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

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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 © 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 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 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 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. coness Medical Center (Boston, MA). Animal use and care conformed to All samples were analyzed using a FACSort equipped with CellQuest ϩ ϩ Downloaded from the guidelines established by the animal care committee of our institution. software (BD Bioscience, Mountain View, CA). CD4 and CD8 T cells were electronically gated, and the cell division profile and the expression Monoclonal Abs of novel costimulatory molecules at distinct division cycles were analyzed. At least 100,000 events were collected for each sample. The following Abs used for surface staining were obtained from BD PharMingen (San Diego, CA). FITC-anti-mouse CD4 (clone GK1.5, rat Intracellular cytokine staining IgG2b), PE-anti-mouse CD8␣ (clone 53-6.7, rat IgG2a), CyChrome anti- mouse CD4 (clone GK1.5), CyChrome anti-mouse CD8␣ (clone 53-6.7), CFSE-labeled cells harvested from the host mice were resuspended in biotin-anti-CD27 (clone LG.3A10, hamster IgG), biotin-anti-OX40 (clone RPMI 1640 medium supplemented with 10% FCS and 1% penicillin/strep- http://www.jimmunol.org/ OX86, rat IgG1), biotin-anti-4-1BB (clone 1AH2, rat IgG1), biotin-anti- tomycin at 5 ϫ 106/ml. Cells were stimulated in vitro with PMA (50 ng/ml) ICOS (clone 7E.1799, rat IgG2b), PE-anti-mouse IL-2 (clone JES6-5H4, and ionomycin (500 ng/ml; Sigma-Aldrich) at 37°Cfor4h.Inthelast2h rat IgG2b), PE-anti-mouse IFN-␥ (clone XMG 1.2, rat IgG1), PE-strepta- of culture, GolgiStop (BD PharMingen) was added to the culture at a con- vidin, PE-isotype control Abs, and hamster anti-mouse CD3 (clone 2C11, centration of 1 ␮g/ml. Cells were harvested after the in vitro stimulation, hamster IgG). stained with CyChrome anti-mouse CD4 or CyChrome anti-mouse CD8, Anti-ICOSL mAb (clone HK5.3, rat IgG2a) (23), anti-OX40 ligand (anti- respectively, and fixed, and the cell membrane was permeabilized in Cyto- OX40L) mAb (clone RM134L, rat IgG2b), anti-CD70 mAb (clone FR70, fix/Cytoperm solution (BD PharMingen) at 4°C for 10 min, followed by rat IgG2b), and anti-4-1BB ligand mAb (TKS-1, rat IgG2a) (17) were washing in Perm/Wash solution (BD PharMingen). Cells were then resus- manufactured from their respective hybridomas by BioExpress Cell Cul- pended in Perm/Wash solution (1 ϫ 106) and stained with PE-conjugated ␥ ture Services (West Lebanon, NH) and used for the in vivo experiments. Abs against mouse IL-2 and IFN- on ice for 30 min. PE-conjugated iso- by guest on October 1, 2021 Murine CTLA-4Ig (mCTLA-4Ig) was a gift from Dr. R. Peach (Bristol type control Ab was included in the staining protocol as a control. The Myers Squibb, Princeton, NJ). The isotype control rat IgG used for the in expression of IL-2 and IFN-␥ in vivo at distinct cell division cycles was vivo study was obtained from Sigma-Aldrich (St. Louis, MO). analyzed by FACS. A hybridoma cell line secreting the anti-mouse CD154 mAb (MR1, hamster IgG) was obtained from American Type Culture Collection (Ma- Calculation of cell division frequency nassas, VA). The hybridoma cells were grown in serum-free UltraCulture The responder frequency of CFSE-labeled CD4ϩ and CD8ϩ T cells pro- medium (BioWhittaker, Walkersville, MD), and mAb was purified from liferating in vivo in the allogeneic hosts was calculated as previously re- the culture supernatant with G columns. ported (25). Briefly, distinct rounds of cell divisions were identified by Cell proliferation assay their CFSE profiles. The absolute number of cells in each cell division was calculated using the FACS acquisition software, CellQuest. The number of Splenic leukocytes were prepared as previously described (24). Cells were precursors that proliferated and gave rise to the absolute number of daugh- plated in 96-well plates (2 ϫ 105/well) in RPMI 1640 medium supple- ter cells was extrapolated using the formula: y/2n, where y is the absolute mented with 10% FCS and 1% penicillin/streptomycin (BioWhittaker) and number of cells in each cell cycle, and n is the number of cell divisions. For were stimulated with various concentrations of anti-CD3 (0.6–10 ␮g/ml; example, 16 daughter cells in the third cell division are the progeny of two 2C11; PharMingen) in triplicate. Cells were incubated at 37°C for 72 h, and precursors, each of which have divided three times (16/23 ϭ 2). The fre- for the last 16 h of culture cells were pulsed with [3H]TdR (1 ␮Ci/well; quency of proliferating T cells in the responder population was then cal- Amersham Pharmacia Biotech, Arlington Heights, IL). [3H]TdR uptake culated by dividing the total number of precursors by the sum of total was determined by scintillation counting. CFSE-labeled cells collected. CFSE labeling Skin grafting and treatment protocols Spleen and peripheral lymph nodes were harvested from donor mice, and A full-thickness tail skin graft (ϳ1cm2) from DBA/2 donors (H-2d) was a single-cell suspension was prepared in HBSS. RBC were lysed by hy- transplanted onto the thoracic wall of wild-type (wt) C57BL/6 mice (H-2b) potonic shock. Cells were resuspended in HBSS at 1 ϫ 107 cells/ml for and CD28/CD154 DKO mice (H-2b). The skin graft was secured with an labeling with CFSE (Molecular Probes, Portland, OR) as previously de- adhesive bandage for the initial 5 days. Graft survival was then followed by scribed (25). Briefly, cells were incubated with CFSE at a final concentra- daily visual inspection. Rejection was defined as complete necrosis and tion of 5 ␮M in serum-free HBSS at room temperature for 6 min. The loss of viable skin tissue. labeling was then terminated by the addition of FCS (10% of the total Treatment of transplant recipients with mCTLA-4Ig consisted of 0.5 mg volume). Cells were washed twice in HBSS before i.v. injection. i.p. on days 0, 1, and 3 after skin grafting. Anti-CD154 mAb was given at 0.5 mg i.p. on days 0, 1, 3, and 6. Anti-OX40L, anti-ICOSL, anti-4-1BB, In vivo activation of CFSE-labeled cells or anti-CD70 mAb was given at 0.5 mg i.p. on days 0, 2, 4, and 8 after skin transplantation. Host DBA/2 mice were lethally irradiated (1000 rad) with a Gammacell 7 Exactor (Kanata, Ontario, Canada). Each mouse then received ϳ6 ϫ 10 Histopathology CFSE-labeled donor cells in 0.5 ml of HBSS via the tail vein. Three days later, the host mice were sacrificed, spleens and peripheral lymph nodes The skin graft was removed from recipient mice at specified time points were harvested, and a single-cell suspension was prepared for cell surface after transplantation, fixed in 10% formalin, and embedded in paraffin. The Journal of Immunology 1693

