Copyright 2014 Cognizant Comm. Corp. E-ISSN

Cell Transplantation, Vol. 23, pp. 355 363, 2014 0963-6897/14 $90.00 +.00 Printed in the USA. All rights reserved. DOI: http://dx.doi.org/10.3727/096368912x661346 Copyright 2014 Cognizant Comm. Corp. E-ISSN 1555-3892 www.cognizantcommunication.com Rapid Dendritic Cell Activation and Resistance to Allotolerance Induction in Anti-CD154-Treated Mice Receiving CD47-Deficient Donor-Specific Transfusion Yuantao Wang,* 1 Hui Wang, 1 Roderick Bronson, Yaowen Fu,* and Yong-Guang Yang* *First Hospital of Jilin University, Changchun, China Columbia Center for Translational Immunology, Columbia University College of Physicians and Surgeons, New York, NY, USA Department of Pathology, Harvard Medical School, Boston, MA, USA CD47 SIRPa signaling plays an important role in regulating macrophage and dendritic cell (DC) activation. Here we investigated the role of CD47 expression on donor cells in tolerance induction by combined treatment with donor-specific transfusion (DST) plus anti-cd154 mab in a mouse model of fully MHC-mismatched heart allotransplantation. The majority of BALB/c recipient mice that received anti-cd154 and CD47 +/+ B6 splenocytes (DST) showed indefinite donor heart survival (median survival time, MST > 150 days). Donor heart survival was improved in anti-cd154-treated BALB/c mice that received CD47 +/ (MST = 90 days) or CD47 / B6 DST (MST = 42 days) when compared to the nontreated (MST = 7 days) and anti-cd154 alonetreated (MST = 15 days) controls, but significantly reduced when compared to mice receiving anti-cd154 plus CD47 +/+ B6 DST. Recipient mice treated with anti-cd154 plus CD47 / or CD47 +/ DST also showed significantly increased antidonor, but not anti-third-party, MLR responses compared to those receiving anti-cd154 and CD47 +/+ DST. Furthermore, CD47 / DST induced rapid activation of CD11c hi SIRPa hi CD8a DCs via a mechanism independent of donor alloantigens. These results demonstrated that CD47 expression on donor cells is essential to the success of tolerance induction by combined therapy with DST and CD40/CD154 blockade. Key words: CD47; Costimulatory blockade; Dendritic cells (DC); Donor-specific transfusion (DST); Signal regulatory protein-a (SIRPa); Transplantation INTRODUCTION Chronic rejection and complications associated with nonspecific immunosuppressive medications remain the major factors limiting long-term allograft survival in clinical organ transplantation. Great effort has been invested to develop strategies for overcoming these problems, including protocols for inducing tolerance. Despite limited clinical success, allograft tolerance can now be routinely achieved in animal models (11). Further defining the mechanisms for tolerance induction would help translate the successful protocols of tolerance induction from laboratory animals to humans. Donor-specific transfusion (DST) has been shown to prolong allograft survival or induce tolerance, particularly when used in combination with costimulatory blockade (13,20,21). Treatment with anti-cluster of differentiation 154 (CD154) inhibits alloresponses of CD4 T-cells and, when used in combination with CD8 depletion or DST, results in long-term mixed chimerism and permanent tolerance (27), suggesting that DST has the potential to suppress CD8 T-cell alloresponses. Consistently, marked prolongation of skin allograft survival has been achieved in a major histocompatibility complex (MHC) class I-mismatched B6-to-bm1 (or bm1-to-b6) combination after DST (25). Using this model, we recently assessed the role of CD47, a ligand for an inhibitory receptor signal regulatory protein-a (SIRPa) expressed by monocytes/macrophages and dendritic cells (DCs), in tolerance induction by DST. We showed that CD47 expression on DST donor cells is required for inhibiting antidonor CD8 T-cell responses and improving donor skin graft survival in DST-treated recipients (31). In the present study, we demonstrate that the lack of CD47 expression on donor cells significantly impaired tolerance induction by combined treatment with anti-cd154 mab and DST in a fully MHC-mismatched B6-to-BALB/c combination. Received September 30, 2011; final acceptance December 4, 2012. Online prepub date: January 2, 2013. 1 These authors provided equal contribution to this work. Address correspondence to Yong-Guang Yang, M.D., Ph.D., Columbia University College of Physicians and Surgeons, 650 West 168th St., New York, NY 10032, USA. E-mail: yy2324@columbia.edu or Yaowen Fu, M.D., Ph.D., First Hospital of Jilin University, 71 Xinmin St., Changchun, China. E-mail: fuyaowen@medmail.com.cn IP: 5.10.31.210 On: Mon, 355 09 Apr 2018 09:34:25

