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The Journal of Immunology, 2007, 178: 2221-2228.
Copyright © 2007 by The American Association of Immunologists, Inc.

Abrogation of Antibody-Mediated Allograft Rejection by Regulatory CD4 T Cells with Indirect Allospecificity1

Chris J. Callaghan, Foad J. Rouhani, Margaret C. Negus, Allison J. Curry, Eleanor M. Bolton, J. Andrew Bradley and Gavin J. Pettigrew2

University Department of Surgery, Addenbrooke’s Hospital, Cambridge, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Alloantibody is an important effector mechanism for allograft rejection. In this study, we tested the hypothesis that regulatory T cells with indirect allospecificity can prevent humoral rejection by using a rat transplant model in which acute rejection of MHC class I-disparate PVG.R8 heart grafts by PVG.RT1u recipients is mediated by alloantibody and is dependent upon help from CD4 T cells that can recognize the disparate MHC alloantigen only via the indirect pathway. Pretransplant treatment of PVG.RT1u recipients with anti-CD4 mAb plus donor-specific transfusion abrogated alloantibody production and prolonged PVG.R8 graft survival indefinitely. Naive syngeneic splenocytes injected into tolerant animals did not effect heart graft rejection, suggesting the presence of regulatory mechanisms. Adoptive transfer experiments into CD4 T cell-reconstituted, congenitally athymic recipients confirmed that regulation was mediated by CD4 T cells and was alloantigen-specific. CD4 T cell regulation could be broken in tolerant animals either by immunizing with an immunodominant linear allopeptide or by depleting tolerant CD4 T cells, but surprisingly this resulted in neither alloantibody generation nor graft rejection. These findings demonstrate that anti-CD4 plus donor-specific transfusion treatment results in the development of CD4 regulatory T cells that recognize alloantigens via the indirect pathway and act in an Ag-specific manner to prevent alloantibody-mediated rejection. Their development is associated with intrinsic tolerance within the alloantigen-specific B cell compartment that persists after T cell help is made available.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The immune response to an allograft is complex and consists of a number of heterogeneous mechanisms for graft recognition and destruction. Of these, humoral alloimmunity is being increasingly recognized as an important contributor to acute and chronic graft rejection. For example, the development of alloantibody directed against donor HLA is associated with the early failure of kidney transplants in humans (1), and the passive transfer of alloantibody may, in animal studies, result in graft rejection (2, 3, 4). In addition to producing alloantibody, allospecific B cells may have an important role in the activation of alloreactive CD4 T cells (5). This parallels the role of humoral immunity in autoimmune responses where B cells, acting as both Ab-producing effector cells and APCs for autoreactive T cell activation, contribute to disease development (6). This has resulted in the development of new treatments that reduce the severity of autoimmune disease by targeting the B cell response (7) and, although untested, similar treatments directed against the alloimmune B cell response may be effective in preventing allograft rejection.

An approach for targeting humoral alloimmunity that holds particular appeal is the development of strategies that incorporate regulatory mechanisms, because the humoral alloimmune response is likely to be a dynamic process and regulatory mechanisms will be best able to adapt to changes that occur within the allospecific B cell population during the life of the transplant. Of the regulatory mechanisms that have been described, the CD4+CD25+ T regulatory cell (Treg)3 has been most extensively studied and its ability to suppress the response of CD4 and CD8 naive T cells is well documented (8, 9). More recently, an additional direct suppressor effect on B cells has been reported (10, 11, 12) that depends on epitope-specific recognition of the B cell’s MHC-peptide complex by the Treg, resulting in B cell lysis (10, 12). The ability of regulatory CD4 T cells to prevent alloantibody-mediated transplant rejection has not been studied previously, but one would expect alloantigen-specific Tregs to similarly lyse B cells responding to transplant Ags. There are two main pathways for CD4 T cell allorecognition (13): the "direct" pathway, whereby T cells recognize intact donor MHC on the surface of a donor APC, and the indirect pathway, whereby donor MHC is recognized as processed peptide following internalization and presentation by a recipient APC. Regulatory CD4 T cells that recognize alloantigen via either pathway have been described (14, 15, 16, 17), but only those CD4 regulatory T cells that are specific for the donor MHC peptide would be expected to recognize alloantigen after B cell internalization and presentation. In other words, only Tregs that are activated via the indirect pathway could interact in a cognate fashion with allospecific B cells to effect their lysis.

