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The Journal of Immunology, 2001, 166: 322-329.
Copyright © 2001 by The American Association of Immunologists

Targeting Signal 1 Through CD45RB Synergizes with CD40 Ligand Blockade and Promotes Long Term Engraftment and Tolerance in Stringent Transplant Models1

David M. Rothstein2,3,*, Mauren F. A. Livak2,{dagger}, Koji Kishimoto{ddagger}, Charlotte Ariyan{dagger}, He-Ying Qian{dagger}, Scott Fecteau{dagger}, Masayuki Sho{ddagger}, Songyan Deng*, Xin Xiao Zheng§, Mohamed H. Sayegh{ddagger} and Giacomo P. Basadonna{dagger}

Departments of * Internal Medicine and {dagger} Surgery, Yale Medical School, New Haven, CT 06520; {ddagger} Department of Medicine, Brigham and Women’s Hospital, Boston, MA 02115; and § Department of Medicine, Beth Israel Deaconess Medical Center, and Harvard Medical School, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The induction and maintenance of allograft tolerance is a daunting challenge. Although combined blockade of CD28 and CD40 ligand (CD40L)-costimulatory pathways prevents allograft rejection in some murine models, this strategy is unable to sustain engraftment in the most immunogenic allograft and strain combinations. By targeting T cell activation signals 1 and 2 with the novel combination of anti-CD45RB and anti-CD40L, we now demonstrate potent enhancement of engraftment in C57BL/6 recipients that are relatively resistant to costimulatory blockade. This combination significantly augments the induction of tolerance to islet allografts and dramatically prolongs primary skin allograft survival. Compared with either agent alone, anti-CD45RB plus anti-CD40L inhibits periislet infiltration by CD8 cells, B cells, and monocytes; inhibits Th1 cytokines; and increases Th2 cytokine expression within the graft. These data indicate that interference with activation signals one and two may provide synergy essential for prolonged engraftment in situations where costimulatory blockade is only partially effective.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The hope for long term allograft acceptance in humans, after a brief treatment course, remains unfulfilled. Thus, prevention of allograft rejection in transplant patients requires continuous treatment with potent immunosuppressive agents that have deleterious side effects. Rejection of allografts is a T cell-dependent process. Activation of T cells normally requires both a primary signal (signal 1) through the TCR and a costimulatory signal (signal 2) through the CD28/B7 and CD40/CD40 ligand (CD40L)4 pathways (1, 2). Interference with signal 1 or 2 during T cell activation can induce anergy in CD4+ T cell clones in vitro (3, 4). As such, therapeutic strategies that interfere with T cell activation by targeting T cell-signaling molecules qualitatively alter the immune response and promote engraftment in a number of transplant models (5, 6, 7, 8, 9, 10, 11, 12). The efficacy of individual agents is frequently enhanced by combining them with one another. In this regard, the combined blockade of B7/CD28 and CD40/CD40L pathways with CTLA4-Ig plus anti-CD40L has proved particularly potent (13, 14).

However, in other transplant models, even combined costimulatory blockade is relatively ineffective. For example, CTLA4-Ig plus anti-CD40L is unable to significantly prolong skin allograft survival in C57BL/6 or BALB/c recipients (15). Moreover, although renal allografts in nonhuman primates survive for >1 year after anti-CD40L therapy is discontinued, islet allografts routinely reject within several months (16, 17). Such findings suggest both recipient strain- and graft-specific differences in the rejection response. Although a number of studies have demonstrated the primary role of CD4 cells in allograft rejection (5, 18, 19), rejection of skin and small bowel allografts in several commonly used strains of mice can also be mediated by CD8 cells (15, 20, 21). Furthermore, the generation of cytolytic CD8 cells does not always require costimulation through the CD28 and CD40 pathways (22, 23). Thus, there is concern that in outbred species like humans, robust and long-lived tolerance to immunogenic allografts will require the addition of agents that can synergize with costimulatory blockade.