Serial tissue sections (5 ␮m) were prepared and mounted on SuperFrost Plus glass slides (Fisher Scientific, Pittsburgh, PA), fixed in methanol, and stained in H&E for histological evaluation. Results CD28/CD154 DKO mice promptly reject the skin allograft To definitively study the mechanisms of CD28/CD154 blockade- resistant rejection, we generated a new model in which both CD28 and CD154, two conventional T cell costimulatory molecules, are genetically knocked out (i.e., CD28/CD154 DKO mice). FACS analysis showed that the CD4ϩ and CD8ϩ subsets in the spleens of wt C57BL/6 mice and CD28/CD154 DKO mice were compa- rable (Fig. 1A). A similar finding was observed in blood and pe- ripheral lymph nodes (data not shown). However, splenocytes FIGURE 2. Skin allograft survival in wt C57BL/6 and CD28/CD154 from CD28/CD154 DKO mice, in contrast to wt controls, did not DKO recipients. DBA/2 tail skin was grafted onto the thoracic wall of the mount a proliferative response in vitro to anti-CD3 stimulation recipient mice, and graft survival was determined and presented as a (Fig. 1B), confirming the critical role of CD28 and CD154 signals Kaplan-Meier plot. Each group had six or seven animals. in T cell activation in vitro (26). To determine whether CD28/CD154 DKO mice (C57BL/6 background, H-2b) could mount an allograft rejection response, we Downloaded from transplanted fully MHC-mismatched DBA/2 (H-2d) skin allografts graft survival was slightly prolonged compared with that of the wt onto the CD28/CD154 DKO mice, and graft survival was deter- controls (MST, 8 days; n ϭ 7). Histological analysis of the skin mined and compared with that of wt controls. As shown in Fig. 2, allograft revealed heavy lymphocytic infiltration and extensive tis- CD28/CD154 DKO mice promptly rejected the DBA/2 skin allo- sue damage (data not shown). Thus, the skin allograft can be vig- grafts, with a mean survival time (MST) of 12 days (n ϭ 6), albeit orously rejected in the absence of both CD28 and CD154 costimu- latory molecules. http://www.jimmunol.org/