Previous work in our laboratory has focused on a rat allograft model in which the PVG.R8 donor strain differs from the PVG.RT1u recipient at the RT1.A classical MHC class I Ag only (13, 18, 19). Heart grafts are rejected acutely and adoptive-transfer studies have demonstrated that rejection is CD4 T cell dependent and is mediated by IgG alloantibody (2). The disparate MHC class I alloantigen can only be recognized by recipient CD4 T cells as a processed peptide presented by the recipient MHC class II; we have therefore hypothesized that rejection is due to the provision of B cell help by CD4 T cells that have been activated via the indirect pathway (19, 20, 21). In support, CD4 T cell depletion abrogates the alloantibody response to a heart graft and results in long-term graft survival (2). In other murine transplant models, CD4 T cell modulation by CD4 coreceptor blockade using nondepleting mAbs is a well-recognized strategy for inducing transplant tolerance that is sustained by regulatory CD4 T cells (22, 23, 24). In this study we examine whether CD4 Tregs maintain tolerance to PVG.R8 heart grafts in anti-CD4 plus donor-specific transfusion (DST)-treated PVG.RT1u recipients. If so, this would provide evidence that Tregs recognizing alloantigen via the indirect pathway are responsible for inhibiting alloantibody development as well as an opportunity to examine further the mechanisms responsible for this inhibition.


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

PVG.RT1u, congenitally athymic PVG.RT1u nude (that lack functional T cells but have normal B cells) (25, 26), PVG.R8, and Lewis (RT1l) rats were bred at Central Biomedical Services, University of Cambridge (Cambridge, U.K.) or purchased from Harlan Teklad, Shaw’s Farm. Animals were maintained in specific pathogen-free facilities, and all animal studies were reviewed and approved by the U.K. Home Office under the Animal (Scientific Procedures) Act of 1986.

Anti-CD4 mAb

An OX-38 (mouse anti-rat CD4 mAb) hybridoma was obtained from the European Collection of Animal Cell Cultures. Ab was purified from culture supernatant using a protein A affinity column (PROSEP-A high capacity; Millipore) and quantified by radial immune diffusion assay.

Cardiac transplantation

Primary rat heterotopic cardiac transplantation was performed according to the modified technique of Ono and Lindsey (27). Primary heart transplants were placed heterotopically onto the abdominal aorta and inferior vena cava, whereas second heart transplants were grafted onto the cervical vessels. All grafts were assessed daily and rejection was defined as complete cessation of palpable myocardial contraction.

Anti-CD4 plus PVG.R8 DST standard treatment protocol

Naive euthymic PVG.RT1u rats were injected with 10 mg/kg anti-CD4 mAb (OX-38) i.p. and then with 2 mg/kg OX-38 i.p. daily for the next 3 days. Heparinized PVG.R8 whole blood (1.5 ml) was administered i.v. with the final dose of anti-CD4. PVG.R8 heart transplantation was performed 10 days later.

Histology

Heart grafts were placed in 10% formal saline. Samples were paraffin mounted and sections were stained with H&E. Slides were reviewed by an experienced histopathologist blinded to the study group.

Immunization

A 24-mer peptide, the "{alpha}1 peptide" corresponding to the {alpha}-helical region of the {alpha}1 domain (aa 57–80) of the RT1.Aa molecule (21, 28), was synthesized by standard Fmoc chemistry, purified by HPLC, and assessed by mass spectrometry (Immune Systems; peptide purity of >80%). The lyophilized peptide was solubilized in PBS and stored at –20°C. PVG.RT1u rats were immunized with 100 µg of {alpha}-1 peptide, emulsified in an equal volume of CFA (Sigma-Aldrich), and injected s.c. into the flank.

Production of recombinant soluble RT1.Aa protein

A recombinant soluble RT1.Aa protein was produced as described previously (29). Briefly, pET-22b+ expression plasmids containing the DNA sequences encoding either aa 1–276 of the RT1.Aa H chain ({alpha}1, {alpha}2, and {alpha}3 extracellular domains) or the rat beta2m L chain (a gift from Dr. G. Butcher, Babraham Institute, Cambridge, U.K.) were transformed into Escherichia coli BL21 (DE3) strain bacteria (Novagen) and grown in Luria Broth agar. Recombinant RT1.Aa H chain or beta2m were extracted from inclusion bodies released from E. coli pellets by chemical lysis. Soluble RT1.Aa molecules were generated by refolding the purified H chain and beta2m around a synthetic peptide (ILFPSSERR) using the dilution method of Garboczi et al. (30). Finally, fast protein liquid chromatography purification of the refold mixture was performed and the appropriate fraction was collected, pooled, filter sterilized, and stored in aliquots at 4°C.