356 WANG ET AL. Moreover, we identified that SIRPa hi, but not SIRPa lo/, CD11c hi CD8a DCs show rapid activation after CD47 / DST, and their activation is induced by an alloantigenindependent mechanism and cannot be prevented by treatment with anti-cd154 mab. MATERIALS AND METHODS Animals Female mice (6 10 weeks of age) were used in all experiments. C57BL/6 (B6), BALB/c, B10.A, and green fluorescent protein (GFP)-B6 [C57BL/6-Tg(UBC-GFP) 30Scha/J] mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). CD47 heterozygote (CD47 +/ ) B6 mice were generated by breeding of homozygous CD47 knockout (KO; CD47 / ) B6 (kindly provided by Dr. P-A. Oldenborg, Umeå University, Umeå, Sweden) (16,18) and CD47 +/+ wild-type (WT) B6 mice. GFP-transgenic CD47 +/ (GFP-CD47 +/ ) B6 mice were generated by crossing CD47 / B6 mice with GFP-B6 mice and were used to produce GFP-CD47 / B6. Care of animals was in accordance with the Guide for the Care and Use of Laboratory Animal. Protocols involving animals were approved by the Subcommittee on Research Animal Care of the Massachusetts General Hospital and Columbia University Medical Center, and all of the experiments were performed in accordance with the protocols. DST and Recipient Conditioning Splenocytes from CD47 +/+, CD47 +/, and CD47 / B6 mice were depleted of erythrocytes using ammonium chloride-potassium (ACK) lysing buffer (Cambrex Bio Science Walkersville, Walkarsville, MD, USA) and intravenously injected (DST; 1 10 7 cells per mouse) into fully MHC-mismatched BALB/c recipients 7 days prior to cardiac transplantation. Anti-mouse CD154 mab (MR1; 0.25 mg/mouse; National Cell Culture Center, Minneapolis, MN, USA) was administered intraperitoneally 7 (approximately 2 h preceding DST), 5, and 3 days prior to cardiac transplantation. Heterotopic Cardiac Transplantation Heterotopic cardiac transplantation was performed from WT B6 mice into BALB/c mice according to the previously described technique (8). Briefly, the cardiac grafts were transplanted by suturing (11-0 nylon thread, Ethicon Endo-Surgery, Somerville, NJ, USA) of donor aorta and donor pulmonary artery end-to-side to lower abdominal aorta and inferior vena cava, respectively. Ischemic time averaged 35 min. Graft function and survival were monitored every other day by abdominal palpation. Rejection was defined as complete cessation of a palpable beat and confirmed by direct visualization at laparotomy. Cardiac grafts were harvested from recipient mice on the day of complete cessation of palpable beat or at the indicated times (for surviving grafts), fixed on formalin (Fisher Scientific, Fair Lawn, NJ, USA), sectioned, and stained with hematoxylin and eosin (H&E; Sigma- Aldrich, St. Louis, MO, USA). Three slides of each sample were evaluated by an independent pathologist. Mixed Lymphocyte Reaction (MLR) Splenocytes were prepared from BALB/c recipients at days 7 and 21 post-dst, depleted of erythrocytes, and T-cells were enriched by negative selection using the mouse Pan T-Cell Isolation Kit (Miltenyi Biotec, Auburn, CA, USA) according to the manufacturer s protocol. Purified BALB/c recipient T-cells (responders; 2 10 5 ) were incubated with 2 10 5 irradiated (30 Gy; X-ray irradiator; Red Source Technologies, Inc., Suwanee, GA, USA) naive B6 or B10.A (third party) splenocytes (stimulators) in a total volume of 200 ml in round-bottomed 96-well plates (Costar, Corning, NY, USA, USA) for 3 days. The cultures were then pulsed with 1 mci [ 3 H]thymidine (Sigma-Aldrich) for 16 h, harvested, and counted on a beta scintillation counter (ICN Radiochemicals, Irvine, CA, USA). Data are expressed as stimulation index (cpm of stimulated culture/cpm of unstimulated culture). Flow Cytometric Analysis Cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-h-2d b (KH95), R-phycoerythrin (R-PE)-conjugated anti-mouse CD86 or MHC class II (I-A d ), allophycocyanin (APC)-conjugated anti-mouse CD11c, and propidium iodide (four colors) or with FITC-conjugated anti-mouse CD11c, R-PE-conjugated anti-i-a d, APC-conjugated anti-mouse SIRPa, Pacific Blue- conjugated anti-mouse CD4, APC/cy7-conjugated CD8a, and propidium iodide (six colors). Nonspecific binding of labeled mabs was blocked with 2.4G2 [rat antimouse fragment crystallizable, g receptor (FcgR) mab]. All mabs used were purchased from BD Biosciences (San Diego, CA, USA). Flow cytometric (FCM) analysis was performed on FACSCalibur or FACSCanto flow cytometry (BD Biosciences, Mountain View, CA, USA). Dead cells were excluded by gating out low forward scatter plus high propidium iodide-retaining cells. Statistical Analysis Graft survival data were presented as Kaplan Meier survival curves, and differences between groups were analyzed by log-rank test using GraphPad Prism (San Diego, CA, USA). Differences between group means were evaluated by one-way ANOVA followed by Bonferroni correction for post hoc t test. A value of p < 0.05 was considered to be significant.

