D.
,
Murphy
T. L.
,
Murphy
K. M.
.
2006
.
Distinct characteristics of murine STAT4 activation in response to IL-12 and IFN-alpha.
J. Immunol.
177
:
5195
5203
.
40
Thompson
L. J.
,
Kolumam
G. A.
,
Thomas
S.
,
Murali-Krishna
K.
.
2006
.
Innate inflammatory signals induced by various pathogens differentially dictate the IFN-I dependence of CD8 T cells for clonal expansion and memory formation.
J. Immunol.
177
:
1746
1754
.
41
Taraban
V. Y.
,
Rowley
T. F.
,
Al-Shamkhani
A.
.
2004
.
Cutting edge: a critical role for CD70 in CD8 T cell priming by CD40-licensed APCs.
J. Immunol.
173
:
6542
6546
.
42
Bedoui
S.
,
Whitney
P. G.
,
Waithman
J.
,
Eidsmo
L.
,
Wakim
L.
,
Caminschi
I.
,
Allan
R. S.
,
Wojtasiak
M.
,
Shortman
K.
,
Carbone
F. R.
, et al
.
2009
.
Cross-presentation of viral and self antigens by skin-derived CD103+ dendritic cells.
Nat. Immunol.
10
:
488
495
.
43
Dudziak
D.
,
Kamphorst
A. O.
,
Heidkamp
G. F.
,
Buchholz
V. R.
,
Trumpfheller
C.
,
Yamazaki
S.
,
Cheong
C.
,
Liu
K.
,
Lee
H. W.
,
Park
C. G.
, et al
.
2007
.
Differential antigen processing by dendritic cell subsets in vivo.
Science
315
:
107
111
.
44
van Boxel-Dezaire
A. H.
,
Rani
M. R.
,
Stark
G. R.
.
2006
.
Complex modulation of cell type-specific signaling in response to type I interferons.
Immunity
25
:
361
372
.
45
Aichele
P.
,
Unsoeld
H.
,
Koschella
M.
,
Schweier
O.
,
Kalinke
U.
,
Vucikuja
S.
.
2006
.
CD8 T cells specific for lymphocytic choriomeningitis virus require type I IFN receptor for clonal expansion.
J. Immunol.
176
:
4525
4529
.
46
Swain
S. L.
,
Hu
H.
,
Huston
G.
.
1999
.
Class II-independent generation of CD4 memory T cells from effectors.
Science
286
:
1381
1383
.
47
Williams
M. A.
,
Ravkov
E. V.
,
Bevan
M. J.
.
2008
.
Rapid culling of the CD4+ T cell repertoire in the transition from effector to memory.
Immunity
28
:
533
545
.
48
Hataye
J.
,
Moon
J. J.
,
Khoruts
A.
,
Reilly
C.
,
Jenkins
M. K.
.
2006
.
Naive and memory CD4+ T cell survival controlled by clonal abundance.
Science
312
:
114
116
.
49
Soroosh
P.
,
Ine
S.
,
Sugamura
K.
,
Ishii
N.
.
2007
.
Differential requirements for OX40 signals on generation of effector and central memory CD4+ T cells.
J. Immunol.
179
:
5014
5023
.
50
Gramaglia
I.
,
Jember
A.
,
Pippig
S. D.
,
Weinberg
A. D.
,
Killeen
N.
,
Croft
M.
.
2000
.
The OX40 costimulatory receptor determines the development of CD4 memory by regulating primary clonal expansion.
J. Immunol.
165
:
3043
3050
.

R.M.K. is a founder of ImmuRx Inc., a vaccine company for which intellectual property is based on the combined TLR agonist/anti-CD40 immunization 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 Inc.