Alloantibody determination

Alloantibody responses were measured by ELISA using as the target protein either {alpha}-1 peptide or recombinant conformational RT1.Aa. Briefly, the target protein (4 µg/ml in Na2CO3-NaHCO3 buffer (pH 9.6)) was bound to Immulon 4 HBX ELISA plates. Unbound protein-binding sites were blocked with 1% Marvel dried skimmed milk powder (Premier International Foods) at 150 µl/well. Serial tripling dilutions of test sera were added in duplicate and bound IgG Abs were detected by incubation with a biotinylated rabbit anti-rat IgG polyclonal Ab (Vector Laboratories) and ExtrAvidin peroxidase conjugate (Sigma-Aldrich). SureBlue substrate (Kirkegaard & Perry Laboratories) was then applied, the reaction was stopped by the addition of 0.2 M H2SO4, and absorbance was measured on a Packard SpectraCount ELISA plate reader with PlateReader version 3.0 software (Packard Biosciences). For each duplicate sample, a mean absorbance vs dilution curve was plotted, and the area under the curve was calculated (31). The area of the curve of an experimental sample was expressed as the percentage of positive control (pooled hyperimmune serum).

Lymphocyte subset isolation and adoptive transfer

Spleens were teased through a 70-µm nylon cell strainer and dead cells and RBC were removed by density gradient separation. CD4 T cells and B cells were isolated by positive selection using OX-38 (mouse anti-rat CD4) and OX-33 (mouse anti-rat CD45RA) microbeads, respectively, and an autoMACS cell separator (Miltenyi Biotec). Purity of the separated CD4 and B cell subsets was assessed by flow cytometry after gating on the live lymphocyte population and was typically 90–95%.

Statistical analysis

Graft survival was depicted using Kaplan-Meier analysis and groups were compared using the log-rank test. Ab responses were compared using the Mann-Whitney U test. p < 0.05 was considered statistically significant. Statistical analyses were performed using SPSS 11.0 for Windows (SPSS).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Tolerance to MHC class I-mismatched heart allografts is associated with the abrogation of IgG alloantibody responses

Congenic PVG.RT1u rats respond strongly to RT1.Aa MHC class I alloantigen and, as shown in Fig. 1, naive PVG.RT1u animals rejected RT1.Aa-disparate PVG.R8 heart grafts rapidly (median survival time (MST) 6 days). Recipient pretreatment with a combination of the partially depleting anti-CD4 mAb OX-38 and DST resulted in long-term (>100 days) graft survival in ~70% of animals. Naive recipients of a PVG.R8 heart graft mounted a strong IgG alloantibody response that was directed against the mismatched RT1.Aa donor Ag; in contrast, anti-CD4 plus DST-treated animals that maintained their grafts indefinitely never developed IgG alloantibody (Fig. 2). We have demonstrated previously that passive transfer of immune serum into T cell-depleted PVG.RT1u recipients restores PVG.R8 heart graft rejection (2), and the correlation between the presence of alloantibody and graft rejection in this particular transplant model is reinforced by the observation that all animals that rejected their grafts despite anti-CD4 plus DST pretreatment also developed alloantibody (Fig. 2). Anti-CD4 treatment without concurrent DST was ineffective at prolonging graft survival (Fig. 1). The DST effect was Ag-specific, because all of the anti-CD4 treated PVG.RT1u rats that were injected with third-party Lewis (RT1l) blood rather than PVG.R8 blood rejected PVG.R8 heart grafts rapidly (MST 7 days, n = 5).


Figure 1
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  		FIGURE 1. Anti-CD4 plus DST results in long-term PVG.R8 heart graft survival. Kaplan-Meier survival curves of PVG.R8 heart grafts in untreated PVG.RT1u recipients and recipients treated with anti-CD4 mAb plus DST, anti-CD4 mAb alone, or DST alone. *, p < 0.001 when compared with the no treatment group.

 

Figure 2
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  		FIGURE 2. Serum IgG anti-RT1.Aa alloantibody responses are abrogated in PVG.RT1u rats tolerant to PVG.R8 heart grafts. Naive PVG.RT1u rats or those that received the standard anti-CD4 plus DST protocol were transplanted with a PVG.R8 heart graft. IgG alloantibody responses following transplantation were quantified using an RT1.Aa-specific ELISA. Rats treated with the standard protocol were divided into two groups depending on whether their transplant survived 100 days and their alloantibody responses plotted separately. *, p < 0.01; {dagger}, p < 0.05 when compared with untreated controls at day 28 posttransplantation.

 
In anti-CD4 plus DST-treated animals that rejected their grafts, rejection did not always occur rapidly; several grafts rejected between days 50 and 100 after transplantation (Fig. 1). However, all heart grafts functioning for >100 days that were examined histologically appeared normal with neither disordered myocardial architecture nor allograft vasculopathy (Fig. 3). The presence of active tolerance in recipients with long-term surviving PVG.R8 grafts was further confirmed by the observation that second (cervical) PVG.R8 heart grafts were not rejected (MST >50 days, n = 2) and did not provoke an alloantibody response (data not shown).


Figure 3
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  		FIGURE 3. Histological examination of long-term surviving PVG.R8 heart grafts. H&E-stained paraffin sections of transplanted hearts are shown. A, PVG.RT1u heart harvested from a syngeneic recipient 100 days after transplantation (syngeneic control), demonstrating a normal intramyocardial vessel within normal myocardium. B, PVG.R8 heart transplanted to a PVG.RT1u recipient after treatment with the anti-CD4 mAb plus DST protocol, beating 100 days posttransplant. There is minimal cellular infiltrate and a normal intramyocardial vessel is seen within a normal myocardium. C, PVG.R8 heart transplanted to a naive, untreated PVG.RT1u recipient and excised upon rejection (day 6). A moderate neutrophil infiltrate and myocyte necrosis are present. Fibrinoid changes are evident within the intramyocardial vessel, consistent with alloantibody-mediated rejection.

 
Tolerance to heart grafts is associated with regulatory mechanisms

Anti-CD4 treatment by itself was ineffective at prolonging graft survival, suggesting that without concurrent DST treatment residual T cells are still able to effect graft rejection. We hypothesized that DST promotes the differentiation of residual CD4 T cells to alloantigen-specific Tregs that inhibit the provision of CD4 T cell help for the development of alloantibody. PVG.RT1u rats tolerant to a PVG.R8 heart graft (graft survival >100 days) were injected with naive PVG.RT1u lymphocytes in the expectation that the transferred lymphocytes would effect graft rejection unless their activation and differentiation into effector cells was prevented by regulatory mechanisms. None of three tolerant PVG.RT1u animals given 1 x 108 syngeneic splenocytes developed alloantibody (data not shown) and their heart grafts continued to beat indefinitely (> 200 days). The transfer of either 2 x 107 purified naive CD4 T cells (n = 3) or 4 x 107 B cells (n = 3) also failed to cause rejection. To exclude the possibility that accommodation (32) of the engrafted heart transplants prevented their subsequent rejection by adoptively transferred naive lymphocytes, PVG.R8 heart grafts functioning for >100 days in PVG.RT1u recipients were excised and retransplanted into naive, untreated PVG.RT1u rats. All retransplanted grafts were rejected rapidly (MST 8 days, n = 3).

Characterization of the regulatory lymphocyte population

To identify the regulatory cell population, adoptive transfer studies into CD4 T cell-reconstituted, congenitally athymic nude PVG.RT1u animals were performed. Preliminary experiments confirmed that a minimum of 2 x 107 purified naive splenic CD4 T cells was required to effectively restore alloantibody responses (Fig. 4) and PVG.R8 heart graft rejection in PVG.RT1u nude recipients (control group 2; Table I). PVG.RT1u nude animals that instead received an equivalent number of splenic CD4 T cells purified from tolerant PVG.RT1u rats did not develop alloantibody (data not shown) and did not reject PVG.R8 heart grafts (MST >100 days, n = 3). T regulatory function was assessed by the ability of lymphocyte subsets purified from tolerant PVG.RT1u recipients to prevent CD4 T cell-reconstituted PVG.RT1u nudes from rejecting PVG.R8 heart grafts. The transfer of an equivalent number (2 x 107) of CD4 T cells from tolerant and naive PVG.RT1u animals produced a modest prolongation in graft survival compared with the transfer of naive CD4 T cells only (Table I; MST 19 days vs 8.5 days, p = 0.04). When a higher ratio of tolerant to naive CD4 T cells (2:1) was transferred, long-term PVG.R8 graft survival ensued (Table I; MST >100, p < 0.01). This was associated with abrogation of the alloantibody response (Fig. 4).


Figure 4
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  		FIGURE 4. CD4 Tregs abrogate humoral alloimmunity PVG.RT1u nudes were transplanted with PVG.R8 heart grafts 1 day after having received either 2 x 107 effector syngeneic CD4 T cells only (reconstituted nudes) or effector CD4 T cells in combination with splenic lymphocyte subsets purified from tolerant PVG.RT1u animals. The alloantibody was quantified by RT1.Aa-specific ELISA and is expressed as mean ± SD. *, p < 0.05 when compared with reconstituted nudes at day 28 posttransplantation.

 

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  		Table I. PVG.R8 cardiac allograft survival in athymic PVG.RT1u nude rats following adoptive transfer of tolerant splenocyte subsetsa

 
A number of recent studies have identified several different cell types capable of regulatory function, including B cells (33, 34, 35), CD8 T cells (36, 37), and APCs that act in synergy with regulatory CD4 T cells (38). However, cotransfer of B cells at a ratio of 2:1 to naive CD4 T cells did not significantly prolong graft survival (group 3; Table I). The transfer of unfractionated tolerant splenocytes (containing at least 2 x 107 tolerant CD4 T cells) produced only a modest increase in graft survival, comparable to that seen when 2 x 107 tolerant T cells alone were transferred (groups 1 and 2; Table I). Thus, the presence of additional cells, such as B cells, CD8 T cells and dendritic cells, did not augment the ability of the CD4 Tregs to prevent graft rejection.

To confirm that the transferred tolerant CD4 T cells were regulating the naive CD4 T population in an Ag-specific fashion rather than, for example, acting nonspecifically to prevent their homeostatic proliferation (39, 40), nude PVG.RT1u rats were reconstituted with CD4 T cells from tolerant PVG.RT1u recipients and with naive CD4 effector cells at a ratio of 2:1 and then challenged the next day with a third-party Lewis heart graft. In contrast to similarly reconstituted animals that received a PVG.R8 heart graft, Lewis grafts were rejected rapidly (MST 10 days, n = 2).

Rat CD4 Tregs remain relatively poorly characterized and a number of phenotypes with different cell surface markers have been described (41, 42, 43). The requirement for large numbers of tolerant animals to purify enough CD4 T cell subsets for adoptive transfer studies prohibited definitive identification of the CD4 regulatory cell responsible for preventing alloantibody generation in the present study, although Foxp3 mRNA expression on RT-PCR analysis was greater in the splenic CD4+CD25+ T cell subset than the CD4+CD25– subset in tolerant animals (data not shown).

Intrinsic B cell tolerance contributes to the absence of alloantibody and persists after T cell regulation is abrogated

Tregs could in principle suppress alloantibody responses by either the direct killing of alloantigen-specific B cells (10, 11, 12) or by preventing the provision of effective CD4 T cell help (44). To distinguish intrinsic changes to the allospecific B cell compartment from regulatory T cell-mediated inhibition of helper cell activity, tolerant PVG.RT1u heart-graft recipients were tested for their ability to generate alloantibody after abrogation of the T cell regulatory mechanisms.

Two approaches were used to break T cell regulation. First, tolerant PVG.RT1u heart graft recipients were immunized with a synthetic {alpha}1 peptide derived from the hypervariable region of the {alpha}1 domain of the RT1.Aa H chain. Previous work in our laboratory has established that this peptide encompasses the dominant epitope for helper CD4 T cell recognition and, crucially, that the determinants for B cell recognition of this linear peptide are distinct from the B cell determinants on the surface of the intact, conformationally folded RT1.Aa Ag (21). Of seven tolerant PVG.RT1u animals immunized subcutaneously with {alpha}1 peptide in CFA, four developed Ab directed against the immunizing peptide (Fig. 5A). Because help for both anti-peptide and anti-intact RT1.Aa alloantibody responses is provided by the same CD4 Th cells (recognizing the dominant {alpha}1 determinant), this suggests that the T cell regulatory mechanisms responsible for preventing alloantibody responses to the heart graft were broken in the four animals that developed anti-peptide alloantibody. Nevertheless, only one of these developed Abs was directed against conformationally folded RT1.Aa alloantigen (Fig. 5B). Interestingly, a second animal developed Ab against intact RT1.Aa alloantigen (animal V), but this rat did not mount an anti-peptide Ab response. The heart grafts continued to beat indefinitely in all seven immunized animals.


Figure 5
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  		FIGURE 5. Development of anti-allopeptide and anti-RT1.Aa Ab following allopeptide immunization. Serum IgG anti-{alpha}1 peptide Ab (A) and anti-RT1.Aa alloantibody (B) responses in tolerant and control naive animals after immunization with 100 µg of the immunodominant {alpha}1 peptide. The individual responses of seven PVG.RT1u rats tolerant to PVG.R8 heart grafts (MST >100 days) and the mean ± SD of three control animals are depicted. Naive animals that were immunized with CFA alone did not develop an anti-allopeptide or anti-RT1.Aa Ab response (n = 3).

 
The second approach was to re-treat tolerant PVG.RT1u heart graft recipients (MST >100 days) with anti-CD4 mAb (10, 2, 2, and 2 mg/kg i.p. on successive days) in the expectation that this would deplete or disable existing Tregs. Treated animals were then reconstituted 7 days later with 4 x 107 naive, syngeneic CD4 T cells along with a further challenge with 1.5-ml PVG.R8 DST. Flow cytometric analysis of peripheral blood and serum from anti-CD4-treated tolerant animals confirmed the depletion of CD4+ T cells and CD4+CD25+ cells and the absence of unbound OX-38 before CD4+ T cell transfer (data not shown). Only one of four animals treated in this fashion developed Ab (Fig. 6), and this animal rejected its heart graft 20 days after cell transfer. The remaining rats retained functioning grafts (MST >75 days, n = 4). These two experiments thus demonstrate that additional mechanisms occurring intrinsically within the alloantigen-specific B cell compartment contribute to the absence of alloantibody in tolerant PVG.RT1u animals and that these mechanisms are robust and persist after the abrogation of T cell regulation.


Figure 6
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  		FIGURE 6. Effect of breaking T cell regulation on the generation of alloantibody. Tolerant PVG.RT1u rats (n = 4) were treated with an anti-CD4 mAb followed 7 days later by adoptive transfer of naive syngeneic CD4+ T cells and PVG.R8 DST challenge. Animal II rejected its PVG.R8 graft 20 days after cell transfer (120 days after PVG.R8 heart graft transplantation).

 
A possible explanation for the B cell tolerance observed is that alloantigen-specific B cells have been deleted; the DST component of the initial treatment protocol signals alloantigen specific B cells through their BCRs, but these B cells then undergo apoptosis in the absence of T cell help (the anti-CD4 mAb depletes peripheral CD4 T cells to ~10% of normal). In a final set of experiments, we challenged naive, T cell-deficient, athymic PVG.RT1u animals with 1.5-ml PVG.R8 DST, reasoning that this would mirror the tolerogenic B cell signal occurring in anti-CD4 plus DST-treated euthymic animals. Nine days later DST-treated nude rats were reconstituted with 2 x 107 naive PVG.RT1u CD4 T cells and challenged with a PVG.R8 heart graft the following day. Despite DST pretreatment the alloantibody response (Fig. 7) and the kinetics of graft rejection were similar to those of control nude animals that had been reconstituted but not pretreated (Table II). These results suggest that the amelioration of the alloantibody response to heart grafts following DST plus anti-CD4 treatment is not, at least in the initial stages, due to B cell depletion.


Figure 7
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  		FIGURE 7. Recovery of the alloantibody response to PVG.R8 heart grafts after CD4 T cell adoptive transfer to DST-treated athymic rats. Athymic PVG.RT1u rats treated 9 days previously with 1.5-ml PVG.R8 DST were reconstituted with 2 x 107 naive syngeneic CD4 T cells and challenged with a PVG.R8 heart graft the following day (DST plus effectors). As control, unmodified nudes and nudes receiving syngeneic CD4 T cells alone (reconstituted nudes) were also transplanted. The alloantibody response following transplantation was measured by ELISA and expressed as mean ± SD. *, p = 0.33 when compared with reconstituted nudes at day 28 posttransplantation.

 

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  		Table II. PVG.R8 heart graft survival in PVG.RT1u nude recipientsa

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
This study examines the mechanisms responsible for tolerance to heart grafts in a MHC class I-disparate model in which alloantibody is the principal effector mechanism responsible for rejection. Two major findings emerged. First, following anti-CD4 plus DST treatment, the CD4 T cells that recover contain a subset with regulatory function that acts in an Ag-specific fashion to inhibit the development of alloantibody. Second, the absence of alloantibody in tolerant animals is not due to Treg-mediated inhibition of helper T cell activity alone; intrinsic tolerance within the allospecific B cell compartment persists after regulation of the CD4 Th response is broken.

The CD4 T cell response is critical to the recognition and eventual destruction of an allograft, and in many transplant models CD4 T cell depletion results in indefinite graft survival (2, 45). CD4 T cells can recognize MHC alloantigen either as an intact protein on the surface of donor APCs, the so-called "direct" pathway, or as processed peptide fragments presented "indirectly" by recipient APCs in the context of self-MHC. Once activated, allospecific CD4 T cells play a fundamental role in the development of the main effector mechanisms of graft destruction, initiating delayed type hypersensitivity reactions within the graft and providing help for either cytotoxic CD8 T cell activation or the development of alloantibody (46). Of these, the humoral alloimmune response is probably most dependant upon the indirect pathway of CD4 T cell activation. Although "noncognate" T cell help has been described, help provided by "cognate" interaction between the TCR of the CD4 T cell and the MHC/peptide complex of the B cell is much more efficient (47). Because the peptide presented by the B cell is generated from processed Ag that has been internalized through the BCR, only the CD4 T cells that recognize alloantigen via the indirect pathway can provide cognate help for alloantibody generation.

Seminal studies by Steele et al. (48) using genetically modified knockout mice confirmed the importance of the indirect pathway. CD4-replete but MHC class II-deficient recipients whose recognition of alloantigen is restricted to the direct pathway develop only a limited IgM response and no IgG alloantibody following skin grafting. Nevertheless, on the basis that treatment with mAb directed against donor MHC class II Ag abrogated anti-MHC class I alloantibody, earlier studies suggested that CD4 T cells with direct allospecificity may provide effective, noncognate help for alloantibody (49). The conflicting results from these two studies have not been resolved. In the experimental model used in the present study, CD4 T cell recognition of the isolated MHC class I disparity can only occur via the indirect pathway, as previously confirmed by DNA transfer studies (20). The development of alloantibody can therefore be used as a marker for CD4 T cell activation via the indirect pathway. This close correlation between indirect allorecognition and alloantibody has permitted the helper T cell epitopes involved in the recognition of RT1.Aa Ag to be mapped by assaying alloantibody rather than T cell proliferation (21).