CD28/CD154 deficiency and in vivo activation of CD4ϩ and CD8ϩ T cells The prompt rejection of skin allografts by CD28/CD154 DKO mice suggests that the in vivo T cell activation program is rela- tively normal despite impaired T cell activation in vitro. To crit- ically examine the impact of CD28/CD154 deficiency on T cell activation in vivo, we labeled splenic leukocytes from CD28/ by guest on October 1, 2021 CD154 DKO mice (H-2b) with the tracking dye CFSE and injected them into lethally irradiated DBA/2 hosts (H-2d). Proliferation of CD28/CD154-deficient T cells in vivo was determined and com- pared with that of CFSE-labeled wt control cells. This model al- lows quantitative analysis of T cell activation in vivo at the single- cell level (5, 25). As shown in Fig. 3A, both CD4ϩ and CD8ϩ T cells from wt C57BL/6 mice divided vigorously in the allogeneic hosts, and up to eight cell divisions could be clearly identified 3 days after adop- tive cell transfer. Calculation of division frequency revealed that ϳ25% of CD4ϩ T cells and as much as 38% of CD8ϩ T cells recovered from the host spleen entered the cell cycle. Interestingly, CD28/CD154 deficiency preferentially affected the in vivo divi- sion of CD4ϩ T cells, and the division frequency was reduced by Ͼ2-fold (ϳ11%) compared with that in wt controls (ϳ25%). Nonetheless, CD4ϩ T cell division was not completely abolished despite genetic deficiency of both CD28/CD154; ϳ10% of CD4ϩ T cells recovered from the host spleen still entered the cell cycle and divided multiple times (more than eight times), suggesting that not all CD4ϩ T cells rely on CD28 and CD154 signals for acti- vation. Up-regulation of cell surface expression of the CD4 mol- ecule is often associated with robust activation of CD4ϩ T cells

ϩ ϩ (27). Consistent with this, the overwhelming majority of activated FIGURE 1. A, Comparison of CD4 and CD8 T cell subsets in the CD28/CD154-deficient CD4ϩ T cells was confined to the CD4high periphery of wt C57BL/6 and CD28/CD154 DKO mice. Spleen cells were fraction (Fig. 3B). Intracellular cytokine staining revealed that ac- prepared and stained with FITC-anti-CD4 and PE-anti-CD8 and were an- alyzed by FACS. B, Proliferation of wt C57BL/6 and CD28/CD154-defi- tivation of such a T cell subset was associated with expression of high levels of IL-2 and IFN-␥ effector (Fig. 3C). Thus, cient T cells in vitro. Spleen cells from wt and DKO mice were prepared ϩ and stimulated in vitro with anti-CD3 mAb for 72 h. Cell proliferation was levels of CD4 expression by activated CD4 T cells may be a determined by [3H]TdR uptake and is presented as the mean counts per useful tool to examine the CD28/CD154-independent fraction of ϩ minute of triplicate assays. activated CD4 T cells. 1694 OX40 IN CD28/CD154 BLOCKADE-RESISTANT REJECTION

