The Role of the CD134-CD134 Ligand Costimulatory Pathway in Alloimmune Responses in Vivo

The Role of the CD134-CD134 Ligand Costimulatory Pathway in Alloimmune Responses In Vivo

This information is current as Xueli Yuan, Alan D. Salama, Victor Dong, Isabela Schmitt, of September 27, 2021. Nader Najafian, Anil Chandraker, Hisaya Akiba, Hideo Yagita and Mohamed H. Sayegh J Immunol 2003; 170:2949-2955; ; doi: 10.4049/jimmunol.170.6.2949

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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 © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

The Role of the CD134-CD134 Ligand Costimulatory Pathway in Alloimmune Responses In Vivo1

Xueli Yuan,* Alan D. Salama,* Victor Dong,* Isabela Schmitt,* Nader Najaﬁan,* Anil Chandraker,* Hisaya Akiba,† Hideo Yagita,† and Mohamed H. Sayegh2*

The CD134-CD134 ligand (CD134L) costimulatory pathway has been shown to be critical for both T and B cell activation; however, its role in regulating the alloimmune response remains unexplored. Furthermore, its interactions with other costimulatory pathways and immunosuppressive agents are unclear. We investigated the effect of CD134-CD134L pathway blockade on allograft rejection in fully MHC-mismatched rat cardiac and skin transplantation models. CD134L blockade alone did not prolong graft survival compared with that of untreated recipients, and in combination with donor-speciﬁc transfusion, cyclosporine, or rapamycin, was less effective than B7 blockade in prolonging allograft survival. However, in combination with B7 blockade, long-term allograft survival was achieved in all recipients (>200 days). Moreover, this was synergistic in reducing the frequency Downloaded from of IFN-␥-producing alloreactive lymphocytes and inhibiting the generation of activated/effector lymphocytes. Most impressively, this combination prevented rejection in a presensitized model using adoptive transfer of primed lymphocytes into athymic heart transplant recipients. In comparison to untreated recipients (mean survival time (MST): 5.3 ؎ 0.5 days), anti-CD134L mAb alone modestly prolonged allograft survival (MST: 14 ؎ 2.8 days) as did CTLA4Ig (MST: 21.5 ؎ 1.7 days), but all grafts were rejected (within 24 days. Importantly, combined blockade further and signiﬁcantly prolonged allograft survival (MST: 75.3 ؎ 12.7 days

and prevented the expansion and/or persistence of primed/effector alloreactive T cells. Our data suggest that CD134-CD134L is http://www.jimmunol.org/ a critical pathway in alloimmune responses, especially recall/primed responses, and is synergistic with CD28-B7 in mediating T cell effector responses during allograft rejection. Understanding the mechanisms of collaboration between these different pathways is important for the development of novel strategies to promote long-term allograft survival. The Journal of Immunology, 2003, 170: 2949Ð2955.

t is now ﬁrmly established that costimulatory signals, primar- CD70-CD27, which may play a greater role in the maintenance ily through the ligation of B7-CD28 and CD40-CD154, are phase of the immune response (13). Of course, these mechanisms necessary for full T cell activation and play a crucial role in may not be mutually exclusive.