A key question in interpreting our data is, in a model where CD4 T cell help for humoral alloimmunity is provided exclusively through the indirect pathway, does the demonstration that CD4 Tregs abrogate alloantibody prove unequivocally that the Tregs are also restricted to the indirect pathway? The major consideration in answering this question is whether the Tregs are natural or adaptive (50, 51). Natural Tregs are typically CD25+ CD4 T cells that develop during early fetal and neonatal development in response to the recognition of self-Ag expressed in the thymus (52). They play a major role in the prevention of autoimmune responses in the periphery and, although they may also be involved in regulating the response to a foreign Ag, this function may not necessarily be Ag specific. Adaptive regulatory cells are distinguished from natural Tregs by their requirement to differentiate in the periphery upon Ag encounter and, hence, act in an Ag-specific fashion (53, 54). Tregs that have been described in transplantation are considered generally to be Ag-specific, because it is unlikely that natural Tregs will encounter alloantigen as they develop in the thymus (55). In support of this, Karim et al. (24) have described CD4 Tregs capable of preventing skin graft rejection that develop in thymectomized and CD25 T cell-depleted mice. Two observations suggest that the CD4 Tregs described in the present study are adaptive and Ag-specific; first, substituting a third party for PVG.R8 DST did not induce tolerance to PVG.R8 heart grafts and, second, CD4 Tregs transferred into CD4 T cell-reconstituted, athymic nude rats failed to prevent rejection of third-party heart grafts. The last experiment is particularly apposite, because it examines how Tregs from tolerant animals act upon the cotransferred naive CD4 T cells and confirms that their action is directed against alloantigen-specific CD4 T cells rather than the prevention of graft rejection by inhibiting nonspecific homeostatic proliferation of the naive effector population (39, 40, 56).

Despite the extensive literature on the role of Tregs in transplantation, their Ag specificity has not generally been examined and our results add to the limited number of murine studies that suggest that Tregs recognize alloantigen via the indirect pathway (15, 16, 17). These other studies warrant discussion, because different techniques were adopted to examine the Ag specificity of the Tregs. Hara et al. (16) suggested that the mouse Tregs responsible for tolerance to skin grafts were restricted to the indirect pathway because they inhibited the in vitro proliferation of naive T cells when cultured with donor x recipient F1 APCs (direct and indirect pathway), but not when cultured with donor strain APCs (direct pathway). Yamada et al. (15) demonstrated that unlike wild-type recipients, the blockade of costimulatory pathways in MHC class II-deficient mice (which cannot recognize alloantigen via the indirect pathway) failed to prolong skin graft survival. Finally, by using a transplant model in which challenge grafts share the same minor histocompatibility Ags as the initial tolerizing graft but differ at the major loci, Wise et al. (17) verified that linked epitope suppression can operate through the indirect pathway. So, despite providing evidence of Tregs operating through the indirect pathway, these studies have identified neither the precise allopeptide epitopes that the Tregs recognize nor the target alloantigen from which they are derived.

In our study the Tregs can only recognize the peptide epitopes generated from the disparate MHC class I RT1.Aa donor Ag. We have previously mapped the dominant peptide epitope for T cell recognition as residing within the hypervariable region of the {alpha}1 domain of the donor RT1.Aa Ag (21). These studies were functional, based on the ability of synthetic peptides to prime T cell help for IgG alloantibody production, and essentially indicate that the dominant epitope identified is presented by alloantigen-specific B cells upon internalization of the RT1.Aa Ag. The most reasonable assumption with regard to the peptide specificity of the Tregs is that they are also restricted for the dominant epitope, because that would permit interaction with and direct control of allospecific B cells. Absolute confirmation of the peptide specificity of alloantigen-specific Tregs would, however, require the use of TCR transgenic models, preferably on a RAG-knockout background, thereby restricting T cell recognition to a monoclonal population of CD25– CD4 T cells as has been described recently for nontransplant Ags (53). There is only one published transplant study by Sanchez-Fueyo et al. (14) on the use of TCR transgenic animals as a source of Tregs, and it is notable that the transgenic CD4 Tregs could only recognize intact alloantigen via the direct route. However, this mouse model is unusual in that the donor (bm12) and recipient (C57BL/6) strains only differ by three amino acid residues located within the MHC class II I-A Ag, and this disparate sequence is not processed to generate peptide for effective indirect allorecognition (57). At a theoretical level, only Tregs with indirect allospecificity can provide long-term inhibition of the alloimmune response, because direct allorecognition, which is dependant upon the egress of donor APC from the transplant, only happens in the first few weeks following transplantation. Perhaps as a consequence, in the Sanchez-Fueyo study (14) the directly restricted CD4 transgenic Tregs were able to prolong survival of bm12 skin grafts indefinitely, but F1 grafts that expressed additional third-party alloantigens were eventually rejected.

