The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Salvalaggio, P. R. O.
Right arrow Articles by Rothstein, D. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Salvalaggio, P. R. O.
Right arrow Articles by Rothstein, D. M.
The Journal of Immunology, 2006, 176: 2292-2298.
Copyright © 2006 by The American Association of Immunologists

Antigen Exposure during Enhanced CTLA-4 Expression Promotes Allograft Tolerance In Vivo1

Paolo R. O. Salvalaggio2,*, Geoffrey Camirand2,{dagger}, Charlotte E. Ariyan*, Songyan Deng{dagger}, Linda Rogozinski{dagger}, Giacomo P. Basadonna3,{ddagger} and David M. Rothstein3,4,{dagger}

* Department of Surgery and {dagger} Department of Internal Medicine, Yale School of Medicine, New Haven, CT 06510; and {ddagger} Department of Surgery, University of Massachusetts Medical School, Worcester, MA 01655


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The role of CTLA-4 in tolerance is primarily inferred from knockout and blocking studies. Anti-CD45RB mediates allograft tolerance in mice by inducing CTLA-4 expression on CD4 cells, providing a novel opportunity to determine how therapeutic enhancement of CTLA-4 promotes tolerance. We now show that induced CTLA-4 expression normally resolves by day 17. Although thymectomy prolongs enhanced CTLA-4 expression, long-term engraftment is unaffected. To address the temporal relationship between increased CTLA-4 expression and engraftment, transplantation was delayed for various times after anti-CD45RB treatment. Delaying transplantation for 7 days (when CTLA-4 expression had peaked but treatment mAb was no longer detectable), resulted in long-term engraftment comparable to transplantation with no delay (day 0). Delaying transplantation from 10 to 18 days led to a progressively poorer outcome as CTLA-4 expression returned to baseline. This suggested that Ag exposure while CTLA-4 expression is enhanced is sufficient to induce long-term engraftment. To substantiate this, on day 0, anti-CD45RB-treated mice received BALB/c vs unrelated alloantigen, followed by transplantation of BALB/c islets 10 days later. Whereas recipients exposed to unrelated Ag experienced acute rejection, recipients exposed to donor Ag achieved long-term engraftment. Anti-CD45RB-treated mice exposed to alloantigen exhibited anergic CD4+CD25– effector cells and regulatory CD4+CD25+ cells. Moreover, CD25 depletion in the peritransplant period prevented anti-CD45RB-mediated engraftment. Thus, exposure of CD4 cells expressing CTLA-4 to donor Ag is necessary and sufficient to induce long-term engraftment which appears to be mediated by both regulation and anergy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Immunological tolerance promises to enhance allograft survival while reducing risks inherent in chronic immunosuppression. Yet, in the clinical setting, tolerance remains elusive and new insight is needed. T cells play an obligatory role in allograft rejection. Concordantly, interference with positive costimulatory signals required for normal T cell activation, such as CD28/B7 or CD40/CD154, can prevent allograft rejection in various animal models (1).

T cells also express CTLA-4, a potent inhibitory costimulatory molecule that plays an essential role limiting the immune response and maintaining peripheral tolerance (2, 3). The importance of this pathway is exemplified by CTLA-4-deficient mice, which die of rampant systemic autoimmunity by several weeks of age (4, 5). Unfortunately, it has been difficult to target CTLA-4 to promote tolerance. CTLA-4 is primarily expressed on effector cells only after T cell activation, and no soluble agonist ligands exist (3, 6, 7, 8). Indeed, mAbs against CTLA-4 block its negative signal, thereby enhancing autoimmunity and inhibiting tolerance induced by various experimental strategies (2, 9, 10, 11, 12, 13). CTLA-4 is also expressed by natural and induced regulatory T cells (Treg),5 and may play a key role in their function (13, 14, 15).

We have shown that as a single agent, anti-CD45RB induces long-term islet allograft survival in 50% of recipients across immunogenic murine strain combinations (16). Anti-CD45RB also promotes tolerance in murine cardiac and renal transplantation (17, 18). Anti-CD45RB acts through a unique mechanism involving enhanced CTLA-4 expression on CD4 cells (19). This occurs without depletion or overt T cell activation. Importantly, multiple lines of evidence indicate that CTLA-4 induction plays a specific and essential role in anti-CD45RB activity (19, 20). These include the loss of tolerogenic activity: when anti-CD45RB is combined with anti-CTLA-4 or CTLA4-Ig (which interfere with CTLA-4:B7 interaction), when anti-CD45RB is combined with cyclosporin A (which prevents CTLA-4 up-regulation), or when anti-CD45RB is used in CTLA-4-deficient recipients (19, 20).

Most of what is known about the role of CTLA-4 in the immune response comes from studies where CTLA-4 is absent or blocked. Therefore, anti-CD45RB treatment provides a unique opportunity to determine how therapeutic enhancement of CTLA-4 expression promotes tolerance. Specifically, we now show that prolonging the window of enhanced CTLA-4 expression decreases acute rejection of islet allografts, but does not enhance long-term engraftment. In contrast, exposure to donor-specific Ag while CTLA-4 expression is enhanced is necessary and can promote long-term allograft survival of allografts placed even after the complete disappearance of the anti-CD45RB treatment mAb. These results demonstrate the critical nature of CTLA-4 induction per se and indicate that specific exposure of CTLA-4-bearing CD4 cells to donor-Ag is essential to shut down effector responses in vivo.


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

Seven- to 10-wk-old male BALB/c (H-2d), DBA/1 (H-2q), and C57BL/6 (H-2b) mice (National Cancer Institute) were housed with free access to food and water. All studies were done in compliance with National Institutes of Health and Yale Animal Care and Use Committee guidelines.

Abs and immunofluorescence

Purified anti-CD45RB mAb MB23G2 (American Type Culture Collection) and anti-CD154 (MR1) were obtained from Bioexpress. Fluorochrome-conjugated mAbs against CD4, CD25(7D4), CD45RB, CTLA-4, and hamster Ig and rat Ig control Abs were from BD Pharmingen. Anti-CD25 (PC61) was purified from hybridoma supernatants using protein G according to the manufacturer’s recommendation (Pharmacia). CD45RB expression was analyzed by staining with directly conjugated anti-CD45RB (clone 16A), which does not cross-react with the epitope bound by MB23G2 (16, 19). Intracellular CTLA-4 expression was determined by permeabilization with 0.5% saponin after fixation (2% paraformaldehyde) followed by incubation with directly conjugated anti-CTLA-4 or control hamster Ig, as we described (19). For multicolor analysis, cells were stained with surface markers before permeabilization. For phenotyping, 5000 cells/sample were analyzed on a FACStar cytometer (BD Biosciences). Negative controls used appropriate rat or hamster IgG fluorochrome conjugates.

