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Solid organ transplantation (SOT) and hematopoietic cell transplantation (HCT) have developed over the past 5 decades largely independent from each other despite sharing biologic principles, an almost identical repertoire of immunosuppressive (IS) drugs, and biologic response modifiers to overcome barriers of immune rejection between host and donor. Long-term graft function and recipient survival are the same primary endpoints for SOT and HCT. It is remarkable that allogeneic tissues can exhibit physiologic function in a host environment that may display disparate HLA antigens, blood type, and other immune response molecules. Long-term graft function after SOT in most cases requires life-long IS medications to prevent rejection in sharp contrast with HCT where most patients after 1 to 2 years post-HCT will be able to become independent of pharmacologic agents to avoid rejection or graft-versus-host-disease (GVHD) and therefore reach a clinical state of tolerance. The hallmark of tolerance is specific unresponsiveness between host and graft tissues in the absence of any IS drugs; however, an equally significant tenet is the prerequisite freedom from infections, reflecting immunocompetence that is identifiable by protective immune responsiveness against pathogens. The established mechanisms of transplantation tolerance include immunologic ignorance, central (thymic) and peripheral clonal deletion, anergy, and immune regulation . In the following three sections, clinical and mechanistic studies highlight some of these mechanisms, as significant progress has been achieved after HCT, kidney, and liver transplantation. Excitingly, immunomodulatory strategies have recently translated into clinical success by combining HCT with living-donor SOT, using HCT as a means to achieve tolerance .
Allogeneic blood and marrow transplantation is performed with growing success worldwide as highlighted by several centers reporting comparable outcome after unrelated donor bone marrow transplantation (BMT) and cord blood transplantation that match the outcome of genotypically HLA-matched sib recipients [3–5]. Although for many, chronic GVHD remains a major barrier to achieving a sufficiently high quality of life, those patients who are successfully weaned off systemic IS not only demonstrate freedom of underlying malignancy, marrow failure, or primary immunodeficiency but also achieve a state of transplantation tolerance . Full-donor chimerism as a way to “protect from relapse” is an oft-stated goal of transplanters caring for leukemia patients. Nevertheless, not all patients require 100% of their hematopoietic and immune cells to be of donor origin, in particular, those with nonmalignant disorders. Long-term stable coexistence of host and donor cells without clinical evidence of immune-mediated pathology is often referred as persistent mixed chimerism. It existed long before the advances of reduced-intensity conditioning. Despite undergoing myeloablative conditioning, a sizeable proportion of patients with β-thalassemia  or sickle cell disease  reconstitute with mixed host and donor hematopoietic cells, each represented in excess of 10% for many years. Similarly, infants with severe combined immunodeficiency receiving HLA-disparate marrow grafts have demonstrated a sustained mixture of donor and host cells in the absence of GVHD or graft rejection. The concept of split chimerism is best illustrated by ex-severe combined immunodeficiency patients whose fractionated blood chimerism may display predominantly host B lymphocytes and myeloid cells in contrast with overwhelmingly donor-derived T cells, because of selective advantage of donor cells over genetically defective host cells. The underlying immune mechanisms responsible for the absence of alloreactive immunopathology in both full-donor and in persistent mixed chimerism is increasingly being characterized. The presence of circulating T cells with immune reactivity against host tissues even in the tolerant state points to incomplete clonal deletion of newly emerging thymocytes and/or indicating long-term survival of adoptively transferred host-reactive T cells. Nevertheless, peripheral regulatory mechanisms are operational, as highlighted by the identification of IL-10–secreting T regulatory cells (Tregs) and other regulatory T- and B-lymphocytes . Recently, CD4+/CD25+/FoxP3+ Tregs have entered the clinical arena with an encouraging safety profile; however, their efficacy in restoring immune tolerance, that is, successful treatment of GVHD, has not yet been established .
A novel approach for achieving tolerance and immune competence is illustrated by the recent unique case of a 17-year-old patient with combined immunodeficiency disease who required bilateral orthotopic lung transplantation to treat pulmonary failure caused by recurrent bacterial (Stenotrophomonas, Escherichia coli) and mycobacterial pneumonia. Chronic hypoxia and recurrent infectious gastroenteritis led to severe growth failure necessitating total parental nutrition for years. She presented in 2009 with the clinical necessity for lung and hematopoietic transplantations to correct pulmonary insufficiency and the underlying combined immunodeficiency disease. At this age, her lymphocytes had declined to a stage of extreme lymphopenia of T and B cells (30–70 CD3+ T cells/mm3) but normal natural-killer cell numbers.
