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The Journal of Immunology, 2006, 176: 2799-2807.
Copyright © 2006 by The American Association of Immunologists

Antibody-Induced Transplantation Tolerance That Is Dependent on Thymus-Derived Regulatory T Cells1

Shaoping Deng*, Daniel J. Moore{dagger}, Xiaolun Huang*, Mohammad Mohiuddin*, Major K. Lee, IV*, Ergun Velidedeoglu*, Moh-Moh Lian*, Meredith Chiaccio*, Samsher Sonawane*, Anton Orlin*, Jing Wang*, Haiying Chen*, Andrew Caton{ddagger}, Robert Zhong§ and James F. Markmann2,*

* Harrison Department of Surgical Research, Department of Surgery, Hospital of the University of Pennsylvania, Philadelphia, PA 19104; {dagger} Department of Pediatrics, Vanderbilt Children’s Hospital, Nashville, TN 37232; {ddagger} The Wistar Institute, Philadelphia, PA; and § Department of Surgery, University of Western Ontario, London, Ontario, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Targeting of the CD45RB isoform by mAb (anti-CD45RB) effectively induces donor-specific tolerance to allografts. The immunological mechanisms underlying the tolerant state remain unclear although some studies have suggested the involvement of regulatory T cells (T-regs). Although their generative pathway remains undefined, tolerance promoting T-regs induced by systemic anti-CD45RB treatment have been assumed to originate in the peripheral immune system. We demonstrate herein that separable effects on the peripheral and central immune compartments mediate graft survival induced by anti-CD45RB administration. In the absence of the thymus, anti-CD45RB therapy is not tolerogenic though it retains peripheral immunosuppressive activity. The thymus is required for anti-CD45RB to produce indefinite graft survival and donor-specific tolerance, and this effect is accomplished through thymic production of donor-specific T-regs. These data reveal for the first time an Ab-based tolerance regimen that relies on the central tolerance pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Since the description of classical immunological tolerance by Billingham, Brent, and Medawar (1) in the 1950s, induction of donor-specific tolerance has been held as the paramount goal of transplant biologists. Avoidance of maintenance immunosuppression by tolerance induction could eliminate much of the morbidity associated with transplantation and would thereby expand the indications for transplantation in situations where the benefit to risk ratio is currently unfavorable. Despite frequent success in experimental animal transplant models, tolerance induction in the clinical setting has remained elusive and chronic immunosuppression remains necessary to sustain survival in virtually all cases of solid organ allotransplantation.

mAb disruption of the immunological synapse has shown promise as a tolerance-inducing strategy. Ab targeting of a variety of T cell surface proteins or their ligands such as costimulatory molecules, TCR-associated proteins (CD3), coreceptor molecules (CD4, CD8, CD2), adhesion molecules, and cell surface proteins associated with T cell activation have resulted in transplantation tolerance in experimental systems (2, 3, 4, 5). In these cases, the tolerant state has logically been assumed to result through the agent’s effect on the peripheral immune system (6, 7, 8, 9, 10). This basic characteristic may distinguish these therapies from classical immunological tolerance that recapitulates both central and peripheral pathways of self-tolerance. As the thymus typically inactivates or deletes >95% of candidate T cells, it is likely that peripheral tolerance mechanisms are more suited to fine tuning of the repertoire rather than to global restructuring. Thus, the putatively peripherally based mechanism of Ab therapy in general might predict that it would not be as effective in eliminating or inactivating donor-reactive specificities as a centrally based mechanism. This in fact seemed likely, especially considering the high frequency with which alloreactive specificities are generated.

Importantly, most Ab-mediated tolerance-inducing regimens have not been evaluated from the perspective of their effect on the central vs peripheral immune compartments. A relevant and isolated exception is the demonstration that tolerance induced in a rat cardiac allotransplant setting by anti-CD4 therapy requires an intact host thymus (11). The thymic requirement in this model was later shown to be exerted by an effect on recent thymic emigrants (RTE) and not through a direct intercession of anti-CD4 on thymopoiesis of donor-specific clones (11).

In the current work, we provide evidence that treatment with mAb against the CD45RB molecule induces tolerance through pathways that are thymus-dependent. The CD45RB molecule, a restricted isoform of CD45, is expressed on cells of the B and T lymphocyte lineages and has been used to identify specific T cell subsets including putative regulatory T cells (T-regs)3 which express the low m.w. isoform (12, 13). In addition to its use as a molecular marker, the CD45 molecule has also been reported to modulate TCR sensitivity; T cells deficient in this molecule exhibit a markedly diminished capacity to respond to antigenic stimulation (14, 15). Certain Abs against CD45RB have the ability to induce permanent tolerance to allografts, an effect that involves a switch in CD45 isoform expression, apoptosis of CD45RB-expressing cells and perhaps up-regulation of CTLA-4 (16, 17, 18, 19, 20). Although there has been significant success with tolerance induction following a short course of anti-CD45RB, most reports indicate that this therapeutic regimen is not successful in all recipient animals; in fact, in most studies, only 50–75% of treated animals demonstrate a robust state of transplantation tolerance even when all animals experience extended graft survival (18, 19, 20). We therefore considered whether the thymus played a role in limiting the ability of anti-CD45RB and perhaps other peripherally targeted therapies to result in tolerance induction by the continued production of nascent graft-reactive cells. Thus, we investigated whether thymus extirpation would augment tolerance induction as might be expected for a purely peripherally acting agent. We unexpectedly discovered instead that there is a requisite thymic contribution to CD45RB-induced transplantation tolerance as thymectomy before transplantation completely prevents tolerance induction by anti-CD45RB therapy. Furthermore, the role of the thymus appears to be the generation of donor-specific T-regs that secure graft acceptance in the periphery that is concomitantly suppressed by a peripheral action of anti-CD45RB.