Islet isolation and transplantation

Diabetes was induced in C57BL/6 mice with streptozotocin (200 mg/kg i.p.; Sigma-Aldrich) and confirmed by persistent hyperglycemia (blood glucose >350 mg/dl on 2 days) (16). After in situ digestion with collagenase V (Sigma-Aldrich; 1 mg/ml), islets were purified by filtration through a 100-um nylon cell strainer (BD Biosciences) and then hand-picked under a stereomicroscope, as we described (21). Four hundred islets were transplanted under the left kidney capsule of C57BL/6 recipients (16, 19). Blood glucose of <200 mg/dl within 2 days after transplantation and >250 mg/dl (after initial engraftment) defined primary graft function and graft loss, respectively. After day 120 of graft function, left nephrectomy was performed to rule out recovery of native islet function.

Thymectomy

After induction of diabetes some mice underwent thymectomy as described (22). Briefly, a Pasteur pipette was introduced into the mediastinum through a sternotomy and the thymus was removed by suction.

Treatment protocols

Based on previous findings, C57BL/6 recipients received three doses of anti-CD45RB (MB23G2; 100 µg; i.v.) on days –1, 0, and 5 (16, 19). Control recipients were untreated. In some experiments, islet transplantation was delayed until days 7–18. Some recipients were exposed to alloantigen by transplanting 50 islets under the right kidney capsule on day 0 followed by transplantation with a usual dose of 400 islets under the left kidney capsule on day 10. As indicated, thymectomy was performed on day –7, and in other experiments, mice were redosed with anti-CD45RB (100 µg; i.p.) every 15 days after initial treatment (days –1, 0, 5). In some experiments, recipients received anti-CD154 (0.5 mg i.p.; days –7, –4, 0, 4) and donor-specific transfusion (DST) with 107 BALB/c splenocytes on day –7 (23). As indicated, recipients received PC61 (100 µg i.p.) either on day –1 or 10, resulting in >95% depletion of CD25+ cells for >14 days.

In vitro anergy and Treg assays

C57BL/6 recipients received alloantigen (107 BALB/c splenocytes i.p.) and anti-CD45RB (days –1, 0, 5; as above). On day 10, CD4 cells or CD25+ and CD25– CD4 subsets were isolated from spleens of treated and naive control mice by cell sorting. These CD4 cells (105) were examined for proliferation in response to 2 x 105 irradiated BALB/c splenocytes alone (anergy assay) or were examined for their ability to inhibit (105) fresh CD25– C57BL/6 responders (1:2 Treg:effector ratio) stimulated by 2 x 105 irradiated BALB/c splenocytes (Treg assay). Proliferation was assessed on day 5 by [3H]thymidine uptake.

Data analysis

Actuarial curves of graft survival were compared by using the log rank test. Other statistical analyses used the unpaired Student t test. Differences were considered to be significant at p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Redosing anti-CD45RB does not improve long-term graft acceptance

We previously demonstrated that anti-CD45RB treatment (100 µg; days –1, 0, 5) induces a shift in CD45 isoform expression such that 90–95% of CD4 cells express the smaller (CD45RBlow) isoforms (Fig. 1A and Refs.16 , 19 , and 20). This is associated with a 2-fold increase in the number of CD4 cells expressing CTLA-4 (Fig. 1A). The role of altered CD45 isoform expression in this setting is unclear. Although the isoform shift can occur in the absence of augmented CTLA-4 expression, the converse has not been demonstrated (19, 20). Moreover, the shift in CD45 isoforms is not sufficient to enhance allograft survival in the absence of enhanced CTLA-4 expression.


Figure 1
View larger version (34K):
[in this window]
[in a new window]
  		FIGURE 1. Intracellular CTLA-4 and CD45RB expression and circulating mAb after anti-CD45RB treatment. A, Representative phenotype of CD4 cells before and 7 days after anti-CD45RB treatment. The percentage of cells in each quadrant is shown. Cursor placement was based on isotype- and fluorochrome-matched controls (data not shown). B, Residual anti-CD45RB Ab in the circulation and cell surface of naive mice assessed 3 and 7 days after treatment with anti-CD45RB (100 µg i.v, days –1, 0, and 5). Serum, Undiluted serum from treated mice was incubated with splenocytes from untreated mice followed by secondary anti-rat IgG-FITC. Cells were then stained with anti-CD3-PE. Splenocytes, Isolated from treated mice were incubated with secondary anti-rat IgG-FITC followed by anti-CD3-PE. Cells were analyzed by flow cytometry. Data are representative histograms from four treated mice and were compared with cells and serum from untreated mice (n = 2; data not shown). Isotype controls were used to set the cursors (data not shown).

 
As shown previously, the changes in CD45RB and CTLA-4 expression peak around day 7 and return to baseline by day 17 (see Fig. 3A) (20). Nonetheless, this phenotypic change is of key importance, because induction of CTLA-4 expression is essential for anti-CD45RB activity (19, 20). Despite three doses, anti-CD45RB treatment mAb was no longer detectable in the serum or on the surface of splenocytes by day 7 (Fig. 1B). Thus, while graft survival can be long-lasting, both exposure to the treatment mAb and the required phenotypic changes are short-lived.


Figure 3
View larger version (34K):
[in this window]
[in a new window]
  		FIGURE 3. Time course of CD45RB and intracellular CTLA-4 expression on CD4 cells after anti-CD45RB treatment in euthymic and thymectomized (ThyX) mice. Relative expression of CD45RBlow and CTLA-4 on splenic CD4+ cells was determined at various time points by three-color immunofluorescence. Results are shown as the mean percent of positive CD4 cells (+SD) normalized to baseline expression in untreated mice. Upper panel, A, Effect of anti-CD45RB (100 µg i.v.; days –1, 0, and 5) on expression of CD45RBlow (left) and CTLA-4 (right) in euthymic mice after initial treatment (open symbols) and after redosing on day 17 (100 µg i.p.; closed symbols). Lower panel, B, Effect of anti-CD45RB (100 µg i.v.; days –1, 0, and 5) on expression of CD45RBlow (left) and CTLA-4 (right) in euthymic vs thymectomized mice (open and closed symbols, respectively). Control values were established using three to five mice per group and for experimental time points, n = 3–12 mice/group. *, p < 0.05 compared with baseline values for each marker.