In contrast to CD4ϩ T cells, proliferation of CD28/CD154-de- ficient CD8ϩ T cells in vivo was not affected by the genetic de- ficiency of both CD28 and CD154 molecules. CD8ϩ T cells from wt C57BL/6 and CD28/CD154 DKO mice divided with similar kinetics in vivo (Fig. 3A), further supporting the idea that activa- tion of CD8ϩ T cells in certain transplant models is independent of CD28 and CD154 signals (28, 29). In the CFSE model used in the present study, adoptive transfer of CFSE-labeled syngeneic cells into irradiated hosts did not in- duced marked T cell proliferation (Fig. 3A, bottom panel), sug- gesting that the large number of cells transferred (6 ϫ 107 cells/ mouse) and the time point examined (3 days after cell transfer) preclude homeostatic cell expansion, and cell division in this model is driven primarily by the host alloantigens.

Expression of novel costimulatory molecules by CD28/CD154- deficient T cells in vivo To examine the possible role of novel costimulatory molecules (i.e., ICOS, OX40, 4-1BB, and CD27) in the activation of CD28/ Downloaded from CD154-deficient T cells in vivo, we first examined the cell surface expression of such costimulatory molecules upon in vivo activa- tion of CD28/CD154-deficient T cells. CFSE-labeled, CD28/ CD154-deficient T cells were stimulated in vivo, and the expres- sion of such novel costimulatory molecules on T cells at distinct division cycles was examined and compared. As shown in Fig. 4, CD27 was constitutively expressed at high levels by both CD4ϩ http://www.jimmunol.org/ and CD8ϩ T cells regardless of the number of cell divisions. 4-1BB was not detectable on CD4ϩ T cells, but was expressed at low levels on CD8ϩ T cells, especially after multiple cell divi- sions. Interestingly, for both CD4ϩ and CD8ϩ T cells, levels of ICOS expression increased progressively after each consecutive cell division, and virtually all dividing T cells stained positively for ICOS after five or six cell divisions (Fig. 4, B and D). A similar

pattern of OX40 expression was observed. However, levels of by guest on October 1, 2021 OX40 expression were consistently higher on CD4ϩ T cells than on CD8ϩ T cells, especially at later cell divisions (Fig. 4).

Effect of blocking novel costimulatory pathways on in vivo activation of CD28/CD154-deficient T cells To further determine the role of such novel costimulatory mole- cules in the activation of CD28/CD154-deficient T cells in vivo, we again labeled CD28/CD154-deficient cells with CFSE and adoptively transferred them into lethally irradiated allogeneic hosts. The host mice were treated with saturating doses of blocking mAbs to block the OX40/OX40L, ICOS/ICOSL, 4-1BB/4-1BBL, or CD27/CD70 costimulatory pathway, and the proliferation of the CD4high fraction and CD8ϩ T cells was determined and compared with that in control Ab-treated animals. As shown in Fig. 5, A and C, blocking the CD27/CD70 costimulatory pathway did not inhibit the in vivo proliferation of CD28/CD154-deficient CD4high cells and CD8ϩ T cells, and the CD4high fraction and CD8ϩ T cells FIGURE 3. A, Effect of CD28/CD154 deficiency on in vivo T cell pro- liferation. CFSE-labeled cells from either wt C57BL/6 or CD28/CD154 DKO mice (6 ϫ 107) were allowed to proliferate in lethally irradiated DBA/2 hosts. The division history of CD4ϩ and CD8ϩ T cell subsets in the host spleen was analyzed 3 days later by FACS. CD4ϩ and CD8ϩ T cells R1. The CD4high (R2) and CD4low (R3) subsets were identified based on were identified and selectively gated by staining with CyChrome-anti-CD4 the CD4 levels and the FSC, and their division histories were analyzed and or anti-CD8 mAb after recovery from the host mice. The division fre- compared simultaneously. C, Intracellular IL-2 and IFN-␥ staining of quency of each subset was calculated as described in Materials and Meth- CD28/CD154-deficient CD4ϩ T cells. CFSE-labeled CD28/CD154-defi- ods and was shown in each panel. Similar data were obtained in three cient cells were stimulated in vitro with PMA and ionomycin for 4 h after independent experiments. B, High division frequency and increased CD4 recovery from irradiated DBA/2 hosts. Cells were briefly stained with Cy- expression in CD4ϩ T cells. CFSE-labeled CD28/CD154-deficient cells Chrome-anti-CD4, then fixed, permeabilized, and further stained for intra- were recovered from irradiated DBA/2 hosts 3 days after adoptive transfer cellular IL-2 and IFN-␥. Analysis was performed based on cells stained and stained with CyChrome-anti-CD4. The CD4ϩ population was plotted with the PE-isotype control mAb. Representative data from three experi- against the forward scatter (FSC). The total CD4ϩ population was gated as ments are shown. The Journal of Immunology 1695

