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* Department of Medicine, Harvard Medical School, and Division of Immunology, Beth Israel Deaconess Medical Center, Boston, MA 02215;
Laboratory of Immunogenetics and Transplantation, Brigham and Women’s Hospital, and Nephrology Division, Children’s Hospital, Harvard Medical School, Boston, MA 02215; and
Juntendo University School of Medicine, Tokyo, Japan
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The apparent limitation of CD28/CD154 blockade in tolerance induction invites vigorous investigation of the cellular and molecular mechanisms involved in CD28/CD154-independent rejection. Depending on the models studied, activation of CD8+ T cells and NK cells is believed to mediate the CD28/CD154 blockade-resistant rejection (8, 9). Thus, gross depletion of CD8+ T cells or targeting certain cytokine pathways required for activation of CD8+ T cells and NK cells has been shown to synergize with CD28/CD154 blockade in preventing transplant rejection (8, 10, 11, 12). However, cell surface molecules with costimulatory properties are not confined to CD28 and CD154, and multiple alternative T cell costimulatory molecules have recently been identified (13). Indeed, engagement of inducible costimulator (ICOS),3 OX40 (CD134), 4-1BB (CD137), or CD27 during TCR stimulation can costimulate T cell activation, cytokine production, and effector cell function (14, 15, 16, 17, 18). Understanding precisely the role of such novel costimulatory molecules in the activation of diverse alloreactive T cells and their relationship to conventional CD28 and CD154 costimulation is critically important in transplantation. It has been shown that such novel costimulatory pathways can indeed affect the nature of the rejection response. For example, blocking the ICOS/ICOS ligand (ICOSL) pathway delayed, albeit it did not prevent, cardiac allograft rejection (19, 20). Also, allograft survival was markedly prolonged in mice deficient in both CD28 and 4-1BB, although a deficiency of either CD28 or 4-1BB alone did not affect the rejection response (21). Similarly, long term cardiac allograft survival can be achieved by blocking both CD28 and OX40 costimulatory pathways (22). Nonetheless, the identities of cells affected by such novel costimulatory pathways and their precise role in mediating the rejection response in the absence of both CD28 and CD154 signals have not been studied.
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 and used this CD28/CD154 double-knockout (DKO) model to critically examine the cellular basis of skin allograft rejection as well as the role of alternative costimulatory molecules in supporting the rejection response. We found that a skin allograft can be vigorously rejected in the absence of both CD28 and CD154 molecules, and rejection in this model is critically dependent on OX40 costimulation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DBA/2 (H-2d) and C57BL/6 (H-2b) mice, 8- to 10-wk-old, were purchased from The Jackson Laboratory (Bar Harbor, ME). Breeding pairs for CD28-/- and CD154-/- mice, both of which are on the C57BL/6 background, were also obtained from The Jackson Laboratory.
CD28-/- CD154-/- DKO mice were generated by crossing the CD28-/- and CD154-/- single-knockout mice. PCR-assisted genotyping using primer sets spanning the CD28 and CD154 genes as well as the neomycin cassette was performed to identify the genotype of CD28 and CD154 mutations in their offspring. Mice deficient for both CD28 and CD154 (i.e., CD28-/-CD154-/- DKO) were selected and used for this study.
All animals were housed in the animal facility at the Beth Israel Deaconess Medical Center (Boston, MA). Animal use and care conformed to the guidelines established by the animal care committee of our institution.
Monoclonal Abs
The following Abs used for surface staining were obtained from BD PharMingen (San Diego, CA). FITC-anti-mouse CD4 (clone GK1.5, rat 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), biotin-anti-CD27 (clone LG.3A10, hamster IgG), biotin-anti-OX40 (clone OX86, rat IgG1), biotin-anti-4-1BB (clone 1AH2, rat IgG1), biotin-anti-ICOS (clone 7E.1799, rat IgG2b), PE-anti-mouse IL-2 (clone JES6-5H4, rat IgG2b), PE-anti-mouse IFN-
(clone XMG 1.2, rat IgG1), PE-streptavidin, PE-isotype control Abs, and hamster anti-mouse CD3 (clone 2C11, hamster IgG).