 
Based on the hypothesis that maintaining this desired phenotype would promote engraftment, we asked whether retreatment of islet allograft recipients with additional doses of anti-CD45RB every 15 days would enhance the rate of long-term allograft survival beyond that achieved with only three doses of mAb. As seen in Fig. 2, untreated recipients rejected their islet allografts in the second week after transplantation. In agreement with previous results, 47% of the mice treated with a standard three-dose treatment of anti-CD45RB (days –1, 0, 5) exhibited long-term engraftment. A third set of mice received the initial doses of anti-CD45RB followed by a booster dose (100 µg i.p.) every 15 days. This regimen decreased early rejection, however, long-term graft acceptance was not improved.


Figure 2
View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 2. Retreatment with anti-CD45RB decreases acute rejection, but does not improve long-term graft survival. Kaplan-Meier plot of cumulative islet allograft survival vs time. Treatment groups include untreated controls (n = 5); anti-CD45RB-treated recipients (100 µg i.v. days –1, 0, and 5; n = 7); and recipients that received an additional booster dose of anti-CD45RB (100 µg i.p.) every 15 days (n = 7). As shown, p < 0.01 at day 30 but the difference was not significant (NS) at 100 days.

 
In attempts to explain the apparent lack of improvement in long-term engraftment, we reassessed our initial assumption that "booster" doses of anti-CD45RB would reproduce the desired phenotype. The phenotype of CD4+ splenocytes was examined at various time points after initial treatment and after retreatment with anti-CD45RB. As noted above, CD45RBlow and CTLA-4 expression return to baseline within 17 days after standard anti-CD45RB therapy (days –1, 0, 5) (Fig. 3A). Retreatment with anti-CD45RB on day 17, recapitulated increased CD45RBlow expression. In contrast, CTLA-4 expression no longer increased. These results are in general agreement with our previous findings that CTLA-4 up-regulation is essential for the tolerogenic effects of anti-CD45RB (19, 20). For example, administration of cyclosporin A at the time of anti-CD45RB treatment allows the shift in CD45 isoforms, yet prevents CTLA-4 induction and results in acute rejection of islet allografts (19). Nonetheless, the present findings do suggest that chronic administration of anti-CD45RB does have some capacity to inhibit acute rejection.

Thymectomy prolongs increased CTLA-4 expression

Most CD4 cells emerging from the thymus express higher Mr (CD45RBhigh) isoforms and lack CTLA-4 (14, 24, 25). Peripheral conversion from this "naive" phenotype toward expression of the lower (CD45RBlow) Mr CD45 isoforms normally occurs with T cell activation and is purported to be unidirectional (24, 26, 27). Although in the setting of anti-CD45RB treatment, conversion from CD45RBhigh to CD45RBlow expression occurs in the absence of CD4 cell activation (16, 19). Regardless, reappearance of CTLA-4– CD45RBhigh CD4 cells starting 7–10 days after anti-CD45RB treatment can only occur through emergence of naive CTLA-4–CD45RBhigh CD4 cells from the thymus, or through previously unrecognized phenotypic reversion in the periphery. To the extent that the former predominates, thymectomy should prolong the phenotypic changes induced by anti-CD45RB.

To test this, thymectomy was performed 7 days before initiating treatment with anti-CD45RB. As before, anti-CD45RB induced a shift toward CD45RBlow isoforms and an increase in CTLA-4 expression and this is prolonged past the 17 days seen in euthymic mice (Fig. 3B). Interestingly, despite thymectomy, expression of CD45RBlow and CTLA-4 both return to baseline, although this is significantly delayed (day 29). Thus, conversion from higher to lower Mr CD45 isoforms by peripheral murine CD4 cells is not unidirectional and CD4 cells regarded as having a "memory" (CD45RBlow) phenotype can revert back to CD45RBhigh cells in the periphery. However, the delay in re-expression of higher Mr isoforms after thymectomy indicates that the phenotypic changes induced by anti-CD45RB normally also involve emergence of naive cells from the thymus.

Thymectomy does not affect long-term engraftment by anti-CD45RB

Because thymectomy prolongs the enhanced CTLA-4 expression induced by anti-CD45RB and also prevents subsequent emergence of potentially alloreactive naive T cells long after disappearance of therapeutic mAb, we predicted that this should enhance the long-term allograft survival. Diabetic mice were subjected to thymectomy 7 days before anti-CD45RB treatment and islet transplantation (Fig. 4). Similar to euthymic controls, untreated thymectomized recipients promptly reject islet allografts. In thymectomized recipients treated with anti-CD45RB, there was a significant decrease in early rejection, with an 80% graft survival until 75 days.


Figure 4
View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 4. Thymectomy decreases acute rejection, but does not improve long-term graft survival. Kaplan-Meier plot of cumulative islet allograft survival vs time. Treatment groups include: untreated (euthymic) controls (n = 5); anti-CD45RB-treated (euthymic) recipients (100 µg i.v. days –1, 0, 5; n = 7); ThyX (thymectomized) untreated controls (n = 5), and ThyX anti-CD45RB-treated recipients (n = 10). Anti-CD45RB treated ThyX vs anti-CD45RB-treated euthymic recipients, p < 0.05 at day 30; NS at 100 days.

 
Surprisingly, despite thymectomy, late rejection occurred and long-term engraftment was not different from that seen in euthymic recipients treated with anti-CD45RB. Thus, despite prolonging the "window" of enhanced CTLA-4 expression, thymectomy did not increase long-term graft survival. Combined with previous results, this suggests that the initial increase in CTLA-4 expression induced by anti-CD45RB is necessary and already optimal for long-term allograft survival.