Basic pharmacological principles would suggest that agents that interfere with T cell activation signal one should synergize with those blocking activation signal 2. However, this has not been previously demonstrated. In fact, earlier reports indicate that therapies that interfere with signal one, such as anti-CD4 or cyclosporin A, either have no additive effect or actually inhibit the effectiveness of costimulatory blockade (13, 14, 15, 24, 25). Nonetheless, the precise nature of interference with T cell activation signals by such agents may be paramount. In this regard, CD45 is a family of transmembrane protein tyrosine phosphatases that is expressed by leukocytes and plays a critical role in regulating T cell activation through the TCR (26). Multiple CD45 isoforms, differing only in their extracellular domain, are generated by alternative splicing of three exons. The larger and smaller CD45 isoforms are differentially expressed on subsets of CD4 cells having distinct functional repertoires. In mice, these subsets can be differentiated from one another based on their level of reactivity to anti-CD45RB mAbs, which recognize the higher Mr isoforms. For example, CD4 cells expressing the lower Mr (CD45RBlow) isoforms preferentially secrete Th2-type cytokines and have been shown to down-regulate autoimmune mediated colitis (27, 28, 29). Although their precise role is unclear, individual CD45 isoforms preferentially regulate certain signaling pathways and appear to directly contribute to the distinct functions of these T cells (30, 31).

We and our collaborators have previously shown that several doses of anti-CD45RB (MB23G2) can induce long term engraftment of renal and islet allografts in mice (6, 7). Interestingly, treatment with this anti-CD45RB mAb is associated with a shift in expression from higher to lower Mr CD45 isoforms on T cells (7). On the basis of its promising activity as a single agent and distinct mechanisms of action, we now examine the ability of anti-CD45RB to synergize with anti-CD40L and promote allograft survival in rigorous models of transplantation.

Specifically, using an immunogenic strain combination of BALB/c donors and C57BL/6 recipients, we now show that the novel combination of anti-CD45RB plus anti-CD40L significantly boosts long term engraftment of islet allografts and augments tolerance to islet retransplantation. In this same strain combination, combined costimulatory blockade with CTLA4-Ig plus anti-CD40L has only a minimal effect on skin graft survival compared with untreated controls. In contrast, anti-CD45RB plus anti-CD40L markedly prolongs skin graft survival (median survival of 69 days). In concert with the efficacy of this combination in costimulatory blockade-resistant rejection, anti-CD45RB partially depletes CD8 cells from lymph nodes and inhibits perigraft infiltration of CD8 cells. Moreover, compared with individual agents, anti-CD45RB plus anti-CD40L significantly decreases periislet infiltration by APCs, markedly inhibits intragraft expression of Th1 cytokines and augments Th2 expression. Thus, we demonstrate for the first time that agents that selectively alter T cell activation signals 1 and 2 may be effectively combined to enhance graft survival and tolerance in stringent allograft models.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Male C57BL/6 (H-2b) recipient and BALB/c (H-2d) donor mice (Charles River, Boston, MA), 7–10 wk old, were individually housed after transplantation with free access to food and water.

Abs and immunofluorescence

Anti-CD45RB mAb MB23G2 (American Type Culture Collection (ATCC), Manassas, VA) was purified on protein G columns according to the manufacturer’s instructions (Pharmacia, Piscataway, NJ). The MR1 hybridoma producing anti-CD40L (32) was the kind gift of Dr. Randolph Noelle (Dartmouth University, Hanover, NH). MR1 mAb was purified on protein G columns as above. mCTLA4-Ig was kindly provided by Dr. Robert Peach (Bristol Meyer Squibb, Princeton, NJ). mAbs reactive with CD4, CD8, CD11b, CD45RB, and B220 (CD45RA) were from PharMingen (San Diego, CA). Anti-CD45 (TIB 122, ATCC) was purified on protein G columns.

Islet isolation and transplantation

Diabetes was induced in C57BL/6 mice with streptozotocin (200 mg/kg i.p.) and confirmed by persistent hyperglycemia (blood glucose >400 mg/dl). After in situ digestion with collagenase P (Sigma, St. Louis, MO), islets were separated by density gradient centrifugation and hand-picked under a stereomicroscope, as described (7). Then 400 islets/recipient were transplanted under the left kidney capsule. Glycemia <200 mg/dl by day 2 posttransplantation and >250 mg/dl (after initial engraftment) defined primary graft function and graft loss, respectively.

Skin grafts

Full thickness abdominal skin from BALB/c donors was grafted to the dorsum of C57BL/6 recipient mice. Skin grafts were sutured in place and covered with Vaseline gauze; a bolster dressing was applied for 7 days. Graft survival was followed by daily visual inspection, with rejection defined as complete loss of viable skin.

Treatment protocols

Islet transplantation. Based on previous studies (7), C57BL/6 islet allograft recipients received 100 µg anti-CD45RB (MB23G2) i.v. on days -1, 0, and 5; anti-CD40L, three 250-µg i.p. doses on days 0, 2, and 4 (13); or a combination of both mAbs (each at the doses above). Control allograft recipients were untreated.