CD47 IN TRANSPLANTATION TOLERANCE 357 RESULTS Allograft Acceptance in Mice Treated With Anti-CD154 mab and DST Requires CD47 Expression on DST Donor Cells The role of CD47 expression on donor cells in tolerance induction by combined treatment with anti-cd154 mab and DST was assessed in a fully MHC-mismatched (B6àBALB/c) heterotopic cardiac transplantation model. In untreated control BALB/c mice, B6 cardiac allografts were rapidly rejected with a median survival time (MST) of 7 days (Fig. 1A). While treatment with anti- CD154 mab alone significantly delayed allograft rejection (MST = 15 days) compared to untreated controls (p < 0.001), none of these mice showed long-term graft acceptance (Fig. 1A). The rejected cardiac allografts in both groups were histologically characterized by severe mononuclear cell infiltration, a feature of cellular rejection (Fig. 1B-a, b). By contrast, the majority of mice treated with anti-cd154 plus CD47 +/+ DST showed long-term allograft survival (Fig. 1A) (MST > 150 days; p < 0.0001 compared to untreated and anti-cd154 alone controls) with normal heart histology (Fig. 1B-e). However, anti- CD154 plus CD47 / DST was significantly less effective in prolonging allograft survival. Although allograft rejection was significantly delayed in mice treated with anti- CD154 plus CD47 / DST compared to those treated with anti-cd154 alone (p < 0.05), none of these mice showed long-term graft survival (MST = 42 days) and nearly half of the mice rejected allografts as rapidly as those treated with anti-cd154 alone (Fig. 1A). The allograft survival in recipient mice treated with anti-cd154 plus CD47 +/ DST (MST = 97 days) was significantly better than anti- CD154 plus CD47 / DST-treated (p < 0.05), but worse than anti-cd154 plus CD47 +/+ DST-treated (p < 0.05) mice (Fig. 1A). Since CD47 +/ cells express a lower level of CD47 than CD47 +/+ cells (19,31), these data indicate a Figure 1. Donor-specific transplantation (DST) using cluster of differentiation 47 heterozygous and knockout (CD47 +/ and CD47 / ) splenocytes significantly impairs allograft survival in anti-cd154 mab-treated recipients. Naive BALB/c mice (nontreated) and BALB/c mice that were treated with three injections (IP) of anti-cd154 (MR1; 0.25 mg/injection at days 7, 5, and 3) (anti-cd154 alone) or with anti-cd154 plus intravenous injection of 1 10 7 splenocytes (DST) from CD47 +/+, CD47 +/, or CD47 / B6 donors at day 7 were grafted with CD47 +/+ B6 hearts at day 0. (A) Donor heart graft survival. (B) Hematoxylin and eosin (H&E) staining of representative heart allografts from (a) nontreated (rejected at day 7), (b) anti-cd154 alone (rejected at day 16), (c) CD47 / DST/ anti-cd154 (rejected at day 22), (d) CD47 +/ DST/anti-CD154 (rejected at day 63), (e) CD47 +/+ DST/anti-CD154 (a surviving graft harvested at day 150) groups, and (f) a naive mouse (scale bar: 100 mm; original magnification: 200 ). (C) Antidonor (B6) and thirdparty (B10.A) mixed lymphocyte reaction (MLR) responses measured 7 and 21 days post-dst/1 st anti-cd154 injection (i.e., days 0 and 14 after heart transplant, respectively). Three mice from each group at each time point were analyzed. **p < 0.01 and ***p < 0.001 between the indicated groups.

358 WANG ET AL. positive correlation between the survival times of heart allografts and the levels of CD47 expression on transfused donor cells. The rejected allografts in mice treated with anti- CD154 plus CD47 / or CD47 +/ DST also showed severe mononuclear cell infiltration at histology (Fig. 1B-c, d). These data demonstrate that the lack of or reduced CD47 expression on DST cells can significantly impair tolerance induction by treatment with anti-cd154 and DST. MLR assay revealed that CD47 expression on DST donor cells is required for suppressing anti-donor T-cell responses in mice treated with anti-cd154 plus DST. Compared to T-cells from CD47 +/+ DST-treated mice, a significant greater antidonor MLR was seen in mice receiving anti-cd154 alone or anti-cd154 plus CD47 / DST at day 7 post-dst (prior to donor heart transplantation) (Fig. 1C). Although a similar level of suppression was seen between anti-cd154-treated mice receiving CD47 +/+ versus CD47 +/ DST at day 7 post-dst, T-cells from the latter group mediated significantly stronger antidonor MLR than those from the former group at day 21 post- DST (i.e., 14 days after heart transplantation) (Fig. 1C). The comparable levels of anti-third-party MLR between these groups