Exposure of CTLA-4-bearing CD4 cells to donor Ag is essential for graft survival

To directly assess the relationship between augmented CTLA-4 expression, Ag exposure, and graft survival, we treated mice with anti-CD45RB (days –1, 0, 5), and then delayed transplantation for various lengths of time (0–18 days). The disappearance of treatment mAb by day 7 allows us to dissociate the direct effects of the mAb from the requirement for increased CTLA-4 expression.

Delay in transplantation until day 18, when CTLA-4 expression has returned to baseline, fails to prolong islet allograft survival compared with untreated controls (Fig. 5). In contrast, when islet transplantation is delayed by only 7 days, CTLA-4 expression is at its peak and long-term engraftment is achieved in 33% of recipients. This approaches the long-term graft survival seen in transplants performed on day 0 (47%). Finally, when transplantation was delayed for 10 days, at which time CTLA-4 expression is starting to fall, only an occasional allograft survived long-term. However, in the setting of thymectomy, where enhanced CTLA-4 expression mediated by anti-CD45RB is prolonged, transplantation after a 10-day delay now results in survival of 40% of the islet allografts past 85 days (data not shown). Thus, transplantation during the "window" of optimal CTLA-4 expression is necessary and sufficient for prolonged allograft survival, even in the absence of anti-CD45RB treatment mAb.


Figure 5
View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 5. Long-term engraftment requires transplantation during optimal CTLA-4 expression: Kaplan-Meier plot of cumulative islet allograft survival vs time. Treatment groups include: untreated controls (n = 5) vs anti-CD45RB (100 µg i.v. on days –1, 0, and 5) followed by transplantation (Txpl) on day 0 (D0; n = 7) vs delayed transplantation (Txpl) until day 7 (D7; n = 6); day 10 (D10; n = 10); or day 18 (D18; n = 6); *, p < 0.05 day 7 and day 0 vs untreated, day 10, and day 18.

 
To substantiate this issue, we asked whether initial exposure to donor Ag would promote survival of subsequently placed allografts. C57BL/6 recipients were treated with a standard three-dose regimen of anti-CD45RB, and received either no Ag, or alloantigen in the form of 50 islets placed under the right kidney capsule. This subtherapeutic islet dose did not affect glycemia in any of the recipients (data not shown).

As noted above, delaying transplantation until day 10 in mice not receiving alloantigen resulted in long-term islet allograft survival in just 1 of 10 recipients (Fig. 6). In contrast, mice receiving donor alloantigen (BALB/c) on day 0 exhibited ~45% long-term engraftment when transplanted 10 days later with a standard dose of 400 (BALB/c) islets. This is similar to the rate of engraftment seen after anti-CD45RB treatment (days –1, 0, 5) and islet transplantation on day 0 (Fig. 4). Importantly, exposure to third-party alloantigen (DBA/1) on day 0 offered no protection to subsequently transplanted BALB/c islets. This not only rules out a nonspecific effect of transplanting a subtherapeutic dose of islets on day 0, but also indicates that exposing mAb-treated cells expressing CTLA-4 to Ag inhibits the alloresponse in a donor-specific fashion.


Figure 6
View larger version (23K):
[in this window]
[in a new window]
  		FIGURE 6. Long-term graft survival mediated by anti-CD45RB depends on exposure to donor Ag: Kaplan-Meier plot of cumulative islet allograft survival vs time. All recipients received anti-CD45RB (100 µg i.v.; days –1, 0, and 5). On day 0, recipients received no Ag (n = 10), donor Ag (BALB/c; n = 7); or unrelated Ag (DBA/1; n = 6) in the form of 50 islets. On day 10, recipients were transplanted with 400 BALB/c islets. *, p < 0.05 vs other groups.

 
Regulation and anergy are both involved in anti-CD45RB-mediated engraftment

CTLA-4 limits the response of effector cells to TCR stimulation and plays an important role in the activity of Treg. We previously showed that anti-CD45RB augments CTLA-4 expression on CD4 cells (19, 20) and this is expressed on cells that are both CD25+ and CD25– (data not shown). To begin to address the mechanisms by which anti-CD45RB-mediated CTLA-4 expression promotes tolerance, we examined the in vitro activity of CD4, CD4+CD25+, and CD4+CD25– T cells isolated from mice immunized with alloantigen and treated with anti-CD45RB compared with the activity of the same cell subsets from naive mice.

CD4 cells from treated/immunized mice are anergic, exhibiting only 20% of the response of naive CD4 cells to allogeneic stimulation (Fig. 7A). However, these same (treated/immunized) CD4 cells do not suppress the response of naive CD25– cells to allogeneic stimulation (Fig. 7B). This lack of apparent Treg activity may be due to nonregulatory cells within the CD4 population. Therefore, we divided the CD4 population into CD25+ and CD25– subsets and compared their activity. CD25+CD4 cells were highly anergic and regulatory (Fig. 7, A and B). In contrast, CD25– cells from treated/immunized mice were anergic yet not regulatory, clearly distinct from naive CD25– control cells. Thus, anti-CD45RB induces anergy in CD25–CD4 cells.


Figure 7
View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 7. Anti-CD45RB induces anergy in nonregulatory CD4 CD25– cells and requires CD25+ Treg for prolonged graft survival. A and B, CD4+, CD4+CD25+, or CD4+CD25– splenocytes were isolated from naive C57BL/6 mice vs those treated with anti-CD45RB and immunized with BALB/c splenocytes (anti-CD45RB + Allo). A, CD4 cells or subsets were cultured with BALB/c splenocytes (*, p < 0.0001 vs naive). B, CD4 cells or subsets were added as putative Treg at a 1:2 ratio to fresh CD25– C57BL/6 responders plus BALB/c splenocytes (**, p < 0.005 vs other groups). In both A and B, proliferation on day 5 was standardized relative to the proliferation of naive CD4 cells (37–41 x 103 cpm). n = 4–10 mice/group. C, Kaplan-Meier plot of cumulative islet allograft survival vs time. Diabetic C57BL/6 recipients of BALB/c islets were treated with anti-CD45RB (100 µg i.v. on days –1, 0, and 5) vs DST plus anti-CD154 (0.5 mg i.p.; days –7, –4, 0, 4) with or without CD25 depletion on day –1 or day 10 after transplantation.

