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CD40/CD154 Blockade Inhibits Dendritic Cell Expression ofInflammatory Cytokines but not Costimulatory Molecules1

Ivana R. Ferrer*, Danya Liu*, David F. Pinelli, Brent H. Koehn, Linda L. Stempora, andMandy L. FordEmory Transplant Center, Emory University, Atlanta GA 30322

AbstractBlockade of the CD40/CD154 pathway remains one of the most effective means of promotinggraft survival following transplantation. However, the effects of CD40/CD154 antagonsim ondendritic cell (DC) phenotype and functionality following transplantation remain incompletelyunderstood. To dissect the effects of CD154/CD40 blockade on DC activation in vivo, wegenerated hematopoietic chimeras in mice that expressed a surrogate minor antigen (OVA).Adoptive transfer of OVA-specific CD4+ and CD8+ T cells led to chimerism rejection, which wasinhibited by treatment with CD154 blockade. Surprisingly, CD154 antagonism did not alter theexpression of MHC and costimulatory molecules on CD11c+ DC compared to untreated controls.However, DCs isolated from anti-CD154 treated animals exhibited a significant reduction ininflammatory cytokine secretion. Combined blockade of inflammatory cytokines IL-6 andIL-12p40 attenuated the expansion of antigen-specific CD4+ and CD8+ T cells and transientlyinhibited the rejection of OVA-expressing cells. These results suggest that a major effect ofCD154 antagonism in vivo is an impairment in the provision of signal three during donor-reactiveT cell programming, as opposed to an impact on the provision of signal two. We conclude thattherapies designed to target inflammatory cytokines during donor-reactive T cell activation maybe beneficial in attenuating these responses and prolonging graft survival.

IntroductionIn both bone marrow and solid organ transplantation models, specifically targeting graft-reactive T cell responses to prevent transplant rejection remains an important goal. Manystudies in murine and non-human primate models have shown that blockade ofcostimulatory signals promotes survival of bone marrow, skin, kidney, heart, and islettransplants (1–5). Blockade of the CD40/CD154 costimulatory pathway remains one of themost effective means of inhibiting alloreactive T cell responses and inducing long-term graftsurvival following transplantation. However, monoclonal antibodies designed to targetCD154 resulted in thromboembolic events in early pilot studies in humans (6). Renewedinterest in blockade of this pathway for the prevention of graft rejection has been sparked bypromising results from several recent studies in non-human primate transplant models usingmonoclonals directed against CD40 (7–9), and clinical trials using CD40 blockers in renaltransplant recipients are now underway (10). Thus, the therapeutic potential of targeting thispathway is high, and understanding the effects of CD154/CD40 blockade in transplantmodels may uncover other novel downstream targets for therapeutic intervention.

1This work was supported by NIH/NIAID AI073707 and AI079409 to MLF

Corresponding Author: Mandy L. Ford, Ph.D., Assistant Professor, Emory Transplant Center, Dept of Surgery, Emory University, 101Woodruff Rd Suite 5105, Atlanta, GA 30322, 404-727-2900 (office), 404-727-5854 (lab), 404-727-3660 (fax),[email protected].*Authors contributed equally

NIH Public AccessAuthor ManuscriptJ Immunol. Author manuscript; available in PMC 2013 November 01.

Published in final edited form as:J Immunol. 2012 November 1; 189(9): 4387–4395. doi:10.4049/jimmunol.1201757.

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Despite the incontrovertible efficacy of blockade of this pathway, the mechanismsunderlying its effect are incompletely understood. While one report indicated that anti-CD154 monoclonal antibodies may impact the outcome of graft rejection by specificallybinding to and depleting antigen-specific CD4+ T cells which express CD154 followingactivation (11), subsequent studies using anti-CD40 monoclonal antibodies showed similarefficacy in both bone marrow and solid organ transplant models in mouse and non-humanprimates (7–9, 12–14). Thus, it is likely that blockade of the CD40/CD154 pathway, ratherthan antibody-mediated depletion of antigen-specific cells, plays a major role in theobserved attenuation of graft rejection. In dendritic cells (DCs), ligation of CD40 by CD154expressed on activated CD4+ T cells leads to the activation of downstream signalingpathways, resulting in several key events that promote the generation of effective T cellresponses. These include 1) increasing MHC expression which would enhance the strengthof TCR signals (15, 16), 2) inducing costimulatory molecule expression (e.g., CD80, CD86,OX40L) thus enhancing the strength of “second signals” (17, 18), 3) increased production ofpro-inflammatory cytokines (IL-12, IL-6, IL-1) sometimes referred to as “signal three” (19),and 4) increasing DC longevity (20, 21), thereby enhancing T cell priming via of all of theabove mechanisms. However, the impact of CD154/CD40 blockade on these aspects of DCbiology remains incompletely characterized.

