Blockade Acceptance Induced by CD40-CD40L OX40 Costimulation

OX40 Costimulation Prevents Allograft Acceptance Induced by CD40-CD40L Blockade

This information is current as Bryna E. Burrell, Guanyi Lu, Xian C. Li and D. Keith of September 25, 2021. Bishop J Immunol 2009; 182:379-390; ; doi: 10.4049/jimmunol.182.1.379 http://www.jimmunol.org/content/182/1/379 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 © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

OX40 Costimulation Prevents Allograft Acceptance Induced by CD40-CD40L Blockade1

Bryna E. Burrell,*† Guanyi Lu,* Xian C. Li,‡ and D. Keith Bishop2*†

Disrupting the CD40-CD40L costimulation pathway promotes allograft acceptance in many settings. Herein, we demonstrate that stimulating OX40 overrides cardiac allograft acceptance induced by disrupting CD40-CD40L interactions. This effect of OX40 stimulation was dependent on CD4؉ T cells, which in turn provided help for CD8؉ T cells and B cells. Allograft rejection was associated with donor-reactive Th1 and Th2 responses and an unconventional granulocytic inﬁltrate and thrombosis of the arteries. Interestingly, OX40 stimulation induced a donor-reactive IgG class switch in the absence of CD40-CD40L interactions, and the timing of OX40 stimulation relative to transplantation affected the isotype of donor-reactive Ab produced. Inductive OX40 stimulation induced acute graft rejection, which correlated with both IgG1 and IgG2a deposition within the graft. Once graft

acceptance was established following CD40-CD40L blockade, delayed OX40 stimulation did not induce acute allograft rejection Downloaded from despite priming of graft-reactive Th1 and Th2. Rather, chronic rejection was induced, which was characterized by IgG1 but not IgG2a deposition within the graft. These studies reveal both redundancy and key differences in function among costimulatory molecules that manifest in distinct pathologies of allograft rejection. These ﬁndings may help guide development of therapeutics aimed at promoting graft acceptance in transplant recipients. The Journal of Immunology, 2009, 182: 379–390.

Ϫ/Ϫ Ϫ/Ϫ

ostimulation is central to T cell activation, survival, and CD40 BALB/c donors, CD40 BALB/c recipients acutely http://www.jimmunol.org/ effector differentiation (reviewed in Refs. 1–3). Unlike reject CD40Ϫ/Ϫ C57BL/6 allografts (18). In C3H mice, blocking CD28, CD40L and OX40 are expressed almost exclu- both CD28 and CD40L interactions results in long-term skin al- C ϩ sively by activated CD4 T cells and to a lesser degree by acti- lograft survival (19, 20), whereas C57BL/6 mice deﬁcient in both vated CD8ϩ T cells (reviewed in Refs. 2, 3). These molecules CD28 and CD40L acutely reject skin grafts (20, 21). However, interact with CD40 and OX40 ligand (OX40L)3 on the APC, re- blocking OX40-OX40L interactions in CD28- and CD40L-deﬁ- spectively (reviewed in Ref. 1). Stimulation through OX40 induces cient C57BL/6 recipients promotes long-term skin graft survival expansion of T cells activated by TCR engagement (4, 5). Whereas (21), and both CD4ϩ and CD8ϩ T cell-mediated rejection is sen- CD40-CD40L interactions support a Th1 response by inducing sitive to this blockade (22).

APC to produce IL-12 (reviewed in Refs. 6, 7), OX40 signaling Similarly, differences in costimulatory pathway usage and cy- by guest on September 25, 2021 promotes Th2 responses (8, 9) or may paradoxically further drive tokine profiles have been observed in the model of Leishmania the development of a Th1 response (5, 10, 11). infection (8, 23). In response to infection, BALB/c mice mount a Disrupting CD40-CD40L interactions results in prolonged allo- dominant Th2 response resulting in the “non-healer” phenotype graft survival (reviewed in Refs. 2, 12) (13–17). However, there and succumb to progressive lesions (8, 23). This Th2 response is are differences in the requirement for CD40-CD40L interactions driven by OX40-OX40L interactions (8). Conversely, C57BL/6 among different strains of mice, and graft protection resulting from mice infected with Leishmania mount a dominant Th1 response, CD40-CD40L inhibition is in large part dictated by the model in resulting in the “healer” phenotype and clearance of the pathogen which it is studied. For example, whereas C57BL/6 mice that are (23). Indeed, when the BALB/c recipient response is skewed to- Ϫ Ϫ deficient in CD40 (CD40 / ) fail to reject cardiac allografts from ward a Th1 response by neutralizing IL-4 (24), the infection is cleared and the mice recover. Additionally, if the immune response of C57BL/6 mice is skewed away from a Th1 response by genetic † *Section of General Surgery and Graduate Program in Immunology, University of deficiency in IFN-␥ (25), T-bet (26), or CD40L (27), or by over Michigan School of Medicine, Ann Arbor, MI 48109; and ‡Department of Medicine, Harvard Medical School, Boston, MA 02215 expression of OX40L (28), the infection is not cleared and the Received for publication September 15, 2008. Accepted for publication November 2, mice respond similarly to their unmodified BALB/c counterparts. 2008. Interestingly, BALB/c mice infected with Leishmania are able to The costs of publication of this article were defrayed in part by the payment of page clear the disease (8) or significantly delay disease pathology (28) charges. This article must therefore be hereby marked advertisement in accordance following OX40-OX40L blockade. Thus, preferential usage of the with 18 U.S.C. Section 1734 solely to indicate this fact. OX40-OX40L costimulatory pathway may influence Th1/Th2 bal- 1 This work was supported by National Institutes of Health Grants R01 AI061469 (to D.K.B.), R01 HL070613 (to D.K.B.), and R01 AI070315 (to X.C.L.), and The Her- ance and impact the effectiveness of the immune response, but the man and Dorothy Miller Fund Award for Innovative Immunology Research (to exact relevance of this notion in transplant models is unknown. B.E.B.). The present study demonstrates that cardiac allograft acceptance 2 Address correspondence and reprint requests to Dr. D. Keith Bishop, Transplant resulting from disrupting CD40-CD40L interactions in C57BL/6 Immunology Research Laboratory, Section of General Surgery, A560 MSRB II, Box 0654, University of Michigan Medical Center, Ann Arbor, MI 48109. E-mail address: mice is prevented by OX40 stimulation. Acute rejection driven by [email protected] OX40 stimulation was associated with donor-reactive T cell prim- 3 Abbreviations used in this paper: OX40L, OX40 ligand; AP, alkaline phosphatase; ing and the generation of a donor-reactive IgG Ab response, with Ϫ Ϫ Ϫ Ϫ CD40 / , CD40 deficient; CD40L / , CD40L deficient; GIC, graft-infiltrating cell; IgG2a detectable within the graft. Once allograft acceptance was WT, wild type. established, OX40 stimulation failed to induce acute rejection but Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 promoted the progression of chronic rejection and deposition of www.jimmunol.org 380 OX40 COSTIMULATION OVERRIDES CD40-CD40L BLOCKADE