 
To determine whether Treg are actually involved in prolonged allograft survival, we depleted CD25+ cells either just before or after islet transplantation (day –1 or day 10) using anti-CD25. This resulted in rejection by 11 of 12 anti-CD45RB-treated recipients (Fig. 7C). In sharp contrast, islet allograft survival after treatment with anti-CD154 and DST was unaffected by CD25 depletion (in agreement with Ref.23). At the very least, these data reveal a marked difference in the requirement for CD25+ Treg for the induction of long-term engraftment between anti-CD45RB and anti-CD154 and DST, even though both regimens ultimately give rise to Treg cells detectable after 100 days (28, 29).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
CTLA-4 signaling is fundamental to the regulation of peripheral tolerance. However, a lack of soluble agonist ligands has prevented direct study of CTLA-4 signaling in vivo and most of what is known about its role in vivo has been inferred through studies involving CTLA-4-deficient mice or the use of anti-CTLA-4-blocking mAbs. We have shown that anti-CD45RB acts through enhanced CTLA-4 expression (19, 20). This provides a novel opportunity to determine how therapeutic augmentation of CTLA-4 expression can promote tolerance in vivo. We now show that exposure to donor-specific Ag, while CTLA-4 expression is enhanced, is necessary and sufficient to induce long-term donor-specific allograft survival, even after the complete disappearance of the anti-CD45RB treatment mAb.

The present study also provides new information relevant to the regulation of CD45 isoform expression. These isoforms, generated by regulated alternative splicing, differ in the size of their extracellular domains (30). Although their role remains poorly understood, the higher and lower Mr isoforms are differentially expressed on subsets of CD4 cells with distinct functions (31, 32, 33). Moreover, T cell activation causes a shift from larger to smaller (CD45RBlow) isoforms. This led to a long-standing hypothesis that the larger (CD45RBhigh) isoforms are markers for naive cells (24, 26, 27). Underlying this hypothesis is the assertion that the shift in isoform expression is unidirectional. As a result, CD45 isoform expression has been widely used as a surrogate marker for naive vs memory cells in murine studies (25, 34, 35).

We have shown that anti-CD45RB treatment induces a shift from larger to smaller CD45 isoforms. Reappearance of CD45RBhigh cells after anti-CD45RB treatment can only come from naive T cells emigrating from the thymus, or from peripheral conversion. In thymectomized mice, peripheral CD4 cells induced to express lower Mr isoforms ultimately convert back to baseline CD45 isoform distribution. This provides direct evidence that the shift toward expression of smaller CD45 isoforms in murine CD4 cells is not unidirectional. Although the role of the CD45 isoforms remains unclear, this finding suggests that CD45 is not the most reliable marker for maturational status.

On nonregulatory T cells, CTLA-4 is induced after activation where it acts to limit the immune response and promote tolerance. Until recently, there has been no therapeutic means to use this potent inhibitory pathway to promote tolerance and prolong allograft survival in vivo. Anti-CD45RB enhances CTLA-4 expression without overt T cell activation (16, 19). Although CTLA-4 induction plays an essential role in anti-CD45RB activity, it is relatively short-lived. Our data now reveal that potentially alloreactive T cells must be exposed to donor Ag during this window of enhanced CTLA-4 expression. This is in general agreement with our understanding of CTLA-4 function in vitro. Here, CTLA-4 expression is induced in resting T cells by strong stimulation through the TCR and CD28 (3, 6, 7). Such cells are then susceptible to inhibition through mAb-mediated CTLA-4 cross-linking (7, 8, 36). Moreover, coligation of CTLA-4 and the TCR by the same cell or particle (i.e., ligation in "cis-") is most effective at inducing negative signaling (36, 37). In this regard, after CTLA-4 expression is induced on potentially alloreactive CD4 cells by anti-CD45RB, Ag presentation in the transplant setting would be expected to inhibit responsiveness only of those cells that are specifically alloreactive. Indeed, this is in agreement with our findings. Rejection of allografts placed after a 10-day delay was only prevented if CD4 cells expressing CTLA-4 were previously exposed to donor-specific Ag (Fig. 6). Exposure to unrelated alloantigen during this same time frame was unable to prevent allograft rejection. In this case, transplantation on day 10 exposes CD4 cells to a new Ag at a time when CTLA-4 expression is waning. This is likely to stimulate different clones of T cells from those inactivated by unrelated Ag on day 0. Thus, CTLA-4 directs inhibition of nonregulatory T cells in vivo.

The exact consequences of CTLA-4 signaling remain uncertain. In vitro studies indicate that CTLA-4 cross-linking may inhibit activation by interfering with lipid raft formation and/or by sequestering the TCR and other signaling components outside of the rafts (36, 38). This inhibits cell cycle progression and proliferation but has no effect on subsequent activation (36, 39, 40). In fact, anergy can be induced in vitro in the complete absence of CTLA-4 (41).

In contrast to the situation in vitro, CTLA-4 may promote anergy in vivo in some settings (e.g., after i.v. Ag) (2, 42). However, this finding is not generalized. For example, when Ag was expressed as an "endogenous" tissue Ag in islets (a setting more relevant to transplantation), an intact CTLA-4 locus prevented diabetes by reducing the accumulation of "autoreactive" TCR-transgenic CD4 cells, but did not induce anergy in terms of proliferative response ex vivo (43). Transgenic overexpression of CTLA-4 appears to inhibit autoimmunity in IL-2-deficient mice by limiting effector cell responsiveness (44). Similarly, transplantation in the face of CTLA-4 blockade suggests that CTLA-4 normally limits activation of alloreactive CD4 and CD8 cells, thereby decreasing their frequency and differentiation (12). Our studies indicate that anti-CD45RB treatment does induce anergy in a nonregulatory (CD25–) effector CD4 population that is normally alloreactive. Notably, decreased responsiveness occurs in the absence of CD4 deletion (16, 19), after the disappearance of treatment mAb from the cell surface and circulation, and in the presence of increased CTLA-4 expression.

CTLA-4 is also expressed by both natural and induced Treg and may play an important role in their suppressive function (13, 14, 15). Augmented expression of CTLA-4 may suggest that anti-CD45RB actually induces Treg. In this regard, CD25+ but not CD25– cells from anti-CD45RB-treated mice exhibit Treg activity. Importantly, anti-CD45RB-mediated engraftment is highly dependent on the presence of CD25+ cells. Whether this indicates a requirement for induced Treg vs a requirement for natural Treg is not yet known and is the subject of current investigation. We speculate that CTLA-4+ Treg induced by anti-CD45RB contribute to induction of tolerance and that Ag exposure during the window of enhanced CTLA-4 expression is required for the differentiation and/or proliferation of clones bearing the correct Ag specificities (45).

