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Comparison of OX40 Ligand and CD70 in the Promotion of CD4 + T Cell Responses Jonathan S. Kurche, Matthew A. Burchill, Phillip J. Sanchez, Catherine Haluszczak and Ross M. Kedl This information is current as of September 25, 2021. J Immunol 2010; 185:2106-2115; Prepublished online 16 July 2010; doi: 10.4049/jimmunol.1000172 http://www.jimmunol.org/content/185/4/2106 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Comparison of OX40 Ligand and CD70 in the Promotion of CD4+ T Cell Responses

Jonathan S. Kurche,* Matthew A. Burchill,† Phillip J. Sanchez,* Catherine Haluszczak,* and Ross M. Kedl*

The TNF superfamily members CD70 and OX40 ligand (OX40L) were reported to be important for CD4+ T cell expansion and differentiation. However, the relative contribution of these costimulatory signals in driving CD4+ T cell responses has not been addressed. In this study, we found that OX40L is a more important determinant than CD70 of the primary CD4+ T cell response to multiple immunization regimens. Despite the ability of a combined TLR and CD40 agonist (TLR/CD40) stimulus to provoke appreciable expression of CD70 and OX40L on CD8+ dendritic cells, resulting CD4+ T cell responses were substantially reduced by Ab blockade of OX40L and, to a lesser degree, CD70. In contrast, the CD8+ T cell responses to combined TLR/CD40 immunization were exclusively dependent on CD70. These requirements for CD4+ and CD8+ T cell activation were not limited Downloaded from to the use of combined TLR/CD40 immunization, because vaccinia virus challenge elicited primarily OX40L-dependent CD4 responses and exclusively CD70-dependent CD8+ T cell responses. Attenuation of CD4+ T cell priming induced by OX40L blockade was independent of signaling through the IL-12R, but it was reduced further by coblockade of CD70. Thus, costimulation by CD70 or OX40L seems to be necessary for primary CD4+ T cell responses to multiple forms of immunization, and each may make independent contributions to CD4+ T cell priming. The Journal of Immunology, 2010, 185: 2106–2115. http://www.jimmunol.org/

D4+ T cells are important in controlling CMV and other storage and transport; attenuated vaccines are complicated by the chronic infections (1–6) and are involved in protection capacity to revert to virulent form. Although subunit-based vac- C from Mycobacterium tuberculosis (7–9). They are a ma- cines are arguably safer than those formulated around killed or jor target of HIV infection, and their loss is diagnostic of the attenuated pathogens, all such vaccines in the United States use acquired immunocompromised state. Additionally, CD4+ T cells alum as an adjuvant, which is generally recognized as inadequate are important for formation of CD8+ T cell memory populations for generating cellular immune responses (15, 16). Unfortunately, (10), and they may be important for CD8+ T cell priming to efforts to molecularly engineer subunit vaccines using TLR ago- self-Ag, a prerequisite for effective tumor immunity (11). Further- nists have also been disappointing (17). by guest on September 25, 2021 more, they were found to be important for distinct types of B cell Ideally, it would be possible to manipulate individual molecular responses (12, 13). Thus, there is an ongoing need to understand pathways governing adaptive immune responses to program a par- the processes governing CD4+ T cell activation, expansion, and ticular outcome. To this end, there are many potential costimulatory memory formation to comprehend their role in immune responses markers expressed by activated APCs, including those of the Ig su- and to develop clinically useful, CD4+ T cell-directed vaccines. perfamily and those of the TNF superfamily (TNFSF), which may Modern vaccines are based on one of two principal platforms: have the capacity to promote or inhibit T cell responses. The mo- subunit based or based on killed or attenuated pathogens. Of these, lecular processes governing expression of these markers by APCs, killed and attenuated pathogens provide an intrinsic adjuvanticity and the precise impact of one costimulatory marker or another on based around inherent molecular activators of innate immunity. T cell responses is unclear. We chose to focus on the role of TNFSF Generally speaking, these vaccines bear intrinsic ligands for pat- signaling in CD4+ T cell priming in an attempt to resolve the tern-recognition receptors, such as the TLR family, which have the relative contribution of different TNFSF ligands to CD4+ T cell capacity to mobilize innate immune cells (14). Unfortunately, ef- immune responses. Although it may be expected that CD4-specific fective vaccines formulated around killed or attenuated pathogens TNFSF signals exist or that different patterns of TNF ligand sig- can be difficult to derive and they are limited by problems with naling may convey information on the nature of threats to the adaptive immune system, there is a great deal of overlap in the literature regarding TNFSF dependency of CD4+ T cells (18–27). *Integrated Department of Immunology, University of Colorado Denver and In particular, receptor–ligand interactions, such as CD27–CD70 † Integrated Department of Immunology, National Jewish Health, Denver, CO 80206 (20–22) and OX40–OX40 ligand (OX40L) (23–27), are reported Received for publication January 20, 2010. Accepted for publication June 13, 2010. to be important for CD4+ T cell priming; however, we could find Address correspondence and reprint requests to Dr. Ross M. Kedl, Integrated De- no studies directly assessing the relative contributions of CD70 partment of Immunology, National Jewish Health and University of Colorado Denver, and OX40L to CD4+ T cell responses in a given model system. We 1400 East Jackson Street, Denver, CO 80206. E-mail address: ross.kedl@ucdenver. + edu became interested in the details of how CD4 T cells may be The online version of this article contains supplemental material. codependent on two TNFSF members. We reasoned that a better + Abbreviations used in this paper: CD40KO, CD40-deficient; DC, dendritic cell; KO, understanding of the relevant signals conveyed to CD4 T cells by knockout; MFI, mean fluorescence intensity; MHC-II, MHC class II; OX40L, OX40 priming APCs could accelerate the development of alternative ligand; Pam-3-Cys, Pam-3-Cys-K4; poly(I:C), polyinosinic:polycytidylic acid; TNFSF, vaccine platforms. TNF superfamily; WT, wild-type. We previously showed that combined TLR/CD40 stimulus pro- + Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 motes exponential CD8 T cell responses in mice. These responses www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000172 The Journal of Immunology 2107 are obligately dependent on the TNF ligand superfamily member blockade for immunization was facilitated by i.p. injection of 250 mg CD70 (28, 29) and independent of CD4+ T cells (17). However, blocking Ab in PBS on days 21 and 0. On day 0, blocking Abs were we had not investigated what impact this combined TLR/CD40 mixed with prepared vaccines, and both were delivered in a single injec- + tion. For pathogen challenge, TNF blockade was facilitated by i.p. injec- stimulus would have on CD4 T cells, to what degree it would tion of 250 mg on days 21, 0, and 2. promote their exponential expansion, whether it would promote cytokine production or memory formation, and upon what costi- Flow cytometry mulatory pathways it might depend. We demonstrate in this study For T cell assays, mice were sacrificed 7 d following immunization, pe- that Ag-specific CD4+ T cells are synergistically induced by com- ripheral blood was harvested from the abdominal aorta, and spleens were bined TLR/CD40 stimulus relative to either agonist alone. Induced harvested and minced with forceps in HBSS containing 5 mM EDTA and + 2% heat-inactivated FCS. Single-cell suspensions were made by passing CD4 T cells produce IFN-g and can mount potent anamnestic minced spleens through nylon mesh filters. RBCs were lysed in peripheral responses following a single vaccination. Furthermore, we found blood samples using ACK lysis solution (BioSource, Rockville, MD). All that costimulation for CD4+ T cell priming was dependent on samples were resuspended in RPMI 1640 medium containing 2.5% FCS, CD70 and OX40L. We did not find a role for IL-12 signaling in 2-ME, L-glutamine, nonessential amino acids, HEPES, sodium pyruvate, this system. We observed that simultaneous blockade of OX40L penicillin, and streptomycin. Cells were stained with PE-labeled, tetramer- ized MHC molecules bearing Ags of interest. Drosophila S2 cells trans- and CD70 reduced responses below that of single blockade, infected with 2W1S-IAb monomers were a gift of Marc Jenkins, University dicating that either TNFSF member can play a role in CD4+ T cell of Minnesota (31). Secreted 2W1S-IAb was harvested from supernatants, priming. Using a bone marrow chimera approach, we observed as described (32), but it did not require additional biotinylation because of b that complete priming of CD4+ T cells required expression of cotransfection of the BirA enzyme. K reagents were purified as described (33) and were loaded with OVA (SIINFEKL) or B8R (TSYKFESV) Ags