indicate that the immunosuppressive effect of DST plus anti-cd154 mab is donor specific. CD47 /, But Not CD47 +/, B6 Donor Cells Are More Rapidly Cleared Than CD47 +/+ B6 Donor Cells After Injection Into Allogeneic BALB/c Mice Previous studies have shown that, although CD47 +/ cells are not susceptible to phagocytosis under steady conditions, these cells appeared more sensitive than CD47 +/+ cells to phagocytosis under inflammatory conditions (14). However, we have shown that splenocytes of CD47 +/ B6 mice survived similarly as those from CD47 +/+ B6 mice after injection into MHC class I-mismatched bm1 mice (31). To determine whether CD47 +/ B6 splenocytes have disadvantages in survival compared to CD47 +/+ B6 splenocytes in fully MHC-mismatched BALB/c mice treated with anti-cd154 mab, we injected CD47 /, CD47 +/, and CD47 +/+ GFP-Tg B6 mouse splenocytes into anti-cd154- treated BALB/c mice and followed the survival of the injected donor cells. Unlike CD47 / B6 splenocytes that were significantly more rapidly rejected than CD47 +/+ splenocytes, no difference in survival was detected between CD47 +/ and CD47 +/+ B6 splenocytes after injection into fully MHC-mismatched BALB/c mice (Fig. 2). It is possible that CD47 / DST may not mediate efficient deletion of donor-reactive T-cells due to their rapid rejection by macrophages and DCs and thereby fails to induce immune suppression. However, the similar survival of donor cells in anti-cd154-treated mice receiving CD47 +/ versus CD47 +/+ DST and reduced allograft survival in the former group indicate that the role of CD47 in DST is more than just preventing phagocytosis of donor cells. Figure 2. Clearance of CD47 /, CD47 +/, and CD47 +/+ B6 splenocytes in BALB/c mice. BALB/c mice were treated with anti-cd154, followed about 2 h later by intravenous injection of 1 10 7 splenocytes from CD47 +/+, CD47 +/, or CD47 / green fluorescent protein (GFP)-B6 mice. Levels (%) of surviving GFP + donor cells in white blood cells were measured by flow cytometry at the indicated times. Results (mean ± SD) shown are normalized with the values at 1 h after cell transfer as 100%. CD47 / DST Results in Rapid Activation of Recipient CD11c hi SIRPa hi CD4 + CD8a DCs Independently of Alloresponses CD47 / DST induces rapid activation of recipient CD11c hi DCs in a MHC class I-mismatched B6àbm1 model (31). Here we observed that CD47 / DST mediates a similar effect on recipient DCs in anti-cd154-treated fully MHC-mismatched allograft recipients. A significant increase in CD11c hi CD86 + and CD11c hi I-Ad hi cells (i.e., activated CD11c hi DCs) was detected in BALB/c recipients of CD47 / DST compared to those receiving CD47 +/+ DST 24 h after DST/anti-CD154 mab treatment (Fig. 3). However, despite the reduced ability of CD47 +/ DST to prolong allograft survival in anti-cd154-treated recipients, CD11c hi DC activation was not seen in these mice 24 h (Fig. 3) or 7 days (data not shown) after DST compared to those receiving CD47 +/+ DST. It is possible that DC changes in response to CD47 +/ DST were below the sensitivity of FCM analysis or our assessments might have missed the peak responses. Alternatively, the data may suggest that CD47 expression can also facilitate tolerance induction via mechanisms other than preventing DC activation in mice treated with DST plus anti-cd154. The rapid (by 24 h) elevation of CD86 and I-Ad expression on CD11c hi DCs from mice treated with anti- CD154 and CD47 / DST suggests that DC activation in these mice is likely induced by mechanisms independent of alloresponses. To confirm this, we compared CD86 and I-A b expression on CD11c hi DCs in CD47 +/+ B6 mice receiving CD47 +/+ or CD47 / B6 splenocytes. Similar to allogeneic combinations, a significant increase in CD11c hi CD86 + and CD11c hi I-Ab hi cells was also seen