I by guest on September 27, 2021 mediating allograft rejection. In several models, CD28-B7 block- Recent data have demonstrated that numerous members of the ade alone or in combination with donor-specific transfusion TNF superfamily are capable of providing costimulatory signals (DST),3 has been shown to prevent acute rejection, induce long- and regulating T cell immune responses (13, 14). The most studied term allograft survival (1Ð4), and prevent the development and of these pathways is CD40-CD154 (15), blockade of which is syn- progression of chronic rejection (5, 6). However, the same degree ergistic with B7 blockade in preventing allograft rejection (7, 16, of efficacy is not seen when more stringent transplantation models 17). Newer members of the superfamily are CD134 and CD134L have been used (7Ð10). Moreover, CD28- or CD154-deficient an- (also termed OX40 and OX40 ligand), which are expressed, re- imals are still capable of rejecting allografts acutely, albeit at a spectively, on activated T cells (18Ð21) and APC (22, 23) (includ- slower tempo, as well as developing chronic rejection (9, 11, 12). ing activated B cells and dendritic cells) as well as vascular en- Therefore, it appears that CD28-B7- and CD40-CD154-indepen- dothelial cells (24). In vitro and in vivo evidence has demonstrated dent mechanisms can mediate full T cell activation. These include that the CD134-CD134L interaction can provide a costimulatory effector cells that are less reliant on CD28 and CD154 signaling for signal to T cells, increasing T cell proliferation and cytokine pro- ϩ activation, such as CD8 cells, NK cells, and memory cells, or duction (25, 26) as well as influencing B cell proliferation and Ig activation using alternative costimulatory pathways, such as in- production (27, 28). CD134ϩ T cells have been implicated in var- ducible costimulator-B7h, CD134-CD134 ligand (CD134L), and ious immune-mediated diseases, such as experimental autoimmune encephalomyelitis (29, 30), rheumatoid arthritis (31, 32), inflammatory skin disease (33), as well as graft-vs-host disease (34). In *Laboratory of Immunogenetics and Transplantation, Brigham and Women’s Hospi- experimental autoimmune encephalomyelitis, administration of a tal, Harvard Medical School, Boston, MA 02115; and †Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan neutralizing anti-CD134L mAb or OX40-Fc fusion protein ame- Received for publication September 9, 2002. Accepted for publication January liorates disease (35, 36), while in animals lacking CD28 costimu- 3, 2003. lation, CD134-CD134L blockade can completely abrogate disease The costs of publication of this article were defrayed in part by the payment of page (37). These data suggest that CD134-CD134L costimulation may charges. This article must therefore be hereby marked advertisement in accordance be in part responsible for the CD28-B7-independent activation of T with 18 U.S.C. Section 1734 solely to indicate this fact. cells, at least in an autoimmune model. In the present study, we used 1 This work was supported by National Institutes of Health Grant RO1 AI34965. a newly developed anti-rat CD134L mAb, ATM-2 (38), in allogenic 2 Address correspondence and reprint requests to Dr. Mohamed H. Sayegh, Labora- cardiac and skin transplantation models, as well as in a more stringent tory of Immunogenetics and Transplantation, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115. E-mail address: [email protected] sensitized model, to explore the role of the CD134-CD134L pathway 3 Abbreviations used in this paper: DST, donor-specific transfusion; CD134L, CD134 in mediating allograft rejection and its interaction with the CD28-B7 ligand; MST, mean survival time; hCTLA4Ig, human CTLA4Ig; CsA, cyclosporine. pathway.

Materials and Methods Abs and immunosuppressive agents Animals Hybridoma cell lines producing mAbs directed against rat CD134L (ATM-2) (38), rat B7-1 (3H5), and B7-2 (24F) (41) were generous gifts Eight- to 10-wk-old inbred male Wistar-Furth (WF, RT1u) or ACI (RT1a) from Dr. H. Yagita (Juntendo University, Tokyo, Japan) and were pro- rats were used as heart or skin allograft donors and Lewis (LEW, RT1l) animals as recipients (all from Harlan Sprague-Dawley, Indianapolis, IN). duced by Bioexpress (West Lebanon, NH). hCTLA4Ig was a kind gift from Athymic LEW/Mol-rnu rats were purchased from M&B (Ry, Denmark) Dr. R. Peach (Bristol-Myers Squibb, Princeton, NJ). Anti-CD134L Ab (1 and bred in our facility. All animals were housed and cared for under mg) was given by injection (i.p.) on the day of transplantation and on days National Institutes of Health guidelines. 2, 4, 6, 8, 10, and 12 following transplantation. Anti-rat B7-1 or B7-2 mAb (0.5 mg), or hCTLA4Ig (0.5 mg) were administrated as a single i.p dose on day 2, in cardiac transplant recipients, or as multiple doses (0.5 mg each) Cardiac transplantation on days 0, 2, 4, and 6 in the skin transplant recipients and in athymic nude Heterotropic vascularized cardiac transplants were performed by standard rats. Cyclosporine (CsA; 10 mg/kg/day; Bedford Laboratories, Bedford, microvascular techniques (39, 40). Donor hearts were engrafted into the OH) and rapamycin (0.3 mg/kg/day; Wyeth-Ayerst, Madison, NJ) were recipient’s abdomen using an end-to-side anastomosis of the donor ascend- given for 4 consecutive days following transplantation. ing aorta and pulmonary artery with the recipient’s abdominal aorta and Statistics vena cava. Transplantation tolerance was confirmed by engrafting long- term transplant survivors with a second original donor or third-party donor The Kaplan-Meier test was used to calculate the graft survival and the heart. Graft function was assessed by daily palpation, and rejection was log-rank (Mantel-Cox) test was applied to compare differences in survival confirmed at autopsy. between groups. A value of p Ͻ 0.05 was considered significant.