FIGURE 4. A, Expression of novel co- stimulatory molecules by CD28/CD154-de- ficient CD4ϩ T cells. CFSE-labeled CD28/ CD154-deficient cells were allowed to proliferate in irradiated DBA/2 hosts for 3 days. The expression of 4-1BB, CD27, OX40, and ICOS on CD4ϩ T cells at distinct division cycles was stained, analyzed, and presented. B, Comparison of levels of 4-1BB, CD27, OX40, and ICOS expression on CD4ϩ T cells at individual division cy- cles. The CD4ϩ population was gated, and division history was identified based on the Downloaded from CFSE profile. Each individual cell division was gated, and the expression of 4-1BB, CD27, OX40, and ICOS molecules was cal- culated. The percentage of positive cells was plotted against the number of cell divisions. C and D, The expression of 4-1BB, CD27, OX40, and ICOS on CD28/CD154-deficient http://www.jimmunol.org/ CD8ϩ T cells. Parallel analysis was per- formed as described in A and B. Representa- tive data from three experiments are shown. by guest on October 1, 2021

divided with a similar kinetics as the controls. Blocking the ICOS/ whereas the control mice rejected the skin allograft with a MST of ICOSL costimulatory pathway or the 4-1BB/4-1BBL pathway ex- 12 days (n ϭ 5). Treatment of CD28/CD154 DKO mice with anti- erted some inhibitory effect on the late expansion of CD8ϩ T cells, ICOSL, anti-4-1BBL, or anti-CD70 failed to prevent skin allograft but had no effect to block the proliferation of CD4high T cells. In rejection, and all skin allografts were rejected within 20 days after stark contrast, blocking the OX40/OX40L pathway nearly abol- transplantation. ished in vivo proliferation of the CD4high fraction (Fig. 5A). In To prove that the critical role of OX40 signals in allograft re- fact, the entire CD4ϩ population recovered from the anti-OX40L- jection is not restricted to the CD28/CD154 DKO model, we trans- treated hosts showed no apparent up-regulation of the CD4 mol- planted the DBA/2 skin allograft onto wt C57BL/6 mice, which are ecule and remained undividing (Fig. 5B). Interestingly, blocking often regarded as the toughest strain in transplantation (4). The the OX40/OX40L pathway also markedly inhibited the in vivo recipient C57BL/6 mice were treated with anti-OX40L (0.5 mg on proliferation of CD28/CD154-deficient CD8ϩ T cells (Fig. 5C). days 0, 2, 4, and 8) to block the OX40/OX40L pathway along with These findings suggest that OX40 costimulation plays a key role in mCTLA-4Ig (0.5 mg on days 0, 1, and 3) and anti-CD154 (MR1; the activation of CD28/CD154-deficient T cells. 0.5 mg on days 0, 1, 3, and 6) to block both /CD28 and CD40/ CD154 costimulatory pathways. As shown in Fig. 7, untreated Role of OX40 in CD28/CD154-independent rejection mice promptly rejected the skin allograft, with a MST of 8 days The critical role of OX40 costimulation in the activation of CD28/ (n ϭ 7). Neither mCTLA-4Ig and anti-CD154 nor anti-OX40L CD154-deficient T cells suggests that OX40 signals may play a treatment alone inhibited skin allograft rejection, and all treated key role in mediating the skin allograft rejection in CD28/CD154 mice rejected DBA/2 skin within 15 days after transplantation. In DKO mice. To test this possibility, we transplanted the DBA/2 stark contrast, C57BL/6 mice treated with anti-OX40L plus skin allograft onto CD28/CD154 DKO recipients. The recipient mCTLA-4Ig and anti-CD154 experienced long term skin allograft mice were treated with anti-OX40L mAb (0.5 mg i.p. on days 0, 2, survival (MST, Ͼ100 days; n ϭ 5). Histological analysis of the 4, and 8 after skin grafting) and skin allograft survival was deter- skin graft 100 days after transplantation revealed a grossly normal mined. As shown in Fig. 6, treatment of CD28/CD154 DKO re- skin graft with minimal lymphocytic infiltration compared with the cipients with anti-OX40L mAb markedly prolonged skin allograft controls (data not shown), further demonstrating the critical role of survival, and four of seven transplants survived for Ͼ100 days, OX40 costimulation in CD28/CD154-independent rejection. 1696 OX40 IN CD28/CD154 BLOCKADE-RESISTANT REJECTION