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

Wild-type C57/B6 (B6, H2b), B6-SCID, C3H (H2c), and BALB/c (H2d) mice were purchased from The Jackson Laboratory. TCR-transgenic (tg) mice (TS1) were used as recipients of cardiac grafts from hemagglutinin (HA)-tg donor mice (HA104) as previously described by our laboratory (21). TS1 mice express a tg TCR that is specific for the site 1 peptide (S1) of viral HA in the context of MHC class II IEd molecules. Approximately 30% of peripheral CD4 T cells express high levels of the HA-specific TCR, and this receptor can be detected with a clonotypic Ab. The HA104 line has diffuse tissue expression of the HA transgene due to its regulation by the SV40 promoter. This mouse serves as an ideal graft donor for experiments examining the response to HA+ grafts by HA-specific (6.5+) TS1 T cells. Importantly, each line has been extensively backcrossed to the BALB/c background making transplant experiments possible without concern of confounding influences of residual minor antigenic disparities. All mice were housed under specific pathogen-free barrier conditions at the University of Pennsylvania.

Heart transplantation

Experiments were performed according to a protocol approved by the Institutional Animal Care and Use Committee at the University of Pennsylvania. Transplantation was performed according to the Ono-Lindsey model as adapted for mice (22). Recipient mice were anesthetized using i.p. injection of ketamine (50 mg/kg) and xylazine (10 mg/kg) and a midline abdominal incision was made in the donor mouse which was then heparinized through the inferior vena cava (50 U). The incision was extended cephalad to open the chest through a median sternotomy. The heart was rapidly harvested after arrest with potassium cardioplegia solution administered via the inferior vena cava (1 ml, 20 mEq/L), and the coronary arteries were flushed (0.5 ml of preservation solution) and placed into lactated Ringer’s solution for 30–60 min at 4°C. The recipient’s abdominal aorta and inferior vena cava was exposed and transplantation was achieved by anastomosis, end to side, of the donor aorta and pulmonary artery to the recipient’s abdominal aorta and inferior vena cava, respectively, using 10-0 nylon suture. To test tolerance, a second heart graft either from the same C3H or BALB/c strain was transplanted in the neck of B6 mice bearing long-term functioning cardiac allograft without any additional treatment. Graft function was monitored by daily palpation, and grafts were removed for histological analysis at the time of rejection, defined as complete cessation of contractility.

Anti-CD45RB therapy

Animals were treated with i.p. injection of 100 µg of rat anti-mouse CD45RB Ab (clone: MB23G2; American Type Culture Collection) on days 0, 1, 3, 5, and 7 following transplantation. Control animals were left untreated.

Adoptive transfer model

To determine whether T-regs play a role in anti-CD45RB-induced tolerance, we used an adoptive transfer model in which C3H hearts were transplanted into immunodeficient (Scid) B6 mice followed by injection of naive B6 lymphocytes (106 splenocytes) alone or a mixture of naive B6 splenocytes and tolerant splenocytes or thymocytes isolated from the spleen or the thymus of B6 mice bearing long-term functioning C3H heart. In some adoptive transfer experiments, CD4+CD25+ T cells from the splenocytes or thymocytes of tolerant mice were sorted by FACS using positive selection method as described in our previous studies (23, 24).

Flow cytometry

One million cells were suspended in biotin-free RPMI 1640 containing 0.1% azide and 3% FCS and surface-stained in 96-well plates with the following anti-mouse Abs: anti-CD4-PE, anti-CD4-allophycocyanin, anti-CD8-FITC, anti-CD8-PE, anti-CD25-FITC, anti-CD25-PE, anti-CD45RB-PE, and anti-CD62L-PE (BD Pharmingen). In addition, mAb (6.5)-biotin (21) were used to detect the tg TCR of TS1 T cells. Biotin-conjugated mAbs were subsequently stained with streptavidin-RED670 (Invitrogen Life Technologies); cells were washed no fewer than two times before the addition of the secondary reagent. Anti-rat IgG Ab conjugated with PE (Southern Biotechnology Associates) was used as secondary Ab. All samples were analyzed on a FACSCalibur flow cytometer (BD Biosciences) using CellQuest software.