IgG1 within the graft. These data suggest that despite the impor- tance of CD40-CD40L engagement in the C57BL/6 recipient response to allografts, perturbation through an alternative costimulatory molecule pathway may supersede the necessity for these interactions in the progression of both acute and chronic rejection.

Materials and Methods Mice Female C57BL/6 (H-2b) mice, CD40LϪ/Ϫ C57BL/6 mice, and BALB/c (H-2d) mice were purchased from The Jackson Laboratory. Breeder pairs of CD40Ϫ/Ϫ C57BL/6 mice were purchased from The Jackson Laboratory. Breeder pairs of CD40Ϫ/Ϫ BALB/c mice were provided by Dr. Randy Noelle (Dartmouth College, Lebanon, NH). Colonies of CD40Ϫ/Ϫ mice were established, and all mice were housed under specific pathogen-free conditions maintained by the Unit for Laboratory Animal Medicine at Uni- versity of Michigan. Mice used were between 6 and 12 wk of age. These experiments were approved by the University Committee on Use and Care of Animals at the University of Michigan. Culture medium Downloaded from RPMI 1640 (for ELISPOT) or DMEM (for cell culture) was supplemented with 2% FCS, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 ␮g/ml streptomycin, 1.6 mM L-glutamine, 10 mM HEPES buffer (all from In- vitrogen), 0.27 mM L-asparagine, 1.4 mM L-arginine HCl, 14 ␮M folic acid, and 50 ␮M 2-ME (all from Sigma-Aldrich). Flow cytometry for OX40 http://www.jimmunol.org/ One million splenocytes per milliliter were cultured for times indicated with 1 ␮g/ml Con A (Sigma-Aldrich). After separating viable cells by FIGURE 1. Comparison of OX40 surface expression between C57BL/6 Ficoll-Hypaque gradient, splenocytes were triple labeled with FITC-con- and BALB/c cells after stimulation. Splenocytes from either WT or jugated anti-OX40 mAb (OX86, Cedarlane Laboratories), PE-conjugated CD40Ϫ/Ϫ C57BL/6 and BALB/c mice were stimulated with Con A for 0, anti-CD4 mAb (GK1.5, BD Pharmingen), and Cy5-conjugated anti-CD8 24, 48, or 72 h before analysis of OX40 surface expression by flow cy- mAb (2.43, BD Pharmingen). Cell analyses were performed on a FACS- tometry. Data are represented as the mean channel fluorescence of CD4ϩ Calibur (BD Biosciences) using forward vs side scatter to gate on T cells stained for OX40, reflecting OX40 surface density (A), or the per- lymphocytes. ϩ ϩ centage of CD4 splenocytes that are OX40 (B). All data are represen- Heterotopic cardiac transplantation tative of at least three separate experiments. by guest on September 25, 2021 C57BL/6 wild-type (WT), CD40LϪ/Ϫ, or CD40Ϫ/Ϫ mice were transplanted with intact WT or CD40Ϫ/Ϫ BALB/c cardiac allografts, as described (29). In this model, the donor heart is anastomosed to the great vessels of the merated by trypan blue exclusion. For differential enumeration, GIC were abdomen, perfused with the recipient mouse’s blood, and it resumes con- placed on slides with a cytocentrifuge and stained with Wright stain. traction. Transplant function was monitored by abdominal palpation. In vivo treatment with mAb ELISPOT assay for in vivo-primed, donor-reactive cytokine-producing cells The hybridomas secreting anti-CD4 (clone GK1.5) and anti-CD8 (clone 2.43) were obtained from the American Type Culture Collection (ATCC). ELISPOT assays were performed as described (36). Capture and detection The hybridoma secreting anti-CD40L (clone MR1) was provided by Dr. mAbs specific for IFN-␥ (RA-6A2, XMG1.2), IL-4 (BVD4-1D11, BVD6- Randy Noelle (Dartmouth, Lebanon, NH). Anti-CD4, anti-CD8, anti- 24G2), and IL-17 (TC11-18H10, TC11-BH4.1) were purchased from BD CD40L, and anti-OX40 (clone OX86) (30) mAbs were purified and resus- Pharmingen. Polyvinylidene difluoride-backed microtiter plates (Milli- pended in PBS by Bio X Cell. To deplete CD4ϩ or CD8ϩ T cells, allograft pore) were coated with unlabeled mAb and blocked with 1% BSA in PBS. 5 6 recipients were injected i.p. with 1 mg of anti-CD4 or anti-CD8 mAb on Irradiated (1000 rad) donor splenocytes (4 ϫ 10 ) and 10 recipient spleno- days Ϫ1, 0, and 7 relative to transplant (31–33). For inductive anti-CD40L cytes were added to each well. After washing, biotinylated detection mAbs therapy, mice were injected i.p. with 1 mg of anti-CD40L on days 0, 1, and were added to the plates. After washing, a 1/1000 dilution of anti-biotin 2 posttransplant (14, 32). To induce OX40 stimulation, recipients were alkaline phosphatase (AP) conjugate (Vector Laboratories) was added to injected with 0.25 mg i.p. of anti-OX40 mAb on days 0, 1, 3, and 7 post- IFN-␥ and IL-17 plates and a 1/2000 dilution of HRP-conjugated strepta- transplant, a regimen similar to what has been published regarding islet vidin (SA-HRP; Dako) was added to IL-4 plates. Plates were washed and transplantation (34). To induce delayed OX40 stimulation, recipients were spots visualized by addition of NBT (Bio-Rad)/5-bromo-4-chloro-3-in- injected with 0.25 mg i.p. of anti-OX40 mAb on days 30, 31, 33, and 37 dolyl phosphate (BCIP; Sigma-Aldrich) to IFN-␥ and IL-17 plates or relative to transplant. 3-amino-9-ethylcarbazole (AEC; Pierce) to IL-4 plates. Color development continued until spots were visible and was stopped by adding H2O. Plates Histology were dried and spots quantified using an ImmunoSpot series 1 ELISPOT analyzer (Cellular Technology). Allografts were recovered at the times indicated posttransplantation, fixed in formalin, and embedded in paraffin. Sections were stained with H&E to In vitro T cell subset depletion assess myocyte viability (presence of cross-striation and nuclei) and the ϩ ϩ nature and intensity of graft-infiltrating cells (GIC). As described (35), CD4 or CD8 cells were depleted using Dynal beads (Invitrogen) as per Masson trichrome stain was used to identify collagen deposition. the manufacturer’s protocol. Single-cell suspensions of splenocytes were incubated with anti-CD4- or anti-CD8-coated beads for 30 min. Bead- Recovery of GIC bound cells were removed magnetically. Unbound cells were confirmed to be depleted by flow cytometry (ϳ1% contamination; FACSCalibur). Three transplanted hearts were removed, pooled, minced, and digested with 1 mg/ml collagenase A (Roche) for 30 min at 37°C. Tissue debris was Morphometric analysis of intragraft collagen deposition allowed to settle at 1 ϫ g, and the suspension containing GIC was harvested by pipette. RBC were lysed by hypotonic shock, GIC were passed Quantitative analyses of areas of intragraft collagen deposition were per- through a 30-␮m pore size nylon mesh, and viable leukocytes were enu- formed as previously described (37). Briefly, images of 10 areas of each The Journal of Immunology 381 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 2. Stimulatory anti-OX40 mAb act on CD4ϩ T cells. A, WT C57BL/6 allograft recipients were transiently depleted of CD4ϩ or CD8ϩ cells (1 mg i.p. anti-CD4 mAb or anti-CD8 mAb on days Ϫ1, 0, and 7 relative to transplant) concurrent with agonistic anti-OX40 mAb (filled symbols, solid line) or control rat IgG treatment (open symbols, dashed line) (0.25 mg i.p. on days 0, 1, 3, and 7 relative to transplant). B, Recipients treated as described in A were sacrificed either at time of rejection or 30 days posttransplant. Donor-reactive Th1 (IFN-␥) and Th2 (IL-4) splenocyte responses were quantified by ELISPOT. Individual datum points represent the number of primed donor-reactive Th1 or Th2 cells in individual allograft recipients. Bars are indicative of the average number of donor-reactive splenocytes per experimental group. C, WT recipients inductively depleted of CD4ϩ T cells were treated with stimulatory anti-OX40 mAb (F, solid line) or control rat IgG (E, dashed line) 30 days posttransplantation (0.25 mg i.p. on days 30, 31, 33, and 40 relative to transplant) and monitored for function. D, Recipients treated as described in C were sacrificed either at the time of allograft rejection or 30 days after the initiation of treatment. Th1 (IFN-␥) and Th2 (IL-4) splenocyte responses were quantified by ELISPOT. Individual datum points represent the donor- reactive Th1 and Th2 responses of individual allograft recipients. Bars are indicative of the average number of donor-reactive Th1 or Th2 cells per experimental group.