Skin transplantation. Based on the vigor of the response against skin allografts, the treatment regimen was empirically intensified. Recipient mice received: CTLA4-Ig (250 µg i.p. on days 0, 2, 4, 6, and 8); anti-CD40L (250 µg i.p. on days 0, 2, 4, 6, and 8); anti-CD45RB (100 µg i.v. on days -1, 0, 1, 2, 5, and 8); combined costimulatory blockade with anti-CD40L plus CTLA4-Ig (five doses of each given as above); or combination therapy with anti-CD45RB plus anti-CD40L (six and five doses, respectively, as detailed above). Control animals were untreated.

Data analysis

Actuarial curves of graft survival were compared by the log rank test. Other statistical analyses used the unpaired Student t test.

Isolation of lymphocytes/mononuclear cells

RBCs were removed from single-cell suspensions of spleen by hypotonic lysis. Splenic mononuclear cells were separated into Ig+ (B cells) and Ig- cells with anti-IgG- and anti-IgM-coated immunomagnetic beads (PerSeptive Diagnostics, Cambridge, MA). T cells were enriched from the Ig- population by nonadherence to plastic plates.

Cell lysis and Western blotting

Cell populations from treated and untreated animals were lysed in 1% Nonidet P-40 lysis buffer containing 25 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM aminoethylbenzenesulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin, as described (31). Postnuclear supernatants (4 x 106 cell equivalents/lane) were boiled in Laemmli sample buffer (nonreducing), separated by 6% SDS-PAGE, and transferred to nitrocellulose. Membranes were immunoblotted with pan anti-CD45 and developed with enhanced chemiluminescence (7, 31).

Histology

Allografts were excised 8 days posttransplantation and were snap frozen in liquid nitrogen, cryosectioned, fixed in paraformaldehyde, and either stained with hematoxylin and eosin or incubated with primary mAb followed by peroxidase-conjugated secondary Ab, as we previously described (7). Quantitative analysis was performed similar to that previously described (7). In brief, the entire area of the allograft in each tissue section was analyzed using a grid (>50 squares/sample). Infiltrating cells within the islets and those surrounding the islets were analyzed separately. For cells invading the islets, the total number of positively stained cells per islet was calculated for each tissue section. Positively stained cells within the periislet region were assessed as total positive cells per number of squares counted (positive cells per square).

Quantitative RT-PCR for cytokines was performed as detailed in Refs. 7, 33 . RNA isolated from snap-frozen islets obtained 6 days posttransplantation was reverse transcribed into cDNA. For each cytokine, cDNA was coamplified with a specific competitive template and a single pair of specific primers in a "same tube reaction." Sample and competitive template PCR products were then separated by electrophoresis, and the relative amount of each band was determined by OD. To control for the efficiency of each individual reverse transcription reaction, expression of the housekeeping gene, GAPDH, was determined by the same PCR technique. The ratio of picograms target cDNA to picograms GAPDH cDNA was calculated for each sample. PCR primer sequences: IL-2, CTT GGC ATG CTT GTC AAC AGC GCA CCC ACT and GTG TTG TAA GCA GGA GGT ACA TAG TTA; IFN-{gamma}, CAC GGC ACA GTC ATT GAA AGC C and CTT ATT GGG ACA ATC TCT TCC C; IL-4, CCC AGC TAG TTG TCA TCC TGC and CGA GTA ATC CAT TTG CAT GAT GCT C; IL-10, CTG CCT GCT CTT ACT GAC TGG C and AAT CAC TCT TCA CCT GCT CC.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Combined therapy with anti-CD45RB and anti-CD40L augment islet engraftment and induction of tolerance

Parker et al. (10) previously reported that 40% of C57BL/6 recipients demonstrated long term engraftment of BALB/c islet allografts after treatment with anti-CD40L. Using the same high response strain combination, we showed that three doses of anti-CD45RB induce long term islet engraftment in ~50% of the recipients (7). On the basis of the premise that combining agents that act on distinct T cell activation pathways should act in synergy, we examined the efficacy of combining anti-CD45RB and anti-CD40L. As shown in Fig. 1Go, untreated control animals promptly reject their islets, with all recipients becoming hyperglycemic by day 12. Animals treated with anti-CD40L exhibited a relatively low incidence of early allograft rejection. However, because of later rejection episodes, only 56% of mice treated with anti-CD40L alone ultimately displayed long term engraftment (>120 days). This compares favorably with the results of Parker et al. noted above. Similar to our previous results, 44% of the recipients treated with anti-CD45RB alone experienced long term engraftment. Compared with anti-CD40L treatment, animals treated with anti-CD45RB sustained a higher rate of early rejection but enjoyed more stable engraftment thereafter. Treatment with anti-CD40L plus anti-CD45RB in combination resulted in 83% long term engraftment, surpassing the efficacy of either agent alone (p <= 0.05).