Statistical analysis

All data are presented as mean ± SD. Statistical analysis was done by a Student’s t test or ANOVA using n-1 custom hypotheses tests as appropriate. Significance was defined as a p value <0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Donor-specific tolerance induced by anti-CD45RB therapy

Our experience with anti-CD45RB is similar to previous reports using islet or kidney grafts with respect to the efficiency of tolerance induction (16, 17, 18, 19, 20). In our model, vascularized cardiac grafts from C3H (H-2k) mice were transplanted to the abdominal cavity of C57BL/6 (B6, H-2b) mice. After transplantation, recipient mice were treated with a short course of anti-CD45RB Ab (100 µg i.p., days 0, 1, 3, 5, and 7). In untreated control mice (n = 9) and mice (n = 6) treated with control Ab, cardiac allografts were rejected acutely with a mean survival time (MST) of 8.3 ± 1.9 and 15.2 ± 2.0 days, respectively; whereas in mice treated with anti-CD45RB, the majority (11 of 16) of grafts survived indefinitely (>100 days, p < 0.001) with a MST >106 ± 47 days (Fig. 1).


Figure 1
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  		FIGURE 1. Survival of cardiac allografts in normal C57BL/6 (B6) mice. Hearts from C3H mice were transplanted into the abdominal cavity of normal B6 mice treated with or without anti-CD45RB Abs (100 µg, i.p. on days 0, 1, 3, 5, 7). All grafts (n = 9) were rapidly rejected in untreated and control Ab-treated mice, but the majority (11 of 16) of grafts survived permanently (>100 days, p < 0.001) in mice that received anti-CD45RB therapy.

 
To confirm that donor-specific tolerance was induced in the B6 recipient mice bearing long-term functioning C3H cardiac allografts, a second C3H cardiac allograft was transplanted to the neck of these tolerant mice without additional therapy. The antigenic specificity of the tolerant state was verified with control grafts in other putatively tolerant hosts from third-party BALB/c donors (H-2d). All hearts from the BALB/c mice were rejected rapidly (MST = 7.5 days, n = 4) while second hearts from the C3H strain survived indefinitely (n = 3) without any evidence of rejection (data not shown). These data demonstrated that donor-specific tolerance was established in recipient mice treated with anti-CD45RB Ab.

Anti-CD45RB-induced tolerance requires an intact thymus

Although the majority of treated mice exhibited indefinite graft survival, a subset (5 of 16) ultimately rejected their grafts and did not become tolerant (MST = 62 ± 25.9 days). One plausible explanation for this inconsistency is that the host’s thymus continues to generate nontolerant donor-reactive clones that mediate late graft rejections. To address this possibility, we evaluated whether pretransplant thymectomy improved the rate of tolerance induction following anti-CD45RB therapy. Unexpectedly, we found that prior recipient thymectomy (2–4 wk pretransplant) not only did not improve the effectiveness of therapy but instead completely abrogated its tolerogenic effects (Fig. 2A). In six thymectomized C57BL/6 mice, anti-CD45RB treatment prolonged survival to an average of 55.7 ± 22.3 days (vs 9.3 ± 1.3 days in the nontreated group (n = 4); however, all mice ultimately rejected their grafts documenting an absence of tolerance to donor Ags. These results suggest that anti-CD45RB administration exerts two distinct and separable effects: a direct suppressive effect on the peripheral immune cells that in itself is not tolerogenic, and a thymus-dependent effect that is required for transplantation tolerance to develop. The requirement for the thymus suggested a number of mechanistic possibilities for the action of anti-CD45RB and, in particular, the generation of T-regs which has been associated with this therapy. We first verified that thymic selection was required for tolerance and not merely that the tolerant state results by action on RTE as seen with anti-CD4-mediated tolerance which would be eliminated by this thymectomy protocol (11).


Figure 2
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  		FIGURE 2. Survival of cardiac allografts in thymectomized B6 mice. Hearts from C3H mice were transplanted into the abdominal cavity of mice thymectomized 2–4 wk before cardiac transplantation and treated with or without anti-CD45RB Abs. Although the majority of allografts were accepted permanently in normal euthymic B6 mice (n = 16) treated with anti-CD45RB (Fig. 1), no grafts survived permanently in the thymectomized mice whether they were treated with anti-CD45RB or not (A). However, graft survival was significantly prolonged in treated thymectomized mice (MST = 55.7 ± 22.3 days, n = 6) as compared with untreated, thymectomized mice (MST = 9.3 ± 1.3 days, n = 4, p < 0.01). To discern the role of RTEs, hearts from C3H mice were transplanted into the abdominal cavity of B6 mice that were then thymectomized at either 1 or 4 wk posttransplant. Thymectomy (not sham operation) at 1 wk (n = 8) abrogated the induction of tolerance while the same treatment at 4 (n = 6) wk permitted indefinite graft survival in four of six mice (B).

 
The window for CD45RB-induced thymic tolerance

If the thymus-dependent nature of tolerance was related to the availability of thymic emigrants, then thymectomy after the administration of therapy should not interfere with tolerance induction, as the pool of RTEs would be maintained. To ascertain whether there was a point after which the thymus was no longer required for tolerance to develop, anti-CD45RB-treated B6 recipients of allogeneic C3H heart grafts were thymectomized at 1 or 4 wk posttransplant (Fig. 2B). When the thymus was removed at 1 wk, tolerance was not observed though graft survival (MST = 44.3 ± 3.5, n = 8) was prolonged similar to when thymectomy was performed pretransplant. In striking contrast, thymectomy at 4 wk posttransplant did not interfere with the development of tolerance (four of six) indicating that the role of the thymus in tolerogenesis was executed during the window between 1 and 4 wk posttransplant. Sham operation did not have any impact on long-term survival and tolerance induction in mice (n = 6) treated with anti-CD45RB (Fig. 2B). These data suggest that the action of anti-CD45RB on RTEs is insufficient to account for generation of the tolerant state and that the tolerogenic effect we observed is dependent on the direct actions of the thymus on either nascent or recirculating lymphocytes.

