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CUTTING EDGE |
Departments of
*
Surgery and
Medicine, Yale Medical School, New Haven, CT 06520; Departments of
Medicine and
Surgery, University of Massachusetts Medical School, Worcester, MA 01655; and Departments of
¶ Pathology and
|| Medicine, Brigham and Women’s Hospital, and Harvard Medical School, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CTLA-4 is a potent T cell down-regulatory molecule that appears essential for the development of peripheral tolerance. For example, mice lacking CTLA-4 develop fatal systemic autoimmunity resulting from unopposed T cell activation and proliferation in response to environmental and self Ags (1, 2). Blocking mAbs against CTLA-4 (anti-CTLA-4) prevent tolerance to i.v. administered Ag and can exacerbate (or even induce de novo) autoimmunity (1, 2). Unfortunately, targeting CTLA-4 to promote tolerance has been a formidable challenge. Firstly, CTLA-4 is not expressed by most T cells until after activation and entry into the cell cycle (2, 3). Secondly, no soluble agonist ligands are available. mAbs against CTLA-4 require extensive cross-linking to trigger negative signaling (2, 4). As noted above, administration of soluble anti-CTLA-4 actually inhibits negative signaling and augments the immune response. Until recently, these issues have frustrated attempts to use this potent down-regulatory pathway for therapeutic inhibition of the immune response.
CD45 is a family of transmembrane tyrosine phosphatases intimately involved in T cell activation (5). Multiple alternatively spliced CD45 isoforms differ in the size of their extracellular domains and may differentially influence signaling. Although individual T cells express multiple CD45 isoforms, the higher and lower Mr isoforms are differentially distributed on CD4 cells with distinct functions. In mice, these cells can be differentiated by their high and low level expression of isoforms containing exon B (respectively designated CD45RBhigh and CD45RBlow). Interestingly, a subset of CD4 cells expressing the smaller isoforms (CD45RBlow) constitutively expresses CTLA-4 (6).
CD45 is also a potent tolerogenic target. For example, anti-CD45RB can induce tolerance to murine islet and renal allografts (7, 8). We have shown that the tolerogenic anti-CD45RB mAb, MB23G2, causes a rapid shift in CD45 isoform expression from high to low Mr isoforms that is not accompanied by activation or depletion of CD4 cells (7, 9). Interestingly, this increase in CD45RBlow expression was associated with a 2-fold increase in the number of CD4 cells expressing CTLA-4, raising the possibility that anti-CD45RB actually acts through CTLA-4 up-regulation (9). In agreement, treatment with a blocking mAb against CTLA-4 induced acute rejection of islet allografts despite anti-CD45RB therapy (9). However, anti-CTLA-4 has also been shown to augment allo-responsiveness and prevents the induction of transplant tolerance by costimulatory blockade (10). Thus, results obtained using Ab-mediated blockade of CTLA4 might be due to the more global role of CTLA-4 in regulating immune responsiveness and the exact relationship between CTLA-4 up-regulation and anti-CD45RB activity remains to be established.
We now demonstrate a critical and specific role for CTLA-4 and its up-regulation in inducing tolerance mediated by anti-CD45RB. This is important not only for understanding the mechanisms of anti-CD45RB action, but also in demonstrating that CTLA-4 negative signaling can be specifically enhanced to induce long-term allograft survival.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male BALB/c (H-2d) and C57BL/6 (H-2b) mice (6–8 wk old) were from Taconic Farms (Germantown, NY). Triple knockout (TKO)4 B7-1 (CD80) -/-; B7-2 (CD86) -/-; CTLA-4-/- mice and double knockout (DKO) B7-1-/-; B7-2-/- mice (129 svJae; H-2b) were as described (11).
Abs and reagents
Anti-CD45RB mAb MB23G2 (ATCC) was from Bioexpress (Lebanon, NH). Murine CTLA4-Ig was kindly provided by Bristol-Myers Squibb (Princeton, NJ). All fluorochrome-conjugated mAbs were from BD PharMingen (San Diego, CA).