Our studies indicate that enhanced CTLA-4 expression during transplantation is necessary and sufficient for prolonged allograft survival. However, the time frame of enhanced CTLA-4 expression after anti-CD45RB is limited and Ag (transplantation) must be introduced within the first 7 days to ensure that effector or regulatory cells are adequately exposed. Apparently, once CTLA-4 expression is lost, these cells revert back to their basal state and Ag exposure after this time results in full-blown immune responsiveness.

Thymectomy significantly prolongs the window of enhanced CTLA-4 expression, yet does not enhance long-term graft survival. These results, combined with the inability of anti-CD45RB redosing to enhance the rate of long-term engraftment, suggest that CTLA-4 exposure is already optimal and further improvements will not come from altered anti-CD45RB dosing, but will require the addition of other agents. In this regard, we have shown that anti-CD154 is synergistic with anti-CD45RB in islet, skin, and cardiac allograft survival (12, 46, 47).

The results obtained in thymectomized recipients are also important in that they reveal that preventing emergence of potentially alloreactive T cells from the thymus has no beneficial effect on long-term outcome. Because the treatment mAb and the phenotypic changes induced are short-lived in vivo, this finding suggests that Treg observed in long-term allograft survivors (29) must maintain tolerance, and moreover, are sufficiently potent to overcome any future alloreactivity. As a corollary, graft loss after anti-CD45RB treatment does not appear to result from a failure of Treg activity (in which case thymectomy would provide at least some advantage in long-term survival). These studies add to our understanding of the capacity of Treg in the maintenance of allograft tolerance (29, 48, 49).

In summary, the unique ability of anti-CD45RB to induce CTLA-4 expression on CD4 cells allows us to further define the role of augmented CTLA-4 expression in tolerance induction which appears to be mediated through both Treg and effector cell anergy. Specifically, we show that exposure to donor Ag while CTLA-4 expression is enhanced is essential for long-term engraftment, which occurs even in the absence of residual anti-CD45RB. Exposure to third-party Ag is not protective. These findings are consistent with the known requirement for TCR signaling for inhibition of T cell activation by CTLA-4, and the ability of Ag to induce proliferation of Treg in vivo, thereby shaping the Treg repertoire in an Ag-specific fashion (14, 45, 50). In addition to a critical role in induction of tolerance by anti-CD45RB, Treg must maintain tolerance in the face of new thymic emigrants that see neither anti-CD45RB nor altered phenotype. These studies provide new insight into the role of CTLA-4 and raise the prospect that agents useful in promoting clinical tolerance in transplantation and autoimmunity may ultimately capitalize on this potent endogenous inhibitory signal.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by the National Institutes of Health Grant AI45485 (to D.M.R.). Back

2 P.R.O.S. and G.C. contributed equally to this work. Back

3 G.P.B. and D.M.R. contributed equally to this work. Back

4 Address correspondence and reprint requests to Dr. David M. Rothstein, Department of Internal Medicine, Yale Medical School, P.O. Box 208029, New Haven, CT 06520. E-mail address: david.rothstein{at}yale.edu Back

