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Editorials and Perspectives: Overview

Approaching the Promise of Operational Tolerance in Clinical Transplantation

Bishop, G. Alex1,8; Ierino, Francesco L.2; Sharland, Alexandra F.1; Hall, Bruce M.3; Alexander, Stephen I.4; Sandrin, Mauro S.5; Coates, P. Toby6; McCaughan, Geoffrey W.7

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doi: 10.1097/TP.0b013e318215e742
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Abstract

Transplantation is the treatment of choice for end-stage organ failure, including the kidney, pancreas, liver, heart, lung, cornea, and intestine. Tens of thousands of patients worldwide are sustained by a transplant that may be life saving, or in the case of kidney failure, improves the quality of life and life expectancy compared with dialysis. One of the major complications of transplantation is loss of allograft function secondary to rejection, an immune process that destroys the grafted organ but which may be controlled by immunosuppressive drugs. Despite continued improvements in these drugs, considerable side effects remain, including increased susceptibility to infection and malignancy. For these reasons, there has been decades of research to understand mechanisms of immune tolerance to the transplanted organ. This review will focus on approaches to achieve operational tolerance in clinical organ transplant recipients.

In animal models, rejection has been shown to depend on recipient T cells (1). There are multiple pathways of inducing T-cell tolerance including deletion, anergy, immune deviation, and suppression (Fig. 1). Transplant tolerance in animal models is defined as the inability of the recipient to reject an allograft in the complete absence of any ongoing immunosuppression. These tolerant animals should accept subsequent transplants of donor origin but reject third-party grafts that are unrelated to the donor or recipient. This should be associated with an absence of graft pathology indicative of rejection or immune injury. Transplant tolerance is often an “active” process rather than a failure to respond to the allograft.

FIGURE 1.
FIGURE 1.:
Mechanisms of inducing tolerance in T cells. The primary pathways for induction of T-cell tolerance involve: death (deletion) of the responding T cells, either of immature T cells in the thymus or of mature T cells in the periphery; anergy, where the antigen-activated T cell becomes unresponsive and refractory to further stimulation; immune deviation, where the activated T cell makes the inappropriate cytokine response and suppression (regulation), where the activated T cell not only fails to make an immune response but also prevents the response of other potentially antigen-reactive T cells. IL, interleukin; IFN, interferon; TGF, transforming growth factor; Treg, regulatory T cell.

In clinical transplantation, all the criteria used to define transplant tolerance in animal models cannot be achieved. Thus, the definition of “operational transplant tolerance” in patients is prolonged survival of a transplanted organ in the absence of immunosuppression, without evidence of a destructive response. In addition, the operationally tolerant transplant recipient should be able to respond normally to immune stimuli such as infection and tumors. Operational tolerance is difficult to achieve, especially if it is defined as lasting for the lifetime of the recipient without evidence of chronic rejection. The maintenance of a functioning graft in the complete absence of immunosuppression poses some risk of graft damage or loss as a result of low-grade chronic processes, especially as these cannot be effectively monitored at present. There is also the possibility that infection may induce crossreactive antidonor responses termed “heterologous immunity” that potentially disrupts a previously stable state of operational tolerance (2).

In 1999, the Immune Tolerance Network was formed by the National Institutes of Health to promote clinical tolerance trials in transplantation and autoimmunity. It has provided funding and oversight for many of the trials discussed here. Initially, tolerance trials were confined to kidney transplant patients, as failure to induce tolerance and consequent rejection of the transplanted kidney is not life threatening because of the availability of dialysis. Subsequently, liver transplants were also included because of the relative ease with which they are accepted, both in animal models (3) and in the clinic (4), and the liver's ability to regenerate in response to damage such as rejection.

The following review divides approaches to operational tolerance according to the organ transplanted and the strategy by which tolerance is attempted. Renal transplant patients have had a number of approaches tested, including induction of tolerance by creation of mixed allogeneic chimeras and by peripheral deletion of T cells. Liver transplant patients have been weaned from conventional immunosuppression or have been treated with alemtuzumab (Campath 1H, a monoclonal antibody to CD52 that massively depletes peripheral T cells). It also examines some novel immunosuppressive agents that could be used alone or in combination for future trials and approaches for monitoring of tolerance in kidney or liver transplant patients.