Anti-ICOSL mAb (clone HK5.3, rat IgG2a) (23), anti-OX40 ligand (anti-OX40L) mAb (clone RM134L, rat IgG2b), anti-CD70 mAb (clone FR70, rat IgG2b), and anti-4-1BB ligand mAb (TKS-1, rat IgG2a) (17) were manufactured from their respective hybridomas by BioExpress Cell Culture Services (West Lebanon, NH) and used for the in vivo experiments. Murine CTLA-4Ig (mCTLA-4Ig) was a gift from Dr. R. Peach (Bristol Myers Squibb, Princeton, NJ). The isotype control rat IgG used for the in vivo study was obtained from Sigma-Aldrich (St. Louis, MO).
A hybridoma cell line secreting the anti-mouse CD154 mAb (MR1, hamster IgG) was obtained from American Type Culture Collection (Manassas, VA). The hybridoma cells were grown in serum-free UltraCulture medium (BioWhittaker, Walkersville, MD), and mAb was purified from the culture supernatant with protein G columns.
Cell proliferation assay
Splenic leukocytes were prepared as previously described (24). Cells were plated in 96-well plates (2 x 105/well) in RPMI 1640 medium supplemented with 10% FCS and 1% penicillin/streptomycin (BioWhittaker) and were stimulated with various concentrations of anti-CD3 (0.6–10 µg/ml; 2C11; PharMingen) in triplicate. Cells were incubated at 37°C for 72 h, and for the last 16 h of culture cells were pulsed with [3H]TdR (1 µCi/well; Amersham Pharmacia Biotech, Arlington Heights, IL). [3H]TdR uptake was determined by scintillation counting.
CFSE labeling
Spleen and peripheral lymph nodes were harvested from donor mice, and a single-cell suspension was prepared in HBSS. RBC were lysed by hypotonic shock. Cells were resuspended in HBSS at 1 x 107 cells/ml for labeling with CFSE (Molecular Probes, Portland, OR) as previously described (25). Briefly, cells were incubated with CFSE at a final concentration of 5 µM in serum-free HBSS at room temperature for 6 min. The labeling was then terminated by the addition of FCS (10% of the total volume). Cells were washed twice in HBSS before i.v. injection.
In vivo activation of CFSE-labeled cells
Host DBA/2 mice were lethally irradiated (1000 rad) with a Gammacell Exactor (Kanata, Ontario, Canada). Each mouse then received 
6 x 107 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 were harvested, and a single-cell suspension was prepared for cell surface staining and intracellular cytokine staining. The large number of cells transferred (6 x 107 cells/mouse) and the time point examined (3 days) preclude homeostatic expansion of CFSE-labeled cells in the irradiated hosts, and cell division in this model is driven primarily by the host alloantigens (5).
Treatment of irradiated host mice
In the CFSE model, treatment of irradiated hosts with anti-OX40L, anti-CD70, anti-4-1BBL, or anti-ICOSL mAb consisted of 0.5 mg i.p. daily for 3 consecutive days starting with i.v. injection of CFSE-labeled cells. Mice treated with rat IgG (Sigma-Aldrich) were included as controls.
Cell surface staining and flow cytometry
CFSE-labeled cells were recovered from the host mice 3 days after adoptive cell transfer. Cells were resuspended in PBS/0.5% BSA (2 x 106/ml) and stained with CyChrome-conjugated anti-CD4 and CyChrome-anti-CD8 on ice for 30 min, followed by staining with biotinylated anti-OX40, anti-4-1BB, anti-ICOS, and anti-CD27. Cells were washed in PBS/BSA and were further stained with PE-streptavidin. Cells stained with PE-conjugated isotype control Ab were included as a control. After staining, cells were fixed in 1% formaldehyde before analysis.
All samples were analyzed using a FACSort equipped with CellQuest software (BD Bioscience, Mountain View, CA). CD4+ and CD8+ T cells were electronically gated, and the cell division profile and the expression of novel costimulatory molecules at distinct division cycles were analyzed. At least 100,000 events were collected for each sample.