5 Abbreviations used in this paper: Treg, regulatory T cell; DST, donor-specific transfusion. Back

Received for publication May 26, 2005. Accepted for publication November 25, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Sayegh, M. H., L. A. Turka. 1998. The role of T-cell costimulatory activation pathways in transplant rejection. N. Engl. J. Med. 338: 1813-1821. [Free Full Text]
  2. Perez, V. L., L. Van Parijs, A. Buckians, X. X. Zheng, T. B. Strom, A. K. Abbas. 1997. Induction of peripheral T cell tolerance in vivo requires CTLA-4 engagement. Immunity 6: 411-417. [Medline]
  3. Chambers, C. A., M. S. Kuhns, J. G. Egen, J. P. Allison. 2001. CTLA-4-mediated inhibition in regulation of T cell responses: mechanisms and manipulation in tumor immunotherapy. Annu. Rev. Immunol. 19: 565-594. [Medline]
  4. Tivol, E., F. Borriello, A. Schweitzer, W. Lynch, J. Bluestone, A. Sharpe. 1995. Loss of CTLA-4 leads to massive lymphoproliferation and fatal multiorgan tissue destruction, revealing a critical negative regulatory role of CTLA-4. Immunity 3: 541-547. [Medline]
  5. Waterhouse, P., J. M. Penninger, E. Timms, A. Wakeham, A. Shahinian, K. P. Lee, C. B. Thompson, H. Griesser, T. W. Mak. 1995. Lymphoproliferative disorders with early lethality in mice deficient in Ctla-4. Science 270: 985-988. [Abstract/Free Full Text]
  6. Alegre, M. L., P. J. Noel, B. J. Eisfelder, E. Chuang, M. R. Clark, S. L. Reiner, C. B. Thompson. 1996. Regulation of surface and intracellular expression of CTLA4 on mouse T cells. J. Immunol. 157: 4762-4770. [Abstract]
  7. Walunas, T. L., D. J. Lenschow, C. Y. Bakker, P. S. Linsley, G. J. Freeman, J. M. Green, C. B. Thompson, J. A. Bluestone. 1994. CTLA-4 can function as a negative regulator of T cell activation. Immunity 1: 405-413. [Medline]
  8. Krummel, M. F., J. P. Allison. 1995. CD28 and CTLA-4 have opposing effects on the response of T cells to stimulation. J. Exp. Med. 182: 459-465. [Abstract/Free Full Text]
  9. Karandikar, N. J., C. L. Vanderlugt, T. L. Walunas, S. D. Miller, J. A. Bluestone. 1996. CTLA-4: a negative regulator of autoimmune disease. J. Exp. Med. 184: 783-788. [Abstract/Free Full Text]
  10. Judge, T. A., Z. Wu, X. G. Zheng, A. H. Sharpe, M. H. Sayegh, L. A. Turka. 1999. The role of CD80, CD86, and CTLA4 in alloimmune responses and the induction of long-term allograft survival. J. Immunol. 162: 1947-1951. [Abstract/Free Full Text]
  11. Zheng, X. X., T. G. Markees, W. W. Hancock, Y. Li, D. L. Greiner, X. C. Li, J. P. Mordes, M. H. Sayegh, A. A. Rossini, T. B. Strom. 1999. CTLA4 signals are required to optimally induce allograft tolerance with combined donor-specific transfusion and anti-CD154 monoclonal antibody treatment. J. Immunol. 162: 4983-4990. [Abstract/Free Full Text]
  12. Sho, M., A. Yamada, N. Najafian, A. D. Salama, H. Harada, S. E. Sandner, A. Sanchez-Fueyo, X. X. Zheng, T. B. Strom, M. H. Sayegh. 2002. Physiological mechanisms of regulating alloimmunity: cytokines, CTLA-4, CD25+ cells, and the alloreactive T cell clone size. J. Immunol. 169: 3744-3751. [Abstract/Free Full Text]
  13. Takahashi, T., T. Tagami, S. Yamazaki, T. Uede, J. Shimizu, N. Sakaguchi, T. W. Mak, S. Sakaguchi. 2000. Immunologic self-tolerance maintained by CD25+CD4+ regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen 4. J. Exp. Med. 192: 303-310. [Abstract/Free Full Text]
  14. Sakaguchi, S.. 2004. Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses. Annu. Rev. Immunol. 22: 531-562. [Medline]
  15. von Boehmer, H.. 2005. Mechanisms of suppression by suppressor T cells. Nat. Immunol. 6: 338-344. [Medline]
  16. Basadonna, G., L. Auersvald, C. Khuong, X. Zheng, N. Kashio, D. Zekzer, M. Minozza, H.-Y. Qian, L. Visser, A. Diepstra, et al 1998. Antibody mediated targeting of CD45 isoforms: a novel immunotherapeutic strategy. Proc. Nat. Acad. Sci. USA 95: 3821-3826. [Abstract/Free Full Text]
  17. Lazarovits, A., S. Poppema, Z. Zhang, K. Khandaker, C. LeFeuvre, S. Singhal, B. Garcia, A. Jevnikar, M. White, G. Singh, et al 1996. Prevention and reversal of renal allograft rejection by antibody against CD45RB. Nature 380: 717-720. [Medline]
  18. Kishimoto, K., S. Sandner, J. Imitola, M. Sho, Y. Li, P. B. Langmuir, D. M. Rothstein, T. B. Strom, L. A. Turka, M. H. Sayegh. 2002. Th1 cytokines, programmed cell death, and alloreactive T cell clone size in transplant tolerance. J. Clin. Invest. 109: 1471-1479. [Medline]
  19. Fecteau, S., G. Basadonna, A. Freitas, C. Ariyan, M. Sayegh, D. Rothstein. 2001. Upregulation of CTLA-4 expression mediates tolerance induced through CD45. Nat. Immunol. 2: 58-63. [Medline]
  20. Ariyan, C., F. S., P. Salvalaggio, D. Mandelbrot, A. Sharpe, M. Sayegh, G. Basadonna, and D. Rothstein. 2003. Cutting edge: transplantation tolerance through enhanced CTLA-4 expression. J. Immunol. 171: 5673–5677.
  21. Salvalaggio, P. R., S. Deng, C. E. Ariyan, I. Millet, W. S. Zawalich, G. P. Basadonna, D. M. Rothstein. 2002. Islet filtration: a simple and rapid new purification procedure that avoids Ficoll and improves islet mass and function. Transplantation 74: 877-879. [Medline]
  22. Sjodin, K., A. P. Dalmasso, J. M. Smith, C. Martinez. 1963. Thymectomy in newborn and adult mice. Transplantation 29: 521-525.
  23. Banuelos, S. J., T. G. Markees, N. E. Phillips, M. C. Appel, A. Cuthbert, J. Leif, J. P. Mordes, L. D. Shultz, A. A. Rossini, D. L. Greiner. 2004. Regulation of skin and islet allograft survival in mice treated with costimulation blockade is mediated by different CD4+ cell subsets and different mechanisms. Transplantation 78: 660-667. [Medline]
  24. Tedder, T. F., L. T. Clement, M. D. Cooper. 1985. Human lymphocyte differentiation antigens HB-10 and HB-11. I. Ontogeny of antigen expression. J. Immunol. 134: 2983-2988. [Abstract]
  25. Metz, D. P., D. L. Farber, T. Taylor, K. Bottomly. 1998. Differential role of CTLA-4 in regulation of resting memory versus naive CD4 T cell activation. J. Immunol. 161: 5855-5861. [Abstract/Free Full Text]
  26. Akbar, A. N., L. Terry, A. Timms, P. C. L. Beverley, G. Jannosy. 1988. Loss of CD45R and gain of UCHL1 reactivity is a feature of primed T cells. J. Immunol. 140: 2171-2178. [Abstract]