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  		FIGURE 1. Anti ({alpha})-CD45RB combined with anti-CD40L augments islet allograft survival. Kaplan-Meier plot of cumulative allograft survival vs time. Treatment groups include: untreated controls (n = 5); anti-CD45RB, 100 µg i.v. on days -1, 0, and 5 (n = 39); anti-CD40L, 250 µg i.p. on days 0, 2, and 4 (n = 25); and anti-CD45RB plus anti-CD40L, each dosed as above (n = 23) (p < 0.05 compared with all other groups).

 
To help determine whether this improvement was quantitative or qualitative, we examined long term survivors in each group for tolerance to new islet allografts from the same donor strain without further treatment. For this purpose, long term graft survivors (>120 days) in each treatment group underwent left nephrectomy to remove the islet allograft. This resulted in recurrence of diabetes in each case. Animals were then retransplanted with freshly isolated BALB/c islet grafts (under the right kidney capsule) without further therapy. Whereas 50% of recipients originally treated with anti-CD45RB and 66% of animals originally treated with anti-CD40L accepted and maintained new islet allografts, seven of eight animals (88%) treated with combined therapy exhibited tolerance to islets (Table IGo). These data suggest that these two agents act in synergy, augmenting the frequency with which both long term engraftment and tolerance were achieved.


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  		Table I. Augmentation of tolerance in animals treated with combined anti-CD45RB and anti-CD40L1

 
Combination therapy with anti-CD45RB and anti-CD40L dramatically prolongs skin graft survival

To further test of the potency of targeting signals 1 and 2 with anti-CD45RB and anti-CD40L, we examined survival of BALB/c skin grafts on C57BL/6 recipients, a highly stringent allograft model. We directly compared this regimen to combined costimulatory blockade with CTLA4-Ig plus anti-CD40L. Used individually, CTLA4-Ig, anti-CD40L, or anti-CD45RB only minimally prolonged engraftment (ranging from a median survival of 8 days in untreated animals to 14 days in anti-CD40L-treated animals; see Fig. 2Go). Despite efficacy in other models (13), combined costimulatory blockade (CTLA4-Ig plus anti-CD40L) was relatively ineffective in prolonging skin graft survival in this immunogenic strain combination, with a median survival of only 18 days. In marked contrast, the combination of anti-CD40L and anti-CD45RB demonstrates potent synergy and significantly prolonged graft survival, resulting in a median survival of 69 days.



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  		FIGURE 2. Prolonged skin graft survival induced by anti ({alpha})-CD45RB plus anti-CD40L. Kaplan-Meier plot of cumulative allograft survival vs time. Treatment groups: untreated controls (n = 2); CTLA4-Ig, 250 µg i.p. on days 0, 2, 4, 6, and 8 (n = 5); anti-CD40L, 250 µg i.p. on days 0, 2, 4, 6, and 8 (n = 5); anti-CD45RB, 100 µg i.v. on days -1, 0, 1, 2, 5, and 8 (n = 6); costimulatory blockade with anti-CD40L plus CTLA4-Ig (n = 5); and combination therapy with anti-CD45RB plus anti-CD40L (n = 8). Control animals were untreated. *, p < 0.01 vs control; **, p <= 0.015 vs any individual agent; ***, p <= 0.0015 vs all other treatment groups.

 
Combination therapy with anti-CD45RB and anti-CD40L alters islet allograft infiltration by B cells, monocytes, and CD8 cells

To begin to define the synergistic effect of these agents on the alloimmune response, we compared the histology of islet allografts 8 days after transplantation in mice treated with anti-CD40L alone, anti-CD45RB alone, or both agents in combination. Control animals exhibited diffuse mononuclear infiltrates with significant invasion of mononuclear cells into the islets ("insulitis"), frequently making the margins of individual islets difficult to distinguish (Fig. 3Go). Both periislet and intraislet infiltrates contained CD4 and CD8 cells, monocytes/macrophages (CD11b+ cells) and B cells (B220+ cells). Compared with untreated control animals, all three treatment regimens markedly inhibited insulitis (Figs. 3Go and 4GoA). However, because of variability within the untreated control group, reduction of intraislet infiltration by B cells achieved statistical significance only in the animals receiving combination therapy (Fig. 4GoA). Combination therapy with anti-CD40L and anti-CD45RB also tended to be somewhat more effective at reducing islet infiltration by CD8 cells than either individual agent.