Islet isolation and transplantation
C57BL/6, DKO, or TKO recipients (all H-2b) received 180 mg/kg i.p. streptozotocin (Sigma-Aldrich, St. Louis, MO). Blood glucose was >350 mg/dl for 5–7 days before transplantation. Pancreati from BALB/c donors were digested in situ with collagenase P (Sigma-Aldrich) and islets were purified with a 100-µM cell strainer (BD Biosciences, Mountain View, CA), as we described (12). Four hundred hand-picked islets were then transplanted under the left kidney capsule (7, 9). All recipients had glycemia <200 mg/dl by day 2 posttransplant. Blood glucose >250 after engraftment was defined as rejection. In some experiments, the original allograft was excised (by nephrectomy), and freshly isolated BALB/c islets were transplanted under the right kidney capsule (7). All in vivo studies followed National Institutes of Health and Yale Animal Care Committee guidelines.
Treatment protocols
Recipients received anti-CD45RB (MB23G2; 100 µg i.v.; days -1, 0, and 5) (7, 9), alone or in combination with CTLA-4-Ig (200 µg i.p.; day 2) (20), unless otherwise noted. Control allograft recipients were untreated.
Immunofluorescence
Surface CD45RB was examined as we described (7, 9), using anti-CD45RB-FITC (clone 16A) which recognizes a different epitope than the treatment mAb, MB23G2. Intracellular CTLA-4 expression was examined by fixing cells in 2% paraformaldehyde, permeablization with 0.5% saponin, and incubation with anti-CTLA-4-PE, as we described (9). Negative controls used rat or hamster IgG fluorochrome conjugates of appropriate isotype.
Statistics
Kaplan-Meier plots of allograft survival were compared by log rank test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The efficacy of therapeutic agents is frequently enhanced by using them in combination, as exemplified by combining anti-CD40L with CTLA-4-Ig or anti-CD40L with anti-CD45RB (13, 14). The CTLA-4-Ig fusion protein can promote allograft survival by interfering with B7(CD80 and CD86):CD28-mediated costimulation. However, in binding to B7 with high affinity, CTLA-4-Ig can also partially block B7:CTLA-4 signals (15). If CTLA-4 up-regulation and signaling plays an essential role in anti-CD45RB-mediated engraftment, we would predict that by binding to B7, CTLA-4-Ig will interfere rather than synergize with anti-CD45RB.
Consistent with previous results, three doses of anti-CD45RB induces >120-day engraftment of BALB/c islets in 
57% of C57BL/6 recipients (Fig. 1). When combined, CTLA-4-Ig and anti-CD45RB, may decrease early rejection. However, long-term graft survival does not occur (median survival time (MST) 56 days), consistent with the notion that CTLA-4-Ig can interfere with augmented CTLA-4 signaling by anti-CD45RB.
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To more directly assess the role of CTLA-4 in anti-CD45RB-mediated engraftment, we examined mice lacking CTLA-4 expression. To avoid confounding systemic autoimmunity, we used CTLA-4-deficient TKO mice that also lack B7-1 and B7-2 (11). These were compared with B7-deficient (B71-/-, B7-2-/-) DKO control mice with an intact CTLA-4 locus. As shown in Fig. 2, untreated TKO and DKO mice (H-2b) both rapidly reject islet transplants from BALB/c (H-2d) donors. Direct comparison reveals that TKO mice reject wild type (wt) allografts somewhat more rapidly than do DKO mice (Fig. 2, A vs B; MST 8 vs 14 days, respectively). Because DKO and TKO recipient mice lack B7 and differ only in CTLA-4 expression, this difference in graft survival suggests that B7 is expressed by the allograft and influences the immune response. We confirmed the presence of both B7-1 and B7-2 expression on APCs within wt (BALB/c) islets 3–4 days after transplantation into TKO recipients (data not shown).
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In marked contrast, in the absence of CTLA-4 (TKO recipients), anti-CD45RB is unable to prolong graft survival (Fig. 2A; MST 12 day). However, in DKO recipients where the CTLA-4 locus is intact, anti-CD45RB treatment induces long-term engraftment in 50% of the recipients (Fig. 2B), similar to that achieved in wt mice (Fig. 1 and Ref. (7)). These results indicate that whereas CTLA-4 is not required for long-term engraftment induced by CTLA-4-Ig, CTLA-4 plays an essential and specific role in anti-CD45RB mediated engraftment.