A single-patient protocol proposing the use of a lung and BMT from the same donor to reduce the probability of pulmonary graft rejection or GVHD of the lungs was approved by Duke University institutional review board and FDA (IDE #14206). A 4 of 8-HLA-matched unrelated cadaveric donor was identified by the United Network for Organ Sharing, who underwent iliac crest marrow harvest yielding 5.4 × 108 cells/kg. Lungs were procured and transplanted following the marrow harvest. The marrow was cryopreserved following CD3 and CD19 depletion. Bilateral orthotopic lung transplantation was performed in December 2009 following pulse steroids and basiliximab. Within days, the patient became independent of supplemental oxygen. Three months later, while receiving FK506 and low-dose prednisone, she underwent conditioning with rituximab, alemtuzumab, antithymocyte globulin, hydroxyurea, a dose of Thiotepa, and a single fraction of total-body irridiation (lung shielding) before infusion of the T cell-depleted thawed bone marrow. She was discharged on day +20 on FK506 monotherapy with outstanding performance status and full-donor chimerism in whole blood, CD3+ T cell, and CD15+ myeloid cell fractions. She has remained without detectable host leukocytes—last tested at 15 months post-BMT. Serial repeat lung biopsies have shown absence of immune rejection. Severe gut GVHD developed in September 2010 following a bout of norovirus gastroenteritis and an associated drop in FK506 levels. GVHD resolved after a single dose of infliximab and low-dose steroid therapy; however, her course was complicated by enterococcus sepsis and DIC. FK506 wean was started ~1 year post-BMT and completed by ~15 months after BMT. The pretransplantation lymphopenia has resolved, and since age 9 months post-BMT, her CD4+ cells are >250 cells/mm3. CD8+ cells have been >500 cells/mm3 since ~3 months post-BMT with associated significant anticytomegalovirus proliferative responses (stimulation index >10). Functional B cell recovery is evident with normal serum IgA levels. Thymic output has been slow and became detectable only at last testing (15 months post-BMT) when the percentage of CD4+ T cells dually expressing CD45RA and CD62L rose to 4%. Biological studies were performed at 12 months posttransplantation to test for alloreactivity. Highly purified peripheral blood (donor) T cells showed hyporeactivity (proliferation, IL-2 and tumor necrosis factor secretion) against host-derived Epstein-Barr virus-transformed B cell lines compared with Epstein- Barr virus-lymphoblastic cell lines generated from haplotype mismatched maternal (<7%) or fully mismatched unrelated donor (<5%). This “tolerant” state could not be broken by in vitro Treg depletion and neither HLA class I or II blockade led to further attenuation of self-reactivity. There is no detectable IL-10 in coculture supernatants. Taken together, in this T cell-depleted BMT setting, the ancillary immune data support a central clonal deletion mechanism to explain the lack of antirecipient reactivity. In sum, this unique case shows for the first time, the feasibility and immune consequences of tandem cadaveric lung and T cell-depleted marrow transplantation from the same HLA-mismatched unrelated donor to create solid organ and recipient-specific tolerance and fully functional donor-derived hematopoietic, immune, and pulmonary organ function in the absence of systemic immunosuppression. However, with all leukocytes of donor origin, it cannot be determined if there is a threshold prerequisite for donor cell chimerism to achieve tolerance.
Human kidney transplantation for end-stage renal disease was facilitated by existence of two kidneys per individual. Because just one can be life-sustaining, kidney transplantation could originate as a living donor procedure. The first successful transplantations were done in identical twins in the mid-1950s, and later, as corticosteroids (Pred) and azathioprine (Aza) began to be applied in the early 1960s, transplantations were extended to HLA-identical siblings and to HLA-mismatched family members. Although cadaveric kidney transplantations were made possible by the Aza-Pred immunosuppressive drug combination, the advent of the T cell inhibitory agent cyclosporine A in the 1980s, greatly increased the volume and success rate of transplantations from deceased donors. The problem of chronic rejection, known variously as chronic allograft nephropathy and interstitial fibrosis and tubular atrophy (IF/TA), persisted into the cyclosporine and tacrolimus era and remains the major cause of late graft failure for both live-donor and deceased-donor transplantations. Major histocompatibility complex (MHC) typing is routinely performed in kidney transplantation, and patient and graft survival have benefited from high degrees of HLA matching, in particular for HLA class II (DR and DQ) antigens. Because of late graft losses and return to the transplantation waiting list for a second or third transplantation, anti-HLA antibody formation and its sequelae, antibody-mediated rejection, have emerged as major clinical problems, requiring careful attention to HLA mismatches and various strategies to suppress or eliminate antibody-forming cells. Finally, it appears that the long-term reliance on calcineurin inhibitors for chronic IS is a recipe for nephrotoxicity and end-stage renal disease. A solution to the three-headed problem of chronic IS toxicity, donor-specific HLA antibody formation, and chronic rejection lies in the generation of stable immune tolerance, the long-sought “Holy Grail” of transplantation immunology. However, even short of this lofty goal, one might envision “leveraging” some degree of tolerance at the T and B cell level to achieve long-term stable graft function under minimal IS therapy.