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  		FIGURE 3. Comparison of islet allograft histology from animals in each treatment group (displayed in vertical columns). Top row, hematoxylin/eosin (H&E). Other rows show immunoperoxidase staining for anti ({alpha})-CD4 (row 2), anti-CD8 (row 3), CD11b (macrophage/monocytes) (row 4), and B220 (B cells) (row 5). Histological sections for each marker are representative of three to four animals from each treatment group.

 


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  		FIGURE 4. Combination therapy inhibits periislet infiltration by CD8 cells, B cells, and monocytes/macrophages. Quantitative analysis was performed by counting immunoperoxidase-positive cells in periislet infiltrates from animals in each treatment group using a grid. Data are displayed as the number of cells per square. Cells actually invading the islets were counted separately and are presented as the total number of positive cells per islet counted. Data are depicted as the mean (+SD) of tissue sections from three to four animals in each treatment group. *, p < 0.05 vs control; **, p = 0.09).

 
Although similar in reducing insulitis, these treatment regimens had differing effects on the composition of periislet infiltrates (Figs. 3Go and 4GoB). Interestingly, none of the treatment regimens had a statistically significant effect on CD4 infiltration. Whereas individual agents had no effect on periislet infiltration by monocytes and macrophages, combined therapy significantly reduced infiltration by these cells. A similar but less consistent effect on B cell infiltration was observed for combined therapy. Thus, when used in combination, anti-CD45RB and anti-CD40L decrease the influx of APCs to the region of the allograft.

Although anti-CD40L itself had little effect on CD8 cells, these cells were significantly reduced by treatment with anti-CD45RB and were reduced almost 4-fold by anti-CD45RB plus anti-CD40L. Given the importance of CD8 cells to costimulatory blockade-resistant rejection of skin grafts in this strain combination (15), anti-CD45RB-mediated inhibition of CD8 infiltration may be one means by which this agent synergizes with anti-CD40L in prolonging graft survival. To further address this issue, we examined the effect of anti-CD45RB treatment on CD4 and CD8 cells in secondary lymphoid tissue. Although anti-CD45RB had no effect on relative expression of CD4 and CD8 cells in peripheral blood or spleen, it causes a selective 2-fold reduction in the percentage and absolute number of CD8 cells in lymph nodes (data not shown). Thus, anti-CD45RB may specifically affect CD8 homing to lymph nodes or induce partial depletion of this T cell subset. The significance of this finding may be underscored by the absolute dependence of skin graft rejection on lymph nodes (34).

Anti-CD40L does not affect altered CD45 isoform expression induced by anti-CD45RB

Although anti-CD45RB appears to limit the CD8 alloresponse this agent has additional effects that are likely to contribute toward long-term engraftment when combined with anti-CD40L. For example, one means by which tolerogenic anti-CD45RB mAbs alter the immune response is by inducing a shift in the expression of CD45 from higher to lower Mr isoforms, which may alter T cell activation signaling and functional repertoire, skewing the immune response toward tolerance (7, 27, 28, 29, 30, 31). Whether this shift in CD45 isoforms is unique to anti-CD45RB treatment or is a feature of other strategies that promote Th2 cytokines is completely unknown. Moreover, the addition of other agents that interfere with T cell signaling could either augment or interfere with, the shift in isoforms induced by anti-CD45RB. To address this issue, we examined the effect of each treatment regimen on CD45 isoform expression by splenic T cells from otherwise naive animals (Fig. 5Go). As we have previously shown, T cells from untreated control animals express relatively large amounts of CD45 containing a single alternative exon (190 kDa), lesser amounts of the CD45R0 isoform that lacks alternative exons (180 kDa), and small amounts of larger isoforms that contain two or three alternative exons (7). Treatment with anti-CD45RB causes a decrease in the expression of isoforms containing alternative exons and a concomitant increase in the CD45R0 isoform. Treatment with anti-CD40L alone had no effect on CD45 isoform expression, and addition of anti-CD40L did not alter the shift in isoforms induced by anti-CD45RB. Thus, this proposed mechanism may be unique to anti-CD45RB or at least is not shared by anti-CD40L. In contrast, anti-CD40L does not interfere with this mechanism of anti-CD45RB activity.