CTLA-4-Ig induces ignorance rather than tolerance in TKO mice
CTLA-4 is thought to play a requisite role in the generation of peripheral tolerance (10). However, our data reveal that in TKO recipients, CTLA-4-Ig is able to induce long-term engraftment. It is possible that the more limited expression of B7 (by donor cells) in this setting might predispose the immune response toward either tolerance or ignorance despite the absence of CTLA-4 expression. To further address this issue, TKO recipients with long-term allograft survival, underwent left-sided nephrectomy. In each case, allograft excision led to hyperglycemia. When these TKO mice were re-transplanted with fresh islet allografts of original donor strain, acute rejection occurred (Fig. 2C). Of note, graft survival was very similar to that observed in untreated naive control (TKO) recipients (Fig. 2C). This indicates that although long-term engraftment can indeed be induced by CTLA-4-Ig in the absence of CTLA-4, ignorance rather than tolerance is achieved. These data support the notion that tolerance induction does require the presence of CTLA-4.
Anti-CD45RB does not prolong graft survival in the absence of CTLA-4 up-regulation
Next, we sought to obtain evidence that the up-regulation of CTLA-4 expression per se, is important for tolerance mediated by anti-CD45RB. As we previously showed, 7–10 days after initiating anti-CD45RB therapy, the number of CD4 cells expressing low Mr CD45 isoforms and CTLA-4 increases 2-fold (Ref.9 and Fig. 3, A and B). We now show that the increase in CTLA-4 expression begins to decline after 10 days, returning to baseline by day 17 (Fig. 3B). Re-expression of baseline CD45RBlow levels occurs in a parallel time frame. Treatment of mice with a second course of anti-CD45RB starting on day 17, reproduces the shift toward lower Mr CD45 isoforms, but surprisingly, no longer augments CTLA-4 expression (Fig. 3, A and B). If the increase in CTLA-4 expression is key, then transplantation at a time when treatment with anti-CD45RB no longer augments CTLA-4 expression, should not prolong islet allograft survival. Moreover, this allows us to assess the relative importance of the shift in CD45 isoforms vs CTLA-4 up-regulation in prolongation of graft survival by anti-CD45RB.
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50% long-term allograft survival (Figs. 1 and 3C). A second set of recipients was pretreated with three doses of anti-CD45RB 17 days before transplantation. Mice were then transplanted and retreated with a standard three dose regimen of anti-CD45RB (days -1, 0, and 5). In this situation, where CTLA-4 expression could not be induced, anti-CD45RB was ineffective in prolonging allograft survival (MST 19 days). Therefore up-regulation of CTLA-4 expression appears critical for long-term engraftment mediated by anti-CD45RB. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously showed that anti-CD45RB treatment rapidly augments CTLA-4 expression on CD4 cells (9). However, a specific role for CTLA-4 in anti-CD45RB activity was not established. Combined with previous work, five lines of evidence now demonstrate that CTLA-4 and its up-regulation play a specialized and requisite role mediating engraftment by anti-CD45RB: 1) anti-CTLA-4 not only prevents long-term engraftment induced by anti-CD45RB, but precipitates acuterejection, with no allograft surviving >26 days (9). Although anti-CTLA-4 also inhibits long-term engraftment by CTLA4-Ig, 37% of these allografts survived >80 days (10). This difference suggests that anti-CD45RB is significantly more sensitive to CTLA-4 mediated signaling than is CTLA4-Ig; 2) cyclosporin A prevents CTLA-4 up-regulation and also prevents anti-CD45RB-mediated engraftment resulting in acute rejection (MST 22 days) (9). Although cyclosporine also blocks tolerance induction by costimulatory blockade, this may to be due to a distinct mechanism that involves inhibition of apoptosis (16); 3) Anti-CD45RB cannot prolong graft survival in CTLA-4-deficient (TKO) mice. In contrast, CTLA4-Ig can induce ignorance and long-term engraftment in this setting; 4) When CTLA-4 up-regulation at the time of transplantation is prevented by pretreatment, anti-CD45RB is unable to prolong engraftment; and finally, 5) CTLA4-Ig can interfere with anti-CD45RB-mediated tolerance, presumably by binding B7 and limiting its access to CTLA-4. An increase in early graft survival is apparent when these two agents are combined (Fig. 1) and could be related to short-lived immunosuppressive effects of CTLA-4-Ig.