Skin transplantation Results Downloaded from Full thickness skin grafts were prepared from the lateral thoracic skin of the The effect of CD134-CD134L and CD28-B7 blockade on donor, cut into 2.5-cm circular pieces, and then maintained at 4¡C until cardiac allograft rejection transplantation. Donor skin was engrafted onto the recipient’s lumbar re- gion and checked daily for signs of rejection (39, 40). First, we compared the effects of B7 vs CD134L blockade on vascularized cardiac allograft rejection. In the WF into LEW rat trans- ELISPOT plant model, untreated control cardiac allografts were all rejected

promptly, with a mean survival time (MST) of 8.6 Ϯ 1.2 days (n ϭ http://www.jimmunol.org/ Immunospot plates (Cellular Technology, Cleveland, OH) were coated 8). Following treatment with a single injection of hCTLA4Ig on with purified anti-rat IFN-␥ Ab (BD PharMingen, San Diego, CA) and stored at 4¡C overnight. The next day, they were washed three times with posttransplant day 2, allograft survival was significantly prolonged PBS and blocked for at least 2 h with PBS containing 1% BSA. After (MST: 35.7 Ϯ 13.1 days; n ϭ 6, p Ͻ 0.001) (Fig. 1), while the further washing with PBS, irradiated WF spleen cells (5 ϫ 105/well) and administration of anti-CD134L mAb alone had no significant effect ϫ 5 splenocytes (5 10 /well) from naive LEW rats, or those of LEW recip- on allograft survival (MST: 9.3 Ϯ 3.5 days; n ϭ 6, p ϭ NS com- ients of skin transplants (7, 14, or 30 days posttransplantation), which received treatment with or without anti-CD134L Ab, CTLA4Ig, or a compared with allograft controls) (Fig. 1). bination of both, were added to some wells, each in 100 ␮l of complete As we have previously described (39), administration of DST on RPMI 1640 medium containing 10% FCS (Sigma-Aldrich, St. Louis, MO), the day of transplant followed by a single injection of hCTLA4Ig 2mML-glutamine, 100 U/ml penicillin/streptomycin (BioWhittaker, on day 2 resulted in all of the allografts achieving long-term sur- by guest on September 27, 2021 Walkersville, MD), and 50 mM 2-ME (Sigma-Aldrich). Control wells con- vival (MST Ͼ 200, n ϭ 6, p Ͻ 0.001 compared with untreated taining stimulator cells alone or responder cells alone were also plated out. The plate was incubated at 37¡C for 36 h, and then washed three times with controls and hCTLA4Ig alone). However, DST showed no signif- PBS and then PBS containing 0.05% Tween. Biotin-labeled secondary anti- icant additional effect on graft survival when combined with anti- IFN-␥ mAb, diluted in PBS-1% BSA-Tween, was added to each well, and CD134L Ab (MST: 15.5 Ϯ 1.732 days; n ϭ 4, p ϭ NS) (Fig. 1). the plate was incubated overnight at 4¡C. After further washing, HRP con- A short course of CsA (10 mg/kg i.p. on days 0, 1, 2, and 3) jugate (DAKO, Carpinteria, CA) was added for2hatroom temperature. Development was performed with 3-amino-9-ethylcarbazole (Sigma-Aldrich). resulted in some prolongation of allograft survival but all grafts The resulting spots were counted on a computer-assisted ELISASpot Image were ultimately rejected (MST: 20.5 Ϯ 6.5 days; n ϭ 6, p ϭ Analyser (Cellular Technology). The results were then calculated as cytokine- producing cells per half million splenocytes.

Flow cytometry Splenocytes were isolated 14 or 30 days following skin transplantation from animals treated with human CTLA4Ig (hCTLA4Ig) alone, anti- CD134L alone, or combined therapy, or from untreated allograft controls (three animals in each group), and were stained with anti-CD3-PE and anti-CD44-FITC mAb (BD PharMingen). Cells were analyzed on a FACS- Calibur (BD Biosciences, San Jose, CA) using CellQuest software (BD Biosciences). The percentages of splenocytes expressing a CD3ϩCD44high phenotype were then calculated. The expansion and survival of T cells in the peripheral blood and spleen of the athymic heart recipients, adoptively transferred with sensitized splenocytes and treated with the different regi- mens, was examined by staining for T cells using an anti-CD3-PE mAb 18 days after transplantation and adoptive transfer. FIGURE 1. Effect of CD28-B7 and CD134-CD134L blockade on graft rejection. CD28 blockade with hCTLA4Ig (0.5 mg i.p. on day 2) signiﬁ- Sensitization and adoptive transfer of sensitized spleen cells cantly prolonged allograft survival, while CD134-CD134L blockade with anti-CD134L mAb (1 mg i.p. on days 0, 2, 4, 6, 8, 10, and 12) showed no Fourteen days following skin transplantation of a WF graft onto a LEW effect compared with untreated allograft controls. Transfusion of 4 ϫ 107 animal, the recipients were sacriﬁced. Spleens were removed, and single- donor splenocytes into the recipients (DST) on the day of transplantation cell suspensions were obtained. RBC were removed by incubating with ACK lysing buffer (BioWhittaker) for 10 min at room temperature. After was synergistic with CD28-B7 blockade in prolonging allograft survival, Ϯ several washes, cells were counted and resuspended in PBS. Immediately but not with anti-CD134L Ab. (MST: allograft controls, 8.6 1.2 days, following transplantation of a WF heart into an athymic nude rat of LEW n ϭ 8; anti-CD134L mAb, 9.3 Ϯ 3.5 days, n ϭ 6; anti-CD134L mAb plus background, 4 ϫ 107 of the sensitized splenocytes were adoptively trans- DST, 15.5 Ϯ 1.7 days, n ϭ 6; hCTLA4Ig, 35.7 Ϯ 13.1 days, n ϭ 6; and ferred by i.v. injection into the nude rats. hCTLA4Ig plus DST, Ͼ200 days, n ϭ 6). The Journal of Immunology 2951

0.0004 compared with control allografts). When CsA was used in The effect of CD134-CD134L and CD28-B7 blockade on skin combination with hCTLA4Ig, all of the grafts achieved long-term allograft rejection Ͼ ϭ Ͻ survival ( 200 days, n 6, p 0.001 compared with either CsA Mirroring the results from the cardiac transplant model, prolonga- or hCTLA4Ig treatment alone). However, the combination of CsA tion of skin transplantation was best achieved with a combination with anti-CD134L mAb resulted in no further survival beneﬁt of anti-CD134L and hCTLA4Ig. All of the untreated allograft con- (MST: 16.5 Ϯ 0.5 days; n ϭ 6, p ϭ NS compared with CsA alone) trol animals rejected their grafts within 10 days (MST: 8.2 Ϯ 1.0; (Fig. 2A). n ϭ 6), while anti-CD134L mAb alone had no effect on graft Rapamycin (0.3 mg/kg i.p. on days 0, 1, 2, and 3) treatment survival (MST: 8.7 Ϯ 1.2; n ϭ 6, p ϭ NS) and hCTLA4Ig mar- alone signiﬁcantly prolonged graft survival (37.4 Ϯ 11.7 days, n ϭ ginally delayed graft rejection (MST: 13.5 Ϯ 1.4 days; n ϭ 6, p ϭ 5, p ϭ 0.001 compared with allograft controls). When recipients 0.001 compared with allograft controls). However, the combina- were treated with both rapamycin and hCTLA4Ig, allograft rejection of anti-CD134L mAb and hCTLA4Ig dramatically prolonged tion was further delayed (MST: 87.5 Ϯ 10.5 days; n ϭ 6, p ϭ graft survival in this stringent transplant model, with 33% of al- 0.0005 and 0.0007, compared with hCTLA4Ig or rapamycin alone) lografts surviving over 100 days (MST: 59.1 Ϯ 32.7 days; n ϭ (Fig. 2B). Moreover, rapamycin in combination with anti-CD134L 6, p Ͻ 0.0007 compared with control and hCTLA4Ig groups) mAb also prolonged allograft survival more than rapamycin alone (Fig. 3C). (MST: 56.5 Ϯ 6.3; n ϭ 4, p ϭ 0.0049 compared with rapamycin alone) (Fig. 2B). In vivo blockade of CD134-CD134L and CD28-B7 diminishes

Synergy between CD134-CD134L and CD28-B7 blockade Downloaded from the frequency of alloreactive IFN-␥-producing lymphocytes and Although CD134-CD134L blockade alone had no effect on allo- prevents the development of activated cells graft survival, the combination of anti-CD134L mAb and ␥ hCTLA4Ig demonstrated signiﬁcant synergy (Fig. 3A) with indef- Using ELISPOT analysis, the frequency of alloreactive IFN- -pro- inite survival being achieved in all animals (MST Ͼ 200 days, n ϭ ducing lymphocytes from skin transplant recipients, treated with or 8, p Ͻ 0.001 when compared with hCTLA4Ig alone or anti- without anti-CD134L mAb, hCTLA4Ig, or their combination, was