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  		FIGURE 5. Anti ({alpha})-CD45RB, but not anti-CD40L, alters CD45 isoform expression on T cells. Anti-CD45 immunoblot on day 8 using lysates from splenic T cells from animals that were either untreated or treated with anti-CD40L, anti-CD45RB, or the combination of anti-CD45RB plus anti-CD40L. Numbered arrowheads, number of alternative exons used to generate CD45 isoforms (bands) of each size, as defined using lysates from transfectants expressing single CD45 isoforms (provided by Dr. Kim Bottomly, Yale University, New Haven, CT) (7 ).

 
Combined therapy alters cytokine expression within the allografts

We have previously shown that the shift toward the lower Mr isoforms induced by anti-CD45RB treatment is associated with an increase in IL-4 expression within islet allografts (7). In contrast, anti-CD40L used alone had no effect on cytokine expression in cardiac allografts (13). However, the effects of an individual agent on cytokine expression may be altered when used in combination with other agents (13, 35). For example, the reduction of both APC and CD8 cell infiltration into the allograft produced by the combination of anti-CD45RB plus anti-CD40L may have distinct effects on cytokine expression. We therefore compared intragraft cytokine expression in each treatment group 8 days posttransplantation using quantitative RT-PCR (Fig. 6Go). Compared with untreated control animals, anti-CD40L had a small effect on IL-2 secretion that did not quite attain statistical significance (p = 0.056). Anti-CD40L had essentially no effect on other cytokines. Anti-CD45RB treatment was associated with an increase in IL-4 and a trend toward increased IL-10 expression but was without consistent effect on IL-2 or IFN-{gamma}. However, when combined, anti-CD40L plus anti-CD45RB cause a significant decrease in Th1 cytokines in addition to a significant increase in both IL-4 and IL-10.



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  		FIGURE 6. Anti ({alpha})-CD45RB plus anti-CD40L inhibits Th1 and increases Th2 cytokine expression. Allograft tissue obtained 8 days posttransplantation was analyzed by quantitative RT-PCR for each cytokine mRNA using competitive templates and then normalized to the amount of GAPDH (GAP) mRNA in each sample. Three to four animals were analyzed for each treatment group. Data are depicted as the mean (+SEM) of the ratio of picograms cytokine mRNA to picograms GAPDH mRNA. *, p <= 0.05 and **, p <= 0.015 compared with untreated control group. {dagger}, p = 0.056 and {dagger}{dagger}, p = 0.076 vs untreated control group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
T cell-signaling molecules are promising immunomodulatory targets. In a number of transplant models, the efficacy of an individual agent can be augmented by combination with other agents (5, 8, 9, 24, 35). However, not all combinations enhance engraftment, and some are clearly antagonistic (13, 24). Considerable excitement was generated when agents targeting the CD40L- and CD28-costimulatory pathways were combined and shown to be extremely effective in promoting allograft survival in murine and nonhuman primate models. However, these initial reports were tempered by subsequent studies demonstrating that the efficacy of this combination is both strain and organ/tissue dependent (12, 13, 15, 21). Although a prolonged course of anti-CD40L appears to allow 1- to 2-year treatment-free survival of renal allografts in rhesus monkeys, islet allografts are uniformly rejected several months after therapy is withdrawn (12, 16, 17). Moreover, in monkeys, the addition of CTLA4-Ig to block CD28 adds little to treatment with anti-CD40L alone (12, 14). Finally, in mice, combined costimulatory blockade is relatively ineffective in prolonging skin graft survival in immunogenic strain combinations where CD8 cells participate in the rejection process (15). Thus, it is likely that the generation of robust tolerance to various organs and tissues in outbred large animal species such as humans will require the addition of new agents that can effectively synergize with costimulatory blockade. Here we demonstrate for the first time that agents that target T cell activation signal 1 and 2 may be effectively combined. Anti-CD45RB plus anti-CD40L demonstrate potent synergy in costimulatory blockade-resistant transplant models, through mechanisms that include inhibition of CD8 cells.