The thymic effect is not mediated by action on recirculating cells during anti-CD45RB-mediated tolerance induction

Discrimination of the thymic effect of Ab therapy on nascent vs recirculating lymphocytes is not readily achieved in a typical allotransplant setting as it would require the elimination of all bone marrow activity to prevent the generation of new alloreactive specificities. Although such studies can be accomplished by cellular transfer to immunodeficient recipients, the effects of the empty host and of homeostatic proliferation on the generation and function of T-regs confound interpretation of studies in these systems. Because of these limitations, we have evaluated the efficacy of anti-CD45RB in a tg model of cardiac rejection that we have previously reported (21). TS1 TCR tg mice carry TCR {alpha}- and beta-chain transgenes that result in an expanded population of class II (IE-d) restricted, CD4+ T cells specific for the immunodominant site 1 (S1) peptide of viral HA. Analysis of these mice is facilitated as S1-reactive T cells can be readily detected by a clonotypic Ab (6.5). Because of endogenous TCR {alpha}-chain rearrangement in TS1 mice, the CD4 T cell population expresses a spectrum of 6.5 receptor density (Fig. 3A) that we have classified into high, intermediate, and low level expression.


Figure 3
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  		FIGURE 3. Effect of anti-CD45RB treatment on donor Ag-reactive T cells. The presence of the tg TCR was detected with an anti-idiotypic Ab (6.5), and the distribution of 6.5 high, 6.5 intermediate, and 6.5 low T cells (A). Detection of anti-CD45RB Ab bound to thymocytes of mice in vivo was accomplished with a conjugated anti-rat Ig secondary Ab, showing the binding of anti-CD45RB Abs reaches the peak on day 7 but quickly disappears within 1 wk after secession of anti-CD45 treatment (B–E). To detect changes in donor-specific T cells in CD45RB-treated TS1 hosts, on days 3 and 7 in PBLs (F and G) and on day 10 in thymocytes (H and I), were analyzed from the same animals. In PBLs, a significant but incomplete reduction in the percentage of CD4+ T cells bearing the highest levels of the tg receptor was detected in anti-CD45RB-treated graft recipients ({cjs2108}, n = 6) on both day 3 (F, p < 0.002 vs untreated graft recipients ({square})) and day 7 (G, p < 0.003 vs untreated graft recipients and p < 0.001 vs CD45 treatment alone). No significant alteration was detected in comparisons between untreated graft recipients ({square}, n = 5), CD45RB treated ungrafted animals ({blacksquare}, n = 3), and naive tg animals ({cjs2100}, n = 5). Analysis of thymocytes revealed no significant alterations in comparison among any of the groups for either the percentage of Ag-reactive cells (H) or the absolute number of these cells (I). All data presented here represent at least two separate experiments.

 
TS1 mice reject cardiac grafts from BALB/c mice expressing an HA transgene (HA104), and this process is dependent on both graft HA expression and host 6.5+ T cells. TS1 hosts grafted with HA104 hearts and treated with anti-CD45RB reliably accept their grafts and exhibit donor strain tolerance paralleling the results in B6 mice that possess a normal T cell repertoire (Fig. 4A). Moreover, also similar to the C3H to B6 model, pretransplant thymectomy averts tolerance in all cases, again confirming the thymic-dependent nature of anti-CD45RB-induced tolerance (Fig. 4B).


Figure 4
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  		FIGURE 4. Thymic-dependent anti-CD45RB induced tolerance in a tg allotransplant model. TS1 mice tg for an HA-reactive TCR received intra-abdominal cardiac transplants from HA-expressing donors (HA104 mice). A, In the absence of anti-CD45RB (n = 8), tg grafts are reliably rejected with an MST of 12.5 ± 6.3 days. Treatment with anti-CD45RB resulted in indefinite survival in the majority of recipients (n = 5 of 8, 3 died with functioning graft). B, Thymectomy of TS1 mice at 2 wk before transplant abrogated tolerance induction. Untreated thymectomized (n = 4) mice rejected with the same tempo as seen for euthymic mice (MST = 16 ± 4.2 vs 12.5 ± 6.3). However, tolerance was not induced in thymectomized mice (n = 5) despite a marginal prolongation in graft survival (MST = 25.4 ± 4.3, p < 0.01). C, Adoptive transfer (1–3 days before heart transplantation) of 1 million TS1 lymph node cells into BALB/c mice results in the consistent rejection of HA-expressing allografts (n = 5, MST = 22.2 ± 11.5). In this system, the addition of anti-CD45RB (n = 3) resulted in only modest prolongation (MST = 40.7 ± 20.8, p < 0.05) of graft survival without tolerance induction.