graft’s myocardium were captured and analyzed using the application pro- IgG-HRP (SouthernBiotech) or either IgG1-AP or IgG2a-AP followed by gram IP Lab Spectrum-R4 (BD Biosciences). The color wavelengths of incubation with 1/600 streptavidin-HRP (Vector Laboratories). Incubation image copies were transformed into digital readings. Using the original with AEC revealed Ab binding. image for comparison, the color spectrum of the copied image was adjusted until areas positive for collagen turned green, while unaffected surrounding Alloantibody assay myocytes remained black. Percentage collagen was calculated by dividing 6 d the total pixel area of the field by the pixel area within areas of collagen As described (31, 38), 10 P815 (H-2 ) cells (ATCC) were incubated with 1/50 dilutions of sera collected from CD40Ϫ/Ϫ - deposition. C57BL/6 recipients at spec ified time points after transplantation. Cells were then washed and incu- Immunohistochemistry bated with FITC-conjugated, affinity-purified rabbit anti-mouse IgM or IgG Ab (Binding Site). The samples were analyzed on a FACSCalibur cytom- To detect IgG deposition within the graft, frozen sections of grafts were eter equipped with CellQuest software using forward vs side scatter for fixed in cold acetone and incubated with 1/150 dilution of goat anti-mouse appropriate gating. Data are reported as the mean channel fluorescence. 382 OX40 COSTIMULATION OVERRIDES CD40-CD40L BLOCKADE

ELISA for total serum IgG levels pleted of CD4ϩ cells and given anti-OX40 (Fig. 2B). Thus, in the absence of CD4ϩ cells, OX40 stimulation had no measurable ef- Total concentrations of IgG present in serum samples were determined by ϩ ELISA as previously described (39, 40). Briefly, a mixture of unlabeled fect on the CD8 T cell response. anti-IgG isotype-specific Abs (IgG1, IgG2b, IgG2c, IgG3; SouthernBio- We next asked whether OX40 stimulation would stimulate re- tech) were diluted to 10 ␮g/ml in a carbonate buffer and used to coat jection once CD4ϩ cells returned following transient depletion. Immulon 2, 96-well flat bottom ELISA plates (Costar/Corning) overnight. Following initial depletion, CD4ϩ T cells begin to repopulate the After washing, wells were blocked by the addition of 100 ␮l PBS, 10% FCS, and 0.4% azide per well. Following washing, 100 ␮l per well of periphery between 3 and 4 wk posttransplantation (35, 38). Indeed, samples diluted in PBS, 10% FCS, and 0.4% azide was added. After wash- when recipients in this study were sacrificed 39–60 days post- ing, a mixture of AP-tagged anti-IgG isotype-specific Abs (SouthernBio- transplantation, 7–15% of the splenocytes were CD4ϩ (data not tech) was diluted 1/1000 in Tris buffer and added at 100 ␮l per well. After shown). Recipients received delayed anti-OX40 mAb 30 days washing, plates were developed by the addition of 100 ␮l per well of ϩ substrate (Sigma Fast pNPP and Tris buffer tablets, diluted in distilled posttransplant as CD4 T cells returned. All recipients treated with water; Sigma-Aldrich) and read at 405 nm by an EL 800 microtiter plate anti-OX40 mAb rejected their allografts by day 44 posttransplant, reader (BioTek Instruments). All incubations were performed at room tem- or 14 days after receiving anti-OX40 mAb (Fig. 2C, p ϭ 0.0101). perature. Data are reported as the mean absorbance (ϮSEM) of three to In contrast, recipients given delayed control rat IgG maintained seven mice per experimental group. their grafts, verifying that donor-reactive CD4ϩ T cells were hy- Statistical analysis poresponsive (Fig. 2C). These observations indicate that the rejec- ϩ Data were analyzed with GraphPad Prism 4.0c software using Student’s tion mediated by OX40 stimulation is dependent on CD4 T cells. unpaired t tests with Welch’s correction or log-rank tests as appropriate. p ELISPOT analysis revealed primed, donor-reactive Th1 and values of Յ0.05 were considered statistically significant. Th2 responses in recipient splenocytes following delayed treat- Downloaded from ment with anti-OX40 (Fig. 2D). While unmodified recipients pro- Results ␥ ϩ duce elevated levels of donor-reactive IFN- , levels of donor-re- OX40 is up-regulated following CD4 T cell activation active IL-4 are comparatively rare (31). Hence, once CD4ϩ T cells This study assessed the effects of stimulation via an alternative repopulated the periphery, OX40 stimulation reversed their state of costimulatory molecule on allograft acceptance induced by CD40- hyporesponsiveness and induced both a Th1 and an atypical Th2