The interaction of T cell-based CD40L with CD40 provides signals critical for activation, maturation, and function of APCs and B cells (36). For example, CD40-mediated signals are essential for B cells to undergo Ig class switching and for germinal center formation (32, 37). On APCs, CD40 ligation up-regulates expression of class II MHC, B7-costimulatory receptors, adhesion molecules (e.g., ICAM and CD44), and cytokines (e.g., IL-12 in dendritic cells; NO, TNF-{alpha}, and IL-1 in macrophages) (38, 39, 40, 41, 42, 43, 44). Thus, CD40L-CD40 interactions play a critical role in up-regulating the Ag-presenting capacity of these cells, which in turn enhances their ability to provide costimulatory signals required for CD4-dependent immune responses (2). Moreover, CD40-CD40L interaction during Ag presentation to CD4 cells can "arm" dendritic cells with the capacity to directly activate CD8+ CTL without further CD4 participation (45, 46).

Given the importance of CD40L in a number of immunological processes, it is not surprising that blockade of CD40L, with or without additional blockade of CD28 costimulation, has proved effective in inhibiting allograft rejection and autoimmunity in several murine models (13, 47, 48). Nonetheless, mice lacking CD28 or CD40 are capable of generating virus-specific CD8+ CTL after infection with lymphocytic choriomeningitis virus (22, 23). Additionally, 2C TCR-transgenic mice, which exclusively develop Ag-specific CD8+ T cells, can reject allogeneic tumors that express relevant peptide Ag but lack B7 molecules (49). These findings suggest that CD8+ CTL generation against certain viruses and allogeneic tumors can occur independently of these costimulatory pathways. Recent studies have directly extended these findings toward allograft rejection. For example, rejection of skin and small bowel allografts occurs despite blockade of CD28 and/or CD40L pathways and is mediated by alloreactive CD8+ cells that are resistant to costimulatory blockade (15, 21). Prolongation of allograft survival in these models required CD8 depletion (by gene ablation or mAb therapy) in addition to combined costimulatory blockade.

CD45 plays a critical role in proximal signal transduction through the TCR (signal one) by regulating the activity of Src family kinases Lck and Fyn, and regulating dephosphorylation of the TCR-{zeta} chain (26, 50). Accordingly, T cell lines lacking CD45 are unable to signal through the TCR, whereas costimulatory signals through CD28 remain intact (31). Although much is known about the role of CD45 in T cell activation, relatively little is known about the mechanism(s) by which anti-CD45RB induces tolerance. The tolerogenic anti-CD45RB mAb used in our studies has been shown to increase activation-induced phosphorylation of phospholipase C{gamma}1 in cell lines, suggesting that CD45RB ligation can directly alter TCR-mediated signaling (6). Similar changes in phospholipase C{gamma}1 phosphorylation have been associated with anergy in T cell clones (51). In addition to the direct effect of CD45RB ligation on signal 1, we have shown that administration of this anti-CD45RB mAb causes a rapid shift in CD45 isoform expression on T cells (7). The expression of different CD45 isoforms alters TCR-mediated signaling through key signaling intermediates such as Vav and SLP-76 and ultimately results in altered IL-2 production in vitro (31, 50). Exactly how this promotes long term engraftment is not yet clear. However, the shift in CD45 isoforms does seem to alter cytokine expression in a manner that promotes tolerance (Fig. 6Go and discussion below).

The combination of anti-CD40L and anti-CD45RB enhances permanent engraftment and tolerance in islet transplantation. More substantial synergy is demonstrated in the prolongation of BALB/c to C57BL/6 skin grafts. The important contribution of CD8 cells to costimulatory blockade-resistant rejection of skin grafts in this strain combination (15) suggests that the addition of anti-CD45RB to anti-CD40L results in more effective inhibition of alloreactive CD8 cells than does the combination of CTLA4-Ig plus anti-CD40L. This is supported by the demonstration that anti-CD45RB (alone and in combination with anti-CD40L) significantly inhibits infiltration of CD8 cells into the graft. Moreover, anti-CD45RB treatment itself induces partial depletion of CD8 cells in lymph nodes. Although these effects could be the result of selective depletion by this mAb, a role for CD45 in regulating integrin-mediated adhesion in macrophages and T cells has recently been described (52, 53, 54). Thus, anti-CD45RB ligation, or the associated shift in isoform expression, could alter adhesion and homing, selectively interfering with entry of CD8 cells into the graft and lymph nodes.