 
The TS1 rejection model affords the opportunity to investigate the effect of anti-CD45RB treatment on the activity of peripheral donor-specific T cells in the absence of the expanded population of tg donor-specific thymocytes. Thus, tolerance development in this setting could suggest that the thymic contribution to tolerance was dependent on mature donor Ag-specific peripheral T cells returning to the thymus rather than the development of nascent donor-reactive T cells by the organ. For these studies, TS1 lymph node cells were transferred to naive syngeneic BALB/c hosts that were grafted with HA104 hearts within 1 wk after cell transfer. Whereas unmodified BALB/c hosts do not spontaneously reject HA104 heart grafts (presumably due to an inadequate frequency of HA-reactive T cells), transfer of TS1 T cells endowed the BALB/c hosts with the ability to promptly reject HA-expressing heart grafts. This model thus permits examination of the effect of anti-CD45RB on peripheral HA-reactive T cells in the presence of a normal thymus but absent thymopoiesis of HA-specific tg T cells. In contrast to the tolerogenic activity of CD45RB in naive TS1 hosts, transfer of TS1 lymph node cells to BALB/c hosts grafted with HA104 hearts and treated with anti-CD45RB did not reveal the development of tolerance in any case (Fig. 4C). These results suggest that anti-CD45RB-induced tolerance in naive TS1 mice requires maturation of donor-reactive T cells in the thymic environment during anti-CD45RB exposure.

Effect of anti-CD45RB therapy on donor Ag-specific T cells

The thymus may produce a state of long-term tolerance through action on nascent thymocytes either through deletion of Ag-reactive cells or by the transformation of these cells into a regulatory phenotype (T-regs). The TS1 tg model of anti-CD45RB tolerance induction was uniquely suited to determine both the degree of deletion of Ag-specific T cells induced by this therapy and to ascertain phenotypic changes in the central and peripheral T cell compartments.

A prerequisite for this effect to occur is that the administered agent has access to developing thymocytes when administered peripherally. To assess this directly, we examined the thymocytes of anti-CD45RB-treated and untreated mice for evidence of cell surface-bound anti-CD45RB Ab. We found that the majority of thymocytes from treated mice had detectable Ab bound on their surface as detected by an anti-rat secondary reagent. The binding of anti-CD45RB Abs to thymocytes reached a peak at the end (day 7) of anti-CD45 therapy, but only maintained for a few days and quickly disappeared thereafter (Fig. 3, B–E). No appreciable staining was observed in control Ab-treated mice.

To evaluate the impact of anti-CD45RB on donor Ag-specific T cells, HA104-grafted TS1 mice that were treated with anti-CD45RB were examined for the presence of HA-specific T cells in central and peripheral immune compartments. On days 3 and 7, PBLs were analyzed for the presence of Ag-reactive cells with the anti-idiotypic Ab 6.5. In animals receiving a heart graft and anti-CD45 Ab therapy, there was a statistically significant though incomplete reduction in cells expressing the highest levels of the tg receptor (Fig. 3, F and G). This diminution in peripheral 6.5-staining was completely recovered by 60 days posttherapy (data not shown). We anticipated that this reduction might be a combination of a peripheral and central depleting effect or TCR down-modulation. However, measurement of the CD4 single-positive thymocytes expressing the tg receptor on day 10 showed no reduction in the percentage or absolute number of Ag-reactive cells during Ab therapy (Fig. 3, H and I). The absolute number of thymocytes as well as the proportion of double-negative, double-positive, and CD4 single-positive thymocytes was also unaltered (data not shown). Overall, these data suggest only a minimal contribution of cell deletion to this mechanism and support the possibility of a temporary reduction in receptor intensity among those cells expressing the highest Ag-specific receptor density.

Phenotypic analysis of thymic and peripheral T cells was also performed as we had previously characterized a decrease in receptor staining intensity and an increase in CD25+ tg T cells during tolerance induced by intrathymic Ag inoculation. Treatment with anti-CD45 did not alter the 6.5 staining intensity or the detected level of CD25+, CD4+, CD8– thymocytes, even in the presence of the Ag-bearing graft (Fig. 5). The expected effect of Ab treatment was evident however by the observed reduction in the level of CD62L on thymocytes in all animals treated with anti-CD45RB (Fig. 5C), a finding previously characterized by Sutherland et al. (25). In addition, we found that anti-CD45RB administration was accompanied by a consistent down-regulation of thymocyte coreceptor expression compared with untreated controls. This effect was evident in both single-positive CD4 and CD8 T cells as assessed by mean channel fluorescence comparing untreated (n = 3) and treated (n = 4), respectively (CD4 mean fluorescence intensity (MFI) = 680 ± 12.7 vs 392 ± 20.2 (p < 0.001), and CD8 MFI = 688.3 ± 26.2 vs 515 ± 91.8 (p < 0.05)). Interestingly, there was no difference evident in the coreceptor levels of double-positive thymocytes (CD4 MFI = 536 ± 29.7 vs 510 ± 75.9 (p = 0.58), CD8 MFI = 702 ± 94.7 vs 763.5 ± 121.7 (p = 0.55)) suggesting that the key tolerogenic alterations induced by anti-CD45RB may occur during single-positive selection stage of thymocyte development.