  27. Lee, W. T., E. S. Vitetta. 1990. Limiting dilution analysis of CD45Rhi and CD45Rlo T cells: further evidence that CD45Rlo cells are memory cells. Cell. Immunol. 130: 459-471. [Medline]
  28. Graca, L., K. Honey, E. Adams, S. P. Cobbold, H. Waldmann. 2000. Cutting edge: anti-CD154 therapeutic antibodies induce infectious transplantation tolerance. J. Immunol. 165: 4783-4786. [Abstract/Free Full Text]
  29. Gao, Z., R. Zhong, J. Jiang, B. Garcia, J. J. Xing, M. J. White, A. I. Lazarovits. 1999. Adoptively transferable tolerance induced by CD45RB monoclonal antibody. J. Am. Soc. Nephrol. 10: 374-381. [Abstract/Free Full Text]
  30. Hermiston, M. L., Z. Xu, A. Weiss. 2003. CD45: a critical regulator of signaling thresholds in immune cells. Annu. Rev. Immunol. 21: 107-137. [Medline]
  31. Lee, W., X.-M. Yin, E. Vitetta. 1990. Functional and ontogenetic analysis of murine CD45hi and CD45lo CD4+ T cells. J. Immunol. 144: 3288-3295. [Abstract]
  32. Bottomly, K., M. Luqman, L. Greenbaum, S. Carding, J. West, T. Pasqualini, D. Murphy. 1989. A monoclonal antibody to murine CD45R distinguishes CD4 T cell populations that produce different cytokines. Eur. J. Immunol. 19: 617-623. [Medline]
  33. Morimoto, C., N. L. Letvin, A. W. Boyd, M. Hagen, H. Brown, M. M. Kornacki, S. F. Schlossman. 1985. The isolation and characterization of the human helper inducer subset. J. Immunol. 134: 3762-3769. [Abstract]
  34. Watson, A. R., W. T. Lee. 2004. Differences in signaling molecule organization between naive and memory CD4+ T lymphocytes. J. Immunol. 173: 33-41. [Abstract/Free Full Text]
  35. Ahmadzadeh, M., S. F. Hussain, D. L. Farber. 2001. Heterogeneity of the memory CD4 T cell response: persisting effectors and resting memory T cells. J. Immunol. 166: 926-935. [Abstract/Free Full Text]
  36. Martin, M., H. Schneider, A. Azouz, C. E. Rudd. 2001. Cytotoxic T lymphocyte antigen 4 and CD28 modulate cell surface raft expression in their regulation of T cell function. J. Exp. Med. 194: 1675-1681. [Abstract/Free Full Text]
  37. Hwang, K. W., W. B. Sweatt, I. E. Brown, C. Blank, T. F. Gajewski, J. A. Bluestone, M. L. Alegre. 2002. Cutting edge: targeted ligation of CTLA-4 in vivo by membrane-bound anti-CTLA-4 antibody prevents rejection of allogeneic cells. J. Immunol. 169: 633-637. [Abstract/Free Full Text]
  38. Chikuma, S., J. B. Imboden, J. A. Bluestone. 2003. Negative regulation of T cell receptor-lipid raft interaction by cytotoxic T lymphocyte-associated antigen 4. J. Exp. Med. 197: 129-135. [Abstract/Free Full Text]
  39. Greenwald, R. J., M. A. Oosterwegel, D. van der Woude, A. Kubal, D. A. Mandelbrot, V. A. Boussiotis, A. H. Sharpe. 2002. CTLA-4 regulates cell cycle progression during a primary immune response. Eur. J. Immunol. 32: 366-373. [Medline]
  40. Krummel, M. F., J. P. Allison. 1996. CTLA-4 engagement inhibits IL-2 accumulation and cell cycle progression upon activation of resting T cells. J. Exp. Med. 183: 2533-2540. [Abstract/Free Full Text]
  41. Frauwirth, K. A., M. L. Alegre, C. B. Thompson. 2000. Induction of T cell anergy in the absence of CTLA-4/B7 interaction. J. Immunol. 164: 2987-2993. [Abstract/Free Full Text]
  42. Greenwald, R. J., V. A. Boussiotis, R. B. Lorsbach, A. K. Abbas, A. H. Sharpe. 2001. CTLA-4 regulates induction of anergy in vivo. Immunity 14: 145-155. [Medline]
  43. Walker, L. S., L. J. Ausubel, A. Chodos, N. Bekarian, A. K. Abbas. 2002. CTLA-4 differentially regulates T cell responses to endogenous tissue protein versus exogenous immunogen. J. Immunol. 169: 6202-6209. [Abstract/Free Full Text]
  44. Hwang, K. W., W. B. Sweatt, M. Mashayekhi, D. A. Palucki, H. Sattar, E. Chuang, M. L. Alegre. 2004. Transgenic expression of CTLA-4 controls lymphoproliferation in IL-2-deficient mice. J. Immunol. 173: 5415-5424. [Abstract/Free Full Text]
  45. Nishimura, E., T. Sakihama, R. Setoguchi, K. Tanaka, S. Sakaguchi. 2004. Induction of antigen-specific immunologic tolerance by in vivo and in vitro antigen-specific expansion of naturally arising Foxp3+CD25+CD4+ regulatory T cells. Int. Immunol. 16: 1189-1201. [Abstract/Free Full Text]
  46. Rothstein, D., M. Minozza, K. Kishimoto, C. Ariyan, X. Zheng, H.-Y. Qian, S. Fecteaus, M. Sho, M. Sayegh, G. Bassadonna. 2001. Targeting signal one through CD45RB synergizes with CD40L blockade and promotes long term engraftment and tolerance in stringent transplant models. J. Immunol. 166: 322-329. [Abstract/Free Full Text]
  47. Sho, M., H. Harada, D. Rothstein, M. H. Sayegh. 2003. CD45RB-targeting strategy for preventing chronic vasculopathy and establishing peripheral tolerance. Transplantation 75: 1142-1146. [Medline]
  48. Graca, L., A. Le Moine, S. P. Cobbold, H. Waldmann. 2003. Dominant transplantation tolerance. Opinion. Curr. Opin. Immunol. 15: 499-506. [Medline]
  49. Wood, K. J., S. Sakaguchi. 2003. Regulatory T cells in transplantation tolerance. Nat. Rev. Immunol. 3: 199-210. [Medline]
  50. Walker, L. S., A. Chodos, M. Eggena, H. Dooms, A. K. Abbas. 2003. Antigen-dependent proliferation of CD4+ CD25+ regulatory T cells in vivo. J. Exp. Med. 198: 249-258. [Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
K. Y. Jen, M. Campo, H. He, S. S. Makani, G. Velasco, D. M. Rothstein, D. L. Perkins, and P. W. Finn
CD45RB Ligation Inhibits Allergic Pulmonary Inflammation by Inducing CTLA4 Transcription
J. Immunol., September 15, 2007; 179(6): 4212 - 4218.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. Deng, D. J. Moore, X. Huang, M.-M. Lian, M. Mohiuddin, E. Velededeoglu, M. K. Lee IV, S. Sonawane, J. Kim, J. Wang, et al.
Cutting Edge: Transplant Tolerance Induced by Anti-CD45RB Requires B Lymphocytes
J. Immunol., May 15, 2007; 178(10): 6028 - 6032.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
J. Bodor, Z. Fehervari, B. Diamond, and S. Sakaguchi
Regulatory T cell-mediated suppression: potential role of ICER
J. Leukoc. Biol., January 1, 2007; 81(1): 161 - 167.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Salvalaggio, P. R. O.
Right arrow Articles by Rothstein, D. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Salvalaggio, P. R. O.
Right arrow Articles by Rothstein, D. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS