Critical Role of OX40 in CD28 and CD154-Independent Rejection Gülçin Demirci, Farhana Amanullah, Reshma Kewalaramani, Hideo Yagita, Terry B. Strom, Mohamed H. This information is current as Sayegh and Xian Chang Li of October 1, 2021. J Immunol 2004; 172:1691-1698; ; doi: 10.4049/jimmunol.172.3.1691 http://www.jimmunol.org/content/172/3/1691 Downloaded from References This article cites 42 articles, 23 of which you can access for free at: http://www.jimmunol.org/content/172/3/1691.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on October 1, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Critical Role of OX40 in CD28 and CD154-Independent Rejection1 Gu¨lc¸in Demirci,* Farhana Amanullah,* Reshma Kewalaramani,† Hideo Yagita,‡ Terry B. Strom,* Mohamed H. Sayegh,† and Xian Chang Li*2 Blocking both CD28 and CD154 costimulatory pathways can induce transplant tolerance in some, but not all, transplant models. Under stringent conditions, however, this protocol often completely fails to block allograft rejection. The precise nature of such CD28/CD154 blockade-resistant rejection is largely unknown. In the present study we developed a new model in which both CD28 and CD154, two conventional T cell costimulatory molecules, are genetically knocked out (i.e., CD28/CD154 double-knockout (DKO) mice) and used this model to examine the role of novel costimulatory molecule-inducible costimulator (ICOS), OX40, 4-1BB, and CD27 in mediating CD28/CD154-independent rejection. We found that CD28/CD154 DKO mice vigorously rejected compared with the wild-type controls (mean (6 ؍ fully MHC-mismatched DBA/2 skin allografts (mean survival time, 12 days; n Downloaded from OX40 costimulation is critically important in skin allograft rejection in this model, as blocking the .(7 ؍ survival time, 8 days; n OX40/OX40 ligand pathway, but not the ICOS/ICOS ligand, 4-1BB/4-1BBL, or CD27/CD70 pathway, markedly prolonged skin allograft survival in CD28/CD154 DKO mice. The critical role of OX40 costimulation in CD28/CD154-independent rejection is further confirmed in wild-type C57BL/6 mice, as blocking the OX40/OX40 ligand pathway in combination with CD28/CD154 Our study revealed a key cellular mechanism of rejection .(5 ؍ blockade induced long term skin allograft survival (>100 days; n and identified OX40 as a critical alternative costimulatory molecule in CD28/CD154-independent rejection. The Journal of http://www.jimmunol.org/ Immunology, 2004, 172: 1691–1698. D28 and CD154 (also called CD40 ligand) are the pro- NK cells is believed to mediate the CD28/CD154 blockade-resis- totype and extensively studied T cell costimulatory mol- tant rejection (8, 9). Thus, gross depletion of CD8ϩ T cells or ecules, and their role in supporting T cell activation and targeting certain cytokine pathways required for activation of C ϩ acute allograft rejection has been well established (1). However, CD8 T cells and NK cells has been shown to synergize with detailed studies have repeatedly demonstrated that CD28 and CD28/CD154 blockade in preventing transplant rejection (8, 10– CD154 blockade is not always effective in preventing transplant 12). However, cell surface molecules with costimulatory proper- rejection, especially in stringent models (2–4). For example, the ties are not confined to CD28 and CD154, and multiple alternative by guest on October 1, 2021 effect of blocking CD28/CD154 costimulation on allograft survival T cell costimulatory molecules have recently been identified (13). varies considerably among different mouse strain combinations Indeed, engagement of inducible costimulator (ICOS),3 OX40 (4). Moreover, the remarkable effect of CD28/CD154 blockade on (CD134), 4-1BB (CD137), or CD27 during TCR stimulation can permanent cardiac allograft survival is not consistently observed in costimulate T cell activation, cytokine production, and effector cell the stringent skin transplant model (5). Furthermore, targeting function (14–18). Understanding precisely the role of such novel CD28/CD154 costimulation in large animal models consistently costimulatory molecules in the activation of diverse alloreactive T failed to produce stable allograft survival, even after prolonged cells and their relationship to conventional CD28 and CD154 co- treatment (6, 7). stimulation is critically important in transplantation. It has been The apparent limitation of CD28/CD154 blockade in tolerance shown that such novel costimulatory pathways can indeed affect induction invites vigorous investigation of the cellular and molec- the nature of the rejection response. For example, blocking