CD47 IN TRANSPLANTATION TOLERANCE 359 Figure 3. Lack of CD47 expression on DST cells induces rapid activation of CD11c hi DCs. BALB/c recipients (n = 4 per group) were treated with anti-cd154 alone or anti-cd154 plus DST from CD47 /, CD47 +/, or CD47 +/+ B6 donors, and recipient (H-2D d+ ) splenic CD11c hi DC activation was measured 24 h after DST/anti-CD154 treatment by flow cytometric (FCM) analysis of CD86 and major histocompatibility complex class II (I-Ad) expression. (A) Percentages of CD11c hi CD86 + and CD11c hi I-Ad hi cells in total splenic CD11c hi cells. *p < 0.05 and **p < 0.01 between the indicated groups. (B) Representative flow cytometry profiles showing I-Ad and CD86 expression on gated CD11c hi splenocytes. FSC, forward scatter. in CD47 /, but not CD47 +/+, splenocyte-injected B6 mice compared to naive (i.e., non-dst) B6 mice (Fig. 4). If rapid CD11c hi DC activation in mice receiving CD47 / splenocytes was due to the lack of CD47 SIRPa inhibitory signaling, only SIRPa + DCs should be affected. To address this question, we compared the activation of CD11c hi SIRPa hi versus CD11c hi SIRPa low/ DCs in BALB/c mice treated with DST alone or DST plus anti-cd154. Compared to BALB/c mice that received CD47 +/+ syngeneic BALB/c or allogeneic B6 splenocytes, CD47 / B6 DST induced rapid activation (shown by elevated I-Ad expression) of CD11c hi SIRPa hi, but not CD11c hi SIRPa low/ DCs, in both anti-cd154-treated and untreated recipients (Fig. 5A, B). Furthermore, the majority of CD11c hi SIRPa hi DCs were CD4 + CD8a, but almost all CD11c hi SIRPa low/ DCs were CD4 (either CD8a + or CD8a ) (Fig. 5C). DISCUSSION CD47 is a ligand of SIRPa, an inhibitory receptor expressed on myeloid cells including macrophages and DCs (2). CD47 SIRPa interaction was initially

360 WANG ET AL. Figure 4. Rapid activation of CD11c hi DCs in CD47 +/+ B6 mice receiving syngeneic CD47 / DST. Naive WT B6 mice (n = 4 per group) received no treatment (non-dst) or 1 10 7 of CD47 +/+ or CD47 / B6 splenocytes, and splenic CD11c hi DC activation was assessed 24 h later by FCM analysis of CD86 and I-Ab expression. Data shown are percentages of CD11c hi CD86 + and CD11c hi I-Ab hi cells in total splenic CD11c hi cells. **p < 0.01 between the indicated groups. discovered to deliver inhibitory signals in macrophages. Previous studies using CD47 KO mice demonstrated that encounter of macrophages with CD47-deficient hematopoietic cells results in macrophage activation and phagocytosis (18,29). In agreement with this finding, we have shown that the lack of cross-species interaction between CD47 and SIRPa induces phagocytosis of xenogeneic hematopoietic cells (12,28,30). Further studies indicate that CD47 on lymphohematopoietic cells also delivers negative signals to DCs (5,29). We recently observed that CD47 expression on donor cells is required for DST-induced immunosuppression in a mouse model of MHC class I H-2k-mismatched skin transplantation (31). In the present study, we show that the lack of or reduced CD47 expression on DST cells can significantly diminish tolerance to fully MHC-mismatched cardiac allografts in mice treated with anti-cd154 and DST. We also show that the lack of CD47 expression on DST cells induces Figure 5. CD47 / DST results in recipient CD11c hi SIRPa hi CD4 + CD8 DC activation. BALB/c mice received DST from CD47 +/+ B6, CD47 / B6, or CD47 +/+ BALB/c mice with or without anti-cd154, and splenic CD11c hi DC activation was assessed 24 h later by FCM analysis of I-Ad expression (n = 3 per group). (A) Representative flow cytometry profiles showing I-Ad expression on gated CD11c hi SIRPa lo/ and CD11c hi SIRPa hi DCs from BALB/c mice treated with anti-cd154 plus DST (left) or DST alone (right). (B) Percentages of CD11c hi SIRPa hi I-Ad hi (left) and CD11c hi SIRPa lo/- I-Ad hi (right) cells in splenocytes. *p < 0.05, **p < 0.01, ***p < 0.001 between the indicated groups. (C) Representative FCM profiles showing CD4 and CD8a expression on gated CD11c hi SIRPa hi and CD11c hi SIRPa lo/ splenic DCs. SIRPa, signal regulatory protein-a.

CD47 IN TRANSPLANTATION TOLERANCE 361 rapid activation of CD11c hi SIRPa hi CD4 + CD8a DCs via an alloantigen-independent mechanism, which cannot be prevented by treatment with anti-cd154 mab. Moreover, the levels of CD47 expression on DST cells were found to be positively correlated with the strength of DST to suppress antidonor immune responses. These results indicate that the CD47 SIRPa pathway may provide a new therapeutic target for inhibiting alloresponses and inducing transplant tolerance. Furthermore, our data suggest that the lack of cross-species interaction between CD47 and SIRPa (12,26,30) may be a reason for the inefficient immunosuppression or tolerance induction by DST in discordant xenogeneic combinations (34). The failure of DST plus anti-cd154 to suppress alloresponses in recipients lacking antigen-presenting cells (APCs) indicates that the recipient APCs are required for tolerance induction by this protocol (15,33). Consistent with this observation, CD11c-positive, B-cell isoform of 220-kDa-positive, plasmacytoid dendritic