Here, we hypothesized that blockade of the CD40/CD154 pathway in vivo would result inaltered DC phenotype and/or function, which could in turn result in sub-optimal T cellpriming and thus lead to protection of hematopoietic chimerism following bone marrowtransplantation. To test this hypothesis, we generated hematopoietic chimeras that expressedmembrane-bound chicken ovalbumin (OVA) on all hematopoietically-derived cells.Subsequent adoptive transfer of OVA-specific CD8+ OT-I and CD4+ OT-II T cells led to aGVHD- like response as measured by a loss of OVA-expressing cells, a process that wasattenuated by treatment with anti-CD154 mAb (MR-1). In order to dissect the effects ofCD154/CD40 blockade on DC activation in vivo, antigen-bearing splenic DCs were isolatedfrom recipients that had received host-reactive CD4+ and CD8+ T cells in the presence orabsence of anti-CD154 mAb. Results demonstrated that DCs derived from anti-CD154-treated recipients did not differ with regard to their expression levels of MHC orcostimulatory molecules, but instead exhibited impaired secretion of inflammatorycytokines.

Materials and MethodsMice

BALB/c (CD45.2, H2-Kd), C57BL/6 (CD45.2, H2-Kb) and B6-Ly5.2/Cr (CD45.1, H2-Kb)mice were obtained from the National Cancer Institute (Charles River, Frederick, MD). OT-Iand OT-II TCR transgenic mice (C57BL/6 background) were bred to C57BL/6 Thy1.1congenic animals at Emory University. OT-II×RAG−/− TCR transgenic mice werepurchased from Taconic and bred to Thy1.1 congenic animals at Emory University. mOVAmice on a C57BL/6 background (22) were a gift from Dr. Marc Jenkins (University ofMinnesota, Minneapolis, MN) and were maintained at Emory University. All animals werehoused in pathogen-free animal facilities at Emory University. All studies were approved bythe Institutional Animal Care and Use Committee of Emory University.

Bone marrow isolation and establishment and screening of mOVA BM chimeraRecipient B6-Ly5.2/CR (CD45.1) (NCI) mice were treated one day prior to bone marrowadoptive transfer with 500µg of Busulfan (Busulfex, Otsuka America Pharmaceutical, Inc.)intraperitoneally. Bone marrow was flushed from femurs and tibias of mOVA (CD45.2+)mice with saline using a 27g needle and was disrupted through the needle. BM cells were

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subsequently resuspended in saline and adoptively transferred i.v. at a dose of 20×106 cellsper mouse. On the day of bone marrow infusion, recipients were treated with 500µg of bothCTLA-4 Ig (Bristol-Myers Squibb) and anti-CD154 (MR1, BioExpress), followed by thesame dose on days 2, 4, 6, to prevent an immune response against the OVA antigen. mOVAbone marrow chimeras were used in experiments at approximately 8–12 weeks post-transplant. Hematopoietic chimerism was determined by staining peripheral blood withB220-PerCP, CD45.1-PE, and CD45.2-FITC (all from BD Pharmingen). Data were acquiredon a LSR II flow cytometer (Becton Dickinson) and analyzed using FlowJo Software(Treestar, San Carlos, CA).

Adoptive transfers and antibody treatmentSpleens of OT-I and OT-II mice were disrupted with frosted glass slides and processed intoa single cell suspension. Splenocytes were stained with CD4-APC, CD8-FITC, Vα2-PE andThy1.1-PerCP monoclonal antibodies (BD Pharmingen) for flow cytometric analysis. UsingTruCount beads (BD Pharmingen), absolute numbers of each cell population were obtainedand 5×106 OT-I and 106 OT-II T cells were adoptively transferred i.v. into recipient mice.Following adoptive transfer, animals were treated with 250µg of anti-CD154 (MR1,BioXCell, West Lebanon, NJ) or a combination of anti-IL-12/23p40 (clone C17.8,BioXCell) and anti-IL-6R (clone 15A7, BioXCell) on days 0, 2, 4, and 6, in the indicatedgroups, or left untreated in control groups.