+ Downloaded from CD40 on innate cells only. We then observed that splenic CD8 prior to staining. Cells were coincubated with tetramer for 1 h at 37˚C prior dendritic cells (DCs) detectably coexpressed CD70 and OX40L in to staining with surface Abs. Surface Abs were purchased from BioLegend response to combined TLR/CD40 stimulus but not to single ago- (San Diego, CA) or eBioscience (San Diego, CA). For cytokine assays, cells nists alone. To validate our results, we found that the costimula- were incubated for 3.5 h at 37˚C with 6 mg/ml brefeldin A in complete tory requirements of CD4+ and CD8+ responses to combined TLR/ media, as above. Cells were restimulated with 10 mg/ml 2W1S and SIINFEKL or B8R peptides; following the incubation period, cells were CD40 immunization were the same as those for responses to ﬁxed, permeabilized, and stained for intracellular cytokines, as described + vaccinia virus challenge, such that CD4 T cell priming depended (34). DCs were isolated from spleens in EHAA media (Invitrogen http://www.jimmunol.org/ upon OX40L and CD70 but CD8+ priming depended only upon Carlsbad, CA) containing DNAse (Worthington, Lakewood, NJ) and CD70. Intriguingly, CD4+ T cell responses to vaccinia virus were Collagenase D (Roche Diagnostic Systems, Somerville, NJ), as described (33). All cells were stained in FACS buffer containing 10% supernatant from also dependent on CD40–CD40L interactions, suggesting some 2.4G2 cell culture (B cell hybridoma-blocking FcgRs). physiologic overlap between our combined agonist system and vaccinia infection. Thus, CD4+ T cell priming is characterized Mixed bone marrow chimeras by a degree of TNF costimulation plasticity, which may facilitate Recipient mice were lethally irradiated with 900 rad and were grafted via the their role as helpers for multiple arms of the immune response. tail vein with 4 3 106 total T cell-depleted donor bone marrow cells in 200 ml PBS. Most mixed bone marrow chimeras were grafted at a ratio of

1:1; however, Rag1-deficient bone marrow was enriched relative to com- by guest on September 25, 2021 Materials and Methods petitor bone marrow 3:1 to enhance engraftment. Chimeric mice were Mice and infections given a diet containing Septra (trimethoprim/sulfamethoxazole; Harlan Teklad, Madison, WI) for 6 wk following irradiation and rested a minimum Six- to eight-week-old female C57BL/6 mice were obtained through the Na- of 12 wk before being used in experiments. tional Cancer Institute or Harlan Breeders (Indianapolis, IN). Mice deficient in CD40 (B6.129P2-Cd40tm1Kik/J) or IL-12Rb2 (B6.129S1-IL12rb2tm1Jm/J) In vitro culture and CD11c-DTR mice (B6.FVB-Tg[Itgax-DTR/EGFP]57Lan/J) were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice deficient Spleen cells were harvested, as described above, and cultured unfractio- 3 6 for the Rag1 gene (Rag knockout [KO], B6.129S7-Rag1tm1Mom/J) were nated at 1 10 cells/ml in 24-well flat-bottom plates. Cells were stim- ε a gift of Dr. Phillippa Marrack, National Jewish Health. Vaccinia virus ulated with 0.5 mg/ml anti-CD3 (clone 2C11; BioLegend) in complete expressing 2W1S was developed by Thomas Mitchell, as described (30). RPMI 1640 medium supplemented as above and with 10% FCS. At Virus was titered and propagated using BSC-1 cells. Postinfectious BSC-1 indicated time points, cells were harvested by washing and were stained cells and supernatant (stock) were stored at 280˚C and were thawed and as above. sonicated immediately prior to injection. Mice were injected i.p. with 1– Experimental and statistical analysis 2 3 107 PFU virus in 200 ml stock diluted in PBS. Mice were housed at the Biological Resource Center at National Jewish Health. The Institutional Spleen cells were quantified on a Vi-Cell cell viability analyzer (Beckman Animal Care and Use Committee at National Jewish Health approved all Coulter, Fullerton, CA). Cytometry samples were acquired on a CyAn ADP animal procedures. (DakoCytomation, Ft. Collins, CO) using Summit acquisition software. Samples were analyzed using FloJo software (Tree Star, Ashland, OR). Immunizations, depletions, and Ab blockade In most cases, results from FloJo analysis were imported into Prism Unless indicated otherwise, mice were immunized i.p. with 500 mg whole (GraphPad, San Diego, CA), and pairwise statistical analyses were made chicken OVA protein (Sigma-Aldrich, St. Louis, MO), 100 mg2W1S between samples using the Student t test. In vivo experiments were com- peptide (EAWGALANWAVDSA, custom synthesized by Pi Proteomics, pleted independently at least twice, with a minimum of three individuals Huntsville, AL), 50 mg CD40 agonist Ab (clone FGK4.5; Bio X Cell, West per group. In vitro experiments were completed independently at least Lebanon, NH), and 50 mg polyinosinic:polycytidylic acid [poly(I:C), three times. Amersham/GE Healthcare, Piscataway, NJ]. All vaccinations were prepared by mixing each component in PBS and were injected in 200 ml. Poly(I:C) was stored in frozen aliquots in PBS at 220˚C and was recon- Results stituted prior to injection by melting at 56˚C for 10 min and then allowing CD4+ T cells, like CD8+ T cells, respond synergistically to the solution to cool to room temperature to limit concatamerization. As combined TLR/CD40 stimulus indicated, 50 mg Pam-3-Cys-K4 (Pam-3-Cys; InvivoGen, San Diego, CA) was occasionally substituted for poly(I:C). All reagents were found to Our previous results demonstrated that immunizing mice with Ag contain minimal LPS content by Limulus amoebocyte assay (Lonza, Wal- combined with TLR agonists (TLR) and an agonistic Ab to murine kersville, MD). CD11c-DTR mice were depleted of CD11c+ cells by s.c. CD40 (clone FGK4.5) evoked a massive expansion of Ag-specific injection of 100 ng diphtheria toxin (Sigma-Aldrich) 2 d prior to immu- + nization. Blocking Abs for CD70 (FR70), OX40L (RM134L), 41BBL CD8 T cells (17, 28, 29). In this setting, Ag specificity was as- (TKS-1), and CD30L (RM153) were obtained from Bio X Cell. TNF sessed using MHC class I tetramers and revealed that combined 2108 CD4+ T CELLS USE MULTIPLE TNF PATHWAYS

TLR and CD40 stimulus (TLR/CD40) promoted expansion of stimulation elicited a large population of CD4+ T cells producing splenic CD8+ T cells 5–10-fold more than immunization with IFN-g in response to 2W1S restimulation by intracellular cytokine TLR or CD40 agonist alone (17, 28, 29). This effect was apparent staining (Fig. 1D), which was proportional to the size of the for all TLR agonists tested, as well as for several other stimulators tetramer-stained pool. Thus, TLR/CD40 immunization elicits a pre- of innate immunity (29), indicating that it was a generalizable trait sumptive Th1-type of primary CD4+ T cell response. Many of of pattern-recognition receptors. We became curious as to whether these cells were polyfunctional for the cytokines IL-2 and TNF-a the synergism between TLR agonists and CD40 agonists was re- (Supplemental Fig. 1), which was shown to correlate with Th cell stricted to CD8+ T cells or whether it could be extended to other function in certain infections (3, 5, 8, 9, 36). Furthermore, CD4+ cell types of the adaptive immune system. Thus, we chose to T cells elicited by a single injection of combined TLR/CD40 assess CD4+ T cell immune responses in the setting of TLR/ stimulus were long-lived and could mount anamnestic responses CD40 immunization. to vaccinia virus challenge, regardless of whether they were primed We injected C57BL/6 mice with the MHC class II (MHC-II) IAb- with Pam-3-Cys/CD40 or poly(I:C)/CD40 (Fig. 1E). Recall binding peptide 2W1S (EAWGALANWAVDSA) and whole chicken responses were similarly characterized by IFN-g production (data OVAprotein (35), alone or in combination with TLR agonist [poly(I: not shown). Thus, combined TLR/CD40 stimulus is a potent inducer C) for TLR3/mda5 or Pam-3-Cys for TLR1/2] and/or anti-CD40 Ab. of Th1-type, CD4+ T cell differentiation. We became interested in After 7 d, which was found to correspond to the peak of the primary understanding how TLR/CD40 might exert such potent CD4+ T cell immune response (data not shown), mice were sacrificed, and spleen priming, with the hope that we might better understand the systems and peripheral blood were harvested. FACS analysis of MHC class I governing CD4+ T cell immunity. Downloaded from and class II tetramer staining on splenic and peripheral blood cells + revealed that CD4+ and CD8+ T cells developed a population of CD40 expression by CD4 T cells is not necessary for their CD44high Ag-specific responders with all adjuvants (Fig. 1A,1B). response to combined TLR/CD40 immunization However, in the case of TLR/CD40 immunization, elicited popula- CD4+ T cells are reported to express CD40 (37), which is a mem- tions of CD4+ T cells far outnumbered those elicited by either adju- ber of the TNFSF. Given our use of an exogenous CD40 stimulus, vant alone (Fig. 1C), and the numbers were ∼10-fold greater in the we wanted to rule out a role for direct stimulation of CD4+ T cells + spleen than any other adjuvant. Thus, TLR/CD40 stimulation syner- through CD40 in influencing the response of CD4 T cells. In- http://www.jimmunol.org/ gizes to induce the potent expansion of Ag-specific CD4+ T cells in deed, a recent publication suggested that the use of poly(I:C) can the same fashion as for CD8+ T cells, in that the T cell expansion by induce CD40L expression on DCs, leading to direct stimulation of combined agonists is exponentially greater than either agonist alone. CD8+ T cells, presumably through their expression of CD40 (38). To determine whether TLR/CD40 immunization produced func- To address this issue, we generated bone marrow chimeras by tional CD4+ T cells, we immunized mice as above and restimulated reconstituting lethally irradiated CD40-deficient mice with con- splenocytes 7 d later with 2W1S peptide in the presence of brefeldin genically marked CD45.1 wild-type (WT) B6 and CD45.2 A to test for intracellular cytokines. Combined TLR/CD40 CD40-deficient (CD40KO) bone marrow mixed at a 1:1 ratio by guest on September 25, 2021