CD40L blockade. We focused on the OX40-OX40L pathway, as response. http://www.jimmunol.org/ BALB/c mice have been reported to preferentially utilize the OX40-OX40L pathway relative to C57BL/6 mice (28) and CD40 OX40 stimulation overrides CD40-CD40L blockade-induced is not required for the rejection response in BALB/c allograft re- allograft acceptance cipients (18). To verify differential expression of OX40 by Ϫ Ϫ Ϫ Ϫ Since BALB/c CD40 / mice, but not C57BL/6 CD40 / mice, BALB/c and C57BL/6 mice, splenocytes from both WT and reject cardiac allografts (18), and BALB/c mice employ the OX40- CD40Ϫ/Ϫ BALB/c and C57BL/6 mice were stimulated for 0, 24, OX40L pathway to a greater extent than do C57BL/6 mice (8, 28), we 48, and 72 h with Con A and analyzed for OX40 expression by asked whether OX40 stimulation would override graft acceptance in- ﬂow cytometry (Fig. 1). Before stimulation, OX40 expression was

duced by CD40-CD40L blockade in C57BL/6 recipients. To this end, by guest on September 25, 2021 negligible. After stimulation, OX40 was up-regulated on CD4ϩ T CD40-CD40L interactions were disrupted in C57BL/6 allograft re- cells (Fig. 1) but was not detected on CD8ϩ T cells (data not ϩ cipients by treating WT mice with anti-CD40L mAb, or using shown). BALB/c CD4 T cells expressed OX40 to a greater extent Ϫ Ϫ Ϫ Ϫ CD40L / or CD40 / recipients. Allograft recipients were treated than did their C57BL/6 counterparts both in terms of surface den- inductively with agonistic anti-OX40 mAb or rat IgG as a control. sity (Fig. 1A) and percentage of CD4ϩ T cells expressing OX40 Regardless of the method of CD40-CD40L disruption, OX40 stimu- (Fig. 1B). Furthermore, BALB/c CD4ϩ cells maintained OX40 lation induced acute allograft rejection in all recipients by day 14 expression for a longer period of time as compared with their posttransplant (Fig. 3). In contrast, grafts from rat IgG-treated control C57BL/6 counterparts (Fig. 1B). recipients continued to function normally until the termination of the Agonistic anti-OX40 mAb stimulates CD4ϩ T cells and induces experiment at day 30. For reference, otherwise unmodified WT mice a distinct pathology of allograft rejection were treated with either anti-OX40 or control rat IgG. Following ϩ OX40 stimulation, WT recipients rejected their allografts at a signif- In WT allograft recipients, transient depletion of CD4 T cells ϩ icantly accelerated tempo as compared with the rat IgG-treated con- allows for long-term graft survival (32, 33, 35, 38). These CD4 trols ( p ϭ 0.0009, Fig. 3). T cells repopulate the periphery 3–4 wk postdepletion, yet these repopulating cells are hyporesponsive (41). In contrast, depletion of CD8ϩ T cells allows only for a modest increase in graft survival Unconventional pathology induced by OX40 stimulation time (42). Since only CD4ϩ T cells expressed OX40 (Fig. 1) fol- OX40-induced rejection in allograft recipients following CD40- lowing Con A stimulation, we hypothesized that stimulatory anti- CD40L blockade was characterized by thrombosis of arteries OX40 mAb would directly activate CD4ϩ cells, but not CD8ϩ T (Fig. 4A). Arterial thrombosis is not a feature of unmodified cells. To test this hypothesis, WT allograft recipients were induc- acute rejection in WT mice (Fig. 4A, lower left panel). OX40- tively depleted of either CD4ϩ or CD8ϩ T cells and treated with induced rejection was additionally characterized by a granulo- agonistic anti-OX40 mAb or control rat IgG. In recipients induc- cytic infiltration of the graft (Fig. 4B) that is also not a feature tively depleted of CD8ϩ T cells, OX40 stimulation significantly of unmodified rejection (Fig. 4B) (31). Indeed, the few GIC that accelerated allograft rejection when compared with rat IgG-treated were recovered from control mice following CD40-CD40L controls (Fig. 2A, p ϭ 0.0079). Additionally, ELISPOT analysis blockade were primarily mononuclear in nature (Fig. 4B, bot- revealed a significant increase in both donor-reactive Th1 (IFN-␥) tom right panel). The absolute GIC yield recovered from grafts and Th2 (IL-4) cells following OX40 stimulation (Fig. 2B). In in anti-OX40-treated recipients was significantly greater when contrast, OX40 stimulation failed to induce acute graft rejection compared with control recipients following CD40-CD40L following inductive depletion of CD4ϩ T cells (Fig. 2A). Further- blockade (Fig. 4D). more, ELISPOT analysis revealed that donor-reactive Th1 and Th2 A significantly higher percentage of granulocytes was also responses were not induced in recipients that were inductively de- present in the spleens of anti-OX40-treated allograft recipients The Journal of Immunology 383