CD134L alone). Moreover, donor-speciﬁc tolerance was demon- measured 7, 14, and 30 days after transplantation. On day 7, the http://www.jimmunol.org/ strated by the acceptance of a second WF heart graft (Ͼ100 days) allograft control group had an elevated frequency of alloreactive and rejection of a third-party ACI strain heart within 10 days (Ta- IFN-␥-producing lymphocytes compared with that of naive control ble I). To further elucidate the mechanism of this synergy between animals (Fig. 4). Treatment with anti-CD134L mAb alone dimin- hCTLA4Ig and anti-CD134L, blocking anti-B7-1 and -B7-2 mAbs ished the IFN-␥ frequency by 21%, but a greater reduction was were used. Anti-B7-1 and anti-B7-2 mAb alone or in combination achieved with hCTLA4Ig and combined hCTLA4Ig and anti- (0.5 mg for each, i.p on posttransplantation day 2) modestly pro- CD134L mAb (74.3 and 64.2% reduction, respectively). However, longed allograft survival (MST: 13.5 Ϯ 1.3 days, n ϭ 4; 15.4 Ϯ by day 14, when animals treated with hCTLA4Ig were rejecting 1.8, n ϭ 5; and 18 Ϯ 1.8, n ϭ 4, respectively). However, while the their grafts, the frequency of alloreactive IFN-␥-producing cells in addition of anti-CD134L to either anti-B7-1 (MST: 13.3 Ϯ 3.6 this group increased signiﬁcantly, while the combined treatment by guest on September 27, 2021 days, n ϭ 7) or anti-B7-2 (23.1 Ϯ 13.6 days, n ϭ 7) produced no group of hCTLA4Ig and anti-CD134L mAb maintained low allo- further prolongation of allograft survival ( p ϭ NS when compared reactive frequencies consistent with the frequencies on day 7. By with isolated anti-B7-1 or -B7-2 mAb treatment, respectively), co- day 30, a time when most grafts treated with combined blockade administration of anti-CD134L with both anti-B7-1 and -B7-2 are in the process of rejection, the alloreactive T cell frequency in mAbs resulted in long-term allograft survival in all recipients untreated recipients was unchanged, while the frequency in the (MST Ͼ 200 days, n ϭ 6, p Ͻ 0.001 compared with the combi- combined treatment group had increased, compared with day 14 nation of CD134L mAb with either anti-B7-1 or anti-B7-2 alone or (Fig. 4). Furthermore, 14 days posttransplantation, the combined with anti-B7-1 and anti-B7-2 combined) (Fig. 3B). Therefore, treatment group demonstrated the greatest reduction of activated/ blockade of both B7-1 and B7-2 is necessary to achieve long-term effector T cells, as judged by a CD3ϩCD44high phenotype (43% allograft survival with anti-CD134L mAb. reduction) (Fig. 5), while single therapy with hCTLA4Ig (22%

FIGURE 2. CsA and rapamycin differentially affected allograft survival following CD28-B7 and CD134-CD134L blockade. A, CsA (10 mg/kg i.p. on days 0, 1, 2, and 3) was synergistic with CD28-B7 blockade in prolonging allograft survival, but had no additional effect on allograft survival when combined with anti-CD134L mAb. (MST: allograft controls, 8.6 Ϯ 1.2 days, n ϭ 8; CsA, 20.5 Ϯ 6.5 days, n ϭ 6; hCTLA4Ig, 35.7 Ϯ 13.1 days, n ϭ 6; hCTLA4Ig plus CsA, Ͼ200 days, n ϭ 6; anti-CD134L mAb, 9.3 Ϯ 3.5 days, n ϭ 6; and CsA plus anti-CD134L mAb, 16.5 Ϯ 0.5 days, n ϭ 6). B, Rapamycin (0.3 mg/kg i.p. on days 0, 1, 2, and 3) was synergistic with both hCTLA4Ig and anti-CD134L mAb in prolonging allograft survival (MST: allograft controls, 8.6 Ϯ 1.2 days, n ϭ 8; rapamycin, 37.4 Ϯ 11.7 days, n ϭ 5; hCTLA4Ig, 35.7 Ϯ 13.1 days, n ϭ 6; hCTLA4Ig plus CsA, Ͼ87.5 Ϯ 10.5 days, n ϭ 6; anti-CD134L mAb, 9.3 Ϯ 3.5 days, n ϭ 6; and rapamycin plus CD134L mAb, 56.5 Ϯ 6.3 days, n ϭ 4). 2952 CD134-CD134L IN ALLOIMMUNITY

Table I. Survival of second allografts from original or unrelated third-party donors in long-term survived recipients