FIGURE 1. Combined TLR and CD40 agonists promote synergistic immune responses for CD4+ T cells. Mice were immunized i.p. with OVA protein and 2W1S peptide with 50 mg CD40 agonist alone (FGK4.5), 50 mg TLR agonist alone, or both agonists combined; spleens were harvested 7 d later. Shown are representative FACS plots of tetramer stains for single and combined adjuvants with poly(I:C) (A) or Pam-3-Cys (B) as the TLR agonist. OVA-Kb- tetramer–specific CD8+ T cell responses are shown in the top row of each figure, and 2W1S-IAb-tetramer–specific CD4+ T cell responses are shown in the bottom row. 2W1S-specific CD4+ T cells per spleen are quantified for each treatment (C), as are the number of CD4+ T cells staining positive for intracellular IFN-g (D). Statistics represent pairwise comparisons between all samples and CD40 agonist alone (CD4). ppp , 0.01; pppp , 0.005; Student t test. E, CD4+ T cells primed by combined TLR and CD40 promote robust secondary responses. Shown are peripheral blood 2W1S tetramer-specific CD4+ T cells over time. Mice were immunized on day 0 and challenged with recombinant 2W1S-expressing vaccinia on day 50. Statistics represent comparisons with naive mice. ppp , 0.01 for both groups; Student t test. Error bars represent the SEM. Data are representative of at least two independent experiments containing three or more mice per group. The Journal of Immunology 2109

(Fig. 2A). Our prediction was that if direct CD40 stimulation of TLR/CD40 immunization, then we should be able to see a defect CD4+ T cells by the CD40 agonist was required to produce the in the response of CD40KO CD4+ T cells compared with those exponential T cell expansion we observed following combined that are WT for CD40. Reconstituted mice were immunized with 2W1S, OVA, CD40 agonist, and poly(I:C) or Pam-3-Cys. The CD8+ and CD4+ T cell responses were determined by tetramer stain, and the relative responses of the Ag-speciﬁc T cells derived from WT and CD40KO backgrounds were compared using the CD45 congenic marker. We observed that the responding T cells had equal representation from CD40KO and B6 compartments (Fig. 2B). This was true for the Ag-speciﬁc CD8+ T cells as well (data not shown). These data indicate that CD40 is not required on responding T cells for their expansion in response to combined TLR/CD40 immunization, demonstrating that the CD40 agonist does not have a direct costimulatory effect on CD4+ T cells.

CD4+ and CD8+ T cell responses to combined TLR/CD40 depend on DCs

We next hypothesized that combined TLR and CD40 stimulus Downloaded from could support CD4+ T cell priming through indirect effects on APCs. It is generally well established that primary CD4+ and CD8+ T cell responses are dependent upon Ag presentation by DCs, and several studies showed that priming of CD4+ and CD8+ T cells might depend on different DC subsets (39, 40). However, naive B cells also + + express significant levels of CD40 (41), and CD4 (42) and CD8 http://www.jimmunol.org/ (43) T cells can enter B cell follicles. Because our immunization used an agonistic CD40 Ab and a preprocessed CD4+ T cell epitope, this presented the very real possibility that we might be endowing B cells with the capacity to present Ag to naive CD4+ and/or CD8+ T cells. Because CD40 was not necessary on CD4+ T cells for TLR/ CD40 responses to priming, we reasoned that CD40 expression would be necessary on the required APCs. Therefore, we generated mixed bone marrow chimeras using WT B6.SJL bone marrow or RAG-deficient (RagKO) bone marrow mixed with CD40KO bone by guest on September 25, 2021 marrow. In some cases, CD40KO bone marrow alone was used as a control (Fig. 2A). We chose CD40KO mice as recipients because we could be assured that no remnant T cells could express CD40. In addition, the use of CD40KO hosts as bone marrow recipients would determine whether hematopoietic expression of CD40 was required for eliciting the magnitude of the T cell responses observed in response to combined TLR/CD40 immunization. Using this system, we were able to isolate CD40 to expression only on innate immune cells derived from RagKO bone marrow. We found that, consistent with a role for CD40 in combined TLR/CD40 immunization, FIGURE 2. The synergistic effect of combined poly(I:C)/CD40 is in- CD40KO chimeric controls mounted very poor responses to immu- dependent of T cell CD40 but is dependent on innate CD40 expression. A, nization in the CD8+ and CD4+ T cell subsets (Fig. 2C,2D). How- Bone marrow was harvested from CD40-deficient (CD40KO), congenically + ever, B6.SJL and RagKO mixed chimeric mice mounted effective marked CD45.1 CD40-sufficient (B6.SJL), or Rag-deficient but CD40- responses to the combined stimulus. Thus, CD40 is sufficient on sufficient (RAGKO) mice and transplanted into CD40-deficient reci- innate, bone marrow-derived cells for effective CD4+ and CD8+ pients. B, CD4+ T cells do not require surface CD40 to respond to combined TLR/CD40 stimulus. Bone marrow from CD40-deficient (CD40KO, T cell immunization in this system (Fig. 2C,2D). CD45.2) and -sufficient (B6.SJL, CD45.1) mice was mixed 1:1 and injected This result seems to implicate DCs or myeloid cells as the prin- into lethally irradiated CD40KO CD45.2 recipients. Cells were gated on cipal APCs in the combined TLR/CD40 model system. To clarify CD45.1 expression and 2W1S-specific responses were measured in each the identity of the required APCs, we used the CD11c-DTR trans- group. For preimmune samples (d), the ratio of WT (CD45.1+) to CD40KO genic mouse, in which the DCs express the diphtheria toxin recep- 2 (CD45.1 ) CD4+ T cells is shown. For immunized samples (s), the ratio of tor and are selectively depleted following injection of diphtheria + 2 b WT (CD45.1 ) to CD40KO (CD45.1 ) 2W1S-IA tetramer staining is toxin. We found that a low (100 ng) s.c. dose of diphtheria toxin, shown. C and D, CD40KO recipient mice were lethally irradiated and given 2 d prior to immunization, effectively depleted DCs (data not grafted with CD40KO bone marrow, RagKO bone marrow mixed 3:1 with shown). CD11c-DTR mice or littermate controls were selectively CD40KO bone marrow, B6.SJL bone marrow mixed 1:1 with CD40KO bone depleted of DCs and immunized with Ag and combined TLR/ marrow, or RagKO bone marrow mixed 3:1 with B6.SJL bone marrow. All + + chimeras were grafted with 4 3 106 total T cell-depleted bone marrow cells. CD40, as described previously. We found that CD4 and CD8 Mice were rested .12 wk prior to immunization with poly(I:C)/CD40, and responses were attenuated in DC-depleted mice (Supplemental Ag-specific responses by CD8+ T cells (C) or CD4+ T cells (D) were quan- Fig. 2). Because DCs are innate bone marrow-derived cells, we tified. Statistics were calculated by the Student t test and are relative to concluded that classical DCs are likely to be the primary APC for CD40KO-grafted controls. pp , 0.05; ppp , 0.01; pppp , 0.005. T cells following combined TLR/CD40 immunization. 2110 CD4+ T CELLS USE MULTIPLE TNF PATHWAYS