cell antigen-1- positive (CD11c + B220 + PDCA-1 + ) plasmacytoid dendritic cells (pdcs) were found to mediate tolerance to heart allografts in mice treated with DST plus anti-cd154 mab (17). Studies using diphtheria toxin receptor (DTR)-Tg mice have shown that DT treatment that depletes CD11c hi cells [but not pdcs that express a lower level of CD11c (4)] does not compromise allograft survival in mice treated with DST (31) or DST plus anti-cd154 (10), indicating that CD11c hi DCs are not required for the immunosuppressive effect of DST. Our results showed that the lack of CD47 expression on donor cells mainly activated CD11c hi DCs. We have previously shown that depletion of CD11c hi DCs facilitate allograft survival in mice receiving CD47 / DST in a class I (H-2k)-mismatched model (31). Thus, although CD11c hi DCs are not involved in tolerance induction by DST, their activation is likely to be one of the mechanisms for the failure of CD47 KO DST to suppress alloresponses. These studies suggest that CD47 expression on DST cells is required for repressing CD11c hi DC activation in DSTtreated mice and that CD11c hi DC activation may promote antidonor alloresponses and thereby makes the recipients resistant to tolerance induction. This possibility is further supported by the previous observation that addition of CD47 / splenocytes into CD47 +/+ DST inoculum significantly reduced the ability of CD47 +/+ DST to suppress alloresponses (31). Recently, CD11b-positive, granulocyte receptor-1-positive (CD11b + Gr1 + ) myeloid suppressor cells were found to promote allograft survival in mice treated with tolerance induction protocols (1,10). Further studies are needed to examine SIRPa expression on these myeloid-derived suppressor cells and their responses to CD47 KO DST. SIRPa serves as an inhibitory receptor in DCs, and its signaling upon ligation of CD47 suppresses DC endocytosis (29) and activation (6). It has been shown that the interaction of SIRPa with CD47 on the target cells, but not CD47 expressed on the surrounding cells, is required for preventing phagocytosis of the target cells by macrophages and DCs (12,29,30). Because SIRPa is the only known inhibitory receptor expressed on DCs that interacts with CD47, the observed role for CD47 expression on donor cells in DST is likely mediated by SIRPa + DCs. Thymic SIRPa + conventional DCs are CD8a low and capable of inducing the differentiation of Tregs in vitro (22,32). However, unlike thymic SIRPa + conventional DCs, splenic CD11c hi SIRPa hi DCs are mostly CD4 + CD8a (Fig. 5C) and incapable of inducing Tregs in vitro (22). Furthermore, CD8a +, but not CD8a, DCs were found to play an important role in endocytosis of apoptotic cells and tolerance induction (3,9,23). Although the function of SIRPa + DCs in the peripheral lymphoid tissues remains relatively less investigated, these studies suggest that activation of peripheral CD11c hi SIRPa hi CD4 + CD8a DCs will likely facilitate immune responses and impair tolerance induction. Previous studies have shown that DST induces donorspecific immunosuppression (24,31). DST could induce tolerance or significantly prolong allograft survival in MHC class I-mismatched settings, but it needs to be used in combination with an additional treatment to suppress CD4 T-cell alloresponses (e.g., anti-cd4 or anti-cd154) for achieving significant protection against allograft rejection in fully MHC-mismatched transplants (7,13,20,21). It has been shown that allotolerance can be equally induced by treatment with anti-cd154 plus DST or with anti-cd154 plus anti-cd8 mab (27). Although DST may only induce CD8 T-cell tolerance, recipient CD11c hi SIRPa hi CD4 + CD8a DC activation induced by CD47 KO DST will likely promote both CD4 and CD8 T-cell responses. We also noted that the immune stimulatory effect of CD47 KO DST seemed to be nonspecific, as CD47 / DST also induced CD11c hi DC activation in a syngeneic combination (Fig. 4). However, CD47 KO DST only augmented antidonor, but not antithird-party, MLR responses compared to CD47 +/+ DST. These data suggest that the exposure of alloantigens to activated CD11c hi SIRPa hi CD8a DCs is likely important for promoting alloimmune responses, which may explain why CD47 / DST, which induces nonspecific activation of CD11c hi SIRPa hi CD8a DCs, affected only the antidonor, but not anti-third-party, alloresponses. CD47-deficient cells can activate macrophages and DCs and be phagocytosed even when they are surrounded by large numbers of CD47- expressing cells (12,29,30), meaning that it is possible that the activated CD11c hi SIRPa hi CD8a DCs may only efficiently promote immune responses to the alloantigens expressed on CD47-deficient donor cells. ACKNOWLEDGMENTS: The authors thank Dr. Donna Farber and Dr. Adam Griesemer for critical review of this manuscript and Ms. Shumei Wang for technical support. This work was supported by grants from NIH (AI-064569 and AI-045897),