Intracellular cytokine staining
CFSE-labeled cells harvested from the host mice were resuspended in RPMI 1640 medium supplemented with 10% FCS and 1% penicillin/streptomycin at 5 x 106/ml. Cells were stimulated in vitro with PMA (50 ng/ml) and ionomycin (500 ng/ml; Sigma-Aldrich) at 37°C for 4 h. In the last 2 h of culture, GolgiStop (BD PharMingen) was added to the culture at a concentration of 1 µg/ml. Cells were harvested after the in vitro stimulation, stained with CyChrome anti-mouse CD4 or CyChrome anti-mouse CD8, respectively, and fixed, and the cell membrane was permeabilized in Cytofix/Cytoperm solution (BD PharMingen) at 4°C for 10 min, followed by washing in Perm/Wash solution (BD PharMingen). Cells were then resuspended in Perm/Wash solution (1 x 106) and stained with PE-conjugated Abs against mouse IL-2 and IFN- on ice for 30 min. PE-conjugated isotype control Ab was included in the staining protocol as a control. The expression of IL-2 and IFN- in vivo at distinct cell division cycles was analyzed by FACS.
Calculation of cell division frequency
The responder frequency of CFSE-labeled CD4+ and CD8+ T cells proliferating in vivo in the allogeneic hosts was calculated as previously reported (25). Briefly, distinct rounds of cell divisions were identified by their CFSE profiles. The absolute number of cells in each cell division was calculated using the FACS acquisition software, CellQuest. The number of precursors that proliferated and gave rise to the absolute number of daughter cells was extrapolated using the formula: y/2n, where y is the absolute number of cells in each cell cycle, and n is the number of cell divisions. For example, 16 daughter cells in the third cell division are the progeny of two precursors, each of which have divided three times (16/23 = 2). The frequency of proliferating T cells in the responder population was then calculated by dividing the total number of precursors by the sum of total CFSE-labeled cells collected.
Skin grafting and treatment protocols
A full-thickness tail skin graft (1 cm2) from DBA/2 donors (H-2d) was transplanted onto the thoracic wall of wild-type (wt) C57BL/6 mice (H-2b) and CD28/CD154 DKO mice (H-2b). The skin graft was secured with an adhesive bandage for the initial 5 days. Graft survival was then followed by daily visual inspection. Rejection was defined as complete necrosis and loss of viable skin tissue.
Treatment of transplant recipients with mCTLA-4Ig consisted of 0.5 mg 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, or anti-CD70 mAb was given at 0.5 mg i.p. on days 0, 2, 4, and 8 after skin transplantation.
Histopathology
The skin graft was removed from recipient mice at specified time points after transplantation, fixed in 10% formalin, and embedded in paraffin. Serial tissue sections (5 µm) were prepared and mounted on SuperFrost Plus glass slides (Fisher Scientific, Pittsburgh, PA), fixed in methanol, and stained in H&E for histological evaluation.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 CD4+ and CD8+ subsets in the spleens of wt C57BL/6 mice and CD28/CD154 DKO mice were comparable (Fig. 1A). A similar finding was observed in blood and peripheral lymph nodes (data not shown). However, splenocytes from CD28/CD154 DKO mice, in contrast to wt controls, did not mount a proliferative response in vitro to anti-CD3 stimulation (Fig. 1B), confirming the critical role of CD28 and CD154 signals in T cell activation in vitro (26).
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The prompt rejection of skin allografts by CD28/CD154 DKO mice suggests that the in vivo T cell activation program is relatively normal despite impaired T cell activation in vitro. To critically examine the impact of CD28/CD154 deficiency on T cell activation in vivo, we labeled splenic leukocytes from CD28/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 compared with that of CFSE-labeled wt control cells. This model allows quantitative analysis of T cell activation in vivo at the single-cell level (5, 25).
As shown in Fig. 3A, both CD4+ and CD8+ 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 adoptive cell transfer. Calculation of division frequency revealed that 25% of CD4+ T cells and as much as 38% of CD8+ T cells recovered from the host spleen entered the cell cycle. Interestingly, CD28/CD154 deficiency preferentially affected the in vivo division of CD4+ T cells, and the division frequency was reduced by >2-fold (11%) compared with that in wt controls (25%). Nonetheless, CD4+ T cell division was not completely abolished despite genetic deficiency of both CD28/CD154; 10% of CD4+ T cells recovered from the host spleen still entered the cell cycle and divided multiple times (more than eight times), suggesting that not all CD4+ T cells rely on CD28 and CD154 signals for activation. Up-regulation of cell surface expression of the CD4 molecule is often associated with robust activation of CD4+ T cells (27). Consistent with this, the overwhelming majority of activated CD28/CD154-deficient CD4+ T cells was confined to the CD4high fraction (Fig. 3B). Intracellular cytokine staining revealed that activation of such a T cell subset was associated with expression of high levels of IL-2 and IFN- effector cytokines (Fig. 3C). Thus, levels of CD4 expression by activated CD4+ T cells may be a useful tool to examine the CD28/CD154-independent fraction of activated CD4+ T cells.