CD4+ T cells are sensitive to blockade by OX40L or CD70 combined poly(I:C)/CD40 were dependent on IL-12 or CD70, during primary immune responses depending on which DC subset was presenting Ag to the responding T cells. Th1 priming by DEC205+ DCs required CD70 cos- Our previous work indicated that another member of the TNFSF, + CD70, mediated CD8+ T cell responses to combined TLR/CD40 timulation, whereas T cell priming by 33D1 DCs required IL-12 stimulus (28). To determine whether CD70 or other TNFSF (21). Therefore, we examined more directly the difference be- members play a role in CD4+ T cell responses to TLR/CD40, we tween these published results and our demonstration of the impor- administered blocking Abs to the TNF family ligands CD70, tance of OX40L. We reasoned that the published results predict that the elimination of IL-12– and CD70-mediated signaling OX40L, 4-1BBL, and CD30L and assessed the T cell response + + should eliminate the CD4 T cell response to combined poly 7 d later. Consistent with previous data (28), we found that CD8 + + T cells were sensitive to CD70 blockade in response to combined (I:C)/CD40 immunization, because 33D1 DCs and DEC205 poly(I:C)/CD40 (Fig. 3A) or Pam-3-Cys/CD40 (Fig. 3B) immuni- DCs constitute the majority of classical DC subsets. zation. Notably, this pattern was consistent whether whole OVA We immunized WT or IL-12b2R-deficient (IL12b2RKO) mice protein or SIINFEKL peptide was used as an Ag (data not shown). with Ag and poly(I:C)/CD40, with or without CD70 or OX40L However, in contrast to CD8+ T cells, we found that CD4+ T cells blockade. IL12b2RKO mice lack the capacity to signal through IL-12 but retain normal IL-23 signaling. We found that in the ab- were sensitive to CD70 or OX40L blockade for poly(I:C)/CD40 + (Fig. 3C), whereas they were only detectably sensitive to OX40L sence of IL12 signals, CD4 T cell-proliferative (Fig. 4A) and IFN-g responses (Fig. 4B) were modestly, but not significantly, reduced. blockade in the case of Pam-3-Cys/CD40 (Fig. 3D). The reduction + + in CD4+ T cell responses following CD70 blockade of poly(I:C)/ The number of IFNg CD4 T cells was proportional to the number of 2W1S-IAb tetramer+ cells in the absence of IL12b2R, suggesting Downloaded from CD40-primed mice was consistently more modest than that pro- + voked by OX40L blockade. Elicitation of IFN-g–producing CD4+ that loss of IL-12 signaling did not result in a major change in CD4 T cells was similarly impaired by TNFSF blockade (Supplemental T cell differentiation. These data were consistent with other experi- Fig. 3). The loss of Ag-specific CD4+ T cells in TNF ligand- ments using IL-12b1R-deficient mice, as well as mice administered blocked mice was not due to the depletion of APCs, as we (Sup- the IL12p40-blocking Ab C17.8 (data not shown). Furthermore, we found that CD70 blockade did not combine with IL12b2R defi- plemental Fig. 4) and other investigators have demonstrated (28, + + ciency to eliminate CD4 T cell responses. Although CD70 block- http://www.jimmunol.org/ 44). We conclude from these studies that although CD4 and + + ade had an impact on CD4 T cell responses in WT mice, CD70 CD8 T cell responses have overlapping and divergent costimu- + latory requirements, the optimal response of either T cell subset is blockade had little to no impact on CD4 T cell responses in dependent on members of the TNF ligand superfamily. Further- IL12b2RKO mice (Fig. 4A,4B). In contrast to CD70 blockade, more, we find in our system that CD4+ T cell responses are dependent upon CD70 and OX40L but less so on CD30L or 41BBL. CD4+ T cell priming by poly(I:C)/CD40 does not require IL-12 but may be promoted by CD70 or OX40L Our findings were somewhat surprising given the results of a recent by guest on September 25, 2021 publication (21), which suggested that CD4+ T cell responses to

FIGURE 4. Compared with the role of OX40L in CD4+ T cell responses to poly(I:C)/CD40, IL12 and CD70 are dispensable. A and B, IL-12 deficiency has a modest effect on poly(I:C)/CD40 immunization, but OX40L blockade reduces the response significantly. WT B6 or IL12b2R-deficient FIGURE 3. T cell proliferation in response to combined TLR/CD40 (KO) mice were administered blocking Abs for different TNF family mem- stimulus is dependent on TNFSF members. Mice were administered bers i.p. on days 21 and 0 relative to immunization. Spleens were harvested blocking Abs for several different TNF family members i.p. on days 21 7 d after immunization, and splenic 2W1S-specific CD4+ T cell tetramer (A) and 0 relative to immunization. Spleens were harvested 7 d after immu- and cytokine (B) responses to poly(I:C)/CD40 were quantified in the pres- nization, and CD8+ T cell responses to poly(I:C)/CD40 (A) or Pam-3-Cys/ ence of the blocking Abs. C and D, Combined blockade of CD70 and CD40 (B) were quantified in the presence of the blocking Abs. Likewise, OX40L reduces CD4+ T cell responses more than single blockade. B6 mice the number of Ag-specific CD4+ T cells for poly(I:C)/CD40 (C) or Pam-3- were immunized as in A and B. Mice were harvested 7 d after immunization, Cys/CD40 (D) were quantified in the same mice. All statistics represent and 2W1S-specific CD4+ T cell tetramer (C) and cytokine (D) responses in comparisons to unblocked controls (No Rx) and were calculated using the the spleen were quantified. Error bars represent SEM. Data are representa- Student t test. pp , 0.05; ppp , 0.01; pppp , 0.005. Data are represen- tive of at least two independent experiments with at least three mice per tative of at least two independent experiments containing three or more group. All statistics represent comparisons with unblocked controls (No mice per group. Error bars represent the SEM. Rx), unless indicated otherwise. pp , 0.05; ppp , 0.01; pppp , 0.005; Student t test. The Journal of Immunology 2111 we found that OX40L blockade consistently and reproducibly re- Only combined TLR/CD40 promotes CD70 and OX40L duced CD4+ T cell proliferation (Fig. 4A) and IFN-g production expression on CD8+ DCs (Fig. 4B) in WT and IL12b2RKO mice. Our failure to find + Our previous data indicated that combined TLR/CD40 immuni- a CD70-dependent response in IL12b2RKO mice by CD4 T cells zation uniquely elicited a significant increase in CD70 expression was not due to insufficient blockade by the Ab, because CD70 + on lymphoid-resident DC subsets in vivo (28). Although we had blockade reduced CD8 T cell responses in WT and IL12b2RKO also previously observed an increase in DC OX40L expression, mice (Supplemental Fig. 5). These data are consistent with the in- our observation of the dependency of the CD4+ T cell response on terpretation that CD70 and IL-12, rather than being separate and + OX40L compelled us to take a closer look at the expression of this independent means of eliciting CD4 T cells responses, are actually TNF ligand family member. Therefore, we harvested spleens at separate components of the same pathway: a conclusion that is different time points following immunization with combinations supported by other investigators (22). Our data suggest that CD70 + of TLR and CD40 agonists and analyzed the surface expression of and OX40L may act in semiredundant pathways for CD4 T cell different costimulatory molecules on DCs by FACS. priming, because CD70 blockade was insubstantial in the IL-12– + + Detectable CD70 expression was found primarily on CD8 DCs deficient setting, but OX40L blockade inhibited CD4 T cell (Fig. 5A,5B) and only under conditions of combined poly(I:C)/ responses in IL-12–sufficient and -deficient animals. CD40 immunization (Fig. 5B). This confirmed that high CD70 To determine whether this was indeed the case, we blocked expression was unique to combined TLR/CD40 agonist adminis- mice with anti-CD70 and anti-OX40L. Our results did not show a tration and that CD8+ DC activation positively correlated with statistically significant additive effect to dual blockade of CD70 synergistic T cell activation that we find with combined agonists.

+ Downloaded from and OX40L for CD4 T cell expansion (Fig. 4C). However, com- The kinetics of OX40L expression were similar to those observed bined CD70 and OX40L blockade produced a statistically signif- for CD70 expression (Fig. 5A,5B). CD8+ DCs expressed the most icant effect on CD4+ T cell IFN-g production (Fig. 4D). We + OX40L (Fig. 5A,5B), and expression was most signiﬁcant when conclude from these data that although the expansion of CD4 combined poly(I:C)/CD40 was used for immunization (Fig. 5B). T cells driven by TLR/CD40 is largely dependent upon OX40L Interestingly, we found that CD70 and OX40L were coexpressed interactions, CD70 can play a detectable role in the absence of on the same cells within the CD8+ DC subset (Fig. 5A). Thus,

OX40L. This suggests that CD70 and OX40L may operate along http://www.jimmunol.org/ + coordinated CD70 and OX40L expression on the same DCs cor- parallel, but semiredundant, pathways for CD4 T cell costim- relates with the effects of combined TLR/CD40 immunization. We ulation. Therefore, CD70 or OX40L may be used to promote + also found that these same DCs can express high levels of MHC-II CD4 T cell priming. with OX40L (data not shown). CD8+ DCs are normally associated by guest on September 25, 2021