DONOR BONE MARROW-BASED THERAPIES IN RENAL TRANSPLANTATION

The landmark studies in animal models by Owen (5), Medawar and coworkers (6) and Hasek and Hraba (7) showed that exposure of a fetus or neonate to donor blood cells led to hematopoietic chimerism (blood type of donor origin). These hosts had specific transplant tolerance as they accepted skin transplants of blood donor strain. Subsequently, protocols have been developed in adult animal models to induce transplant tolerance by creation of bone marrow (BM) chimeras. Their feasibility in clinical organ transplantation was first demonstrated when patients who had undergone BM transplantation (BMT) for treatment of hematological malignancies subsequently underwent renal transplantation for renal failure, which resulted from therapy associated with BMT (8). The first such report showed that two patients who received human leukocyte antigen (HLA)-identical BMT from sibling donors and who subsequently developed chronic renal failure several years later accepted a kidney transplant from their original BMT donor (9). These results were subsequently confirmed in both HLA-identical and haplotype-mismatched donor/recipient combinations (10–12).

Despite operational tolerance of transplanted kidneys in recipients that have been tolerized to donor antigens by BMT, the toxicity of the myeloablative therapy and the potential of lethal graft-versus-host disease (GVHD) preclude this protocol for routine transplantation associated with nonmalignant conditions. Two approaches have been tried to reduce toxicity by use of nonlethal conditioning regimens to ablate the recipient marrow sufficiently to allow various levels of chimerism to occur. One involves the creation of a mixed allogeneic chimera using cytotoxic drugs and thymic irradiation in combination with a limited course of immunosuppression. The other involves total lymphoid irradiation (TLI).

Nonmyeloablative Regimens to Induce Mixed Allogeneic Chimerism

In animal models, creation of a mixed allogeneic chimera, by performing a BMT with donor BM consisting of both self-cells and donor cells, allowed the subsequent acceptance of a transplanted organ from the BM donor strain (13). Subsequently, using a nonmyeloablative conditioning regimen that created mixed allogeneic chimerism, tolerance was shown to be induced by donor-derived cells that migrate to the thymus. There they eliminate alloreactive immature T cells by a central deletion mechanism (reviewed in Ref. 14) although there may be additional peripheral mechanisms, including induction of alloantigen-specific regulatory T cells (Tregs) (14). The strength of tolerance induced by creating a mixed allogeneic chimera in animal models (15) led to interest in its application in the clinical setting. However, the toxicity of the conditioning regimen and the problem of GVHD mediated by donor T cells have restricted its clinical application to carefully selected patient groups.

The first trial was in a patient with renal failure secondary to multiple myeloma. In this situation, the HLA-matched BMT was primarily performed to treat the myeloma, and the coexistence of renal failure provided the opportunity to transplant a renal allograft from the BM donor. The kidney function remained normal, and myeloma proteins decreased to undetectable levels at the time of reporting, 174 days after transplantation (16). A follow-up report on this patient, which included a second patient treated similarly, showed that kidney function remained normal despite absence of immunosuppression and that there was no evidence of myeloma or GVHD (17).

After this salutary outcome, a further four patients were treated, and the outcomes for all six were reported (18). Rejection was observed in only one patient, and this was reversed by recommencing immunosuppression, which was later discontinued. Two patients developed GVHD, which required ongoing immunosuppression. These patients were transplanted across a HLA-identical sibling donor barrier; however, a subsequent study by the same group examined kidney transplants in nonmyeloma patients with combined kidney and BMT from one haplotype-mismatched sibling donors (19). All are alive, and four of the five patients have functioning kidneys despite being off all immunosuppression (19). Importantly, in this study of mismatched BMT recipients, there was no evidence of GVHD.