The defining characteristic of regulatory T cells is their ability to inhibit naive T cell responses, and the most obvious explanation for their suppression of alloantibody is by preventing the provision of T cell help (44). Alloantigen-specific B cells that then encounter target alloantigen in the absence of T cell help would either become anergic or undergo apoptosis. In models of autoimmunity self-reactive B cells that persist in the periphery can be rescued from their anergic state by the provision of T cell help (58, 59), and this could explain how alloantibody is generated when CD4 T cells are injected into athymic animals that had recently received DST. Perhaps the most interesting finding from our experiments is that restoring T cell help in long-term tolerant recipients by breaking regulation did not result in an alloantibody response. Two different approaches were used to break regulation. In the first, CD4 T cells in tolerant recipients were depleted with anti-CD4 mAb, the recipients were reconstituted with naive CD4 T cells, and then they were rechallenged with alloantigen. This approach may be criticized for two reasons: either additional non-CD4 T regulatory cells are present in tolerant animals or CD4 Tregs persist after Ab depletion. The former is unlikely, because our initial adoptive transfer experiments did not suggest any population other than CD4 T cells capable of regulation. Although we cannot definitely exclude the presence of a small population of residual CD4 Tregs after anti-CD4 treatment of tolerant animals, we think it unlikely that these would be present in sufficient numbers to inhibit the response of the adoptively transferred naive CD4 T cell population because a minimum ratio of 2:1 tolerant to naive CD4 T cells was required for the indefinite prolongation of PVG.R8 heart graft survival in PVG.RT1u recipients. The second approach to break CD4 T cell regulation was to immunize tolerant animals with the immunodominant {alpha}1 peptide in CFA. In this case the development of anti-peptide Ab demonstrates unequivocally that helper T cells capable of providing help for humoral alloimmunity against intact class I MHC are present but, nevertheless, the majority of animals did not develop alloantibody. Taken in conjunction, these two different approaches thus strongly suggest that as tolerance develops the RT1.Aa-specific B cell population undergoes a more profound change than reversible anergy.

Whether the development of robust and intrinsic B cell tolerance is dependent upon, rather than being simply associated with, the presence of T cell regulatory mechanisms remains speculative, but one possible explanation is that as alloantigen-specific Tregs develop they lyse RT1.Aa-specific B cells that are presenting allopeptide (10, 11, 12). Confirmation of this would require direct examination of population changes within the alloantigen-specific B cell compartment, which would itself necessitate the adoption of a B cell transgenic model. Such a model would also help to address why, irrespective of changes within the mature allospecific B cell compartment, newly formed B cells do not produce alloantibody after T cell regulation is broken. The most reasonable explanation is that immature B cells are uniquely susceptible to tolerance induction (60) and that even with T cell help available their encounter with alloantigen, either within the heart graft or as sequestered Ag within the spleen, results in apoptosis or anergy. Notably, in the two animals that developed alloantibody after T cell regulation was broken (Fig. 5B) the alloantibody response was much slower and weaker than the response of naive recipients to PVG.R8 heart grafts (Fig. 2). This alloantibody response is presumably generated by newly formed B cells, and the delay in the response reflects the additional time required for B cell maturation. This may also explain why these two animals did not reject their heart grafts; in the presence of alloantigen the Ab response generated by newly formed B cells is a suboptimal effector mechanism for graft rejection, either because of its reduced magnitude or perhaps because of skewing to a less cytotoxic isotype.

The role of humoral alloimmunity in acute and chronic human allograft rejection is becoming increasingly apparent. The present study demonstrates for the first time that regulatory T cells recognizing alloantigen via the indirect pathway can prevent alloantibody-mediated allograft rejection and that this results in intrinsic and robust B cell tolerance. Strategies aimed at the development of regulation through the indirect pathway may have clinical potential.


   Acknowledgments
 
We acknowledge the expertise of Dr. Susan Stewart, Consultant Histopathologist, Papworth Hospital, Cambridge, U.K., and the technical assistance of Susanne Negus, Sylvia Rehakova, and Dr. Gabsi Brons.


   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 British Heart Foundation Program Grant RG02/002, a Wellcome Trust Research Training Fellowship, a Beverly and Raymond Sackler Studentship (to C.J.C.), and an Academy of Medical Sciences and Health Foundation Clinician Scientist Fellowship (to G.J.P.). Back

2 Address correspondence and reprint requests to Dr. Gavin J. Pettigrew, University Department of Surgery, Box 202, Level E9, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 2QQ, United Kingdom. E-mail address: gjp25{at}cam.ac.uk Back

3 Abbreviations used in this paper: Treg, T regulatory cell; DST, donor-specific transfusion; MST, median survival time. Back

Received for publication August 30, 2006. Accepted for publication November 21, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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