Antigen-specific induced Foxp3+ regulatory T cells are generated following CD40/CD154 blockade Ivana R. Ferrer, Maylene E. Wagener, Minqing Song, Allan D. Kirk, Christian P. Larsen, and Mandy L. Ford1 Emory Transplant Center and Department of Surgery, Emory University, Atlanta, GA 30322 Edited by James P. Allison, Memorial Sloan-Kettering Cancer Center, New York, NY, and approved November 4, 2011 (received for review April 7, 2011) Blockade of the CD40/CD154 pathway potently attenuates T-cell in vivo accumulation of Treg and deletion of graft-specific ef- responses in models of autoimmunity, inflammation, and trans- fectors after CD40/CD154 blockade has not been investigated. plantation. Indeed, CD40 pathway blockade remains one of the To address these questions, we used an ovalbumin (OVA)- most powerful methods of prolonging graft survival in models of expressing transgenic mouse model (20), which allowed us to di- + + transplantation. But despite this effectiveness, the cellular and rectly track both donor-reactive CD4 and CD8 T-cell responses molecular mechanisms underlying the protective effects of CD40 simultaneously over time. Although previous studies have used pathway blockade are incompletely understood. Furthermore, the transgenic models to assess the degree of T-cell deletion after CD154/CD40 blockade (16, 21), none has systematically inter- relative contributions of deletion, anergy, and regulation have not + been measured in a model in which donor-reactive CD4+ and CD8+ rogated the impact of anti-CD154 on the kinetics of both CD4 and CD8+ T-cell deletion and acquisition of effector function. T-cell responses can be assessed simultaneously. To investigate the fi Our results indicated that although CD40/CD154 blockade alone impact of CD40/CD154 pathway blockade on graft-speci c T-cell delayed donor-reactive CD4+ and CD8+ T-cell expansion and responses, a transgenic mouse model was used in which recipients differentiation, the addition of DST was required for antigen- fi + + containing ovalbumin-speci cCD4 and CD8 TCR transgenic T specific T-cell deletion. Importantly, anti-CD154 combined with − cells were grafted with skin expressing ovalbumin in the presence DST also led to peripheral conversion of Foxp3 donor-reactive or absence of anti-CD154 and donor-specific transfusion. The re- + + + CD4 T cells to Foxp3 CD25 donor-specificTreg. sults indicated that CD154 blockade altered the kinetics of donor- reactive CD8+ T-cell expansion, delaying differentiation into IFN-γ+ Results + IMMUNOLOGY TNF multifunctional cytokine producers. The eventual differenti- Combined DST/CD154 Blockade Prolongs Skin Graft Survival and ation of cytokine-producing effectors in tolerant animals coincided Prevents Cellular Infiltration. Although many groups have studied + hi + with the emergence of an antigen-specific CD4 CD25 Foxp3 the impact of CD40 pathway blockade on graft survival and T-cell T-cell population, which did not arise from endogenous natural responses (16, 19, 21, 22), the precise mechanism by which in- − Treg but rather were peripherally generated from naïve Foxp3 hibition of CD40-mediated signals attenuates donor-reactive T- precursors. cell responses remains incompletely understood. In fully allogeneic models, anti-CD154 mAbs combined with DST has been shown to costimulation blockade significantly delay rejection, but the ability to track alloreactive T cells is dependent on cytokine production. This limits the ability to fi lockade of the CD40/CD154 pathway has long been appreci- study donor-speci c cells that might have become nonresponsive Bated as a potent means of inhibiting alloreactive T-cell due to the absence of CD40/CD154-mediated signals. Therefore, responses and prolonging graft survival. But despite this pro- to assess the contribution of T-cell nonresponsiveness to the pro- tective effect of CD154 blockade on skin graft (SG) survival, naïve nounced effect on allograft survival in both murine and nonhuman donor-reactive OT-I and OT-II T cells were adoptively transferred primate models (1–3), clinical trials using anti-CD154 mAbs in into naïve recipients that were then grafted with OVA-expressing transplantation were halted due to thromboembolic complica- skin (Fig. 1A). Resulting SG survival in this transgenic model tions, likely related to the expression of CD154 on platelets (4, 5). reflected that previously observed in allogeneic systems (13). Un- However, recent studies using anti-CD40 mAbs in both murine treated mice rapidly rejected SGs with a median survival time and nonhuman primate models demonstrated comparable efficacy fi (MST) of 13.5 d, whereas treatment with anti-CD154/DST signif- to CD154 blockade, and thus signi cant interest in the pathway icantly prolonged