CTLA-4 signaling inhibits IL-2 production and cell cycle progression (1). In the allograft setting, CTLA-4 limits the number of CD4 and CD8 cells responding to allogeneic challenge (17). These effects contribute to the critical role of CTLA-4 in the maintenance of self-tolerance and the induction of tolerance to exogenous Ags. However, previous conclusions were all reached by evaluating the effects of blocking mAbs against CTLA-4 or by comparing wt and CTLA-4-deficient mice. In contrast, the current findings demonstrate the effects of augmented CTLA-4 expression on the immune response. We hypothesize that the rapid acquisition of CTLA-4 by resting CD4 cells in the peri-transplant period down-regulates the immune response upon contact with APCs expressing B7 counterligands. Up-regulation of CTLA-4 expression on responding CD4 cells allows the TCR and CTLA-4 to engage their respective ligands in cis during direct and indirect allorecognition. In vitro studies suggest that ligation in cis is required for optimal CTLA-4 activity (18). The notion that CTLA-4 signaling can prevent rejection is supported by demonstration that expression of a single chain anti-CTLA-4 by transfected allogeneic tumor cells inhibits the anti-tumor response in immunodeficient hosts reconstituted with a limited number of TCR-tg CD8 cells (19). In the case of anti-CD45RB, a three-dose regimen capable of inducing short-term up-regulation of CTLA-4 at the time of transplantation actually promotes permanent engraftment and tolerance in immunocompetent, MHC disparate hosts (7, 8). Although anti-CD45RB may have additional mechanisms of action, the present findings indicate that CTLA-4 up-regulation plays an essential role. This should provide impetus for the study of new ways to capitalize on this important inhibitory pathway.
The current studies provide additional insights into costimulation and the alloimmune response. T cells in both TKO and DKO mice develop in the absence of B7-mediated (positive or negative) costimulation. The prolonged survival of grafts in DKO vs TKO recipients is likely due to CTLA-4 expression in the former and points to a role for B7 expressed by passenger leukocytes of donor origin in the alloimmune response by these mice. This role is confirmed by the finding that CTLA-4-Ig prolongs engraftment in the absence of recipient B7 or CTLA-4. CTLA-4-Ig may act through interference with CD28-mediated costimulation, through B7 ligation-induced activation of IDO and tryptophan catabolism by APC’s, or both (20, 21). Although ligation of B7 by CTLA4-Ig may contribute to tolerance by generating a localized tryptophan deficiency, it is currently unknown whether other B7 ligands (CTLA-4, anti-B7, or even CD28) have the same capacity. In this regard, the antagonism of anti-CD45RB by CTLA4-Ig in wild-type mice becomes particularly interesting. Our findings indicate that anti-CD45RB acts through a requisite up-regulation of CTLA-4 expression and signaling. It is unclear how CTLA4-Ig interferes with engraftment mediated by CTLA-4, unless both molecules compete for B7 and primarily act through distinct mechanisms. If CTLA4-Ig and CTLA-4 were to act through the same mechanism, they should be additive (or neutral) rather than antagonistic. Although ligation of B7 by CTLA-4 may induce IDO, our findings support the overriding importance of other mechanisms of action. In this regard, CTLA-4 is known to directly inhibit T cell activation, perhaps through the inhibition of lipid raft formation and/or through sequestration of TCR 
and other signaling components outside of the rafts (22, 23).
Our studies indicate that an easily administered therapeutic agent can induce tolerance through specific up-regulation of CTLA-4. Demonstration that augmented negative signaling through CTLA-4 can induce long-term engraftment provides further insight into CTLA-4 activity and is of practical importance. These results suggest that new efforts aimed at enhancing negative signaling for the induction of tolerance are warranted. Moreover, they provide a rational basis for combination therapy by predicting which therapeutic agents are likely to synergize with rather than antagonize anti-CD45RB activity.
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Footnotes |
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2 G.B. and D.M.R. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. David M. Rothstein, Internal Medicine/Nephrology, Yale Medical School, P.O. Box 208029, New Haven, CT 06520-8029. E-mail address: david.rothstein{at}yale.edu
4 Abbreviations used in this paper: TKO, triple knockout; DKO, double knockout; wt, wild type; MST, median survival time.
Received for publication August 6, 2003. Accepted for publication October 10, 2003.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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0.006 vs other groups; **, p = 0.002 vs control.