T cells profoundly affect the outcome of both solid organ and BMTs. These T cells are of two general types: (1) high-frequency “direct pathway” alloreactive T cells, specific for nominal antigens in the context of self-MHC molecules, but because of cross-reactivity to a foreign MHC/peptide, are able to respond directly to donor antigen-presenting cells (APCs); and (2) low-frequency “indirect pathway” alloreactive T cells, recognizing peptides derived from the donor MHC, but processed and presented by host dendritic cells (DCs). The latter are similar to classic T cells that respond to processed self- and viral antigens, and thus are expected to be highly integrated into the regulatory system of the host. Although many studies have examined direct pathway T cells detected by the in vitro mixed lymphocyte reaction assay, the role of indirect pathway T cells in human tolerance has not been extensively investigated. The following two studies yielded new insights into the host-donor relationship in renal transplantation, based entirely on the indirect pathway T cell response.
(Haynes L. et al., Donor-specific indirect pathway analysis reveals a B cell-independent signature which reflects outcomes in kidney transplant recipients. Am J Transplant. In press).
To investigate the role of donor-specific indirect pathway T cells in renal transplantation tolerance, we used the trans-vivo delayed-type hypersensitivity assay to analyze peripheral blood mononuclear cells of subjects enrolled in the Immune Tolerance Network*-sponsored Registry of Tolerant Kidney Transplant recipients (ITN507ST). The trans-vivo delayed-type hypersensitivity test provides an index of cell-mediated immunity, without exposing the transplantation patient directly to the challenge antigens (Ag). It involves the transfer of human peripheral blood mononuclear cells plus Ag (donor allo-Ag or recall Ag such as tetanus toxoid or cytomegalovirus) into the pinnae or footpads of naive mice. This induces a quantifiable delayed-type hypersensitivity-like swelling response that is Ag-specific and requires prior Ag sensitization. The assay offers a simple, reliable clinical monitoring device and also a model with which to study mechanisms underlying the regulation of host immune responses. Subjects (n = 45) were enrolled into five groups: identical twin, clinically tolerant (TOL), steroid monotherapy (Mono), standard immunosuppression (SI), and chronic rejection (CR), based on transplantation type, posttransplantation immunosuppression, and graft function. The indirect pathway was active in all groups except twins, but distinct inter-group differences were evident, corresponding to clinical status. The antidonor indirect pathway T effector response increased across patient groups (TOL < Mono < SI <CR; P < .0001), whereas antidonor indirect pathway T regulatory response decreased (TOL > Mono = SI > CR; P < .005). This pattern differed from that seen in circulating naive B cell numbers and in a B cell-based cross-platform biomarker analysis. In these studies, some of which were reported previously [10,11], patients on steroid monotherapy were not ranked closest to tolerant patients, but rather, were indistinguishable from chronically rejecting patients. Cross-sectional analysis of the indirect pathway revealed a spectrum in T regulatory:T effector balance, ranging from TOL patients having predominantly regulatory (transforming growth factor-β) responses, to CR patients having predominantly effector (inteferon-γ and IL-17A) responses to donor antigens. Therefore, the indirect pathway measurements reflect a distinct aspect of tolerance from the recently reported elevation of circulating naive B cells , which was apparent only in recipients off immunosuppression.
(Jankowska-Gan et al. Pre-transplant immune regulation predicts allograft outcome: bidirectional regulation correlates with excellent renal transplant function in living-related donor-recipient pairs. Transplantation. In press).
Partially outbred mice with multilineage, multiorgan maternal microchimerism can spontaneously accept heart allografts from a maternal-type donor. We recently found that the “tolerance-prone” and “rejection-prone” mice in a given litter can be predicted by evaluating pretransplantation immune status toward noninherited maternal antigens. To apply this insight to clinical transplantation between family members, we considered two alternative possibilities: that transplantation evokes (1) a “one-way” interaction of host T and B cells with APC and parenchymal cells of a “passive” organ, or (2) a “two-way” interaction in which graft passenger T cells and APC profoundly influence the posttransplantation course. The latter concept was originally proposed by Starzl in 1993 , an era when apoptosis and anergy mechanisms were known, but the relevance of Tregs to transplantation outcome was still unknown.
To test these hypotheses, we conducted a 3-year pilot clinical trial in 25 renal transplantation patients and their living donors under standard (HLA-ID sib) or Campath-1H induction (HLA 1 haplo missmatched and LURD) therapy and calcineurin inhibitor-based maintenance IS regimens. The % inhibition of recall response in the presence of donor or recipient cell lysate was determined 1 day before transplantation by trans-vivo delayed-type hypersensitivity assay, as a measure of familial antigen-specific Tr status.