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In the CFSE model used in the present study, adoptive transfer of CFSE-labeled syngeneic cells into irradiated hosts did not induced marked T cell proliferation (Fig. 3A, bottom panel), suggesting that the large number of cells transferred (6 x 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/CD154-deficient T cells in vivo, we first examined the cell surface expression of such costimulatory molecules upon in vivo activation of CD28/CD154-deficient T cells. CFSE-labeled, CD28/CD154-deficient T cells were stimulated in vivo, and the expression 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 CD4+ and CD8+ T cells regardless of the number of cell divisions. 4-1BB was not detectable on CD4+ T cells, but was expressed at low levels on CD8+ T cells, especially after multiple cell divisions. Interestingly, for both CD4+ and CD8+ 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 OX40 expression were consistently higher on CD4+ T cells than on CD8+ T cells, especially at later cell divisions (Fig. 4).
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To further determine the role of such novel costimulatory molecules 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 CD8+ 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 CD8+ T cells, and the CD4high fraction and CD8+ T cells divided with a similar kinetics as the controls. Blocking the ICOS/ICOSL costimulatory pathway or the 4-1BB/4-1BBL pathway exerted some inhibitory effect on the late expansion of CD8+ T cells, but had no effect to block the proliferation of CD4high T cells. In stark contrast, blocking the OX40/OX40L pathway nearly abolished in vivo proliferation of the CD4high fraction (Fig. 5A). In fact, the entire CD4+ population recovered from the anti-OX40L-treated hosts showed no apparent up-regulation of the CD4 molecule and remained undividing (Fig. 5B). Interestingly, blocking the OX40/OX40L pathway also markedly inhibited the in vivo proliferation of CD28/CD154-deficient CD8+ T cells (Fig. 5C). These findings suggest that OX40 costimulation plays a key role in the activation of CD28/CD154-deficient T cells.
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The critical role of OX40 costimulation in the activation of CD28/CD154-deficient T cells suggests that OX40 signals may play a key role in mediating the skin allograft rejection in CD28/CD154 DKO mice. To test this possibility, we transplanted the DBA/2 skin allograft onto CD28/CD154 DKO recipients. The recipient mice were treated with anti-OX40L mAb (0.5 mg i.p. on days 0, 2, 4, and 8 after skin grafting) and skin allograft survival was determined. As shown in Fig. 6, treatment of CD28/CD154 DKO recipients with anti-OX40L mAb markedly prolonged skin allograft survival, and four of seven transplants survived for >100 days, whereas the control mice rejected the skin allograft with a MST of 12 days (n = 5). Treatment of CD28/CD154 DKO mice with anti-ICOSL, anti-4-1BBL, or anti-CD70 failed to prevent skin allograft rejection, and all skin allografts were rejected within 20 days after transplantation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 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 activation. Clearly, genetic deficiency of both CD28 and CD154 preferentially affects the in vivo activation of CD4+ T cells, but not the CD8+ T cells (Fig. 3A). However, not all CD4+ T cells require CD28 and CD154 signals for activation, and a subset of CD4+ T cells can proliferate extremely well in the absence of CD28 and CD154 molecules (Fig. 3B). Interestingly, OX40 costimulation seems to be critically important in the activation of CD28/CD154-deficient T cells, as blocking the OX40/OX40L costimulatory 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 (>100 days; Figs. 6 and 7). Our study clearly identified OX40 as a key alternative costimulatory molecule in supporting CD28/CD154-independent rejection.