cells did not induce allograft rejection or either Th1 or Th2 responses (Fig. 2, A and B). Delayed OX40 stimulation does not induce acute rejection of accepted allografts but promotes chronic rejection following CD40-CD40L disruption In the previous experiments, our OX40 stimulation protocol was initiated at the time of transplantation. We next asked if stimulation through OX40 would reverse established allograft acceptance following CD40-CD40L blockade. To this end, WT recipients treated with anti-CD40L mAb, or CD40LϪ/Ϫ or CD40Ϫ/Ϫ recipients were transplanted with cardiac allografts and were left otherwise untreated. After 30 days these allograft recipients were treated with either anti-OX40 mAb or a control rat IgG. OX40 stimulation did not induce acute rejection of established allografts (Fig. 6A). Although grafts continued to function in mice that received delayed anti-OX40, contractions of these grafts were no- FIGURE 3. OX40 stimulation overrides allograft acceptance induced ticeably weaker when compared with their rat IgG-treated coun- by inhibition of CD40-CD40L interactions. C57BL/6 WT recipients, WT recipients treated with anti-CD40L mAb (1 mg i.p. on days 0, 1, and 2 terparts as assessed by palpation. Histologically, an increased Downloaded from relative to transplant), CD40LϪ/Ϫ, or CD40Ϫ/Ϫ C57BL/6 mice were trans- density of GIC resulted from delayed OX40 stimulation relative to planted with BALB/c cardiac allografts and treated with agonistic anti- rat IgG-treated controls (Fig. 6B). OX40 mAb (ﬁlled symbols, solid line) or a control rat IgG (open symbols, We also assessed Th1 and Th2 responses by ELISPOT to de- dashed line) and grafts were monitored for function. termine whether delayed anti-OX40 treatment resulted in donor- reactive T cell priming (Fig. 6, C and D). At the termination of the

experiment (day 60 posttransplant), both donor-reactive Th1 and http://www.jimmunol.org/ (Fig. 4C, top panel). Furthermore, splenocytes from anti-OX40- Th2 were readily detectable in recipients receiving delayed OX40 treated recipients exhibited morphology suggestive of cell activa- stimulation but not in the control groups. Hence, despite the pres- tion (abundant cytoplasm and diffuse nuclei) (Fig. 4C, bottom left ence of primed, donor-reactive T cell responses, allografts were panel). Note that these alterations in both graft pathology (throm- not acutely rejected. bosed vessels and granulocyte accumulation) and splenocyte com- Additionally, delayed OX40 stimulation resulted in increased position were not observed in syngeneic graft recipients that were collagen deposition within the grafts as detected by Masson treated with anti-OX40 (data not shown). Thus, these OX40-in- trichrome staining (Fig. 6E, left panel). Morphometric analysis duced pathologies were Ag dependent. was used to quantify the percentage of the graft containing collagen deposition (stained blue) and revealed significantly more col- by guest on September 25, 2021 OX40 stimulation induces donor-reactive Th1 and Th2 lagen in the grafts of mice receiving delayed OX40 stimulation responses relative to rat IgG-treated controls (Fig. 6E, right panel). Since ELISPOT analysis for primed, donor-reactive Th1 and Th2 re- collagen deposition within the graft is a hallmark of chronic allo- sponses revealed that OX40 stimulation induced significant IFN-␥ graft rejection, these data indicate that delayed anti-OX40 treat- (Fig. 5A) and IL-4 (Fig. 5B) responses relative to those observed in ment of mice bearing established allografts promotes the progres- control recipients. Note that donor-reactive Th2 are not character- sion of chronic, rather than acute, rejection. istic of unmodified acute rejection in this model (Fig. 5B) (31), yet OX40 stimulation induced IL-4 production in all groups regardless OX40 stimulation induces donor-reactive IgM and IgG of if or how CD40-CD40L interactions were disrupted. OX40- responses in the absence of CD40 induced donor-reactive Th17 responses were not remarkable (data Since CD40 engagement is reportedly necessary in stimulating not shown). an IgG class switch response (44), we assessed the ability of To determine which T cell subsets were responsible for OX40 stimulation in CD40Ϫ/Ϫ allograft recipients to induce OX40-induced cytokine production, splenocytes from OX40- donor alloantigen-reactive IgM and IgG responses (Fig. 7). stimulated allograft recipients were depleted of CD4ϩ or CD8ϩ OX40 stimulation in CD40Ϫ/Ϫ allograft recipients induced a cells before addition to ELISPOT cultures (Fig. 5, C and D). significant increase in donor-reactive IgM production when Depicted are results obtained from anti-OX40-treated CD40Ϫ/Ϫ compared with rat IgG-treated control recipients (Fig. 7A). Im- allograft recipients, and similar results were generated in WT portantly, OX40 stimulation induced a donor-reactive IgG re- recipients treated with anti-CD40L and in CD40LϪ/Ϫ recipi- sponse in CD40Ϫ/Ϫ allograft recipients (Fig. 7B), indicating ents. Depletion of CD8ϩ cells eliminated the Th1 response (Fig. that OX40 stimulation overrides the reported requirement for 5C), while depleting CD4ϩ cells abrogated the Th2 response CD40 in inducing the IgG isotype switch (44). These data were (Fig. 5D). These data are consistent with reports that CD8ϩ confirmed by a mouse IgG-specific sandwich ELISA that re- cells are the principal source of IFN-␥ in this model (31, 43) vealed significant differences in total IgG levels in the sera of and that OX40 stimulation of CD4ϩ cells induces a Th2 re- anti-OX40 as compared with rat IgG control-treated CD40Ϫ/Ϫ sponse (8, 9, 28). As we were unable to detect OX40 expression allograft recipients (Fig. 7C). on the surface of CD8ϩ T cells (data not shown), and depletion In addition to donor-reactive Ab circulating in the recipient of CD8ϩ T cells concurrent with OX40 stimulation failed to serum, mouse IgG was also detectable by immunohistochemis- prolong graft survival (Fig. 2A), these data suggest that OX40 try within the grafts of CD40Ϫ/Ϫ recipients receiving anti- stimulation induces CD4ϩ T cells to provide help for the primed OX40 at the time of transplantation (ϩ anti-OX40, day 0, Fig. CD8ϩ T cell response. This notion is further supported by the 7D). Interestingly, although donor-reactive Ab was not detect- fact that anti-OX40 treatment of recipients depleted of CD4ϩ T able in the serum of CD40Ϫ/Ϫ recipients treated with anti-OX40 384 OX40 COSTIMULATION OVERRIDES CD40-CD40L BLOCKADE Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 4. Pathology of acute rejection induced by OX40 stimulation following CD40-CD40L blockade. A, Allografts from C57BL/6 WT recipients, WT recipients treated with anti-CD40L mAb, or CD40LϪ/Ϫ or CD40Ϫ/Ϫ recipients treated with either anti-OX40 mAb (top row) or control rat IgG (bottom row) were harvested at time of rejection (anti-OX40 mAb treated) or 10 days posttransplant (rat IgG treated) and stained with H&E. Note the perivascular The Journal of Immunology 385 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 5. OX40 stimulation induces donor-reactive Th1 and Th2 responses following CD40-CD40L blockade. C57BL/6 WT recipients, WT recipients treated with anti-CD40L mAb, CD40LϪ/Ϫ recipients, or CD40Ϫ/Ϫ recipients were transplanted with cardiac allografts and were treated with either anti-OX40 mAb or control rat IgG. Splenocytes were harvested at the time of rejection (recipients treated with anti-OX40 and WT ϩ rat IgG) or 30 days after transplantation (rat IgG-treated recipients in which CD40-CD40L interactions were ablated). Donor-reactive Th1 (IFN-␥, A) and Th2 (IL-4, B) responses were quantiﬁed by ELISPOT. To determine the cell populations producing these cytokines, either CD4ϩ (E)orCD8ϩ (F) cells were depleted from whole splenocyte (f) populations before addition to ELISPOT Th1 (IFN-␥, C) or Th2 (IL-4, D) assays. p values are in comparison to data from whole splenocyte populations. N/A indicates nonapplicable; N/S, not signiﬁcant.