Second Donor Allograft Survival (Days) Number of Animals

WF (RT1u) Ͼ100 3 ACI (RT1a) 10 2

5). Upon adoptive transfer of 4 ϫ 107 lymphocytes from naive LEW rats, allografts were rejected in 12Ð23 days after transplantation (MST: 17 Ϯ 4.6 days; n ϭ 4). If the adoptively transferred cells were obtained from LEW rats presensitized with WF skin grafts, rejection was signiﬁcantly accelerated (MST: 5.3 Ϯ 0.5 days; n ϭ 4, p Ͻ 0.0058 compared with naive lymphocyte trans- fused recipients) (Fig. 6). We used this model to examine the role of B7 and CD134L on transplant rejection mediated by primed/

effector T lymphocytes. Both anti-CD134L mAb and hCTLA4Ig Downloaded from when administered alone delayed the allograft rejection (MST: 14 Ϯ 2.8 days, n ϭ 4, p ϭ 0.009; and 21.5 Ϯ 1.7 days, n ϭ 4, p Ͻ 0.001, for anti-CD134L mAb and hCTLA4Ig, respectively, compared with the untreated control group) mediated by sensitized cells. When anti-CD134L and hCTLA4Ig were combined, allograft survival was further and signiﬁcantly prolonged (MST: http://www.jimmunol.org/ 75.3 Ϯ 12.7 days; n ϭ 6, p Ͻ 0.001 compared with anti-CD134L mAb- or hCTLA4Ig-treated group alone) (Fig. 6). Consistent with the survival data, the percentage of recovered CD3ϩ T cells in the peripheral blood of reconstituted nude recipients 18 days following allogenic cardiac transplantation, was greater in the untreated allograft controls (3.27%) and in the anti-CD134L mAb (4.54%)- and CTLA4Ig (7.51%)-treated nude recipients than in the combination group (0.96%). These data demonstrate that combined B7

FIGURE 3. Synergy between CD28-B7 and CD134-CD134L blockades and CD134L blockade prevented the expansion and/or persistence by guest on September 27, 2021 in prolongation of allograft survival and induction of transplantation tol- of the effector/primed alloreactive T cells in vivo. erance. A and B, Heart model. All of the allografts treated with combined hCTLA4Ig and anti-CD134L achieved long-term survival. Although se- Discussion lective blockade of either B7-1 or B7-2 in combination with anti-CD134L CD134 and CD134L are members of the TNF and TNFR super- was not sufficient to achieve long-term allograft survival, combined anti- families, respectively, and have been recently recognized as effi- B7-1 and -B7-2 mAb with anti-CD134L did (MST: allograft controls, Ϯ ϭ Ϯ ϭ cient T cell costimulatory molecules (25, 42, 43). CD134 is ex- 8.6 1.2 days, n 8; hCTLA4Ig, 35.7 13.1 days, n 6; anti-CD134L ϩ Ϯ ϭ Ͼ pressed on activated T cells, primarily on CD4 cells, although mAb, 9.3 3.5 days, n 6; hCTLA4Ig plus anti-CD134L mAb, 200 ϩ days, n ϭ 8; B7-1 mAb, 13.5 Ϯ 1.3 days, n ϭ 4; B7-2 mAb, 15.4 Ϯ 1.8 CD8 T cell expression is also found but to a lesser degree (18, days, n ϭ 5; B7-1 mAb plus B7-2 mAb, 18 Ϯ 1.8 days, n ϭ 4; anti- 44). CD134L has also been found on activated B cells (22, 23), CD134L mAb plus B7-1 mAb, 13.3 Ϯ 3.6 days, n ϭ 7; anti-CD134L mAb dendritic cells (22), and vascular endothelial cells (24), and has plus B7-2 mAb, 23.1 Ϯ 13.6 days, n ϭ 7; and anti-CD134L mAb plus B7-1 been shown to be up-regulated following the engagement of the mAb plus B7-2 mAb, Ͼ200 days, n ϭ 6). C, Skin model. Only the com- TCRs with MHC-peptide complexes. The expression peaks 24Ð48 bination of hCTLA4Ig and CD134L mAb significantly delayed skin allo- h after T cell engagement with Ag and is down-regulated by 72Ð96 graft rejection, while either agent alone had minimal effect (MST: allograft h (25). CD134L-transfected APC have little ability to induce either controls, 8.2 Ϯ 1.0 days, n ϭ 6; anti-CD134L mAb, 8.7 Ϯ 1.2 days, n ϭ IL-2 secretion or the proliferation of naive T cells; however, en- 6; hCTLA4Ig, 13.5 Ϯ 1.4 days, n ϭ 6; and anti-CD134L mAb combined with hCTLA4Ig, 55.6 Ϯ 31.4 days, n ϭ 7). hanced proliferation is observed when B7 is coexpressed on the APC, and thereafter proliferation persists for several days (25). Thus, signaling through this pathway promotes T cell survival and reduction) or anti-CD134L (26% reduction) resulted in more mod- helps determine the development of CD4ϩ T cell memory by reg- est decreases in the percentage of effector CD44high cells. More- ulating primary clonal expansion (26). Furthermore, signaling over, this effect persisted up to 30 days posttransplantation, when through the CD134-CD134L pathway has been shown to reverse ϩ there was still a significant (27%) reduction in the CD3 CD44high peripheral tolerance (45). CD134ϩ T cells have also been associ- T cells in the combined treatment group compared with the allo- ated with various autoimmune diseases (30, 32Ð34), and targeting graft controls ( p ϭ 0.038, compared with control, by two-tailed of the CD134-CD134L pathway is believed to hold therapeutic Mann-Whitney U test). potential in the treatment of autoimmunity and possibly in enhancing vaccine efficacy (46). Effect of CD134-CD134L and CD28-B7 blockade on primed Although numerous in vitro studies have shown that CD134- alloreactive lymphocytes CD134L interaction plays an important role in T cell activation to LEW background athymic nude rats, lacking T cells, failed to re- alloantigens (25, 47, 48), there is no clear evidence of its effect on ject MHC-mismatched WF cardiac grafts (MST Ͼ 100 days, n ϭ in vivo alloimmune responses. Using our allogenic transplantation The Journal of Immunology 2953

FIGURE 4. Effect of in vivo blockade of CD28-B7 and CD134- CD134L costimulation on alloreactive IFN-␥-producing lymphocyte frequency evaluated by ELISPOT assay. Splenocytes were harvested at 7, 14, and 30 days after transplantation from allogenic skin transplant recipients, treated with anti-CD134L mAb, hCTLA4Ig, or the combination of anti-CD134LmAb and hCTLA4Ig (three animals in each group). The frequency is expressed as the number of IFN-␥-producing alloreactive cells per half million splenocytes (mean Ϯ SD).

ϫ Downloaded from models, we found that, in both heart and skin transplants, CD134- models. Onodera et al. (49), using a LBNF1 (LEW BN F1 hy- CD134L blockade alone had no significant effect on allograft re- brid) donor heart transplanted into a LEW recipients presensitized jection, and its combination with DST, CsA, or rapamycin was not with Brown-Norway skin grafts a week before, demonstrated that as efficacious as CD28-B7 blockade in prolonging allograft sur- grafts were rejected within 36 h. Treatment with CTLA4Ig in the vival. However, the combination of anti-CD134L mAb and sensitization phase resulted in a cardiac graft survival of 4.4 Ϯ 1.3 hCTLA4Ig (or both anti-B7-1 and anti-B7-2) induced donor-spe- days, while treatment in both sensitization and effector phases pro- Ϯ cific tolerance in all cardiac transplant recipients, as evidenced by longed survival to only 10.5 8.5 days. Even the highly effica- http://www.jimmunol.org/ long-term graft survival, acceptance of second donor WF hearts, cious combination of anti-CD40L Ab and DST, which can induce and rejection of third-party ACI allografts. Moreover, it signifi- long-term survival of allografts in fully MHC-mismatched mouse cantly prolonged survival in a more stringent model using fully heart transplant models, failed to prevent cardiac graft rejection in allogenic skin grafts. Following in vivo blockade of both CD134- a presensitized model (50). The demonstration of very low per- CD134L and CD28-B7 pathways, the frequency of alloreactive T centages of recovered CD3ϩ T cells from reconstituted nude re- cells was markedly reduced and was maintained at low levels. cipients treated with the combination of anti-CD134 mAb and Furthermore, the percentage of T cells expressing an activated/ hCTLA4Ig, as compared with untreated controls and animals effector phenotype was also maximally inhibited by combined treated anti-CD134L mAb or hCTLA4Ig alone, suggests that only

CD134-CD134L and CD28-B7 blockade. Interestingly, the de- the combined therapy effectively inhibits T cell expansion and/or by guest on September 27, 2021 crease in activated phenotype persisted, at a time when the fre- decreases cell survival. These findings are consistent with the quency of IFN-␥-producing alloreactive lymphocytes began to re- known functions of CD28-B7 (51) and CD134-CD134L (25, 26) in cover to control values. Taken together, these data demonstrate promoting expansion and survival of Ag-specific T cells in vivo. that the effect of combined blockade is on the development and Specifically, the CD134-CD134L pathway appears to be crucial persistence of effector/memory T cells. for regulating the extent of CD4ϩ T cell expansion in the primary Furthermore, unlike other therapies, this combination was able to suppress transplant rejection mediated by primed/effector T cells from presensitized recipients. The extent of graft survival prolongation achieved has never been reported using other sensitized