OX40 is a member of the TNF receptor superfamily, and unlike CD28, OX40 is not constitutively expressed on naive T cells, but its expression is rapidly induced upon T cell activation (31). OX40L has a much wider tissue distribution than the B7 family proteins. OX40L is expressed not only on APCs, but also on other cell types, including vascular endothelial cells, and its expression is also induced after immune activation (31). The inducible nature of OX40 and its ligand suggests that the OX40/OX40L costimulatory pathway may play a particularly important role when the T cell activation program is successfully launched. In this regard, our data demonstrate several interesting findings. First, costimulatory signals from both CD28 and CD154 are not required for either OX40 expression or its costimulatory function. Clearly, T cells deficient in both CD28 and CD154 can express OX40 upon in vivo activation, and OX40/OX40L costimulatory signals play a key role in supporting CD28/CD154-independent T cell activation. Consequently, blocking OX40 costimulation in CD28/CD154 DKO mice markedly prolonged skin allograft survival (Fig. 6). Second, not all activated T cells express OX40. Instead, OX40 expression is primarily confined to a subset of activated CD4+ T cells that also express higher levels of CD4 on the cell surface (i.e., CD4high fraction). Interestingly, it takes several rounds of cell divisions for the activated T cells to express OX40, as OX40 is highly expressed only after four or five cell divisions (Fig. 4), suggesting that OX40 expression may be regulated by the cell cycle. Third, despite the expression of other alternative costimulatory molecules, OX40 appears to be the key player mediating T cell activation when both CD28 and CD154 signals are blocked. This conclusion is based on the finding that blocking the OX40/OX40L pathways, but not the ICOS/ICOSL, 4-1BB/4-1BBL, or the CD27/CD70 pathway, abolished the activation of CD28/CD154-deficient T cells and induced long term skin allograft survival in the absence of CD28/CD154 costimulation (Figs. 6 and 7). Finally, blocking the OX40/OX40L pathway can also affect the activation of CD8+ T cells in vivo. Consistent with several previous reports (8, 28, 29), genetic deficiency of both CD28 and CD154 had minimal effect on the activation of CD8+ T cells in vivo. However, proliferation of CD28/CD154-deficient CD8+ T cells in the allogeneic hosts was markedly inhibited upon blocking the OX40/OX40L pathway. It remains to be determined, however, whether blocking OX40 inhibits CD8+ T cells directly or indirectly by inhibition of a subset of CD4+ T cells.
In certain models, the most profound effect of blocking OX40 costimulation is the inhibition of the frequency of memory CD4+ T cells generated (14, 15). This finding led to the belief that OX40 signals are required for the generation of CD4+ memory cells. Thus, it is possible that a subset of activated CD4+ 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 blockade (3, 32, 33), and therefore, blocking OX40 signals may be particularly important in control such a unique T cell subset. This is consistent with a recent report that blocking the OX40 pathway is beneficial in a presensitized cardiac transplant model (22). Our study along with a growing number of other reports highlight the critical role of OX40 costimulation in T cell activation (14, 34) as well as in certain cytopathic conditions (35, 36, 37, 38, 39).
The precise role of other alternative costimulatory molecules in activation and effector differentiation of T cells and their relationship to the conventional CD28/CD154 costimulation warrant further study. In vivo analysis clearly demonstrated that CD27 is constitutively expressed at high levels on CD28/CD154-deficient T cells (Fig. 4). It is not clear why blocking the CD27/CD70 pathways completely failed to inhibit the proliferation of CD28/CD154-deficient T cells (Fig. 5). It is possible that blocking the CD27/CD70 pathway may selectively affect the effector function of activated T cells despite a normal proliferative response. However, this is unlikely, as CD28/CD154 DKO mice treated with anti-CD70 mAb can vigorously reject the skin allograft with similar kinetics as the untreated controls. It remains to be determined whether cell types other than the T cells (e.g., B, NK, or NKT cells) are particularly sensitive to the CD27/CD70 costimulatory blockade. Similarly, blocking the ICOS/ICOSL pathway in CD28/CD154 DKO mice also failed to inhibit skin allograft rejection. The lack of effect is not due to the lack of ICOS expression on CD28/CD154-deficient T cells, as ICOS is highly expressed on both activated CD4+ and CD8+ T cells, especially after several cell division cycles in vivo (Fig. 4). This is in contrast with previously reports showing that blocking the ICOS/ICOSL pathway can significantly prolong allograft survival in a mouse heart transplant model (19, 20). The apparent difference is unclear, but is probably due to the different models used (heart vs skin transplant model) or the different treatment protocols used. It is well known that the skin allograft is notoriously more difficult to tolerize than other organ transplants. In a recent report (20), it has been shown that delayed blockade of the ICOS/ICOSL pathway has a far more profound therapeutic effect than the initial blockade. Whether delayed treatment with the anti-ICOSL would prolong skin allograft survival in CD28/CD154-deficient mice remains to be examined.