30 days after transplantation (data not shown), and these grafts cipients treated with anti-OX40 at day 0 vs day 30 differed. were not acutely rejected (Fig. 6A), mouse IgG was also de- While both IgG1 and IgG2a were readily detectable within tectable within the grafts of these recipients (ϩ anti-OX40, day vessels of grafts from recipients treated with anti-OX40 at the 30, Fig. 7D). This finding suggests that the donor-reactive Ab time of transplantation, only IgG1 was detectable within vessels that is produced is absorbed by the graft. Furthermore, this ob- of grafts from recipients treated with anti-OX40 30 days after servation indicates that differential levels of donor-reactive Ab transplantation. Hence, the presence of complement-fixing are produced following OX40 stimulation, dependent on the IgG2a (45) correlated with acute rejection, while the presence timing of OX40 stimulation relative to transplantation. Addi- of non-complement-fixing IgG1 (46) correlated with prolonged tionally, the isotypes of IgG deposited within the grafts of re- graft function and the progression of chronic rejection.

infiltrate and thrombosed vessels (black arrows) in rejecting grafts following OX40 stimulation (magnification, ϫ400). GIC (B) and splenocytes (C) were harvested from recipients treated with anti-OX40 mAb or control rat IgG and Wright stained. One hundred cells were counted in at least four different fields, and bars (mean Ϯ SEM) represent the percentage of granulocytic cells present in either the graft (B) or spleen (C)(n Ն 3 mice). Representative fields from CD40Ϫ/Ϫ recipients treated with anti-OX40 show both granulocytes (red arrows) and mononuclear cells that appear activated (characterized by diffuse nuclei and abundant cytoplasm, black arrows, center panels, ϫ1000). Representative fields of control CD40Ϫ/Ϫ recipients receiving rat IgG are depicted in bottom right of B and C. D, Absolute number of cells recovered from the spleens and allografts of recipients treated with either anti-OX40 (filled bars) or rat IgG (open bars) are depicted. The number of cells present in the spleens of allograft recipients was not dramatically increased, whereas the number of cells present within the allografts of recipients in which CD40-CD40L interactions were ablated was significantly increased after treatment with anti-OX40 mAb (all p values Ͻ0.05). Bars represent the mean (ϮSEM) of cells recovered from at least six recipients per group. 386 OX40 COSTIMULATION OVERRIDES CD40-CD40L BLOCKADE Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 6. Delayed OX40 stimulation promotes chronic, rather than acute, rejection of accepted grafts following CD40-CD40L blockade. A,WT recipients treated with anti-CD40L mAb, CD40LϪ/Ϫ recipients, or CD40Ϫ/Ϫ recipients were transplanted with cardiac allografts and were treated with delayed anti-OX40 mAb (filled symbols, solid line) or control rat IgG (open symbols, dashed line) (0.25 mg i.p. on days 30, 31, 33, and 40 relative to transplant). B, Grafts from recipients described in A were harvested 60 days posttransplantation and stained with H&E. In the anti-OX40 mAb-treated groups areas of increased infiltrate were apparent, particularly around the arteries. Splenocytes from recipients described in A were assessed for donor- reactive Th1 (IFN-␥, C) and Th2 (IL-4, D) by ELISPOT. E, Sections of grafts from recipients described in A were stained with Masson trichrome stain, which stains collagen blue. Frames in both B and E are of grafts from CD40Ϫ/Ϫ recipients and are representative of three to four control rat IgG-treated mice and five to six anti-OX40-treated recipients in which recipient CD40-CD40L interactions were disrupted. The areas of grafts staining positive for collagen deposition were quantified by morphometric analysis (E, right). Bars represent the average percentage (ϮSEM) of graft area positive for collagen in three to four rat IgG-treated recipients and five to six anti-OX40-treated recipients.