FIGURE 6. Effect of OX40/OX40L, 4-1BB/4-1BBL, ICOS/ICOSL, or CD27/CD70 costimulatory blockade on skin allograft survival in CD28/ CD154 DKO mice. CD28/CD154 DKO mice were grafted with DBA/2 skin and treated with blocking mAb directed against OX40L, 4-1BBL, ICOSL or CD70. The Abs were given at 0.5 mg i.p. on days 0, 2, 4, and 8 after skin transplantation, and graft survival was determined and pre- sented as a Kaplan-Meier plot. Downloaded from

Discussion It becomes apparent that blocking CD28 and CD154 costimulatory pathways is necessary, but not sufficient, for preventing transplant rejection. In most stringent transplant models studied, blocking

both B7/CD28 and CD40/CD154 costimulatory pathways often http://www.jimmunol.org/ fails to block transplant rejection, let alone the induction of trans- plant tolerance (4, 5, 30). Thus, identification of mechanisms sup- porting the CD28/CD154-independent rejection and development of means to target such cellular activation are critically important in transplantation research. In the present study we demonstrated, using mice deficient for both CD28 and CD154, that vigorous skin allograft rejection can proceed in the absence of both CD28 and CD154, definitively proving that CD28 and CD154 signals are not absolutely required by guest on October 1, 2021 for rejection in all transplant models. Detailed in vivo analysis revealed that T cells involved in the allograft response exhibit a remarkable heterogeneity in the CD28/CD154 requirement for ac- tivation. Clearly, genetic deficiency of both CD28 and CD154 preferentially affects the in vivo activation of CD4ϩ T cells, but not the CD8ϩ T cells (Fig. 3A). However, not all CD4ϩ T cells require CD28 and CD154 signals for activation, and a subset of CD4ϩ T cells can proliferate extremely well in the absence of CD28 and CD154 molecules (Fig. 3B). Interestingly, OX40 co- stimulation seems to be critically important in the activation of CD28/CD154-deficient T cells, as blocking the OX40/OX40L co- stimulatory pathway, but not the ICOS/ICOSL, 4-1BB/4-1BBL, or CD27/CD70 pathway, abolished the in vivo activation of CD28/ CD154-deficient T cells (Fig. 5). Importantly, blocking OX40/ OX40L pathway in either CD28/CD154 DKO mice or concurrent with transient CD28 and CD154 blockade in wt mice induced long term skin allograft survival (Ͼ100 days; Figs. 6 and 7). Our study

time of cell transfer (0.5 mg i.p. on days 0–2). Analysis was performed on day 3 after cell transfer by gating onto the CD4high fraction. B, Complete inhibition of the CD4high fraction by blocking the OX40 signals. The total CD4ϩ population, identified by staining with CyChrome-anti-CD4, was gated as R1. The CD4high (R2) and CD4low (R3) subsets were identified based on the levels of CD4 expression, and the forward scatter (FSC) as FIGURE 5. A, Effect of blocking the OX40/OX40L, 4-1BB/4-1BBL, described in Fig. 3B, and their division histories were analyzed simulta- ICOS/ICOSL, or CD27/CD70 costimulatory pathway on in vivo prolifer- neously. C, Effect of blocking the OX40/OX40L, 4-1BB/4-1BBL, ICOS/ ϩ ation of CD28/CD154-deficient CD4 T cells. CFSE-labeled, CD28/ ICOSL, or CD27/CD70 pathway on in vivo proliferation of CD28/CD154- CD154-deficient cells were injected into irradiated DBA/2 hosts and deficient CD8ϩ T cells. Parallel analysis was performed as described in A. treated with anti-OX40L, anti-4-1BBL, anti-ICOSL, or anti-CD70 at the Representative data from three experiments are shown. The Journal of Immunology 1697