FIGURE 5. CD8+ DCs uniquely upregulate OX40L and CD70 in response to combined poly(I:C)/CD40 but not to either agonist individually. CD4+ T cells upregulate OX40 late after stimulation in vitro or in vivo. A, Representative FACS plots for the kinetics of TNF ligand expression by CD11c+ CD192 CD32 cells that were either CD11b+ or CD8+. DCs were isolated from mice at various time points following combined poly(I:C)/CD40 immunization. Shown is the gating strategy for separating CD8+ DCs from CD11b+ DCs. B, Expression of CD70 (top row) or OX40L (bottom row) by CD11b (left panels) or CD8+ (right panels) DCs in response to various agonist combinations. Mice were immunized i.p. at the outset, and spleens were harvested at the indicated time points. C– E, CD4+ T cells express OX40 in immunized, but not naive, mice, whereas CD4+ T cells from naive mice express CD27 and only upregulate it modestly upon activation. C, Representative FACS plots showing that activated CD8+ and CD4+ T cells upregulate CD27 upon activation in vitro with 0.5 mg/ml of anti-CD3 (2C11) after 24 h (top row). Naive ex vivo profiles are in gray, and activated profiles are in black. Shown are numerical values for MFI for naive/activated cells. Although CD4+ T cells massively upregulate OX40 in response to CD3 stimulus, CD8+ T cells only modestly change expression (bottom row). Mice were immunized i.p. at t = 0 with poly(I:C)/CD40, harvested at the indicated time points, and cells were stained for CD44 and CD27 (D) or OX40 (E). Data are gated on CD4+ or CD8+ and CD44high cells. Error bars represent SEM. Data are representative of at least two independent experiments with at least three mice per group, except C, which is representative of four independent experiments. MFI, mean fluorescence intensity. 2112 CD4+ T CELLS USE MULTIPLE TNF PATHWAYS with cross-presentation (45) and CD8+ T cell activation (46). Our finding that a population within this DC subset coexpressed OX40L and MHC-II suggests that they may be the dominant APC for promoting Th cell responses to combined TLR/CD40 immunization. Thus, although we could not rule out a role for activation by other APCs, our data suggest that similar CD8+ DCs could mediate the effects of combined TLR/CD40 immunization for CD4+ and CD8+ T cells.

OX40 and CD27 are differentially regulated on CD4+ and CD8+ T cells in vitro and in vivo Because CD4+ and CD8+ T cells may use CD8+ DCs for priming, and CD8+ DCs express CD70 and OX40L, it was curious that CD8+ T cells were exclusively CD70 dependent (Fig. 3A,3B). To explain how CD8+ T cells could be insensitive to OX40L signals, we stimulated unfractionated spleen cells isolated from naive mice for 24 h in culture with anti-CD3 and stained for CD27 and OX40, the receptors for CD70 and OX40L, respectively. We found that Downloaded from unstimulated CD4+ and CD8+ T cells expressed appreciable CD27 (Fig. 5C, top panels, gray line), and that following 24 h of culture, CD4+ and CD8+ T cells upregulated CD27 (Fig. 5C, top panels, black line). In contrast, unstimulated CD4+ and CD8+ T cells expressed low levels of surface OX40 (Fig. 5C, bottom panels, gray line), and only CD4+ T cells appreciably upregulated OX40

+ + http://www.jimmunol.org/ following 24 h of culture (Fig. 5C, bottom panels, black line). FIGURE 6. Responses to vaccinia virus infection by CD4 and CD8 These data suggested that the insensitivity of the CD8+ T cell T cells depend on TNF ligands similar to combined TLR/CD40 immunization. CD8+ T cell responses to vaccinia depend on CD70 (A), whereas response to OX40L-expressing DCs during priming may be due CD4+ T cell responses to vaccinia (B) depend primarily on OX40L, with to their lack of OX40 expression. a secondary dependence on CD70. Mice were infected with 2 3 107 PFU To validate this result, we immunized mice with OVA, poly(I:C), of recombinant vaccinia i.p. 8 d prior to harvest and administered 250 mg and CD40 agonist and harvested splenocytes at different time of blocking Abs against the indicated TNFSF members i.p. on days 21, 0, + + + points following activation. By gating on CD44 CD4 and CD8 and +2 relative to immunization. CD8+ (C) and CD4+ (D) T cell responses T cells, we examined OX40 and CD27 expression in vivo. Similar to vaccinia virus, like TLR/CD40 stimulus, depend on CD40–CD40L sig- + + nals. Mice were infected with 2 3 107 PFU of recombinant vaccinia i.p. to our results in vitro, we found that CD4 and CD8 T cells by guest on September 25, 2021 expressed appreciable CD27 at the time of immunization (Fig. 8 d prior to harvest and were administered the CD40L-blocking Ab MR-1 2 5D). In contrast, only CD4+ T cells expressed appreciable OX40, (anti-CD154), 250 mg i.p., on days 1, 0, and +2 relative to immunization. and this was not fully expressed on activated (CD44high) cells until Shown are combined data for total splenic Ag-specific T cell numbers normalized to the control (No Rx) response for three independent experi- 24 h of stimulation (Fig. 5E). Thus, CD4+ and CD8+ T cells have ments containing at least three mice per group in each experiment. Mean different requirements for TNFSF costimulation, in part because control responses are 2.37e6 cells/spleen for A and C and 1.03e5 cells/ of their different expression patterns for TNFRs. spleen for B and D. Statistics are p values for the indicated group compared with controls, except where indicated. pp , 0.05; ppp , 0.01; pppp , CD4+ immunity to vaccinia demonstrates a dependence on 0.005. All error bars represent SEM. CD40, as well as an overlapping dependence on CD70 and OX40L that, for vaccinia, as for combined TLR/CD40 stimulus, the effects We next wanted to assess whether the important role for CD70 of CD70 and OX40L on CD4+ T cell responses were additive. and OX40L in the CD4+ T cell response was unique to combined Consistency between vaccinia virus infection and poly(I:C)/ TLR/CD40 immunization or was common to primary immune CD40 immunization was not limited to TNFSF costimulation. We responses elicited by other means. Therefore, we enlisted a model found that CD4+ T cells responding to vaccinia virus demonstrated pathogen: recombinant vaccinia virus expressing 2W1S Ag (VV- a requirement for CD40–CD40L signals to promote optimal 2W1S). VV-2W1S also expresses the immunodominant vaccinia priming. Blockade of CD40L (CD154) with the MR1 Ab during virus Ag B8R, which is presented on MHC Kb (47). Mice were vaccinia infection reduced CD8+ and CD4+ T cell responses (Fig. challenged with the recombinant vaccinia virus and sacrificed 6C,6D). Furthermore, CD4+ T cell proliferation and IFN-g pro- 8 d later, which corresponded to the peak of CD4+ and CD8+ duction were also attenuated in CD40KO mice (data not shown), T cell responses in peripheral blood (data not shown). Represen- suggesting that CD4+ T cell responses to vaccinia are CD40– tative mice were concomitantly injected with 250 mg of blocking CD40L dependent. Abs to CD70, OX40L, 4-1BBL, or CD70 and OX40L combined In contrast to combined TLR/CD40 immunization and vaccinia on days 21, 0, and +2 relative to immunization. infection, we found that CD4+ T cell response to Listeria mono- We found that responses to VV-2W1S infection were compara- cytogenes was uniquely dependent on CD70 and we could find no ble to responses to combined TLR/CD40, such that CD8+ T cell role for OX40L (Supplemental Fig. 6A). We also found a greatly responses were dependent on CD70 (Fig. 6A). CD4+ T cell reduced requirement for CD40–CD40L signals for CD4+ T cell responses were dependent on OX40L and CD70, with a greater responses to Listeria (Supplemental Fig. 6C), despite a continued dependence on OX40L (Fig. 6B). Of note, combining CD70 and dependence of the CD8+ T cell response on CD40–CD40L signals OX40L Abs noticeably reduced CD4+, but not CD8+, T cell re- (Supplemental Fig. 6B). Thus, the use of CD27 or OX40 by CD4+ sponses below that of single blocking agents alone. This suggested T cells is highly context dependent, and the role for different The Journal of Immunology 2113