Figure 5
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  		FIGURE 5. Phenotype of central and peripheral Ag-reactive T cells following anti-CD45RB therapy. The effect of anti-CD45RB was assessed by analysis of expression of cell surface markers on both central and peripheral T cells following anti-CD45RB administration to TS1 recipients. In thymocytes (A–C), no change was detected in the intensity of staining with 6.5 (A) or in the levels of CD25 (B). The intrathymic activity of anti-CD45RB was confirmed by reduction in the levels of CD62L (C) in treated animals (black and pink; average 3.5-fold reduction in MFI between groups, p < 0.01). In the periphery (D–H), the levels of 6.5 (D) and CD25 (E) were again unaltered. The presence of anti-CD45RB in treated animals (black and pink) was confirmed by reduction in the detected levels of CD62L (8.2-fold in CD45 alone, p < 0.005, and 4.2-fold in CD45 treated, grafted recipients, p < 0.05) (F) and CD45RB (2.6-fold, p < 0.03) (G). Interestingly, all cells in treated animals demonstrated a reduction in the level of the CD4 coreceptor (H). Groups are identified as follows: anti-CD45RB-treated alone (black line), anti-CD45RB-treatment graft recipients (pink line); untreated, ungrafted (green line); and cardiac graft alone (blue line). In each case, histograms are representative of more than two experiments including more than four animals.

 
Analysis of splenocytes revealed a similar pattern with no detected significant reduction in levels of the tg receptor or increases in CD25+ Ag-reactive cells (Fig. 5, D and H). Reduction in the levels of CD62L was again detected in treated animals, as was a decrease in the level of CD45RB, although whether the latter effect was due to reduction in the extracellular levels or to a blocking effect of the Ab was not confirmed. Interestingly, in animals receiving anti-CD45RB, a significant reduction in the level of the CD4 coreceptor was also noted. This reduction may be further indicative of the altered cellular signaling environment fostered by anti-CD45RB therapy. Similar effects were detected in peripheral lymph nodes as well as the para-aortic nodes that serve as the draining nodes for the abdominal cardiac graft (data not shown).

Formation of T-regs in the thymus following anti-CD45 treatment

As the majority of 6.5+ cells both in the thymus and periphery were not eliminated during tolerance induction by anti-CD45RB, we hypothesized that the thymic generation of cells with regulatory capacity was likely to be involved in the tolerogenic mechanism of action. Although we had not detected a marked increase in Ag-specific CD25+ T cells by flow cytometry, it remained possible that their function might be augmented or that they might continue to mature and expand in the periphery at a later time interval than we studied. To determine whether thymic T cells possessed regulatory capacity following anti-CD45RB therapy, we used an adoptive transfer model to determine whether donor graft survival promoting T-regs existed in the thymus of tolerant mice. We first evaluated whether reconstitution of thymectomized recipients with thymocytes would restore the tolerogenic property of anti-CD45RB therapy (Fig. 6A). C3H hearts were again transplanted to C57BL/6 hosts that had been thymectomized pretransplant and treated with our standard posttransplant regimen of anti-CD45RB. Recipients then received three doses of either naive or tolerant thymocytes (50 x 106) during the period identified above as critical to the thymus’ tolerance promoting effect (i.e., between weeks 1 and 4 posttransplant). We found that reconstitution with thymocytes from naive mice resulted in prompt rejection (MST = 15 ± 1.7). Interestingly, rejection by these hosts was markedly more rapid than in similarly treated mice not receiving any thymocytes (MST = 55.7 ± 22.3 days, p < 0.001). This result may be due to the fact that these naive thymocytes contain a fully immunocompetent cellular fraction, as they were never exposed to anti-CD45RB Abs. To address this possibility, we transferred thymocytes from anti-CD45RB treated but not grafted mice using the same protocol. In this case, prolongation of survival was observed in the recipient of such cells (thymectomized, grafted, and treated); however, tolerance was not observed (MST = 35.8 ± 4.2). This result makes two important points. First is that the administration of anti-CD45RB suppresses the reactivity of thymocytes thereby providing a functional correlate of the phenotypic changes we have observed. In addition, it indicated that for thymocytes to gain tolerance-inducing properties not only does anti-CD45RB have to be administered, but also, donor Ag in the form of a foreign graft needs to be present.