Discussion rejection in this model is associated with a Th1, but not a Th2, In the current study, we present data that demonstrate that OX40 response (31, 32, 47). However, the induction of Th2 by OX40 costimulation overrides cardiac allograft acceptance induced by perturbation in our model correlates with reports from other sys- CD40-CD40L blockade (Fig. 3) and results in the induction of tems that OX40 stimulation is associated with a Th2 response (8, both donor-reactive Th1 and Th2 responses (Fig. 5). Unmodified 9). In transplantation the induction of a Th2 response results in The Journal of Immunology 387 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021 FIGURE 7. OX40 stimulation induces production of donor-reactive alloantibodies in the absence of CD40. Sera from C57BL/6 CD40Ϫ/Ϫ recipients of cardiac allografts treated with anti-OX40 mAb (shaded bars) or rat IgG (open bars) were harvested at either time of rejection or 10 days after transplant. P815 (H-2d) cells were then incubated with a 1/50 dilution of sera, and bound donor-reactive Ab was detected by incubation with FITC-tagged anti-IgM (A) or anti-IgG (B) Abs. The mean channel fluorescence is indicative of the relative amount of donor-reactive Abs. Bars represent the average mean channel fluorescence of three control rat IgG-treated recipient and five anti-OX40-treated recipient samples (ϮSEM). C, Sera of CD40Ϫ/Ϫ allograft recipients treated with anti-OX40 mAb or control rat IgG were diluted 10Ϫ3 and the relative amounts of total mouse IgG present were quantified by sandwich ELISA. Filled bars (anti-OX40 mAb treated) and open bars (rat IgG control treated) represent the average OD (A450 nm) of three control rat IgG-treated recipient and five anti-OX40-treated recipient sera samples (ϮSEM). D, C57BL/6 CD40Ϫ/Ϫ allograft recipients were treated with control rat IgG (left column) or anti-OX40 mAb at time of transplant (inductive, middle column) or 30 days after transplantation (delayed, right column), and grafts were harvested at either time of rejection or functioning grafts were harvested 60 days after transplantation. Depicted are grafts from recipients treated with rat IgG at the time of transplantation, which is representative of results obtained from both inductive and delayed treatment with rat IgG. Graft sections were fixed and incubated with goat anti-mouse IgG, rabbit anti-mouse IgG1, or rabbit anti-mouse IgG2a followed by development with 3-amino-9-ethylcarbazole to visualize mouse Ab deposition. Results are representative of grafts from four to ten recipients. Magnification, ϫ400. an alternative pathology of rejection (31, 32, 47–49) that in- cell depletion, OX40 stimulation failed to result in T cell prim- cludes the vascular involvement and granulocytic infiltration as ing and/or allograft rejection (Fig. 2). While OX40 stimulation is induced by OX40 stimulation (Fig. 4). Hence, we observed a has been reported to promote CD8ϩ T cell effector functions biologic effect of OX40-mediated Th2 induction. In the absence of (52), we were unable to detect CD8ϩOX40ϩ cells by flow cy- CD4ϩ T cells, OX40 stimulation failed to induce this allograft tometry either in the spleen or in the graft (data not shown), pathology in WT recipients (Fig. 2), suggesting that OX40 stim- suggesting that OX40 expression may not be induced on CD8ϩ ulation acts directly on CD4ϩ T cells. Cell selection studies re- T cells in this system. Alternatively, CD8ϩOX40ϩ cells may vealed that these CD4ϩ cells were the principal source of IL-4 in localize in a different compartment in vivo. However, WT re- this system (Fig. 5D) while CD8ϩ cells were the main source of cipients transiently depleted of CD4ϩ T cells and treated with IFN-␥ (Fig. 5C). Interestingly, OX40 stimulation also induced do- delayed agonistic anti-OX40 do reject their allografts. This re- nor-reactive IgG responses in the complete absence of CD40 (Fig. jection is likely dependent on the return of CD4ϩ T cells (30 7). To our knowledge, this role for OX40 in B cell responses has days posttransplant, ϳ5% of splenic cells are CD4ϩ as detected not been described. by flow cytometry, data not shown). Indeed, after CD4ϩ T cell OX40 is expressed preferentially by activated CD4ϩ T cells repopulation, OX40 stimulation induced both IFN-␥- and IL-4- (Fig. 1) (3), and it is likely that in our experiments OX40 stim- producing cells (Fig. 2). Hence, our data indicate that OX40 ulation induced CD4ϩ T cells to provide help to CD8ϩ T cells stimulation affects not only CD4ϩ cells expressing detectable (50, 51) to promote their activation. Indeed, following CD4ϩ T OX40 on their surface, but also induces these CD4ϩ T cells to 388 OX40 COSTIMULATION OVERRIDES CD40-CD40L BLOCKADE provide help for CD8ϩ T cell responses generated in conjunc- The findings discussed above were generated when OX40 stim- tion with alloantigen exposure. ulation occurred at the onset of CD40-CD40L blockade. Hence, Stimulation of OX40 through OX40L on APC is thought to play we assessed the consequences of delayed OX40 stimulation once a critical role in driving Ag-specific T cell responses in vivo (53). allografts had been accepted following CD40-CD40L blockade. In our study, deliberate stimulation of OX40 may be necessary to Inductive CD40-CD40L blockade allowed for continued graft observe these effects, as both CD4ϩ T cells and CD11cϩ dendritic function, and primed donor-reactive cytokine-producing cells were cells from C57BL/6 mice express lower levels of OX40 and rare (Figs. 3 and 5, A and B). However, delayed OX40 stimulation OX40L as compared with their BALB/c counterparts (Fig. 1 and induced graft pathology and Th1 and Th2 cell priming yet did not data not shown). OX40L has a broad tissue distribution, including induce acute graft rejection (Fig. 6). Thus, these findings support B cells, dendritic cells, and endothelial cells (3, 54, 55). Further- previous observations that following CD40-CD40L blockade a more, CD40 engagement up-regulates OX40L surface expression number of recipient lymphocytes retain graft reactivity despite on both B cells (53) and dendritic cells (53, 56), suggesting that graft acceptance (14). As OX40 is only expressed following T cell OX40L expression may be dependent on CD40-CD40L interac- activation (Fig. 1) (3), naive cells should fail to respond to OX40 tions. In our experiments, T cell OX40 expression does not appear stimulation while anergic cells should be responsive. Indeed, anti- to limit OX40-OX40L interactions, as agonistic anti-OX40 mAb is CD40L treatment has been shown to maintain graft-reactive cells sufficient to drive acute allograft rejection (Fig. 3) and induce do- in a quiescent precursor state, and these cells can be activated in nor-reactive cytokine priming (Fig. 5). Rather, low levels of vitro (14). Inhibition of CD40-CD40L interactions favors the gen- OX40L expression may impede contributions of the OX40- eration of regulatory T cells (69–71), and these regulatory T cells OX40L costimulatory pathway to graft pathology under conditions may be adequate to prevent donor-reactive cells from acutely re- Downloaded from where the CD40-CD40L pathway is disrupted. Indeed, Jenkins jecting accepted grafts following delayed OX40 stimulation. Re- et al. (57) demonstrated that stimulation via OX40 restored pro- cent data suggest that after OX40 stimulation regulatory T cells are duction of both Th1 and Th2 responses to Schistosoma mansoni- less efficient suppressors of effector cell proliferation and cytokine soluble egg Ag presented by CD40Ϫ/Ϫ dendritic cells, suggesting production (72, 73), and that OX40 stimulation may reverse reg- that the T cell response was limited by APC OX40L expression as ulatory T cell function (74). opposed to a lack of T cell OX40 expression. Hence, endogenous Differential induction of Ab isotypes by initial vs delayed OX40 http://www.jimmunol.org/ OX40-OX40L interactions may not override CD40-CD40L block- stimulation correlated with acute vs chronic graft rejection. Inter- ade due to limited OX40L, but not OX40 expression, resulting estingly, donor-reactive cytokine production was induced in both from the absence of CD40 stimulation of APC. settings. Indeed, initial vs delayed OX40 stimulation was associ- Disruption of CD40-CD40L interactions is highly effective in ated with differential Ab deposition within the allografts of promoting allograft acceptance (reviewed in Refs. 2, 12) (13–17). CD40Ϫ/Ϫ recipients (Fig. 7D). Hence, deliberate OX40 stimula- However, it is likely that OX40 stimulation mediates multiple ef- tion is sufficient to induce a donor-reactive isotype switch in the fects on CD4ϩ cells in the absence of CD40-CD40L interactions absence of CD40 interactions. The isotypes of these Abs are con-