TNFRs in primary CD4+ T cell immunity may vary, depending on molecules reduces the magnitude of the immune response. Al- the mode of immunization. These data again underscore an intrinsic though we are cautious about overinterpreting our results, these plasticity of CD4+ T cells for different forms of costimulation and data suggest that CD8+ DCs could play a key role in combined reaffirm the generalizable importance of the TNF ligand super- TLR/CD40 vaccination. Furthermore, a minimal role for IL-12 family in mediating potent cellular immunity in response to diverse signals in our system argues against a major role for 33D1+/ stimulus. CD11b+ DC subsets (21). However, we did not exhaustively rule out a role for other APCs, and we remain open to the likelihood Discussion that different APCs may play a role in CD4+ T cell priming under We demonstrated a method for inducing large populations of Ag- other conditions. A recent publication found that migrating DCs specific, IFN-g–producing CD4+ T cells through the coordinate within the draining lymph node of HSV-infected skin could uni- activation of TLR and CD40 pathways. We also showed that formly activate CD4+ T cells, but only CD103+ DCs could cross- a single injection of this vaccine is sufficient to produce long-lived present to CD8+ T cells (40). These data suggest that although memory CD4+ T cells and that the effects of this vaccine on CD4+ CD8+ T cell stimulation may derive from a narrow repertoire of T cells are dependent on the TNF ligands CD70 and OX40L. CD4+ APCs, CD4+ T cell responses may be initiated by a broad variety T cells elicited by combined TLR/CD40 were polyfunctional for of cells. Thus, the reliance of CD4+ T cell responses on one or multiple Th1 cytokines, including IFN-g and TNF-a. Furthermore, another cytokine or TNF family member could be dependent, in we showed that combined TLR/CD40 vaccination uniquely part, on the dominant APC migrating into priming sites following promotes the expression of CD70 and OX40L on CD8+ DCs and infection or immunization, as well as upon the differentiation state that DCs are necessary for productive vaccination. Although not of that APC. Various APCs may or may not be capable of cross- Downloaded from shown, we found that CD8+ DCs can express both TNF ligands in presentation and may express select TNFSF member ligands, conjunction with MHC-II. Furthermore, we demonstrated that the which could focus CD4+ T cell costimulation on one of several role of OX40L or CD70 for CD4+ or CD8+ T cells correlates with TNF pathways. We believe that this may also help to explain the preferential receptor expression upon priming. differences between our results with vaccinia viral infection and We find that the synergistic effect of combining TLR agonists Listeria infection, for which we could not otherwise detect obvi- + with a CD40 agonist is independent of whether poly(I:C) or ous discrepancies in OX40 or CD27 expression on CD4 T cells or http://www.jimmunol.org/ Pam-3-Cys is used for CD4+ T cells, and regardless of whether OX40L or CD70 expression on APCs. poly(I:C) or Pam-3-Cys is used as a TLR agonist, TLR/CD40 pro- Indeed, regulation of TNFSF receptors also seems to be a dy- moted CD4+ memory T cell formation. In this case, we tested poly namic process, because we showed that OX40 is limited on (I:C), which activates TLR3, RigI, and MDA5 (48) but not MyD88, naive cells but increases in an Ag-nonspecific fashion on CD4+ and Pam-3-Cys, which activates MyD88 through TLR1/2 (49). T cells with combined TLR/CD40 stimulus. Our data underscore Although not shown, we also tested agonists for TLR7 and found the fact that CD4+ and CD8+ T cells operate under different rules them to be consistent with the results shown in this study. Our for costimulation. We are not the first to suggest that CD4+ T cells demonstration that CD4+ T cell responses to Pam-3-Cys– or poly are dependent on OX40-OX40L signals (23, 27, 51), whereas (I:C)-based immunizations have a major dependence on OX40L CD8+ T cells are preferentially dependent on CD27-CD70 (52– by guest on September 25, 2021 suggests that the role of OX40–OX40L interactions in CD4+ im- 55). However, we believe that we are among the first authors to munity may be generalizable to other TLR agonists in concert with simultaneously assess the effects of OX40L and CD70 blockade CD40. However, the role of OX40L in CD4+ T cell immunity is on CD4+ and CD8+ T cells in a common host. We confirm that clearly not exclusive, because CD70 can also play at least some role CD4+ T cells are sensitive to CD70 blockade, as several investi- in our system (21), as well as a dominant role in Listeria infection. gators have shown (20–22), and that the roles of OX40L and The discrepancy in TNFSF costimulation for CD4+ T cells be- CD70 in CD4+ T cell priming partially overlap. However, our data tween vaccinia infection and Listeria infection correlates with suggest that the dynamic regulation of OX40 may play an impor- a role for CD40–CD40L interactions in each response. Vaccinia- tant role in this process. Thus, we can envision a model in which provoked CD4+ T cell priming was highly CD40L dependent and TNF signaling pathways used by CD4+ T cells depend on activa- was also reduced in CD40KO mice, whereas Listeria-provoked tion and migration of diverse APCs and the timing and regulation CD4+ T cell responses were not significantly reduced by CD40L of TNFRs on naive CD4+ T cells. blockade. Therefore, a search for the cause of the discrepancy Previous data using BALB/c mice with combined poly(I:C) and must focus on the role of CD40. Given recent and established pub- CD40 agonists with a DC-directed immunization showed that lications suggesting the necessity of CD40 expression in T cells DEC205+ DCs required CD70 to directly stimulate CD4+ T cells for their effective activation (37, 50), it is of interest to note our in the absence of IL-12 (21). The investigators found that IL-12 demonstration that CD4+ and CD8+ T cell responses to combined signals were necessary for priming by 33D1+ DCs, whereas CD70 TLR/CD40 immunization do not require T cell expression of was required by DEC205 DCs (21). These investigators used an CD40. Rather, CD40 expression on innate cells was sufficient to in vitro stimulation approach using naive transgenic CD4+ T cells observe synergy between TLR and CD40 in mediating CD4+ and paired with 24-h activated DCs to demonstrate CD70 dependence, CD8+ T cell expansion. Our demonstration that partial reconstitu- but they did not explore a role for other TNF ligands (21). Given tion of a CD402/2 host with Rag-deficient bone marrow can res- these data, we initially predicted that the absence of CD70 signals cue the T cell response effectively eliminates a role for CD40 on in IL12b2RKO mice would eliminate CD4+ T cell activation, B cells. The direct depletion of CD11c+ cells in the diphtheria because CD8+ DEC205+ DCs and CD11b+ 33D1+ DCs make up toxin transgenic mouse and the corresponding reduced immune the bulk of classical DC subsets. However, we found that CD70 response confirms the key role of DCs in TLR/CD40 priming (21, blockade had a modest to no effect in IL-12–deficient mice, 28, 39). Thus, our data indicate that DC CD40 expression is crit- consistent with a model proposed by other investigators (22), in ical for observing the magnitude of CD4+ and CD8+ T cell ex- which IL-12 acts directly on responding CD4+ T cells in a CD27- pansion elicited by combined TLR/CD40 immunization. dependent manner. We found that OX40L blockade dominantly Combined TLR/CD40 elicits appreciable expression of CD70 reduced responses, even in IL12b2RKO mice. Thus, our data and OX40L predominantly on CD8+ DCs, and blockade of these build upon the work of previous investigators (22) by showing 2114 CD4+ T CELLS USE MULTIPLE TNF PATHWAYS

that OX40L plays an IL-12–independent role for CD4+ T cell on spread and control of cytomegalovirus infection in vivo. J. Gen. Virol. 77: priming in vivo in C57BL/6 mice. 217–225. + 5. Casazza, J. P., M. R. Betts, D. A. Price, M. L. Precopio, L. E. Ruff, J. M. Brenchley, We also found that naive CD4 T cells express very little OX40 B. J. Hill, M. Roederer, D. C. Douek, and R. A. Koup. 2006. Acquisition of direct de novo and upregulate it in vivo in response to combined TLR/ antiviral effector functions by CMV-specific CD4+ T lymphocytes with cellular maturation. J. Exp. Med. 203: 2865–2877. CD40 stimulus in an apparently Ag-independent fashion. This 6. Jonjic´, S., I. Pavic´, P. Lucin, D. Rukavina, and U. H. Koszinowski. 1990. Effi- result may also provide a means to reconcile our observation of cacious control of cytomegalovirus infection after long-term depletion of CD8+ a role for OX40L where previous investigators found a role only T lymphocytes. J. Virol. 64: 5457–5464. + 7. Khader, S. A., G. K. Bell, J. E. Pearl, J. J. Fountain, J. Rangel-Moreno, for CD70 (21) in mediating CD4 T cell expansion following G. E. Cilley, F. Shen, S. M. Eaton, S. L. Gaffen, S. L. Swain, et al. 2007. combined poly(I:C)/CD40 immunization. The data supporting an IL-23 and IL-17 in the establishment of protective pulmonary CD4+ T cell exclusive role for CD70 used DCs that had been stimulated by responses after vaccination and during Mycobacterium tuberculosis challenge. Nat. Immunol. 8: 369–377. poly(I:C)/CD40 in vivo for 24 h paired with T cells from unsti- 8. Forbes, E. K., C. Sander, E. O. Ronan, H. McShane, A. V. Hill, P. C. Beverley, + mulated, transgenic mice. We found that unstimulated CD4 and E. Z. Tchilian. 2008. Multifunctional, high-level cytokine-producing T cells express CD27 but have very little OX40. In contrast, Th1 cells in the lung, but not spleen, correlate with protection against Mycobac- + terium tuberculosis aerosol challenge in mice. J. Immunol. 181: 4955–4964. CD4 T cells, in an Ag-nonspecific fashion, upregulate OX40 as 9. Lindenstrøm, T., E. M. Agger, K. S. Korsholm, P. A. Darrah, C. Aagaard, early as 8 h after stimulus, but do not reach peak OX40 expression R. A. Seder, I. Rosenkrands, and P. Andersen. 2009. Tuberculosis subunit vac- until 24 h after immunization (Fig. 5E). Thus, naive CD4+ T cells cination provides long-term protective immunity characterized by multifunctional CD4 memory T cells. J. Immunol. 182: 8047–8055. paired with activated DCs will have the ability to sense CD70 but 10. Shedlock, D. J., and H. Shen. 2003. Requirement for CD4 T cell help in gen- not OX40L. The inability of naive cells to quickly upregulate erating functional CD8 T cell memory. Science 300: 337–339.