Dendritic cells isolation and flow cytometric analysisSpleens were removed from mice. One millilitre of 2mg/ml collagenase type 3 (WorthingtonBio. Corp, NJ) in HBSS (with Ca+2/Mg+2) was injected into the spleen, which was thenincubated with 2ml collagenase solution at 37°C /5%CO2 for 30 min. Single cellsuspensions were prepared after incubation by mashing the spleen using 3ml syringe plungeron a cell strainer (70µM) and washing cells with PBS. Single cell suspensions were stainedfor flow cytometric analysis with anti-CD11c-APC, anti-CD11b-PerCP, anti-CD8α-APC-Cy7, anti-H-2Kb-FITC, anti-I-Ab-FITC, anti-ICAM-1-APC, anti-CD80-PE, anti-CD86-PE,and anti-CD40-FITC (all from BD Pharmingen). Data were acquired on a LSR II flowcytometer (Becton Dickinson) and analyzed using FlowJo Software (Treestar, San Carlos,CA).

Assessment of ex vivo cytokine production by DCsDCs were isolated as described above and single cell suspensions were enriched by negativeselection using magnetic beads coated with anti-CD19, CD90.1, and CD90.2 monoclonalantibodies (Miltenyi Biotec Inc.) according to the manufacturer's instructions. Purified DCs(purity >70%) were resuspended in RPMI 1640 supplemented with 10% heat-inactivatedfetal bovine serum, 50µM 2-mercaptoethanol, streptomycin (100µg/ml), and penicillin (100units/ml). DCs were then distributed in 200-µl aliquots (6×105 cells/well) to a 96-well plateand cultured for 24 hours at 37°C in 5% CO2 in duplicate. The cultured cell supernatantswere measured for the levels of inflammatory cytokines by a cytometric bead array (CBA)(BD Pharmingen) according to the manufacturer's instructions.

T cell intracellular cytokine stainingTo measure cytokine production by antigen specific T cells, surface and intracellular stainswere performed with monoclonal antibodies to CD8-Pacific Orange (Invitrogen), CD4-Pacific Blue (Invitrogen), Thy1.1-PerCP (BD Pharmingen), TNF-PE-Cy (BD Pharmingen),and IFN-γ-FITC (BD Pharmingen). Spleens of chimeric mice were processed into singlecells suspensions and plated onto 96-well flat-bottom plates at 106 cells per well. Cells werestimulated with 10nM OVA257–264 (Genscript, Inc.) and 10µM OVA323–339 (Genscript,

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Inc.) in the presence of 10µg/mL of Brefeldin A for 5 hours. Cells were processed with anintracellular staining kit (BD Biosciences) according to manufacturer’s instructions. As apositive control, cells were stimulated with 10ng/mL phorbol 12-myristate 13-acetate(PMA) and 1µg/mL ionomycin (Sigma). Data were acquired on a LSR II flow cytometer(Becton Dickinson) and analyzed using FlowJo Software (Treestar, San Carlos, CA).

DC purification, RNA isolation and real time PCR analysisFor DC isolation and purification, single cell suspensions were first enriched by negativeselection using magnetic beads coated with anti-CD19, CD90.1, and CD90.2 monoclonalantibodies as described above (yielding ~70% purity of CD11c+ cells). Followingenrichment, CD11c+ Thy 1.1− cells were further purified by FACS sorting on a BD FACSAria. Following FACS-sorting, DC populations were >90% CD11c+ cells.

RNAs from the sorted DCs were isolated using RNeasy Isolation kit (Qiagen). Reversetranscription of the RNA into cDNA was performed using Taqman reverse transcription kit(Roche). Mouse immune array cards (Applied Biosystems) were used to obtain a real-timePCR analysis of the selected immune related genes. Arrays were run on a 7900HT Real-Time PCR System from ABI.