It should be noted that skin allograft survival in wt mice treated with CTLA-4Ig and anti-CD154 mAb in combination with OX40 blockade is noticeably better than that in CD28/CD154-deficient mice treated with anti-OX40L. This observation raises the possibility that the in vivo effect of CTLA-4Ig and anti-CD154 mAb may be more than just simply blocking CD28 and CD154 costimulatory signals. Recent studies have shown that CTLA-4Ig, which binds to B7 molecules on the surface of APCs with high affinity, can activate the indoleamine 2,3-dioxygenase system; such indoleamine 2,3-dioxygenase activation in APCs mediates tryptophan catabolism that is capable of inhibiting T cell activation and inducing T cell tolerance (40, 41). Furthermore, anti-CD154 (MR1) mAb possesses certain cytolytic activities and can directly kill activated T cells via activation of the complement cascade (42). Thus, these unique features of CTLA-4Ig and anti-CD154 mAb may also contribute to the therapeutic effect in vivo. Clearly, the creation of CD28/CD154 DKO mice certainly provides a unique tool to further unravel this issue.
In conclusion, our study demonstrates that OX40 is critically important in the rejection response when both CD28 and CD154 costimulatory molecules are blocked or genetically deficient. OX40 costimulation plays a key role in supporting the activation of a subset of CD4+ T cells and/or CD8 T cells in the absence of both CD28 and CD154 signals. Clearly, the identification of OX40 as a novel alternative costimulatory molecule involved in CD28/CD154-independent rejection in such a stringent skin transplant model should provide critical insights on the continued development of the costimulatory blockade protocol in the clinic.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Xian C. Li, Department of Medicine, Division of Immunology, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, RN389, Boston, MA 02215. E-mail address: xli{at}bidmc.harvard.edu
3 Abbreviations used in this paper used: ICOS, inducible costimulator; DKO, double knockout; ICOSL, ICOS ligand; mCTLA, murine CTLA; MST, mean survival time; OX40L, OX40 ligand; wt, wild type.
Received for publication September 16, 2003. Accepted for publication November 19, 2003.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain signals, and the alloimmune response. J. Immunol. 168:4382.Related articles in The JI:
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C. C. Kemball, E. D. H. Lee, E. Szomolanyi-Tsuda, T. C. Pearson, C. P. Larsen, and A. E. Lukacher Costimulation Requirements for Antiviral CD8+ T Cells Differ for Acute and Persistent Phases of Polyoma Virus Infection J. Immunol., February 1, 2006; 176(3): 1814 - 1824. [Abstract] [Full Text] [PDF]
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 A. Izawa, K. Sano, M. Takehara, M. Inobe, J.-i. Suzuki, H. Imamura, M. Takahashi, U. Ikeda, M. Isobe, and T. Uede Cre/loxP-mediated CTLA4IgG gene transfer induces clinically relevant immunosuppression via on-off gene recombination in vivo Cardiovasc Res, January 1, 2006; 69(1): 289 - 297. [Abstract] [Full Text] [PDF]
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K. E. Lunsford, M. A. Koester, A. M. Eiring, P. H. Horne, D. Gao, and G. L. Bumgardner Targeting LFA-1 and CD154 Suppresses the In Vivo Activation and Development of Cytolytic (CD4-Independent) CD8+ T Cells J. Immunol., December 15, 2005; 175(12): 7855 - 7866. [Abstract] [Full Text] [PDF]
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S. L. Shiao, J. M. McNiff, and J. S. Pober Memory T Cells and Their Costimulators in Human Allograft Injury J. Immunol., October 15, 2005; 175(8): 4886 - 4896. [Abstract] [Full Text] [PDF]
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