362 WANG ET AL. Chinese Ministry of Education (IRT1133), NSFC (81102237 and 81273334), and American Heart Association (SDG-0930361N). The authors declare no conflict of interest. REFERENCES 1. Adeegbe, D.; Serafini, P.; Bronte, V.; Zoso, A.; Ricordi, C.; Inverardi, L. In vivo induction of myeloid suppressor cells and CD4 + Foxp3 + T regulatory cells prolongs skin allograft survival in mice. Cell Transplant. 20:941 954; 2011. 2. Barclay, A. N.; Wright, G. J.; Brooke, G.; Brown, M. H. CD200 and membrane protein interactions in the control of myeloid cells. Trends Immunol. 23:285 290; 2002. 3. Belz, G. T.; Behrens, G. M. N.; Smith, C. M.; Miller, J. F. A. P.; Jones, C.; Lejon, K.; Fathman, C. G.; Mueller, S. N.; Shortman, K.; Carbone, F. R.; Heath, W. R. The CD8 alpha(+) dendritic cell is responsible for inducing peripheral self-tolerance to tissue-associated antigens. J. Exp. Med. 196:1099 1104; 2002. 4. Bennett, C. L.; Clausen, B. E. DC ablation in mice: Promises, pitfalls, and challenges. Trends Immunol. 28:525 531; 2007. 5. Blazar, B. R.; Lindberg, F. P.; Ingulli, E.; Panoskaltsis- Mortari, A.; Oldenborg, P. A.; Iizuka, K.; Yokoyama, W. M.; Taylor, P. A. CD47 (integrin-associated protein) engagement of dendritic cell and macrophage counterreceptors is required to prevent the clearance of donor lymphohematopoietic cells. J. Exp. Med. 194:541 550; 2001. 6. Braun, D.; Galibert, L.; Nakajima, T.; Saito, H.; Quang, V. V.; Rubio, M.; Sarfati, M. Semimature stage: A checkpoint in a dendritic cell maturation program that allows for functional reversion after signal-regulatory protein-{alpha} ligation and maturation signals. J. Immunol. 177:8550 8559; 2006. 7. Bushell, A.; Morris, P. J.; Wood, K. J. Induction of operational tolerance by random blood transfusion combined with anti-cd4 antibody therapy. A protocol with significant clinical potential. Transplantation 58:133 138; 1994. 8. Corry, R. J.; Winn, H. J.; Russell, P. S. Primarily vascularized allografts of hearts in mice. The role of H-2D, H-2K, and non-h-2 antigens in rejection. Transplantation 16:343 350; 1973. 9. Ferguson, T. A.; Herndon, J.; Elzey, B.; Griffith, T. S.; Schoenberger, S.; Green, D. R. Uptake of apoptotic antigencoupled cells by lymphoid dendritic cells and cross-priming of CD8(+) T cells produce active immune unresponsiveness. J. Immunol. 168:5589 5595; 2002. 10. Garcia, M. R.; Ledgerwood, L.; Yang, Y.; Xu, J.; Lal, G.; Burrell, B.; Ma, G.; Hashimoto, D.; Li, Y.; Boros, P.; Grisotto, M.; van Rooijen, N.; Matesanz, R.; Tacke, F.; Ginhoux, F.; Ding, Y.; Chen, S. H.; Randolph, G.; Merad, M.; Bromberg, J. S.; Ochando, J. C. Monocytic suppressive cells mediate cardiovascular transplantation tolerance in mice. J. Clin. Invest. 120:2486 2496; 2010. 11. Girlanda, R.; Kirk, A. D. Frontiers in nephrology: Immune tolerance to allografts in humans. J. Am. Soc. Nephrol. 18:2242 2251; 2007. 12. Ide, K.; Wang, H.; Liu, J.; Wang, X.; Asahara, T.; Sykes, M.; Yang, Y. G.; Ohdan, H. Role for CD47-SIRPa signaling in xenograft rejection by macrophages. Proc. Natl. Acad. Sci. USA 104:5062 5066; 2007. 13. Iwakoshi, N. N.; Mordes, J. P.; Markees, T. G.; Phillips, N. E.; Rossini, A. A.; Greiner, D. L. Treatment of allograft recipients with donor-specific transfusion and anti-cd154 antibody leads to deletion of alloreactive CD8+ T cells and prolonged graft survival in a CTLA4-dependent manner. J. Immunol. 164:512 521; 2000. 14. Jaiswal, S.; Jamieson, C. H. M.; Pang, W. W.; Park, C. Y.; Chao, M. P.; Majeti, R.; Traver, D.; van Rooijen, N.; Weissman, I. L. CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis. Cell 138:271 285; 2009. 15. Kishimoto, K.; Yuan, X.; Auchincloss, Jr., H.; Sharpe, A. H.; Mandelbrot, D. A.; Sayegh, M. H. Mechanism of action of donor-specific transfusion in inducing tolerance: Role of donor MHC molecules, donor co-stimulatory molecules, and indirect antigen presentation. J. Am. Soc. Nephrol. 15:2423 2428; 2004. 