Figure 6
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  		FIGURE 6. Survival of cardiac allografts in an adoptive transfer model. A, Hearts from C3H or third-party BALB/c donors were transplanted into the abdominal cavity of immunocompetent thymectomized C57BL/6 recipients. Recipients received three doses of 50 million thymocytes from naive or C3H-tolerant B6 donors. Recipients of naive thymocytes (n = 3, MST = 15 ± 1.7) or of third-party BALB/c grafts (n = 3, MST = 34.4 ± 4.0) rejected their transplants. In contrast, tolerant thymocytes replaced thymic function in B6 mice receiving C3H grafts (n = 6, MST = 91.5 ± 13.2, p < 0.001). B, Hearts from C3H mice were transplanted into the abdominal cavity of immunodeficient (B6-Scid) mice (three to six mice in each group) that were reconstituted with 1 million naive B6 splenocytes alone (n = 5) or with 1 million tolerant splenocytes (n = 3) or thymocytes (n = 3) isolated from B6 mice bearing a long-term functioning cardiac allograft. All grafts were rapidly rejected by naive B6 splenocytes (MST = 8.4 ± 1.7), but grafts survived significantly longer in mice receiving a mixture of naive SPCs with tolerant SPCs (MST = 23.3 ± 2.3, p < 0.001) or thymocytes (MST = 31.3 ± 6.1, p < 0.001). C, B6-scid mice receiving C3H abdominal cardiac transplants were reconstituted with a total of 1 million naive splenocytes (n = 5) or with a 1:1 mixture of naive thymocytes with CD25-negative splenocytes (n = 5) or thymocytes (n = 3) or CD25+ splenocytes (n = 5) or thymocytes sorted from C3H tolerant donors (n = 3). Sorted CD25+ cells from spleen or thymus were a ble to promote long-term graft survival in the majority of cases in contrast to CD25– cells from the same donors which induced only modest prolongation. However, sorted CD4+CD25+ thymocytes from naive B6 were unable to prevent naive T cell-mediated rejection in this model.

 
In contrast to these results, transfer of putatively tolerant thymocytes recovered at 1 mo from other C3H heart-grafted anti-CD45RB-treated B6 hosts restored tolerance in four of six recipients (MST = 91.5 ± 13.2). Also, the effect of thymocyte transfer was donor Ag-specific as indicated by fact that thymectomized hosts reconstituted in a similar manner and treated with anti-CD45RB but transplanted with third party heart grafts from BALB/c donors exhibited rejection in all cases (MST = 34.3 ± 4.0). The survival of the BALB/c hearts was significantly prolonged compared with untreated hosts as expected based on the nonspecific peripheral immunosuppressive activity of anti-CD45RB documented above.

We next sought to clarify the phenotype of the regulatory thymocyte population involved in tolerance induction. Because of the large number of transferred cells used in the above experiments for reconstitution of immunocompetent thymectomized mice and the limited number of subpopulations available after cell sorting, we instead relied on cell transfer to immunodeficient hosts. For these studies, C3H hearts were transplanted into the abdominal cavity of B6-Scid mice and, 1 to 2 wk after transplantation, these immunodeficient mice were reconstituted with either 1 million naive B6 splenocytes alone or with B6 splenocytes mixed with an equal number of splenocytes or thymocytes from B6 mice rendered tolerant by anti-CD45RB. As shown in Fig. 6B, all hearts were rapidly rejected in mice that received naive B6 splenocytes (MST = 8.4 ± 1.7 days). However, graft survival was significantly prolonged to a mean survival time of 23 ± 2.3 days and 31 ± 6.1 days in mice receiving a mixture of naive B6 splenocytes with either tolerant splenocytes or thymocytes, respectively.

We hypothesized that permanent tolerance was not induced in these recipients due to an insufficient number or fraction of transferred regulatory cells as the ratio of regulators to naive effector may be important. To enrich the suppressive capacity of the transferred population, CD4+CD25+ T cells were sorted by FACS from animals that had been tolerant for 60–90 days and used in this transfer model. The CD4+CD25+ but not CD4+CD25– T cells from both splenocytes and thymocytes of tolerant mice prevented graft rejection mediated by naive T cells (Fig. 6C). However, the CD4+CD25+ thymocytes from naive B6 mice demonstrated no suppressive effect on naive T cell-mediated rejection in this model (MST = 9.4 ± 1.5 days, n = 5). These data indicate that donor Ag-specific T-regs (CD4+CD25+) exist in both the periphery and the thymus of tolerant mice.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Induced transplantation tolerance has been achieved by Ab targeting of a variety of T cell surface molecules or their ligands and in almost all of these cases, the resulting state of tolerance is thought to result from interaction of the agent with either RTEs or mature peripheral lymphocytes leading to "peripheral tolerance" (11, 26, 27, 28). In these studies, long-term graft survival and tolerance can be induced in adult thymectomized mice treated with a short course of anti-CD154 or anti-CD4 Abs (26, 27). Evidence of formation of T-regs in the periphery had also been demonstrated (26, 27). Furthermore, recent study showed that CD25+CD4+ T-reg can be generated in the periphery from CD25–CD4+ precursors in a pathway distinct to that by which naturally occurring autoreactive CD25+CD4+ T-reg develop (29). In our current work, we demonstrate that transplantation tolerance induced by anti-CD45RB is dependent on the thymus and, specifically, on the thymic generation of donor-specific T-regs that execute tolerance in the periphery that has been simultaneously suppressed by the same agent. Naturally occurring thymic-derived T-regs are essential in controlling the immune response to peripheral self-Ags and in preventing autoimmune disease (29, 30). Because of their TCR-endowed antigenic specificity and potential for self-perpetuation, these cells may also be ideal for regulating the unwanted immune response to transplanted organs and in promotion of transplantation tolerance (31, 32).