survival to more than 100 d (Fig. 1B). remains (6–9). Early studies by Parker et al. (3) described the treatment com- CD154 Blockade/DST Inhibits Antigen-Specific CD8+ T-Cell Expansion. bination of anti-CD154 and donor-specific transfusion (DST) To identify the relative contributions of T-cell deletion and anergy/ for the induction of immune tolerance in transplantation. Several exhaustion, the kinetics of donor-reactive CD8+ T-cell expansion groups have since demonstrated the potent effects of this com- and contraction were measured over time after transplantation bined therapy in the prolongation of islet (3, 10, 11), cardiac (12), and treatment with anti-CD154/DST on days 0, 2, 4, and 6 post- skin (10, 13), and kidney (14, 15) allograft survival in murine and transplantation. Spleens of untreated mice developed a donor- nonhuman primate models. Although costimulation blockade has reactive T-cell response that peaked at days 7–10. CD154 block- been associated with T-cell anergy (16) or deletion (17, 18), elu- ade led to a significant delay (day 10) in the donor-reactive T-cell cidation of the precise independent and synergistic effects of DST response, whereas this response was significantly accelerated (day and anti-CD154 on the kinetics of donor-reactive CD4+ and 4) in DST-treated animals. Finally, combined anti-CD154/DST CD8+ effector T-cell expansion has been limited because of the treatment led to minimal expansion of donor-reactive T cells over inability to identify and track graft-specific T cells in fully allo- geneic models. Importantly, studies by Taylor et al. (19) demonstrated that Author contributions: I.R.F., C.P.L., and M.L.F. designed research; I.R.F., M.E.W., and M.S. + + CD4 CD25 regulatory T cells (Treg) are required for tolerance performed research; I.R.F., M.S., A.D.K., C.P.L., and M.L.F. analyzed data; and I.R.F. and induced via CD40/CD154 pathway blockade in a graft-versus- M.L.F. wrote the paper. host model. Although those studies revealed a requirement for The authors declare no conflict of interest. Treg during tolerance induction, whether the Treg required are This article is a PNAS Direct Submission. preexisting, thymically derived Treg or whether CD40/CD154 1To whom correspondence should be addressed. E-mail: [email protected]. blockade induces the generation of peripherally elicited Treg is This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. not known. Furthermore, the temporal relationship between the 1073/pnas.1105500108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1105500108 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 A D-2: Adoptive transfer A Day 4 Day 7 Day 10 Day 14 0.336 2.26 2.91 0.272 mOVA Assess: Skin Graft SG survival T cell activation Untreated and 0.273 0.422 3.31 0.509 anti-CD154: 0246 B 100 Anti-CD154 Untreated 5.88 0.697 0.346 0.165 -CD154 Thy1.1 75 DST DST/ -CD154 DST 50 1.65 0.192 0.292 0.553 25 Percent survival Percent DST/ 0 Anti-CD154 0 20406080100 CD62L POD Fig. 1. CD154 blockade and DST prolongs SG survival. (A) Mice were Frequency 6 B adoptively transferred with 1.5 × 10 of each OT-I and OT-II T cells 2 d before 7 Untreated -CD154 transplantation. On day 0, mice were transplanted with mOVA SG and were 6 treated with 107 mOVA DST and/or 500 μg of MR-1 where indicated. (B) + DST 5 Untreated mice rejected SGs with an MST of 13.5 d. Anti-CD154 mono- -CD154/ DST 4 therapy led to bimodal survival of SGs, with 50% of mice rejecting grafts Thy1.1 + with an MST of 15 d and 50% demonstrating indefinite survival (P = 0.0027). 3 DST monotherapy resulted in to an MST of 18 d (P = 0.031), whereas com- 2 fi < % CD8 bined anti-CD154/DST led to an inde nite survival of SGs (P 0.0001). Data 1 are cumulative of two independent experiments with five mice per group. 0 051015 POD background at day 4 (Fig. 2 A and B). These effects of CD154 *** *** * *** blockade on donor-reactive T-cell expansion kinetics were fur- C 100 60 * 80 fi 70 ther con rmed in the axillary and brachial draining lymph nodes 80 60 OT-I OT-I (LNs) (Fig S1). OT-I 40 50 60 high low To determine whether CD154/CD40 pathway blockade limits high fi 40 antigen-speci c T-cell differentiation, we analyzed markers of 40 30 20 20 % CD44 activation (CD44 and CD62L) and a marker of short-lived ef- % CD62L 20 % KLRG-1 fector lineage (KLRG-1) (23) in each treatment group on day 7 10 posttransplantation. We observed that CD154 blockade, in both 0 0 0 the presence and absence of DST, decreased the frequencies of DST DST DST high low + -CD154 -CD154 -CD154 CD44 and CD62L antigen-specific CD8 T cells and in- Untreated Untreated Untreated -CD154/ DST -CD154/ DST -CD154/ DST creased the frequency of KLRG-1high antigen-specific CD8+ T cells compared with untreated controls (Fig. 2C).