16. Lindberg, F. P.; Bullard, D. C.; Caver, T. E.; Gresham, H. D.; Beaudet, A. L.; Brown, E. J. Decreased resistance to bacterial infection and granulocyte defects in IAP-deficient mice. Science 274:795 798; 1996. 17. Ochando, J. C.; Homma, C.; Yang, Y.; Hidalgo, A.; Garin, A.; Tacke, F.; Angeli, V.; Li, Y.; Boros, P.; Ding, Y.; Jessberger, R.; Trinchieri, G.; Lira, S. A.; Randolph, G. J.; Bromberg, J. S. Alloantigen-presenting plasmacytoid dendritic cells mediate tolerance to vascularized grafts. Nat. Immunol. 7:652 662; 2006. 18. Oldenborg, P. A.; Zheleznyak, A.; Fang, Y. F.; Lagenaur, C. F.; Gresham, H. D.; Lindberg, F. P. Role of CD47 as a marker of self on red blood cells. Science 288:2051 2054; 2000. 19. Olsson, M.; Bruhns, P.; Frazier, W. A.; Ravetch, J. V.; Oldenborg, P. A. Platelet homeostasis is regulated by platelet expression of CD47 under normal conditions and in passive immune thrombocytopenia. Blood 105:3577 3582; 2005. 20. Parker, D. C.; Greiner, D. L.; Phillips, N. E.; Appel, M. C.; Steele, A. W.; Durie, F. H.; Noelle, R. J.; Mordes, J. P.; Rossini, A. A. Survival of mouse pancreatic islet allografts in recipients treated with allogeneic small lymphocytes and antibody to CD40 ligand. Proc. Natl. Acad. Sci. USA 92:9560 9564; 1995. 21. Preston, E. H.; Xu, H.; Dhanireddy, K. K.; Pearl, J. P.; Leopardi, F. V.; Starost, M. F.; Hale, D. A.; Kirk, A. D. IDEC-131 (Anti-CD154), Sirolimus and donor-specific transfusion facilitate operational tolerance in non-human primates. Am. J. Transplant. 5:1032 1041; 2005. 22. Proietto, A. I.; van Dommelen, S.; Zhou, P.; Rizzitelli, A.; D Amico, A.; Steptoe, R. J.; Naik, S. H.; Lahoud, M. H.; Liu, Y.; Zheng, P.; Shortman, K.; Wu, L. Dendritic cells in the thymus contribute to T-regulatory cell induction. Proc. Natl. Acad. Sci. USA 105:19869 19874; 2008. 23. Qiu, C. H.; Miyake, Y.; Kaise, H.; Kitamura, H.; Ohara, O.; Tanaka, M. Novel subset of CD8{alpha}+ dendritic cells localized in the marginal zone is responsible for tolerance to cell-associated antigens. J. Immunol. 182:4127 4136; 2009. 24. Sandner, S. E.; Clarkson, M. R.; Salama, A. D.; Sanchez- Fueyo, A.; Yagita, H.; Turka, L. A.; Sayegh, M. H. Mechanisms of tolerance induced by donor-specific transfusion and ICOS- B7h blockade in a model of CD4+ T-cell-mediated allograft rejection. Am. J. Transplant. 5:31 39; 2005. 25. Sato, S.; Azuma, T.; Shimizu, J.; Shima, J.; Kitagawa, S.; Hamaoka, T.; Fujiwara, H. Property of class I H-2 alloantigen-reactive Lyt-2+ helper T cell subset. Abrogation of its proliferative and IL-2-producing capacities by intravenous injection of class I H-2-disparate allogeneic cells. J. Immunol. 141:721 727; 1988.

CD47 IN TRANSPLANTATION TOLERANCE 363 26. Takenaka, K.; Prasolava, T. K.; Wang, J. C. Y.; Mortin-Toth, S. M.; Khalouei, S.; Gan, O. I.; Dick, J. E.; Danska, J. S. Polymorphism in Sirpa modulates engraftment of human hematopoietic stem cells. Nat. Immunol. 8:1313 1323; 2007. 27. Takeuchi, Y.; Ito, H.; Kurtz, J.; Wekerle, T.; Ho, L.; Sykes, M. Earlier low-dose TBI or DST overcomes CD8+ T-cellmediated alloresistance to allogeneic marrow in recipients of anti-cd40l. Am. J. Transplant. 4:31 40; 2004. 28. Wang, C.; Wang, H.; Ide, K.; Wang, Y.; Ohdan, H.; Yang, Y. G. Human CD47 expression prevents macrophage-mediated rejection of porcine cells in mice that express SIRPa capable of binding to human CD47. Cell Transplant. 20:1915 1920; 2011. 29. Wang, H.; Madariaga, M. L.; Wang, S.; van Rooijen, N.; Oldenborg, P. A.; Yang, Y. G. Lack of CD47 on nonhematopoietic cells induces split macrophage tolerance to CD47null cells. Proc. Natl. Acad. Sci. USA 104:13744 13749; 2007. 30. Wang, H.; VerHalen, J.; Madariaga, M. L.; Xiang, S.; Wang, S.; Lan, P.; Oldenborg, P. A.; Sykes, M.; Yang, Y. G. Attenuation of phagocytosis of xenogeneic cells by manipulating CD47. Blood 109:836 842; 2007. 31. Wang, H.; Wu, X.; Wang, Y.; Oldenborg, P.-A.; Yang, Y. G. CD47 is required for suppression of allograft rejection by donor-specific transfusion. J. Immunol. 184:3401 3407; 2010. 32. Wu, L.; Shortman, K. Heterogeneity of thymic dendritic cells. Semin. Immunol. 17:304 312; 2005. 33. Yamada, A.; Chandraker, A.; Laufer, T. M.; Gerth, A. J.; Sayegh, M. H.; Auchincloss, Jr., H. Cutting edge: Recipient MHC class II expression is required to achieve long-term survival of murine cardiac allografts after costimulatory blockade. J. Immunol. 167:5522 5526; 2001. 34. Yang, Y. G.; Sykes, M. Xenotransplantation: Current status and a perspective on the future. Nat. Rev. Immunol. 7:519 531; 2007.