Although anti-CD45RB appears to limit the CD8 alloresponse, it is unlikely that this is its sole contribution toward the effectiveness of combined therapy with anti-CD40L. The efficacy of anti-CD40L is clearly enhanced by agents such as CTLA4-Ig that are primarily directed at the CD4 response (13). Moreover, when anti-CD8-mediated depletion of CD8 cells is combined with anti-CD40L, skin graft survival is not prolonged beyond that seen with anti-CD40L alone (M.H.S., unpublished data). Thus, the synergy between anti-CD45RB and anti-CD40L is likely to result from additional effects on the immune response. In this regard, our findings support the notion that when combined, agents exhibit both additive and de novo effects (i.e., those not observed by either agent administered alone). For example, anti-CD45RB alters CD45 isoform expression, decreases CD8 cell infiltration, and augments Th2 type cytokines, all relatively unaffected by anti-CD40L treatment. Yet when combined, anti-CD40L and anti-CD45RB also reduce expression of Th1 cytokines and significantly inhibit periislet infiltration of APCs. Each of these elements might contribute to the potency of this regimen.

The role of altered cytokine secretion remains somewhat controversial. Clearly, Th1 cytokines are not necessary or sufficient for rejection, nor do Th2 cytokines meet these criteria for induction of tolerance (55, 56, 57). Moreover, the presence of at least some IL-2 and IFN-{gamma} is required for tolerance to occur, probably by allowing apoptosis of T cells that proliferate despite signaling blockade (24, 58, 59). However, inappropriate administration of IL-2 can disrupt the generation of tolerance (60). Thus, limiting rather than completely eliminating Th1 cytokines could contribute to tolerance by decreasing CTL generation and activity, while still allowing activation-induced cell death to occur. Moreover, Th2 deviation and, in particular, IL-4 can contribute toward tolerance, as noted when the "hurdle" of the allogeneic disparity is decreased (61). Interestingly, like anti-CD45RB, donor-specific transfusion synergizes with anti-CD40L therapy in murine transplant models, and also promotes strong Th2 deviation (10, 35). Indeed, recent data from our group using STAT4 and STAT6 (important in Th1 and Th2 differentiation, respectively) knockout mice show that CD40L blockade was less effective in a predominantly Th1 environment (STAT6 knockout) unless combined with donor splenocytes (62). Whether inhibition of Th1 cytokines by combined anti-CD45RB and anti-CD40L is caused by decreased infiltration by APCs and CD8 cells or vice versa is not clear. Regardless, this regimen results in decreased capacity to present Ag and generate a cytotoxic response.

The effects of combination therapy on periislet infiltration by CD4 cells was more variable, and in some animals significant numbers of CD4 cells remained. Although it could be argued that such animals were destined to undergo rejection rather than long term engraftment, we have also observed that allografts removed from animals 130 days after transplantation in animals subsequently shown to be tolerant to retransplantation with islets from the original donor strain had large collections of lymphocytes containing CD4 cells (D.M.R., unpublished data). This may be consistent with the induction of CD4+-regulatory cells in anti-CD45RB-treated recipients that can adoptively transfer tolerance into naive hosts (63). Whether tolerance transferred by these CD4 cells depends on IL-4, as is the case with anti-CD4-induced "infectious tolerance," is not yet known (5, 64). These characteristics appear to contrast with combined costimulatory blockade, which strongly inhibits immune reactivity and essentially blocks mononuclear infiltration, cytokine secretion, and the generation of tolerance in cardiac allografts (13, 36).

In many transplant models, the efficacy of agents targeting T cell-signaling molecules is greatly enhanced by combining agents that act on distinct pathways. Although previous attempts to combine agents acting on signals 1 and 2 have been unsuccessful, we now demonstrate that anti-CD45RB plus anti-CD40L can be effectively combined. Among a number of potentially exciting experimental regimens that foster tolerance in murine transplant models, this combination is particularly potent, culminating in enhanced graft survival and augmentation of tolerance in stringent transplant models resistant to other modalities. It now remains to demonstrate the efficacy of this novel combination in trials involving nonhuman primates.


   Acknowledgments
 
We thank Dr. Mark Shlomchick and his laboratory staff, for use of their digital imaging equipment; and Drs. Kim Bottomly, Charlie Janeway, and Randy Noelle, for their gifts of reagents.


   Footnotes
 
1 This work was supported in part by funding through the National Institutes of Health (AI36317 and AI45485 to D.M.R. and AI34965 to M.H.S.) and the Juvenile Diabetes Foundation International (1-1998-242 to G.P.B. and 1-1999-317 to X.X.Z.). Back

2 D.M.R. and M.F.A.L. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. David Rothstein, Department of Medicine, Section of Nephrology, Yale Medical School, 333 Cedar Street, New Haven, CT 06520. Back

4 Abbreviation used in this paper: CD40L, CD40 ligand. Back

Received for publication July 17, 2000. Accepted for publication September 28, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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