FIGURE 6. Combined blockade of CD28-B7 and CD134-CD134L costimulatory pathways delayed cardiac graft rejection mediated by skin allograft-primed alloreactive lymphocytes in an athymic rat adoptive transfer model. Splenocytes (SPC; 4 ϫ 107) from either naive or allogenic skin FIGURE 5. The percentage of T cells expressing an activated/effector graft-sensitized LEW(RT1l) rats were adoptively transferred into LEW- phenotype following in vivo treatment with anti-CD134L mAb, background athymic LEW/Mol-rnu rats on the day of transplantation of a hCTLA4Ig, or the combination of anti-CD134LmAb and hCTLA4Ig, as- WF(RT1u) heart (MST: no treatment controls, Ͼ100 days, n ϭ 5; adoptive sessed by the percentage of CD3ϩCD44high cells. Results are representa- transfer of naive splenocytes, 17 Ϯ 4.6 days, n ϭ 4; adoptive transfer of tive of three experiments. The greatest decrease in CD3ϩCD44high expres- sensitized splenocytes, 5.3 Ϯ 0.5 days, n ϭ 4; adoptive transfer of sensi- sion is seen in those animals treated with combined hCTLA4Ig and anti- tized splenocytes plus anti-CD134L mAb, 14 Ϯ 2.8 days, n ϭ 4; adoptive CD134L mAb therapy on day 14, which persist and remain lower than the transfer of sensitized splenocytes plus hCTLA4Ig, 21.5 Ϯ 1.7 days, n ϭ 4; allograft controls up to day 30 (p ϭ 0.038 compared with allograft and adoptive transfer of sensitized splenocytes plus anti-CD134L mAb controls). plus hCTLA4Ig, 75.3 Ϯ 12.7 days, n ϭ 6). 2954 CD134-CD134L IN ALLOIMMUNITY immune response, and thus the ability of T cells to persist as a 18. Paterson, D. J., W. A. Jefferies, J. R. Green, M. R. Brandon, P. Corthesy, population over time (26). CD134-deficient T cells secrete IL-2 M. Puklavec, and A. F. Williams. 1987. Antigens of activated rat T lymphocytes including a molecule of 50,000 Mr detected only on CD4-positive T blasts. Mol. and proliferate normally during the initial period of activation, but Immunol. 24:1281. cannot sustain this during the latter phases of the primary response 19. Calderhead, D. M., J. E. Buhlmann, A. J. van den Eertwegh, E. Claassen, and exhibit decreased survival over time. Mice lacking CD134 R. J. Noelle, and H. P. Fell. 1993. Cloning of mouse OX40: a T cell activation marker that may mediate T-B cell interactions. J. Immunol. 151:5261. generated lower frequencies of Ag-specific CD4ϩ T cells late in 20. Latza, U., H. Durkop, S. Schnittger, J. Ringeling, F. Eitelbach, M. Hummel, the primary response in vivo and generated lower frequencies of C. Fonatsch, and H. Stein. 1994. The human OX40 homolog: cDNA structure, surviving memory cells as compared with WT mice (26). expression and chromosomal assignment of the ACT35 antigen. Eur. J. Immunol. This study represents the first demonstrating the role of the 24:677. 21. Al-Shamkhani, A., M. L. Birkeland, M. Puklavec, M. H. Brown, W. James, and CD134-CD134L pathway in transplant rejection, and defines the A. N. Barclay. 1996. OX40 is differentially expressed on activated rat and mouse interactions between CD134-CD134L and CD28-B7 pathways in T cells and is the sole receptor for the OX40 ligand. Eur. J. Immunol. 26:1695. naive and primed/effector alloimmune responses in vivo. More- 22. Ohshima, Y., Y. Tanaka, H. Tozawa, Y. Takahashi, C. Maliszewski, and over, it indicates that combined blockade of CD134L and B7 is G. Delespesse. 1997. Expression and function of OX40 ligand on human dendritic cells. J. Immunol. 159:3838. synergistic in promoting allograft survival even in presensitized 23. Brocker, T., A. Gulbranson-Judge, S. Flynn, M. Riedinger, C. Raykundalia, and recipients, and may thus represent one clinically relevant approach P. Lane. 1999. CD4 T cell traffic control: in vivo evidence that ligation of OX40 for overcoming the CD28-B7-independent allograft rejection on CD4 T cells by OX40-ligand expressed on dendritic cells leads to the accu- mechanisms, responsible for hampering the development of toler- mulation of CD4 T cells in B follicles. Eur. J. Immunol. 29:1610. 24. Imura, A., T. Hori, K. Imada, T. Ishikawa, Y. Tanaka, M. Maeda, S. Imamura, ance induction protocols (12). and T. Uchiyama. 1996. The human OX40/gp34 system directly mediates adhe-

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