In certain models, the most profound effect of blocking OX40 costimulation is the inhibition of the frequency of memory CD4ϩ T cells generated (14, 15). This finding led to the belief that OX40 signals are required for the generation of CD4ϩ memory cells. Thus, it is possible that a subset of activated CD4ϩ T cells that are programmed to become memory cells are particularly sensitive to OX40 signals. In this regard, our finding that a subset of activated T cells (i.e., CD4high T cells) is associated with OX40 function is of considerable importance, because such T cells may be destined to become memory T cells. It has been suggested that activation of memory T cells is inherently resistant to CD28 and CD154 block- ade (3, 32, 33), and therefore, blocking OX40 signals may be par- FIGURE 7. Skin allograft survival in wt C57BL/6 mice treated with ticularly important in control such a unique T cell subset. This is anti-OX40L, mCTLA-4Ig, and anti-CD154 mAb. Anti-OX40L mAb was consistent with a recent report that blocking the OX40 pathway is given at 0.5 mg i.p. on days 0, 2, 4, and 8; mCTLA-4Ig at 0.5 mg i.p. on beneficial in a presensitized cardiac transplant model (22). Our days 0, 1, and 3; and anti-CD154 at 0.5 mg i.p. on days 0, 1, 3, and 6 after study along with a growing number of other reports highlight the grafting of DBA/2 skin. critical role of OX40 costimulation in T cell activation (14, 34) as well as in certain cytopathic conditions (35–39). The precise role of other alternative costimulatory molecules in activation and effector differentiation of T cells and their relation- Downloaded from clearly identified OX40 as a key alternative costimulatory mole- ship to the conventional CD28/CD154 costimulation warrant fur- cule in supporting CD28/CD154-independent rejection. ther study. In vivo analysis clearly demonstrated that CD27 is con- OX40 is a member of the TNF receptor superfamily, and unlike stitutively expressed at high levels on CD28/CD154-deficient T CD28, OX40 is not constitutively expressed on naive T cells, but cells (Fig. 4). It is not clear why blocking the CD27/CD70 path- its expression is rapidly induced upon T cell activation (31). ways completely failed to inhibit the proliferation of CD28/ OX40L has a much wider tissue distribution than the B7 family CD154-deficient T cells (Fig. 5). It is possible that blocking the http://www.jimmunol.org/ . OX40L is expressed not only on APCs, but also on other CD27/CD70 pathway may selectively affect the effector function cell types, including vascular endothelial cells, and its expression of activated T cells despite a normal proliferative response. How- is also induced after immune activation (31). The inducible nature ever, this is unlikely, as CD28/CD154 DKO mice treated with of OX40 and its ligand suggests that the OX40/OX40L costimu- anti-CD70 mAb can vigorously reject the skin allograft with sim- latory pathway may play a particularly important role when the T ilar kinetics as the untreated controls. It remains to be determined cell activation program is successfully launched. In this regard, our whether cell types other than the T cells (e.g., B, NK, or NKT data demonstrate several interesting findings. First, costimulatory cells) are particularly sensitive to the CD27/CD70 costimulatory signals from both CD28 and CD154 are not required for either blockade. Similarly, blocking the ICOS/ICOSL pathway in CD28/ by guest on October 1, 2021 OX40 expression or its costimulatory function. Clearly, T cells CD154 DKO mice also failed to inhibit skin allograft rejection. deficient in both CD28 and CD154 can express OX40 upon in vivo The lack of effect is not due to the lack of ICOS expression on activation, and OX40/OX40L costimulatory signals play a key role CD28/CD154-deficient T cells, as ICOS is highly expressed on in supporting CD28/CD154-independent T cell activation. Conse- both activated CD4ϩ and CD8ϩ T cells, especially after several quently, blocking OX40 costimulation in CD28/CD154 DKO mice cell division cycles in vivo (Fig. 4). This is in contrast with pre- markedly prolonged skin allograft survival (Fig. 6). Second, not all viously reports showing that blocking the