We have previously suggested that induction of transplantation tolerance via the central pathway may provide a more durable and complete state of tolerance and thus be desirable for use in the clinical transplant setting (33, 34). Using an intrathymic Ag injection model to gain tolerance, we demonstrated that thymic-derived T-regs were also prominently involved in development of induced tolerance following donor Ag injection in the thymus (23). However, technical issues limit the relevance of this approach to clinical therapy. In fact, few clinically applicable approaches to gain central tolerance have been described short of the mixed chimerism that develops following bone marrow transplantation (35, 36).

That anti-CD45RB prolonged the survival of grafts in thymectomized recipients compared with untreated controls (MST of 55.7 vs 8.3 days) indicates that anti-CD45RB possesses immunosuppressive activity on peripheral lymphocytes but, in the absence of the thymus, is not tolerogenic. Our initial characterization of the mechanism by which this tolerance is induced by the thymus suggests that there is a direct action of anti-CD45RB during T cell development (perhaps at the stage of negative selection) that results in the production of Ag-specific T-regs. The effect of the thymus did not appear to operate on recirculating cells as the generation of new cells in the thymus with Ag specificity for the graft was required.

Importantly the thymus-dependent development of T-regs by anti-CD45RB was Ag-specific. However, the mechanism by which donor Ag specificity is transmitted from the peripherally placed graft to the centrally developed tolerance mechanism remains open to speculation. In other ongoing studies in our laboratory, we have found that B lymphocytes are also required for the development of the anti-CD45RB tolerant state following cardiac transplantation (S. Deng, X. Huang, D. Moore, and J. F. Markmann, manuscript in preparation). As B cells are detected intrathymically (though in small numbers), whether they serve as a vehicle for Ag transport in this system merits further study. Of interest in this regard, Suto et al. (37) have suggested a role for thymic B cells in the generation of spontaneously occurring T-regs in the thymus.

Our recent characterization of the B cell compartment during anti-CD45RB therapy has also demonstrated a number of phenotypic changes within B lymphocytes that may contribute to the promotion of the tolerant state including up-regulation of MHC II and CD54 and down-regulation of CD19 (38). These marked and consistent changes in B cell phenotype along with the fact that B cell-deficient recipients do not become tolerant following anti-CD45RB therapy suggest a crucial role of B cells as APCs in directing the degree and character of the T lymphocyte response. In addition, these data indicate that anti-CD45RB has profound effects on the APC compartment in addition to its reported effects on T lymphocytes. The generation of T-regs is also thought to depend on critical interactions with intrathymic APCs and has been attributed in several instances to the function of the thymic epithelium (39, 40). Whether anti-CD45RB also affects the physiology of these cells and enhances their ability to promote the development of regulatory cells is currently under study. Collectively, these data offer several possibilities to account for the 3 wk window (weeks 1–4) during which the thymus is required for tolerance to occur, including the time necessary for Ag to arrive in the thymus, the time needed to alter the phenotype of the selecting cells, and the time required to accumulate a protective quorum of graft-recognizing T-regs.

The finding that the permanent state of tolerance induced by treatment with anti-CD45RB requires an intact thymus generating nascent graft-reactive cells may be of critical importance to the translation of this therapy and others like it from the experimental to clinical arena. The thymic requirement for anti-CD45RB tolerance to succeed may suggest that such an agent in the clinic could have great potential for tolerance induction. In contrast, whether tolerance based on a central pathway can succeed in the setting of thymic involution remains to be determined. More generally, this raises the question of whether thymic involution and decreased central T cell production rates are obstacles affecting all tolerance-inducing regimens requiring the assessment of the thymic contribution to other preclinical modalities. These questions also relate to accumulating data suggesting that peripheral regulation through homeostatic proliferation may be a barrier to tolerance, as the absence of thymic output may render the host entirely dependent on peripheral expansion for immune reconstitution following lymphocyte depletion (41, 42). Thus, our findings with anti-CD45RB may offer important insight to guide future strategies to optimize the potential for tolerance induction.

In summary, short-term administration of anti-CD45RB Ab effectively prevents cardiac allograft rejection and induces T cell tolerance to donor alloantigens. Posttransplant administration of anti-CD45RB exerts separable effects on the peripheral and central immune compartments. In the periphery, the agent is immunosuppressive, while in the thymus of anti-CD45RB-treated hosts, donor-specific T-regs are generated that are required for the development of tolerance. These findings implicating the central tolerance pathway in tolerance induced by anti-CD45RB suggest that targeting analogous molecules in human transplant recipients may be beneficial for promoting graft survival provided there is adequate thymic function.


   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 by National Institutes of Health Grant RO1-AI48820. Back

2 Address correspondence and reprint requests to Dr. James F. Markmann, Department of Surgery, Hospital of the University of Pennsylvania, Philadelphia, PA 19104. E-mail address: james.markmann{at}uphs.upenn.edu Back

3 Abbreviations used in this paper: T-reg, regulatory T cell; tg, transgenic; HA, hemagglutinin; MST, mean survival time; RTE, recent thymic emigrant; MFI, mean fluorescence intensity. Back

Received for publication September 9, 2004. Accepted for publication November 10, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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