ResultsCD154/CD40 pathway blockade prevents antigen-specific T cell-mediated host celldestruction

To evaluate the impact of CD154/CD40 blockade on the induction and/or abortion of donor-reactive T cell responses, we developed a modified GVHD model in which antigen-specificCD4+ and CD8+ T cells recognized and rejected cognate antigen-bearing hematopoietic cellsfollowing bone marrow transplantation. Using an ovalbumin expressing transgenic mouse(mOVA, B6 background), we generated hematopoietic chimeras in which busulfan-treatedB6 host mice (CD45.1+) received a CD45.2+ bone marrow transplant expressing a singledefined alloantigen (OVA). Adoptive transfer of 5×106 antigen-specific CD8+ (OT-I) and106 antigen-specific CD4+ (OT-II) T cells were sufficient to induce rejection of the CD45.2+

mOVA-expressing bone marrow cells such that by day 6, the number of donor-derived(CD45.2+) peripheral B cells was significantly reduced (Figure 1A). Both CD4+ and CD8+

T cells were required to mediate this effect, as adoptive transfer of either OT-I or OT-IIalone failed to result in rejection (Supplemental Figure 1 A, B). Importantly, treatment ofanimals with a short course of CD154 blockade (MR-1) on days 0, 2, 4, and 6 post-transferresulted in protection of the bone marrow from rejection (Figure 1A, B). These resultsindicated that blockade of the CD154/CD40 pathway alone was sufficient to prevent T cell-mediated destruction of OVA-expressing peripheral leukocytes and preserve hematopoieticchimerism.

CD154/CD40 blockade attenuates antigen-specific T cell expansion and effector functionTo address the potential mechanisms by which this protection occurred, we analyzed thehost-reactive CD4+ (OT-II) and CD8+ (OT-I) T cell responses in anti-CD154-treatedanimals. We observed that while host-reactive CD8+ T cells expanded dramatically inuntreated animals by day 4 post-transfer (Figure 2A–C), treatment with anti-CD154 resultedin a significant diminution in the antigen-specific CD8+ T cell response (p<0.0001). Asimilar result was observed for antigen-specific CD4+ T cell responses, which exhibited amore than two-fold reduction in the presence of CD154/CD40 blockade (Figure 2D–F). Thiswas true for both T cell frequencies and absolute numbers (Figure 2A–F).

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To address whether reduced frequencies of host-reactive T cells were due to reducedactivation and proliferation or increased cell death, host-specific CD8+ T cells were CFSElabeled prior to transfer into OVA.B6 chimeras. Results demonstrated similar levels ofCFSE dilution in anti-CD154 treated animals as compared to PBS-treated animals (Figure2E, F), suggesting that the reduced accumulation of host-reactive CD8+ T cells was likelydue to increased apoptosis, rather than impaired expansion, in the presence of CD40/CD154pathway blockade.

The functionality of the remaining graft-specific CD8+ T cells was further assessed byintracellular cytokine staining following ex vivo restimulation with cognate antigen peptideover time. Results revealed that anti-CD154 treatment delayed the differentiation of antigen-specific CD8+ T cells into cytokine-producing cells compared to untreated controls. Forexample, frequencies of IFN-γ+ producing cells at day 4 were significantly reduced, andfrequencies of IFN-γ+ TNF+ dual cytokine producers at day 10 were significantly reduced(Figure 3A, 3B). Absolute numbers of IFN-γ-producing cells were significantly reduced inanti-CD154 treated animals at day 4, but not at subsequent timepoints (Figure 3C). Takentogether, these results demonstrate that blockade of the CD40/CD154 pathway attenuatesand delays both expansion and differentiation of host-reactive T cells.

CD154 blockade inhibits provision of signal three from DC, but not provision of signal oneor signal two

The DC licensing model of T cell activation suggests that CD154 expressed on activatedCD4+ T cells binds to CD40 expressed on DCs and functions to initiate the upregulation ofclass I and class II MHC and several costimulatory molecules on these DCs, thus enhancingthe priming of antigen-specific T cell responses. Interestingly, however, in this GVHDmodel wherein both CD4+ and CD8+ antigen-reactive T cells are required to precipitaterejection of antigen-bearing cells (Supplemental Figure 1A, B), we observed that both celltypes were required in order to elicit optimum costimulatory molecule expression(Supplemental Figure 1C) and cytokine secretion (Supplemental Figure 1D) from dendriticcells. Because the observed effects of CD40/CD154 blockade on host-reactive T cellresponses were likely a direct result of alterations in DC phenotype and functionality, weendeavored to determine which aspects of DC activation were altered in the presence ofCD40/CD154 blockade. We first characterized the frequency and phenotype of splenic DCsin mOVA bone marrow chimeras following the adoptive transfer of host-reactive T cells.mOVA bone marrow chimeras contained 1.75±0.77×106 total DC per spleen (Figure 4A),and > 90% of those were CD45.2+, expressing the OVA antigen (data not shown). Adoptivetransfer of host-reactive CD4+ and CD8+ T cells did not result in a change in the totalnumber of splenic DCs at day 3 post-transfer, either in the presence or absence of CD154/CD40 blockade (1.59±0.44×106 and 2.04±1.04×106, respectively, Figure 4B). In addition,the relative proportions of CD11c+ CD11b+ CD8α− vs. CD11c+ CD11b− CD8α+DC werenot altered in the presence of CD154/CD40 blockade (Figure 4C–E). Thus, these datademonstrate that blockade of the CD154/CD40 pathway did not impact the overall quantityor myeloid/lymphoid phenotype of DCs in GHVD recipients.

We next examined the impact of CD154 blockade on the expression of MHC (signal one)and costimulatory (signal two) molecules on DCs. The DC licensing model predicts thatCD40 ligation results in upregulation of MHC and costimulatory molecules. This wasconfirmed in our model with experiments in which an agonistic anti-CD40 monoclonalantibody (FGK4.5) was injected into mice and splenic DCs were observed to express highlevels of MHC and costimulatory molecules (data not shown). Next, we confirmed thatCD4+ T cells in our system could provide the CD40 stimulus necessary for DC licensing.First, as predicted, CD154 was upregulated on antigen-specific CD4+ T cells (data notshown). Second, following adoptive transfer of host-reactive CD4+ and CD8+ T cells into

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mOVA chimeric recipients, provision of T cell-derived CD154 signals resulted in astatistically significant increase in class I (H2-Kb) expression on the surface of splenic DCsat day 3 post-transfer (Figure 5). Likewise, expression of CD86 and CD40 were alsoincreased (Figure 5). Surprisingly, however, treatment with CD40/CD154 blockade failed toattenuate the expression of class I or class II MHC, or the expression of costimulatorymolecules on total CD11c+ splenic DCs during graft-specific T cell priming (Figure 5). Wealso assessed the expression of costimulatory molecules on individual CD11c+ DC subsets(data not shown), and on CD11b+ CD11c− myeloid cells (Supplemental Figure 2) in animalsleft untreated or treated with anti-CD154 and again observed no difference in the expressionof class I or class II MHC or CD40, CD80, or CD86. These results therefore indicate thatdiminution in the provision of either signal one or signal two is not the mechanism by whichCD154 blockade attenuates graft-specific T cell responses and promotes long term graftsurvival.

Next, in order to assess the impact of CD154 blockade on the provision of signal three inthis model, we conducted similar experiments in which host-reactive Thy1.1+ CD4+ andCD8+ T cells were adoptively transferred into mOVA chimeric recipients. On day 3 post-transfer, CD11c+ splenic DCs were FACS-sorted and either immediately processed for real-time PCR analysis of mRNA or cultured in vitro for 24h for supernatant assessment ofcytokine production. Results demonstrated that the adoptive transfer of antigen-specific Tcells resulted in a profound increase in the ability of DC to produce pro-inflammatorycytokines. Specifically, DC isolated from T cell adoptive transfer recipients exhibited anincrease in IL-6 and TNF at the protein level, and in IL-12 and IL-1β at the mRNA level(Figure 6). Importantly, DCs isolated from mice that had been treated with CD154 blockadeexhibited significantly reduced IL-6, TNF, IL-12p35 and IL-1β production on day 3 post-Tcell transfer (Figure 6). Neither IL-12p35 nor IL-1β were detected at the protein level in anyof the groups, suggesting that the expression of these cytokines in this in vitro assay wasbelow the limit of detection. These data suggest that a predominant effect of CD154blockade in this system may be to inhibit the provision of inflammatory cytokines (signalthree) to antigen-specific T cells during priming.

Combined IL-6/IL-12p40 blockade attenuates antigen-specific T cell expansion andtransiently protects from GVHD

In order to assess the contribution of reduced provision of signal three on the observedprolongation in graft survival associated with treatment with anti-CD154, we utilized asimilar experimental approach in which adoptive transfer recipients of host-reactive CD4+

and CD8+ T cells were left untreated, treated with anti-CD154 as a positive control, ortreated with a combination of anti-IL-6R and anti-IL-12p40, two inflammatory cytokineswhich were diminished in DC isolated from anti-CD154 –treated recipients. We chose tointerrogate the impact of blockade of IL-6 and IL-12 in these experiments based on previousevidence in the literature indicating that these cytokines can profoundly impact both CD4+

and CD8+ differentiation programs (23–27). Results indicated that while untreated animalsexhibited a significant loss in OVA-expressing CD45.2+ B cells by day 4 post-transfer(Figure 7A, p=0.045), animals treated with either anti-CD154 or the combination of anti-IL-6R and anti-IL-12p40 did not exhibit a significant loss in chimerism. Importantly,combined anti-IL-6R/anti-IL-12p40 protected the animals from the host-reactive immuneresponse to a similar degree as anti-CD154 at this time-point (Figure 7A). We furtherevaluated the host-reactive CD4+ and CD8+ T cell responses in these animals and observedthat the reduction in the expansion of host-reactive CD4+ and CD8+ T cells in animalstreated with combined IL-6/IL-12p40 blockade was comparable to that observed in anti-CD154 treated animals (Figure 7B and 7C, p<0.05 as compared to untreated controls).However, analysis of both survival of OVA-expressing CD45.2+ B cells and expansion of

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host-reactive CD4+ and CD8+ T cells at later time points (days 7, 10, and 14 post-transplant)revealed that the protection afforded by IL-6/IL-12p40 blockade was not durable, in thatOVA-expressing cells were eventually rejected and host-reactive CD4+ and CD8+ T cellsexpanded to frequencies comparable to those observed in untreated animals (data notshown).

DiscussionWe observed that although blockade of CD40/CD154 did not alter the level of expression ofclass I or class II MHC or costimulatory molecules on the surface of dendritic cells, it didsignificantly alter the differentiation of these cells, specifically with regard to their ability tosecrete the inflammatory cytokines IL-6, IL-12p40 and TNF. These results suggest thatblockade of CD40 ligation on DC during the course of graft rejection critically impacts theprovision of signal three to developing donor-reactive T cell populations, with less of animpact on the provision of costimulation (signal two). These data provide a mechanisticbasis for the observed synergy between blockade of the CD40 and CD28 pathways, andsuggest that therapies designed to target the provision of inflammatory cytokines during thegeneration of donor-reactive T cell responses may be beneficial in attenuating theseresponses and prolonging graft survival.

Our results demonstrating that CD154 antagonism (MR-1) does not inhibit CD4+ T cell-induced upregulation of class I or class II MHC on DCs are surprising, given the known rolefor CD154 in DC licensing. Indeed, our results confirmed reports by many other groupsdemonstrating that activated CD154+ CD4+ T cells led to the upregulation of MHC andcostimulatory molecules on DCs (28–30). There are two potential reasons that anti-CD154failed to inhibit this activity. First, it is possible that the antibody does not completely blockcell-associated CD154 binding to CD40 expressed on the surface of DCs. Altered CD154binding could result in a partial signal delivered to the APC, resulting in the upregulation ofMHC and costimulatory molecules but not the elaboration of inflammatory cytokines. CD40signaling in DCs is mediated by binding of individual TRAFs to the intracellular domain,and previous studies have revealed that the CD40 TRAF2/3 binding site is critical forcostimulatory molecule expression, while the TRAF6 binding site is required for productionof inflammatory cytokines (31). Thus, it is possible that binding of anti-CD154 mAb inhibitsthe ability of TRAF6 but not TRAF2/3 to be recruited to the CD40 intracellular domain, andexperiments to test this hypothesis are ongoing. Our results are also consistent with thealternate possibility that there are CD154-dependent mechanisms by which cognate T cellscan upregulate costimulatory molecule expression, but not cytokine production, on DC.

Our results also suggest that this diminution in inflammatory cytokines likely contributes tothe observed ability of anti-CD154 to mitigate anti-host T cell responses, in that treatment ofanimals with a combination of anti-IL-6 and IL-12p40 antibodies partially recapitulated theeffects of anti-CD154. Specifically, antagonism of these cytokine transiently attenuated theexpansion of both antigen-specific CD4+ and CD8+ T cell populations and impaired theirability to reject host cells. These results are supported by evidence in the literaturesuggesting that both IL-12 and IL-6 mediated signals can function to enhance the expansionand differentiation of antigen-specific T cells. With regard to IL-12, seminal studies showedthat this cytokine was as effective as CFA for inducing clonal expansion, differentiation intocompetent effectors, and generation of long-lived memory cells in antigen-specific T cellpopulations (23–25). Importantly, the absence of IL-12 in these studies rendered the T cellstolerant (24), again suggesting that inhibition of inflammatory cytokine signaling may be animportant facet of CD154 antagonism. Likewise, IL-6 has been identified as a predominantinflammatory cytokine associated with the enhancement of alloimmune responses, insofar asneutralization of IL-6 was shown to significantly delay graft rejection, diminish

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differentiation of alloreactive effectors and impact the Th1/Th2 balance during allograftrejection (32, 33). Furthermore, the inhibition of IL-6 and IL-17 was shown to protectcardiac allografts from rapid rejection (34), and the combined reduction of TNF and IL-6has been shown in an in vitro system to reduce allograft specific T cell proliferation anddifferentiation (35).

Another interesting aspect of our study was the finding that adoptive transfer of both CD4+

and CD8+ antigen-specific T cells was required for the optimal upregulation of MHC andcostimulatory molecules and cytokines in this model. Traditional DC licensing modelswould suggest that binding of activated CD4+ T cells would be sufficient to license DC (28–30). However, our data are consistent with other reports in models of viral infection showingthat CD8+ T cells can “license” resting DC (36), and this effect was later shown to bemediated specifically through the elaboration of GM-CSF (37). Thus, future studies on theeffect of CD154 antagonism of CD8+ T cell-derived GM-CSF in this model are warranted.

Taken together, this study demonstrates that while CD40/CD154 pathway blockade does notsignificantly alter the costimulatory receptor profile of dendritic cells, it does alter theircytokine secretion profile. While blockade of two such inflammatory cytokines wasinsufficient to recapitulate the effects of CD40 blockade, the observed attenuation in host-reactive T cell expansion and delay in GVHD progression suggests that decreasedinflammatory cytokine production likely contributes to the observed effects of CD40/CD154blockade. Thus, targeting DC-derived inflammatory cytokines in clinical transplantationcould lead to attenuated alloreactivity and improved outcomes.

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.

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Figure 1. CD40/CD154 pathway blockade protects against T cell-mediated rejection of OVA-expressing host cellsOn Day 0, 5×105 OT-I and 106 OT-II T cells were adoptively transferred into mOVA bonemarrow chimeric mice. At the time of transfer, mice were treated with 500µg anti-CD154mAb (MR-1), with continued treatments on days 2, 4 and 6, in indicated groups. A.Peripheral blood B cell chimerism was measured. Representative flow plots of remainingB220+ cells, gating on the remaining mOVA+ (CD45.2+) cells, over time. B. Total numberof CD45.2+ B220+ mOVA+ B cells in the blood over time. Data are representative of threeexperiments with 4–5 mice per group. Statistics shown are mean±s.e.m. *p≤0.05.,***p≤0.0001.

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Figure 2. CD40/CD154 pathway blockade impairs host- specific CD8+ T cell accumulation butnot entry into cell divisionmOVA chimeric mice were adoptively transferred with 5×106 CFSE-labeled Thy1.1+ OT-Iand 106 CFSE-labeled Thy1.1+ OT-II, and treated with four doses of MR-1 on days 0, 2, 4,and 6, where indicated. A, D. Representative flow plots of antigen-specific Thy1.1+ T cellresponses in the spleens of treated mice. Data displayed are gated on Thy1.1+ CD8+ orCD4+ T cells on day 4 post-transfer. B, E. Frequency of accumulated antigen-specific CD8+

(B) and CD4+ (D) T cells at day 4 post-transfer. C, F. Absolute numbers of accumulatedantigen-specific CD8+ (C) and CD4+ (F) T cells at day 4 post-transfer. G, H. CFSE analysisof cell division of antigen-specific CD8+ T cells at days 2 and 3 post transfer in both

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untreated and anti-CD154 treated recipients. Data shown are gated on Thy1.1+ CD8+ T cellsin spleens of mice. Data are representative of 2–3 experiments with a total of 8–12 mice pergroup. Statistics shown are mean±s.e.m. *p≤0.05.

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Figure 3. CD154/CD40 blockade attenuates antigen-specific T cell expansion and effectorfunctionmOVA chimeric mice were adoptively transferred with 5×106 OT-I and 106 OT-II, andtreated with four doses of MR-1, where indicated. A. Representative flow plots oflongitudinal analysis of intracellular TNF and IFN-γ cytokine staining of antigen-specificThy1.1+ CD8+ T cells following 4-hour ex vivo peptide stimulation. B. Summary data offrequencies of IFN-γ-producing cells (day 4) and IFN-γ+ TNF+ dual producers (days 10,17, 24) as a percentage of antigen-specific Thy1.1+ CD8+ T cells. C. Absolute numbers oftotal IFN-γ producing OT-I T cells over time following adoptive transfer. Data are

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representative of 2–3 experiments with a total of 8–12 mice per group. Statistics shown aremean±s.e.m. *p≤0.05.

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Figure 4. CD40/CD154 pathway blockade does not impact the frequency and number of CD8α+

or CD11b+ dendritic cellsmOVA chimeric mice were adoptively transferred with 5×106 OT-I and 106 OT-II, andtreated with four doses of MR-1, where indicated. A. Representative flow plot of cells gatedon CD11c+ dendritic cells, excluding B cells (CD19), T cells (CD3) and NK cells (NK1.1).B. Total DCs in spleen of mice prior to and post-T cell transfer in the presence or absence ofMR-1, where indicated. C. Representative flow plots of dendritic cell populations, gated onCD11c+ cells. D. Frequency of CD11c+ CD11b+ CD8α− dendritic cells in spleens. E.Frequency of CD11c+ CD11b− CD8α+ dendritic cells in spleens. Data are representative of5 experiments with 3 mice per group. Statistics shown are mean±s.e.m.

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Figure 5. CD40/CD154 pathway blockade does not impact expression of MHC and costimulatorymolecules on dendritic cellsmOVA chimeric mice were adoptively transferred with 5×106 OT-I and 106 OT-II, andtreated with four doses of MR-1 on days 0, 2, 4, and 6, where indicated. Mice weresacrificed on day 3 and spleens were analyzed. A. Representative flow plots of CD11c+ DCsexpression of H-2Kb, CD86, and CD40 (black lines). Surface molecules on CD11c+ DCs inthe absence of T cell transfer (shaded grey histograms). B. Summary data of relativeexpression (MFI) of surface molecules on CD11c+ dendritic cells isolated from spleens ofmice. Data are representative of 5 experiments with 3 mice per group. Statistics shown aremean±s.e.m. **p≤0.01, ***p≤0.0001.

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Figure 6. CD40/CD154 pathway blockade impairs dendritic cell production of inflammatorycytokinesmOVA chimeric mice were adoptively transferred with 5×106 OT-I and 106 OT-II T cellsand were treated with anti-CD154 on days 0, 2, 4, and 6, where indicated. Mice weresacrificed on day 3 post transfer. Splenic dendritic cells were isolated, FACS-sorted andcultured in vitro for 24 hours. Secretion of IL-6 and TNF by isolated DCs was measured insupernatants via cytokine bead array analysis. Production of IL-1β and IL-12p35 transcriptswere measured by real time PCR in sorted CD11c+ DC. Data are representative of twoexperiments with 3 mice per group. Statistics shown are mean±s.e.m. *p≤0.05.

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Figure 7. Combined IL-6/IL-12p40 antagonism attenuates antigen-specific T cell expansion andtransiently protects from GVHDmOVA chimeric mice were adoptively transferred with 5×106 OT-I and 106 OT-II T cellsand were treated with anti-IL-6R and anti-IL12p40 on days 0, 2, 4, and 6, where indicated.A. Total number of CD45.2+ mOVA+ B cells in the blood prior to T cell transfer (day 0) andon day 4 post transfer. B. Representative flow plots of antigen-specific Thy1.1+ T cellresponses in the spleens of treated mice. Data displayed are gated on Thy1.1+ CD8+ orCD4+ T cells on day 4 post-transfer. C. Frequency of antigen-specific CD8+ and CD4+ Tcells at day 4 post-transfer. Data are representative of 2 experiments with a total of 6 miceper group. Statistics shown are mean±s.e.m. *p≤0.05.

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