ICOS/ICOSL pathway activated T cells express OX40. Instead, OX40 expression is pri- can significantly prolong allograft survival in a mouse heart trans- marily confined to a subset of activated CD4ϩ T cells that also plant model (19, 20). The apparent difference is unclear, but is express higher levels of CD4 on the cell surface (i.e., CD4high probably due to the different models used (heart vs skin transplant fraction). Interestingly, it takes several rounds of cell divisions for model) or the different treatment protocols used. It is well known the activated T cells to express OX40, as OX40 is highly expressed that the skin allograft is notoriously more difficult to tolerize than only after four or five cell divisions (Fig. 4), suggesting that OX40 other organ transplants. In a recent report (20), it has been shown expression may be regulated by the cell cycle. Third, despite the that delayed blockade of the ICOS/ICOSL pathway has a far more expression of other alternative costimulatory molecules, OX40 ap- profound therapeutic effect than the initial blockade. Whether de- pears to be the key player mediating T cell activation when both layed treatment with the anti-ICOSL would prolong skin allograft CD28 and CD154 signals are blocked. This conclusion is based on survival in CD28/CD154-deficient mice remains to be examined. the finding that blocking the OX40/OX40L pathways, but not the It should be noted that skin allograft survival in wt mice treated ICOS/ICOSL, 4-1BB/4-1BBL, or the CD27/CD70 pathway, abol- with CTLA-4Ig and anti-CD154 mAb in combination with OX40 ished the activation of CD28/CD154-deficient T cells and induced blockade is noticeably better than that in CD28/CD154-deficient long term skin allograft survival in the absence of CD28/CD154 mice treated with anti-OX40L. This observation raises the possi- costimulation (Figs. 6 and 7). Finally, blocking the OX40/OX40L bility that the in vivo effect of CTLA-4Ig and anti-CD154 mAb pathway can also affect the activation of CD8ϩ T cells in vivo. may be more than just simply blocking CD28 and CD154 costimu- Consistent with several previous reports (8, 28, 29), genetic defi- latory signals. Recent studies have shown that CTLA-4Ig, which ciency of both CD28 and CD154 had minimal effect on the acti- binds to B7 molecules on the surface of APCs with high affinity, vation of CD8ϩ T cells in vivo. However, proliferation of CD28/ can activate the indoleamine 2,3-dioxygenase system; such in- CD154-deficient CD8ϩ T cells in the allogeneic hosts was doleamine 2,3-dioxygenase activation in APCs mediates trypto- markedly inhibited upon blocking the OX40/OX40L pathway. It phan catabolism that is capable of inhibiting T cell activation and remains to be determined, however, whether blocking OX40 in- inducing T cell tolerance (40, 41). Furthermore, anti-CD154 hibits CD8ϩ T cells directly or indirectly by inhibition of a subset (MR1) mAb possesses certain cytolytic activities and can directly of CD4ϩ T cells. kill activated T cells via activation of the complement cascade 1698 OX40 IN CD28/CD154 BLOCKADE-RESISTANT REJECTION

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Peschon, and T. H. Watts. 1999. Analysis of 4-1BB ligand (4-1BBL)-deficient mice and of mice lacking both 4-1BBL and CD28 reveals a OX40 costimulation plays a key role in supporting the activation role for 4-1BBL in skin allograft rejection and in the cytotoxic T cell response to ϩ of a subset of CD4 T cells and/or CD8 T cells in the absence of influenza virus. J. Immunol. 163:4833. both CD28 and CD154 signals. Clearly, the identification of OX40 22. Yuan, X., A. D. Salama, V. Dong, I. Schmitt, N. Najafian, A. Chandraker, H. Akiba, H. Yagita, and M. H. Sayegh. 2003. The role of the CD134-CD134 as a novel alternative costimulatory molecule involved in CD28/ ligand costimulatory pathway in alloimmune responses in vivo. J. Immunol. CD154-independent rejection in such a stringent skin transplant 170:2949. 23. Iwai, H., Y. Kozono, S. Hirose, H. Akiba, H. Yagita, K. Okumura, H. Kohsaka, model should provide critical insights on the continued develop- N. Miyasaka, and M. Azuma. 2002. 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