OX40 may have been a confounder of previous results (21). 11. Antony, P. A., C. A. Piccirillo, A. Akpinarli, S. E. Finkelstein, P. J. Speiss, Downloaded from D. R. Surman, D. C. Palmer, C. C. Chan, C. A. Klebanoff, W. W. Overwijk, et al. However, another intriguing possibility is that nonclassical DC 2005. CD8+ T cell immunity against a tumor/self-antigen is augmented by subsets could play a role in poly(I:C)/CD40 priming when free Ag CD4+ T helper cells and hindered by naturally occurring T regulatory cells. J. is used. The fact that our data for OX40L and CD70 are re- Immunol. 174: 2591–2601. 12. Kim, C. H., L. S. Rott, I. Clark-Lewis, D. J. Campbell, L. Wu, and E. C. Butcher. capitulated in vaccinia viral infection confirms the physiological 2001. Subspecialization of CXCR5+ T cells: B helper activity is focused in relevance of our findings, suggesting that the differences between a germinal center-localized subset of CXCR5+ T cells. J. Exp. Med. 193: 1373–1381. our results and those of other investigators are probably due to http://www.jimmunol.org/ 13. Fazilleau, N., L. Mark, L. J. McHeyzer-Williams, and M. G. McHeyzer- differences in priming conditions, mouse models, or interpretation. Williams. 2009. Follicular helper T cells: lineage and location. Immunity 30: We previously demonstrated the usefulness of the combined TLR/ 324–335. CD40 agonist approach for generating large numbers of Ag-specific 14. Palm, N. W., and R. Medzhitov. 2009. Pattern recognition receptors and control + of adaptive immunity. Immunol. Rev. 227: 221–233. polyfunctional CD8 T cells (17, 28), and we now extend these 15. Pantaleo, G., and R. A. Koup. 2004. Correlates of immune protection in findings to CD4+ T cells. The fact that substantial anamnestic HIV-1 infection: what we know, what we don’t know, what we should know. Nat. Med. 10: 806–810. responses may be generated by single injections of combined ago- 16. Baylor, N. W., W. Egan, and P. Richman. 2002. Aluminum salts in vaccines—US nists should have considerable clinical usefulness. Additionally, perspective. Vaccine 20(Suppl. 3): S18–S23. our work demonstrates the potential of DC engagement without 17. Ahonen, C. L., C. L. Doxsee, S. M. McGurran, T. R. Riter, W. F. Wade,

R. J. Barth, J. P. Vasilakos, R. J. Noelle, and R. M. Kedl. 2004. Combined TLR by guest on September 25, 2021 the requirement of coupling the Ag to targeting Abs. The combi- and CD40 triggering induces potent CD8+ T cell expansion with variable de- nation of two molecular agonists is a considerable improvement pendence on type I IFN. J. Exp. Med. 199: 775–784. over live- or attenuated pathogen-based vaccines, which can con- 18. Croft, M. 2009. The role of TNF superfamily members in T-cell function and diseases. Nat. Rev. Immunol. 9: 271–285. tribute to morbidity in immunocompromised individuals. Further- 19. Watts, T. H. 2005. TNF/TNFR family members in costimulation of T cell more, although there are inherent dose-limiting toxicities to in- responses. Annu. Rev. Immunol. 23: 23–68. jection of TLR agonists alone (56) or CD40 agonists alone (57), 20. Hendriks, J., L. A. Gravestein, K. Tesselaar, R. A. van Lier, T. N. Schumacher, and J. Borst. 2000. CD27 is required for generation and long-term maintenance the available data indicate that the synergistic effect of combining of T cell immunity. Nat. Immunol. 1: 433–440. both agonists creates a wider therapeutic window by reducing the 21. Soares, H., H. Waechter, N. Glaichenhaus, E. Mougneau, H. Yagita, O. Mizenina, toxicity of either agent (58) and by decreasing the effective dose of D. Dudziak, M. C. Nussenzweig, and R. M. Steinman. 2007. A subset of dendritic cells induces CD4+ T cells to produce IFN-gamma by an IL-12-independent either agonist. This is convenient, because we noted that only com- but CD70-dependent mechanism in vivo. J. Exp. Med. 204: 1095–1106. bined agonists promoted robust induction of the potent CD70 and 22. van Oosterwijk, M. F., H. Juwana, R. Arens, K. Tesselaar, M. H. van Oers, OX40L costimulatory molecules on DC subsets. We hope that the E. Eldering, and R. A. van Lier. 2007. CD27-CD70 interactions sensitise naive CD4+ T cells for IL-12-induced Th1 cell development. Int. Immunol. 19: 713– availability of a vigorous, Th1-skewing immunization regimen will 718. prompt further exploration of its therapeutic uses. 23. Kopf, M., C. Ruedl, N. Schmitz, A. Gallimore, K. Lefrang, B. Ecabert, B. Odermatt, and M. F. Bachmann. 1999. OX40-deficient mice are defective in Th cell proliferation but are competent in generating B cell and CTL Responses Disclosures after virus infection. Immunity 11: 699–708. R.M.K. is a founder of ImmuRx Inc., a vaccine company whose intellectual 24. Gramaglia, I., A. Jember, S. D. Pippig, A. D. Weinberg, N. Killeen, and M. Croft. 2000. The OX40 costimulatory receptor determines the development property is based on the combined TLR agonist/anti-CD40 immunization of CD4 memory by regulating primary clonal expansion. J. Immunol. 165: 3043– platform. R.M.K., P.J.S., and C.H. are listed as inventors on patent appli- 3050. cations filed by the University of Colorado and licensed by ImmuRx Inc. 25. Rogers, P. R., J. Song, I. Gramaglia, N. Killeen, and M. Croft. 2001. OX40 promotes Bcl-xL and Bcl-2 expression and is essential for long-term survival of CD4 T cells. Immunity 15: 445–455. 26. Dawicki, W., E. M. Bertram, A. H. Sharpe, and T. H. Watts. 2004. 4-1BB and References OX40 act independently to facilitate robust CD8 and CD4 recall responses. 1. Yi, J. S., M. Du, and A. J. Zajac. 2009. A vital role for interleukin-21 in the J. Immunol. 173: 5944–5951. control of a chronic viral infection. Science 324: 1572–1576. 27. Huddleston, C. A., A. D. Weinberg, and D. C. Parker. 2006. OX40 (CD134) 2. Snyder, C. M., A. Loewendorf, E. L. Bonnett, M. Croft, C. A. Benedict, and engagement drives differentiation of CD4+ T cells to effector cells. Eur. A. B. Hill. 2009. CD4+ T cell help has an epitope-dependent impact on CD8+ J. Immunol. 36: 1093–1103. T cell memory inflation during murine cytomegalovirus infection. J. Immunol. 28. Sanchez, P. J., J. A. McWilliams, C. Haluszczak, H. Yagita, and R. M. Kedl. 183: 3932–3941. 2007. Combined TLR/CD40 stimulation mediates potent cellular immunity by 3. Kannanganat, S., C. Ibegbu, L. Chennareddi, H. L. Robinson, and R. R. Amara. regulating dendritic cell expression of CD70 in vivo. J. Immunol. 178: 1564– 2007. Multiple-cytokine-producing antiviral CD4 T cells are functionally supe- 1572. rior to single-cytokine-producing cells. J. Virol. 81: 8468–8476. 29. McWilliams, J. A., P. J. Sanchez, C. Haluszczak, L. Gapin, and R. M. Kedl. 4. Polic´, B., S. Jonjic´, I. Pavic´, I. Crnkovic´, I. Zorica, H. Hengel, P. Lucin, and 2010. Multiple innate signaling pathways cooperate with CD40 to induce potent, U. H. Koszinowski. 1996. Lack of MHC class I complex expression has no effect CD70-dependent cellular immunity. Vaccine 28: 1468–1476. The Journal of Immunology 2115