that culminate in allograft rejection. After TCR engagement, sistent with the types of cytokines produced. Inductive OX40 stim- by guest on September 25, 2021 OX40 is up-regulated on the CD4ϩ T cell surface (4, 58). OX40 ulation induced both IFN-␥ and IL-4 (Fig. 5) and both IgG2a and stimulation enhances both CD4ϩ T cell cytokine production and IgG1 donor-reactive Abs (Fig. 7) (75). In contrast, delayed OX40 proliferation (59) and can prevent the induction of tolerance to stimulation induced a strong Th2 and dampened Th1 response as peptide (52). Furthermore, OX40 stimulation can promote the ex- compared with the inductively treated recipients (Fig. 6D) and pansion of a pool of Ag-reactive T cells (60, 61). Thus, in our IgG1 production (Fig. 7D). While IgG2 is a potent activator of model inductive OX40 stimulation may serve as the costimulatory complement, IgG1 is not (46). It has been previously reported that signal needed to activate and expand graft-reactive T cells in the IgG2b, but not IgG1 alone, is sufficient to induce acute graft re- absence of CD40-CD40L interactions. Maxwell et al. (60) have jection (76), and that together IgG2a and IgG1 can synergize to established that when initiated concurrent with Ag and “danger induce acute graft rejection and complement deposition (45, 77). signals” such as LPS, OX40 stimulation results in both clonal ex- Additionally, IgG1 alone does not induce acute graft rejection but pansion and survival of Ag-specific T cells. In transplantation, does induce chemokine release by the endothelial cells (77). heat-shock proteins are induced by both ischemia/reperfusion in- Hence, inductive OX40 stimulation may lead to the production of jury and surgery and provide a danger signal (62), which in addi- both IgG1 and IgG2a, synergistically contributing to acute graft tion to the Ag supplied by the graft could synergize with OX40 rejection by activating complement, whereas delayed OX40 stim- stimulation to result in the expansion of the graft-reactive T cell ulation may lead to the production of IgG1 alone and contribute to pool. Additionally, OX40 stimulation in the presence of cytokines chronic rejection. This possibility remains to be formally tested. can reverse the nonresponsiveness of anergic cells and induce fur- Taken together, data herein suggest that OX40 stimulation may ther cytokine production and effector functions (63). While the have multiple effects on immune responses following CD40- actions of anti-CD40L mAb are not fully understood (12, 13, 64– CD40L blockade. These effects may depend on both cell types 67), it is generally thought that deficiency in either CD40L (16) or affected and timing of OX40 stimulation relative to both Ag ex- CD40 (reviewed in Ref. 68) disrupts a crucial step in T cell acti- posure and danger signal presence. Inductive OX40 stimulation vation and renders these cells anergic. These cells could now pro- may affect the cytokine profile of activated cells, prevent the de- vide a target for OX40 stimulation, as anergic cells up-regulate velopment of regulatory cells, and/or rescue graft-reactive cells OX40 on their surface (52). Thus, OX40 stimulation of T cells from anergy induced by CD40-CD40L blockade. Conversely, after may reverse anergy induced by CD40-CD40L blockade, thereby the danger signals associated with transplantation have been re- allowing the emergence of otherwise silenced donor-reactive ef- solved, delayed OX40 stimulation may affect the suppressive ac- fector cells resulting in anti-graft responses. Our findings are in tivities of OX40ϩ regulatory T cells. Furthermore, inductive, but keeping with these possibilities and represent the first report of not delayed, OX40 stimulation correlated with thrombosed arteries OX40 stimulation in the absence of CD40-CD40L interactions in in grafts undergoing acute, but not chronic, rejection. Our findings the transplant setting. should be of interest clinically as they demonstrate redundancy and The Journal of Immunology 389 key functional differences in costimulatory molecule function and 23. Sacks, D., and N. Noben-Trauth. 2002. The immunology of susceptibility and should be considered when developing anti-rejection regimens. resistance to Leishmania major in mice. Nat. Rev. Immunol. 2: 845–858. 24. Sadick, M. D., F. P. Heinzel, B. J. Holaday, R. T. Pu, R. S. Dawkins, and R. M. Locksley. 1990. Cure of murine leishmaniasis with anti-interleukin 4 Acknowledgments monoclonal antibody: evidence for a T cell-dependent, interferon ␥-independent mechanism. J. Exp. Med. 171: 115–127. We thank Dr. Wesley A. Dunnick for insightful discussions regarding Ab 25. Wang, Z. E., S. L. Reiner, S. Zheng, D. K. Dalton, and R. M. Locksley. 1994. Ϫ Ϫ responses in CD40 / mice. CD4ϩ effector cells default to the Th2 pathway in interferon ␥-deficient mice infected with Leishmania major. J. Exp. Med. 179: 1367–1371. 26. Szabo, S. J., B. M. Sullivan, C. Stemmann, A. R. Satoskar, B. P. Sleckman, and Disclosures L. H. Glimcher. 2002. 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