A similar approach to create BM chimerism used TLI. TLI comprises multiple small doses of irradiation, with shielding of the intestines, lungs, central nervous system, and much of the BM, so that the main targets are the thymus and supradiaphragmatic and subdiaphragmatic lymph nodes and spleen (20, 21). Animal studies showed that combination of TLI with BMT led to robust tolerance and indicated that TLI as a form of nonmyeloablative conditioning resulted in markedly reduced incidence of GVHD, compared with total body irradiation, by sparing recipient natural killer (NK) T cells (22). These experimental results have been confirmed in clinical BMT, where conditioning with TLI and antithymocyte globulin (ATG) results in low levels of GVHD even in unmatched recipients (23).

A nonmyeloablative conditioning regimen of TLI followed by combined BM and kidney transplantation induced transplant acceptance in four patients who received HLA-mismatched kidney and bone marrow from parental donors (two patients) or unrelated donors (two patients) (24). One patient developed mixed hematopoietic chimerism and was withdrawn from immunosuppression, one showed evidence of rejection and was maintained on immunosuppression, and two patients were continuing tapering at the time of publication. No GVHD was observed in any patient (24). A follow-up of these four patients indicated that two had undergone mild reversible rejection episodes and all were subsequently maintained on low-dose immunosuppression (21). Consequently, none of these HLA-mismatched kidney transplant recipients was operationally tolerant.

A further study examined a separate cohort of HLA-matched patients, one of whom developed persistent mixed chimerism and was withdrawn from immunosuppression (20, 25). A follow-up of these patients reported that five of the eight had persistent mixed chimerism, which is an indicator that the patient might be a candidate for weaning of immunosuppression (25). Of these five chimeric patients, two were withdrawn from immunosuppression, whereas three were in the process of being weaned at the time of publication. No evidence of GVHD was detected.

Overview of Donor Bone Marrow-Based Therapies

Assessment of any protocol for inducing operational tolerance to solid organs must demonstrate an efficacy and safety profile, which is at least equal to the current standard approaches of modern day long-term immunosuppression. The inherent dangers implicit in creating bone marrow chimeras, namely the toxicity of the conditioning regimen, the danger of nonengraftment, and the likelihood of GVHD have been reduced but not eliminated by the approaches described earlier. It is also apparent that operational tolerance cannot be reliably achieved in HLA-mismatched individuals.

It is possible that both approaches can be modified to yield a more reliable tolerance with minimal risk of GVHD by more stringent control of the donor cell populations injected. Donor CD34 stem cells promote donor engraftment and are unlikely to contribute to GVHD. Conversely, although donor T cells are responsible for GVHD, they can also promote long-term survival of donor stem cells with consequent prolongation of mixed chimerism (20, 21). This raises the possibility that altering the total number or phenotype of T cells infused might be able to control the balance between development of GVHD and development of durable and persisting tolerance. Other factors might contribute such as donor (26) or recipient (22) NK T cells and host Tregs, which can protect against GVHD.

T-CELL DEPLETION STRATEGIES IN RENAL TRANSPLANTATION

Because T cells are central to the rejection response and immunosuppressive drugs target T-cell function, treatments that deplete T cells have long been a component of the immunosuppressive armamentarium. Recently, there has been considerable interest in CAMPATH-1H (alemtuzumab; Millennium Pharmaceuticals, Cambridge, MA) a humanized rat monoclonal antibody to human CD52 that effectively depletes both T and B cells (27, 28). Initial studies of human renal transplant patients treated with a short perioperative course of two doses of alemtuzumab followed by low-dose maintenance therapy with cyclosporine A (CSA) showed the safety and efficacy of treatment (27, 29).

Subsequently, a tolerance trial was initiated, where patients were given three or four doses of preoperative or perioperative alemtuzumab as the only immunosuppressive therapy. Despite the almost total lack of circulating T cells, rejection occurred in all patients from 14 to 28 days after transplantation. This was reversible, and most patients were ultimately weaned to sirolimus monotherapy. Rejection coincided with an infiltrate of monocytes (28), suggesting that the cells of myeloid lineage might have mediated rejection. Thus, a follow-up study combined deoxyspergualin, which inhibits monocytes and macrophages, with alemtuzumab. These patients also developed rejection that was similar in all respects to patients treated with alemtuzumab alone (30).

Although there have been no further tolerance trials of alemtuzumab, there have been many clinical trials of its effect on rejection. These combine alemtuzumab induction therapy with reduced doses of maintenance immunosuppression. Randomized multicenter prospective trials have compared alemtuzumab-treated patients with those on conventional triple drug therapy (31, 32). These show no significant differences in graft or patient survival, incidence of acute rejection, or serum creatinine levels, although in one trial there were more cytomegalovirus infections in the alemtuzumab-treated patients (32).

Despite prolonged, extensive T- and B-cell depletion the incidence of most infections is not greater than with conventional immunosuppression (33), although this might not be the case for hepatitis C (34). There is also the possibility that patients can be weaned onto lower doses of maintenance immunosuppression. Furthermore, its creation of a “window” during the first month after transplantation, where there are low numbers of T or B cells also open possibilities for therapies that could take advantage of the low T-cell precursor frequency in the recipient. For example, it would be possible to reintroduce recipient T cells, harvested before transplant and cultured with donor antigen-presenting cells, to create donor-specific Tregs (35), or to “prune” alloreactive T cells to eliminate donor-reactive T cells (36). Alemtuzumab is now used widely in transplantation, and approximately 10% of all transplant patients in the United States receive it for induction therapy (37).

Mechanism of Alemtuzumab Immunosuppression

Alemtuzumab administration leads to rapid death of T and B lymphocytes with their almost total elimination from peripheral blood in the first hour and marked depletion from peripheral lymphoid tissues over the first 3 to 5 days (28, 37–39). In addition, there was a reduction in the peripheral blood NK cells and monocytes but not in the platelets or neutrophils. Lymphocytes gradually increased after the first month and reached levels comparable with those in transplant patients on conventional immunosuppression after approximately 6 months (28, 38, 40). The effect of alemtuzumab on T-cell subsets was not uniform, and the remnant populations in lymphoid tissues (28) and in peripheral blood (41) after treatment were predominantly of activated memory phenotype (CD3+ HLA-DR+ CD45RO+) possibly due to homeostatic proliferation (42). The relative resistance of memory T cells to alemtuzumab depletion was also demonstrated in functional assays of cytomegalovirus-specific memory T-cell responses in lung transplant patients (43). The memory T cells remaining after depletion seem to be resistant to most immunosuppressive drugs but sensitive to calcineurin inhibitors, with tacrolimus being slightly more effective than CSA for inhibition of proliferation in vitro (41). Alemtuzumab has also been claimed to spare Tregs (44–46) although not all studies support this (41), and the degree of sparing might be dependent on the type of maintenance immunosuppression (40).

“Clonal Deletion” With Donor-Specific Transfusion and Bortezomib

A recent study used pretransplant, donor-specific blood transfusion to activate donor-reactive lymphocytes (47). These activated cells were then targeted with bortezomib, an artificial tri-peptide that inhibits the 26S proteasome and induces apoptotic cell death of rapidly metabolizing plasma cells (48) and proliferating T cells (49). This treatment was claimed to selectively deplete alloactivated cells (47). One or more cycles of donor-specific transfusion followed by bortezomib treatment in combination with methylprednisolone, cyclophosphamide, and ATG were administered pretransplant. After living-related kidney transplantation, patients were treated with ATG, rituximab, and four doses of bortezomib, with all treatment, apart from low-dose prednisone, discontinued 11 days after transplant (47). Patients were monitored for rejection episodes by serum creatinine and increased antidonor HLA antibodies. Of 18 patients, 4 were withdrawn from immunosuppression, and 7 were only maintained on low-dose prednisone monotherapy. This is an encouraging result, and further trials to confirm its potential are required.

LIVER TRANSPLANT TOLERANCE

For many years, it has been known that liver transplants are less likely to reject than transplants of other organs. This was first observed in pigs, where liver transplants between outbred breeds were often accepted without any immunosuppression, whereas heart or kidney transplants were rejected (50). These findings were extended with studies of inbred rat stains, where liver transplants into low-responder strains were consistently accepted without treatment, whereas rejected in high-responder strains (51). In mice, all strains tested accepted the liver transplant without immunosuppression, and no strain was able to consistently reject fully allogeneic liver allografts (52).

There are several mechanisms that account for liver allografts inducing their own acceptance. The liver contains large numbers of passenger leukocytes, and treatment to reduce these by parking in an intermediate host (53) or irradiating the donor (54) prevents tolerance induction. The large size of the liver also contributes to its acceptance, as multiple heart and kidney transplants to a single recipient have markedly prolonged survival, which contrasts to their rapid rejection when transplanted alone (55). Also, the fenestrated endothelium of the hepatic sinusoids allows direct contact between alloreactive T cells and liver parenchyma, leading to T-cell deletion (56). Studies in rodent liver transplant models to identify the mechanism of acceptance have shown that deletion of alloreactive T cells, not ignorance, anergy, or suppression, is the basis of acceptance (57–59).

Operational Tolerance of Liver Transplant Patients

The first report of an immunosuppressive drug-free state after liver transplantation was from Starzl's group in Pittsburg, who described 23 patients. This occurred for three reasons: noncompliance, life-threatening infectious complications requiring termination of immunosuppression, or long-term functioning transplant recipients who were weaned by their physician (60). This report was followed by their prospective study of controlled weaning in liver transplant patients who had stable function with no evidence of rejection at least 5 years after transplantation. Of 59 patients who commenced weaning, 16 (27%) were withdrawn from immunosuppression (61). Subsequently, a number of groups have reported successful weaning of immunosuppression (62–67), with approximately 22% of over 350 patients being weaned. However, these were selected patients who had good graft outcome before weaning and represent something less than 10% of the total number of all liver transplant patients (68).

Another consideration is the safety of weaning immunosuppression. Although many studies have reported that there is little risk involved (62, 67), there are reports of rejection episodes being difficult to control or irreversible (65, 66). Of note, rapid weaning is much less effective than gradual weaning (69, 70). In addition, weaning of patients whose primary liver disease was autoimmune may increase the incidence of disease recurrence (62). Although long-term follow-up of weaned patients has shown its safety (71), there is evidence that weaning leads to increased graft fibrosis that is reversed when immunosuppression is reintroduced (72). Nevertheless, there are some patients, most notably those with chronic viral disease or severe complications of immunosuppression, who may benefit from weaning (67, 71).

Alemtuzumab in Liver Transplants

A number of studies from two centers have reported the results of treatment of liver transplant patients with alemtuzumab (34, 73–76). In hepatitis C-negative recipients, patient and graft survival were similar for both alemtuzumab and control groups. The incidence of rejection was significantly lower in the alemtuzumab-treated group (74, 76). Hepatitis C-positive patients in both alemtuzumab and conventionally treated groups had worse outcomes than patients without hepatitis C. There was similar patient and graft survival in the alemtuzumab compared with the control group, but the authors concluded that alemtuzumab should not be used in patients with hepatitis C (34).

Donor Chimerism and the “Two-Way Paradigm” After Liver Transplantation

The liver is rich in passenger leukocytes, and these have been demonstrated to play a role in liver transplant tolerance in rodent models (53, 54, 77). There is some evidence that donor T cells are the most active donor leukocyte subpopulation in promoting tolerance (55, 78), although a role has also been proposed for dendritic cells (DCs) (79). The liver also contains populations of stem cells, including hematopoietic stem cells that are sufficient to reconstitute lethally irradiated recipients of liver transplants (80). The liver transplant can consequently behave similarly to a BMT, in that it can mount a graft-versus-host reaction mediated by its passenger T cells. It can also reconstitute the recipient with passenger hematopoietic stem cells leading to microchimersim of lymph-hemopoietic cells. This led to the proposal by Starzl's group that a liver transplant establishes tolerance by an active immune attack on the recipient's own immune system. This hypothesis termed the “two-way paradigm” has some clinical support, and cases of clinical GVHD after liver transplantation have been reported (81).

Support for the operation of the two-way paradigm in liver transplantation arises from a recent case report; in which a liver transplant recipient developed complete allogeneic chimerism, with all blood cell lineages becoming of donor origin (82). The recipient was, however, atypical as she had low levels of circulating lymphocytes before transplantation because of a number of factors. This apparently immunocompromised state allowed donor chimerism to develop from the large population of donor hematopoietic stem cells carried in the liver (80). GVHD was avoided in this situation by the lack of mature T cells and the maturation, in the recipient thymus, of T cells derived from donor stem cells. This is an atypical case and is unlikely to occur in immunocompetent recipients.

MONITORING TOLERANCE

Monitoring Kidney Transplant Tolerance

Immune monitoring using bioassays, biomarkers, or surrogate markers of allograft tolerance will be essential for selection of patients for inclusion in transplant tolerance trials and their successful outcome. There have been many attempts to develop such tests including in vitro assays such as the mixed lymphocyte reaction and cell-mediated cytotoxicity. More recently, a trans-vivo assay has been developed in immunodeficient mice where injection of peripheral blood mononuclear cells from the transplant recipient led to a lower delayed type hypersensitivity response than to a recall antigen (83). Mixture of the donor and recall antigen suppressed the recall response in patients who were operationally tolerant because of linked suppression by donor-specific Tregs (83). This suggested that the test could identify patients who might be candidates for weaning of immunosuppression, and there is some support for this (84, 85). Reported problems with its accuracy (86) may be due to requirement for a standardized assay (87).

Analysis and monitoring of operationally tolerant patients compared with nontolerant transplant patients may provide practical clinical approaches to immune monitoring. Several putative markers of tolerance including increased CD4+CD25+ (regulatory) T cells and changes in B cells have been examined (88). Differences in NK cells (89, 90), CD4 T cells (88), and CD8 T cells (91) have also been reported to be associated with operational tolerance. Two recent studies of a number of operationally tolerant renal allograft patients using microarray analysis showed an increase in peripheral blood B-cell markers in tolerance (90, 92). Only two B-cell genes were common to both; TCLIA, which is an oncogene expressed on immature but not mature B cells, and MS4A1 (CD20), a B-cell marker (90, 92).

Monitoring Liver Transplant Tolerance

Flow cytometric analysis of liver transplant patients who were operationally tolerant compared with patients on immunosuppression showed an increase in peripheral blood B cells, γδ T cells, and CD4+CD25+ T cells (93). Another study showed increased CD4+CD25+ T cells and γδ T cells in peripheral blood of tolerant patients (94). A prospective study comparing the outcome of weaning of immunosuppression showed no detectable differences between patients before commencement of weaning but an increase in CD4+CD25+ T cells and Foxp3 expression during weaning in patients who could be successfully withdrawn (95). There was no such increase in patients who underwent rejection (95). A microarray analysis of gene expression in peripheral blood of tolerant recipients showed a gene signature that was closely associated with tolerance. This signature, which mainly involved markers of γδ T cells and NK cells, correctly identified the outcome in a majority of patients in an independent “validation” group who went on to become tolerant or rejected (96). Examination of the phenotype of DCs in peripheral blood showed increased levels of plasmacytoid DCs in patients who could be weaned from immunosuppression (97). A subsequent study confirmed this and showed increased levels of the inhibitory molecule programmed cell death 1 ligand 1 on plasmacytoid DCs (98) although a separate study of peripheral blood DC subsets could not confirm these results (94).

Overview of Tolerance Monitoring

Comparison of the results for kidney and liver transplant patients shows little similarity in the signature for tolerance, apart from an increase in CD4+CD25+ T cells in tolerant recipients and some differences in NK cells. The association between B cells and tolerance in renal transplant patients contrasts to the association with γδ T cells in tolerant liver transplant patients. Nevertheless, these initial studies have established some potential tolerance markers. Future studies should concentrate on prospectively examining patients who undergo weaning to identify markers, which indicate whether weaning will be successfully completed. A search for reproducible and clinically applicable makers of tolerance remains a focus of research.

POSSIBLE FUTURE APPROACHES TO TOLERANCE

Targeting T-Cell Costimulatory and Adhesion Molecules

There are three signaling pathways for T-cell activation, proliferation, and survival (Fig. 2). These pathways, together with the adhesion molecules that stabilize the interaction between T cells and donor antigen-presenting cells or target cells, provide the basis for novel treatment approaches for transplant tolerance. Although these treatments have been used in clinical transplantation, with one exception, they have not yet been tested in tolerance trials.

FIGURE 2.
FIGURE 2.:
Alloreactive T-cell activation. T-cell activation for allograft rejection involves three signaling pathways: Signal 1 comprises the specific recognition of alloantigen by the T-cell receptor (TCR), which is stimulated by donor major histocompatibility antigens; Signal 2 consists of multiple costimulatory pathways (Inset) that provide positive (green arrow) or negative (red arrow) signals for activation; Signal 3 involves signaling through the common γ chain of their cytokine receptors by interleukins (IL)-2, -4, -7, -9, -15, and -21. In addition, adhesion molecules stabilize the interaction of the T cell with the antigen-presenting cell (donor dendritic cell [DC]) during stimulation. Adhesion molecules are also involved in the homing of effector T cells to the transplanted organ and their interaction with target cells within the transplant. MHC, major histocompatibility complex; ICAM-1, intercellular adhesion molecule-1.

Attempts to Induce Transplant Tolerance by Costimulatory Blockade

Interest in preventing rejection by interfering with T-cell signaling was given considerable impetus by the finding that combining a monoclonal antibody to CD40 ligand (CD40L) with CTLA4Ig, induced long-term skin graft acceptance in a mouse model (99). CTLA4Ig is a fusion protein of CTLA4 with immunoglobulin that binds to CD80 and CD86 and blocks CD28 signaling. Non-human primate studies with a humanized antibody to CD40L (Hu5C8) showed that long-term kidney transplant survival resulted from a 6-month treatment course (100). These promising results were complicated when clinical trials (101) and primate transplant studies (102) of monoclonal antibodies to CD40L reported increased thromboembolic events (103). Consequently, all human trials were terminated. A preliminary report of treatment efficacy in renal transplant patients showed that of seven patients treated with a 6-month course of Hu5C8 plus maintenance immunosuppression with mycophenolate, two had mild and two had borderline rejection, whereas all seven grafts had lymphocyte infiltrates on biopsy (104). Thus in humans, anti-CD40L therapy for renal allografts had early, reversible rejection episodes. An alternative approach is to prevent interaction of CD40 and CD40L using an antibody to CD40. Primate transplant trials of several antibodies have been reported including 4D11 (105) and Chi220 (106). Both prolonged transplant survival but were not as effective as Hu5C8 in promoting acceptance.

There is continued interest in blocking the CD28 costimulation pathway with CTLA4Ig. An altered form of CTLA4Ig with 10-fold increased potency (belatacept) has been used successfully in clinical trials in renal transplant patients. Belatacept led to improved kidney function compared with CSA in terms of glomerular filtration rate and less histologic indications of rejection (107). A 5-year follow-up on its safety and efficacy has been published (108). Further trials of belatacept in transplantation were recently reported at the XXIII International Transplant Congress. They showed that switching from CSA to belatacept-based therapy or using belatacept-based therapies from the outset led to improved renal function, although possibly at the expense of more rejection. Also, in Epstein-Barr virus (EBV)-negative recipients, who received renal allografts from EBV-positive donors, there was a significant elevation in EBV-related lymphoproliferative disease.

Blocking Adhesion and Activation Molecules to Induce Transplant Tolerance

Early clinical trials in kidney transplantation used murine monoclonal antibodies to target adhesion molecules such as lymphocyte function-associated antigen (LFA)-1 (109) and intercellular adhesion molecule-1 (110) or the interleukin (IL)-2 receptor CD25 expressed on activated T cells (111) (Fig. 2). Because of induction of neutralizing antibodies by the murine immunoglobulins, these studies were confined to short-term treatments at the time of transplantation or for episodes of acute rejection. Humanization of mouse or rat monoclonal antibodies, or production of fully human antibodies raised in “TransChromo” mice that contain a human immunoglobulin-coding chromosome fragment (112), has produced antibodies that can be used long term for maintenance immunosuppression.

Alefacept is a soluble fusion protein of the adhesion molecule LFA-3 with human IgG1, which binds to its ligand, LFA-2 (CD2) on T cells. This blocks T-cell interactions with antigen-presenting cells and target cells. Alefacept is approved for treatment of psoriasis, and currently, there is a trial underway to examine its effectiveness in renal transplantation (NCT00617604).

Regulatory T Cells

Tregs were identified in animal models of transplantation by their ability to suppress the rejection response of naïve T cells in adoptive transfer assays (113–115). Although many subpopulations of lymphocytes have been shown to mediate suppression of rejection, the most thoroughly characterized and consistently suppressive cell is the CD4+CD25+ T cell (116–118), which are commonly described as Treg. However, alloactiavted effector cells also express CD25, and monoclonal antibodies to CD25 are used to prevent rejection. Tregs express the transcription factor Foxp3, which is responsible for their suppressive functions (119), whereas activated effector T cells do not stably express Foxp3. CD4+CD25+Foxp3+ T cells are present in normal individuals (natural Tregs), which suppress all immune responses equally and are not antigen specific. There has been considerable interest in using Tregs in clinical transplantation; however, natural Tregs are weakly suppressive, requiring a 1:1 ratio with naïve T cells to suppress (35).

Antigen-specific suppression by memory Tregs requires much lower numbers with ratios of one Treg to 10 naïve T cells or lower preventing rejection (35). Although it is possible to grow antigen-specific Tregs in vitro, they proliferate poorly and culture conditions require further optimization before sufficient numbers can be generated for clinical use. In contrast, nonantigen-specific Tregs proliferate well in culture and prevent transplant arteriosclerosis in a humanized mouse model (120). Antigen-specific CD4+CD25+Foxp3+ Tregs develop during transplantation tolerance and are essential for the maintenance of tolerance. One pathway for their development seems to be expansion of alloantigen- specific Tregs from natural CD4+CD25+Foxp3+ T regs as demonstrated in culture (35). Another pathway is for CD4+CD25Foxp3 T cells to be induced to become functional Tregs.

Foxp3 expression can be induced in activated CD4+ Tcells, and transfection with Foxp3 confers a Treg status on these cells (119). Regulatory cytokines such as transforming growth factor-β and IL-10 induce antigen-specific Tregs from normal CD4+ T cells when cultured with antigen, and these are known as induced Tregs. However, IL-10–induced Tregs, known as Tr1 cells, do not express Foxp3 (121). Another approach is to treat the transplant recipient with immunosuppressive drugs that spare Tregs (46, 122) or to treat the patient directly with drugs that promote natural Treg accumulation. The basis for the latter approach has been demonstrated in a mouse model where treatment with IL-2 complexed with antibody to IL-2 leads to massive expansion of Tregs and prevention of islet allograft rejection (123).

CONCLUSIONS

There are many approaches currently being investigated to achieve operational tolerance to organ transplants. The use of donor BM infusion combined with recipient conditioning has consistently led to tolerance across small histocompatibility barriers. However, the procedure presents major safety challenges and is unlikely to be used unless these can be overcome. Massive T-cell depletion of the recipient has not led to tolerance. Neither has modulation of T-cell signaling; however, combination of these approaches might improve the outcome. It is possible to devise a scenario where alemtuzumab or other T-cell–depleting antibodies could eliminate T cells, followed by subsequent modulation of T-cell signaling or costimulatory pathways to skew the development of regenerating T cells toward tolerance, by eliminating or anergizing emerging alloreactive T cells or by promoting natural or alloantigen-specific Tregs.

An alternative might be to take recipient T cells before transplantation and culture them with donor antigen- presenting cells in conditions that would eliminate or tolerize donor-reactive T cells, for example, by generating alloantigen-specific induced Tregs. Reintroduction of these tolerant T cells into recipients that have had their T cells deleted would promote graft acceptance. Liver transplant recipients can be weaned from immunosuppression more successfully than recipients of other organs, although it is difficult to see how this might be applied more generally. The development of “immune monitoring” is essential to determine which patients are developing operational tolerance to select those who can have their immunosuppression reduced or stopped. Although operational tolerance in transplantation has not yet been routinely achieved, development of new agents and improved understanding of transplant immunology increase its likelihood in the near future.

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Keywords:

Organ transplantation; Operational tolerance; Immunosuppression

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