30. Tripathi, P., T. C. Mitchell, F. Finkelman, and D. A. Hildeman. 2007. Cutting 44. Schildknecht, A., I. Miescher, H. Yagita, and M. van den Broek. 2007. Priming Edge: Limiting amounts of IL-7 do not control contraction of CD4+ T cell of CD8+ T cell responses by pathogens typically depends on CD70-mediated responses. J. Immunol. 178: 4027–4031. interactions with dendritic cells. Eur. J. Immunol. 37: 716–728. 31. Moon, J. J., H. H. Chu, M. Pepper, S. J. McSorley, S. C. Jameson, R. M. Kedl, 45. Hildner, K., B. T. Edelson, W. E. Purtha, M. Diamond, H. Matsushita, and M. K. Jenkins. 2007. Naive CD4(+) T cell frequency varies for different M. Kohyama, B. Calderon, B. U. Schraml, E. R. Unanue, M. S. Diamond, et al. epitopes and predicts repertoire diversity and response magnitude. Immunity 27: 2008. Batf3 deficiency reveals a critical role for CD8alpha+ dendritic cells in 203–213. cytotoxic T cell immunity. Science 322: 1097–1100. 32. Crawford, F., H. Kozono, J. White, P. Marrack, and J. Kappler. 1998. Detection 46. Allan, R. S., C. M. Smith, G. T. Belz, A. L. van Lint, L. M. Wakim, W. R. Heath, of antigen-specific T cells with multivalent soluble class II MHC covalent pep- and F. R. Carbone. 2003. Epidermal viral immunity induced by CD8alpha+ tide complexes. Immunity 8: 675–682. dendritic cells but not by Langerhans cells. Science 301: 1925–1928. 33. Kedl, R. M., W. A. Rees, D. A. Hildeman, B. Schaefer, T. Mitchell, J. Kappler, 47. Tscharke, D. C., G. Karupiah, J. Zhou, T. Palmore, K. R. Irvine, S. M. Haeryfar, and P. Marrack. 2000. T cells compete for access to antigen-bearing S. Williams, J. Sidney, A. Sette, J. R. Bennink, and J. W. Yewdell. 2005. antigen-presenting cells. J. Exp. Med. 192: 1105–1113. Identification of poxvirus CD8+ T cell determinants to enable rational design 34. Haluszczak, C., A. D. Akue, S. E. Hamilton, L. D. Johnson, L. Pujanauski, and characterization of smallpox vaccines. J. Exp. Med. 201: 95–104. L. Teodorovic, S. C. Jameson, and R. M. Kedl. 2009. The antigen-specific CD8+ 48. Longhi, M. P., C. Trumpfheller, J. Idoyaga, M. Caskey, I. Matos, C. Kluger, T cell repertoire in unimmunized mice includes memory phenotype cells bearing A. M. Salazar, M. Colonna, and R. M. Steinman. 2009. Dendritic cells require markers of homeostatic expansion. J. Exp. Med. 206: 435–448. a systemic type I interferon response to mature and induce CD4+ Th1 immunity 35. Shiow, L. R., D. B. Rosen, N. Brdickova´, Y. Xu, J. An, L. L. Lanier, J. G. Cyster, with poly IC as adjuvant. J. Exp. Med. 206: 1589–1602. and M. Matloubian. 2006. CD69 acts downstream of interferon-alpha/beta to 49. O’Neill, L. A., and A. G. Bowie. 2007. The family of five: TIR-do- inhibit S1P1 and lymphocyte egress from lymphoid organs. Nature 440: 540– main-containing adaptors in Toll-like receptor signalling. Nat. Rev. Immunol. 544. 7: 353–364. 36. Darrah, P. A., D. T. Patel, P. M. De Luca, R. W. Lindsay, D. F. Davey, B. J. Flynn, 50. Bourgeois, C., B. Rocha, and C. Tanchot. 2002. A role for CD40 expression on S. T. Hoff, P. Andersen, S. G. Reed, S. L. Morris, et al. 2007. Multifunctional CD8+ T cells in the generation of CD8+ T cell memory. Science 297: 2060– TH1 cells define a correlate of vaccine-mediated protection against Leishmania 2063. major. Nat. Med. 13: 843–850. 51. Brocker, T., A. Gulbranson-Judge, S. Flynn, M. Riedinger, C. Raykundalia, and 37. Baker, R. L., D. H. Wagner, Jr., and K. Haskins. 2008. CD40 on NOD CD4 P. Lane. 1999. CD4 T cell traffic control: in vivo evidence that ligation of OX40 Downloaded from T cells contributes to their activation and pathogenicity. J. Autoimmun. 31: 385– on CD4 T cells by OX40-ligand expressed on dendritic cells leads to the accu- 392. mulation of CD4 T cells in B follicles. Eur. J. Immunol. 29: 1610–1616. 38. Johnson, S., Y. Zhan, R. M. Sutherland, A. M. Mount, S. Bedoui, J. L. Brady, 52. Brown, G. R., K. Meek, Y. Nishioka, and D. L. Thiele. 1995. CD27-CD27 E. M. Carrington, L. E. Brown, G. T. Belz, W. R. Heath, and A. M. Lew. 2009. ligand/CD70 interactions enhance alloantigen-induced proliferation and cyto- Selected Toll-like receptor ligands and viruses promote helper-independent cy- lytic activity in CD8+ T lymphocytes. J. Immunol. 154: 3686–3695. totoxic T cell priming by upregulating CD40L on dendritic cells. Immunity 30: 53. Taraban, V. Y., T. F. Rowley, and A. Al-Shamkhani. 2004. Cutting edge: a critical 218–227. role for CD70 in CD8 T cell priming by CD40-licensed APCs. J. Immunol. 173:

39. Dudziak, D., A. O. Kamphorst, G. F. Heidkamp, V. R. Buchholz, C. Trumpfheller, 6542–6546. http://www.jimmunol.org/ S. Yamazaki, C. Cheong, K. Liu, H. W. Lee, C. G. Park, et al. 2007. Differential 54. Rowley, T. F., and A. Al-Shamkhani. 2004. Stimulation by soluble CD70 antigen processing by dendritic cell subsets in vivo. Science 315: 107–111. promotes strong primary and secondary CD8+ cytotoxic T cell responses 40. Bedoui, S., P. G. Whitney, J. Waithman, L. Eidsmo, L. Wakim, I. Caminschi, in vivo. J. Immunol. 172: 6039–6046. R. S. Allan, M. Wojtasiak, K. Shortman, F. R. Carbone, et al. 2009. Cross- 55. Bullock, T. N., and H. Yagita. 2005. Induction of CD70 on dendritic cells presentation of viral and self antigens by skin-derived CD103+ dendritic cells. through CD40 or TLR stimulation contributes to the development of CD8+ Nat. Immunol. 10: 488–495. T cell responses in the absence of CD4+ T cells. J. Immunol. 174: 710–717. 41. Morimoto, S., Y. Kanno, Y. Tanaka, Y. Tokano, H. Hashimoto, S. Jacquot, 56. Aguilar, J. C., and E. G. Rodrı´guez. 2007. Vaccine adjuvants revisited. Vaccine C. Morimoto, S. F. Schlossman, H. Yagita, K. Okumura, and T. Kobata. 2000. 25: 3752–3762. CD134L engagement enhances human B cell Ig production: CD154/CD40, 57. Vonderheide, R. H., K. T. Flaherty, M. Khalil, M. S. Stumacher, D. L. Bajor, CD70/CD27, and CD134/CD134L interactions coordinately regulate T cell- N. A. Hutnick, P. Sullivan, J. J. Mahany, M. Gallagher, A. Kramer, et al. 2007. dependent B cell responses. J. Immunol. 164: 4097–4104. Clinical activity and immune modulation in cancer patients treated with

42. Ansel, K. M., L. J. McHeyzer-Williams, V. N. Ngo, M. G. McHeyzer-Williams, CP-870,893, a novel CD40 agonist monoclonal antibody. J. Clin. Oncol. 25: by guest on September 25, 2021 and J. G. Cyster. 1999. In vivo-activated CD4 T cells upregulate CXC chemokine 876–883. receptor 5 and reprogram their response to lymphoid chemokines. J. Exp. Med. 58. Ahonen, C. L., A. Wasiuk, S. Fuse, M. J. Turk, M. S. Ernstoff, A. A. Suriawinata, 190: 1123–1134. J. D. Gorham, R. M. Kedl, E. J. Usherwood, and R. J. Noelle. 2008. Enhanced 43. Khanna, K. M., J. T. McNamara, and L. Lefranc¸ois. 2007. In situ imaging of the efﬁcacy and reduced toxicity of multifactorial adjuvants compared with unitary endogenous CD8 T cell response to infection. Science 318: 116–120. adjuvants as cancer vaccines. Blood 111: 3116–3125.