the ular mechanisms involved in CD28/CD154-independent rejection. ICOS/ICOS ligand (ICOSL) pathway delayed, albeit it did not Depending on the models studied, activation of CD8ϩ T cells and prevent, cardiac allograft rejection (19, 20). Also, allograft sur- vival was markedly prolonged in mice deficient in both CD28 and 4-1BB, although a deficiency of either CD28 or 4-1BB alone did *Department of Medicine, Harvard Medical School, and Division of Immunology, not affect the rejection response (21). Similarly, long term cardiac Beth Israel Deaconess Medical Center, Boston, MA 02215; †Laboratory of Immuno- genetics and Transplantation, Brigham and Women’s Hospital, and Nephrology Di- allograft survival can be achieved by blocking both CD28 and vision, Children’s Hospital, Harvard Medical School, Boston, MA 02215; and ‡Jun- OX40 costimulatory pathways (22). Nonetheless, the identities of tendo University School of Medicine, Tokyo, Japan cells affected by such novel costimulatory pathways and their pre- Received for publication September 16, 2003. Accepted for publication November cise role in mediating the rejection response in the absence of both 19, 2003. CD28 and CD154 signals have not been studied. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance In the present study we developed a new model in which both with 18 U.S.C. Section 1734 solely to indicate this fact. CD28 and CD154, two conventional T cell costimulatory mole- 1 This work was supported by the Juvenile Diabetic Research Foundation Interna- cules, are genetically knocked out and used this CD28/CD154 dou- tional (to X.C.L.), the National Institutes of Health (to M.H.S. and T.B.S.), and the ble-knockout (DKO) model to critically examine the cellular basis Deutsche Forschungsgemeinschaft (to G.D.). 2 Address correspondence and reprint requests to Dr. Xian C. Li, Department of Medicine, Division of Immunology, Beth Israel Deaconess Medical Center, 330 3 Abbreviations used in this paper used: ICOS, inducible costimulator; DKO, double Brookline Avenue, RN389, Boston, MA 02215. E-mail address: knockout; ICOSL, ICOS ligand; mCTLA, murine CTLA; MST, mean survival time; [email protected] OX40L, OX40 ligand; wt, wild type. Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 1692 OX40 IN CD28/CD154 BLOCKADE-RESISTANT REJECTION of skin allograft rejection as well as the role of alternative costimu- staining and intracellular cytokine staining. The large number of cells latory molecules in supporting the rejection response. We found transferred (6 ϫ 107 cells/mouse) and the time point examined (3 days) that a skin allograft can be vigorously rejected in the absence of preclude homeostatic expansion of CFSE-labeled cells in the irradiated hosts, and cell division in this model is driven primarily by the host al- both CD28 and CD154 molecules, and rejection in this model is loantigens (5). critically dependent on OX40 costimulation. Treatment of irradiated host mice Materials and Methods In the CFSE model, treatment of irradiated hosts with anti-OX40L, anti- Mice CD70, anti-4-1BBL, or anti-ICOSL mAb consisted of 0.5 mg i.p. daily for d b 3 consecutive days starting with i.v. injection of CFSE-labeled cells. Mice DBA/2 (H-2 ) and C57BL/6 (H-2 ) mice, 8- to 10-wk-old, were purchased treated with rat IgG (Sigma-Aldrich) were included as controls. from The Jackson Laboratory (Bar Harbor, ME). Breeding pairs for CD28Ϫ/Ϫ and CD154Ϫ/Ϫ mice, both of which are on the C57BL/6 back- Cell surface staining and flow cytometry ground, were also obtained from The Jackson Laboratory. CD28Ϫ/Ϫ CD154Ϫ/Ϫ DKO mice were generated by crossing the CFSE-labeled cells were recovered from the host mice 3 days after adop- 6 CD28Ϫ/Ϫ and CD154Ϫ/Ϫ single-knockout mice. PCR-assisted genotyping tive cell transfer. Cells were resuspended in PBS/0.5% BSA (2 ϫ 10 /ml) using primer sets spanning the CD28 and CD154 genes as well as the and stained with CyChrome-conjugated anti-CD4 and CyChrome-anti- neomycin cassette was performed to identify the genotype of CD28 and CD8 on ice for 30 min, followed by staining with biotinylated anti-OX40, CD154 mutations in their offspring. Mice deficient for both CD28 and anti-4-1BB, anti-ICOS, and anti-CD27. Cells were washed in PBS/BSA CD154 (i.e., CD28Ϫ/ϪCD154Ϫ/Ϫ DKO) were selected and used for this and were further stained with PE-streptavidin. Cells stained with PE-con- study. jugated isotype control Ab were included as a control. After staining, cells All animals were housed in the animal facility at the Beth Israel Dea- were fixed in 1% formaldehyde before analysis.