Peripheral blood mononuclear cells of 7 HLA-identical D-R pairs, had strong preransplantation (50%–75%) bystander inhibition in both host-versus-graft and graft-versus-host directions, resulting in a mean combined inhibition value of 142%. All had excellent 3-year outcome. In contrast, only half the HLA-haploidentical pairs (9 of 18) had mutual regulation pretransplantation, whereas the rest had either one-way or no regulation. Mutual regulators had 3-year graft survival indistinguishable from that of HLA-identical siblings, with no rejections or donor-specific antibody, whereas nonmutual regulators had relatively poor outcomes in a depletional regimen (Table 1).
The data are consistent with a two-way paradigm, warranting a wider study of pretransplantation regulation and investigation of the regulatory or effector properties of passenger leukocytes in renal transplantations. If confirmed, these results profoundly shift the focus in organ transplantation from posttransplantation, recipient-only toward pretransplantation, recipient and donor immunologic evaluation—including, for example, the relevance of donor autoreactivity to collagen V in lung transplantation.
The Immune Tolerance Network (ITN) is an international clinical research consortium founded by the National Institutes of Health in 2000, whose mission is to accelerate the clinical development of immune tolerance therapies through a unique development model. The mission is broad enough to include tolerance approaches in autoimmune diabetes and in asthma, in addition to solid organ transplantation. In fact, out of the 20 clinical trials and registries currently listed on the ITN Website, http://www.immunetolerance.org/professionals/about-us, five are kidney transplantation related, three are liver-transplantation related, and the remaining 12 are autoimmunity and asthma related. The ITN is currently evaluating new proposals to combine kidney and stem cell transplantations from the same donor to achieve immunologic tolerance in patients with end-stage renal disease. Although results of pilot studies at Massachusetts General Hospital  and Stanford University  utilizing such approaches have already shown great promise in inducing lasting stable tolerance, more research will be needed to ensure safety and efficacy, particularly as these approaches move from HLA-identical siblings to HLA-mismatched live donors and finally to deceased donor transplantations. We feel that our research in the area of indirect pathway alloreactivity and pretransplantation regulation can play a role in patient selection for such trials and in evaluation of other tolerance approaches, such as the current EU-sponsored ONE study http://www.onestudy.org/, that aims to minimize immunosuppression by substituting in vitro-generated regulatory cells for standard immunosuppressive drugs. (Please see supplemental data at the end of Section III listing active ITN studies.)
Human liver transplantation was an experimental procedure, with poor 1-year patient survival (approx 25%) until the 1980s. With the advent of the T cell inhibitory agent cyclosporine A, outcomes improved markedly, and liver transplantation became standard treatment for end-stage liver disease. One-year patient survival is now as high as 85% to 90%, with 50% 15-year patient survival. In liver transplantation, allograft rejection does not contribute to late graft failure to the same extent as with other types of organ graft. The chronic shortage of deceased donor livers justifies the practices of split liver and live donor liver transplantation, the latter including ABO-incompatible transplantation. Unlike with other commonly transplanted organs, MHC typing is not routinely performed in liver transplantation, although some patient populations have benefited from high degrees of HLA matching.
The liver displays a number of features that distinguish it from other organ grafts, including its unique architecture and anatomic location downstream of the gut, its pattern of blood flow, and its regenerative and hematopoietic capacity. It has an unusual constituency of immune cells, including comparatively large numbers of innate immune cells and a range of potentially tolerogenic APCs . Each of these factors contributes to a unique microenvironment that determines the success of liver transplantation. Of special significance, both to basic science and clinical liver transplantation management, is the liver’s inherent tolerogenicity, which is greater than that of other organs. In mice and in certain rat strain combinations, fully MHC-mismatched hepatic allografts are accepted without host immunosuppressive therapy and induce donor-specific tolerance . Liver allografts are also accepted without antirejection therapy in larger animals (pigs); whereas in humans, the liver can afford a protective effect against rejection of other organs transplanted concomitantly or subsequently from the same donor.
There are well-documented reports of human liver allograft recipients (especially those who underwent transplantation as children) who have ceased to take antirejection drugs (because of infectious complications, drug toxicities, patient noncompliance, or physician-controlled weaning) and who have not rejected their transplantations up to many years postdrug withdrawal . Several trials have assessed the feasibility of discontinuing all immunosuppressive drugs under physician supervision. This approach has been successful in approximately 20% of patients . These patients offer valuable investigative material for analysis of the mechanistic basis of clinical organ transplantation tolerance and the potential identification of a tolerance “gene signature” . These studies also offer the prospect of developing an assay(s) that can identify liver transplantation recipients who can discontinue immunosuppressive therapy.
In rodents, the liver is an important hematopoietic organ. Indeed, in lethally irradiated rats, full reconstitution of hematopoietic cell lineages can be achieved by syngeneic liver transplantation . In humans, hematopoietic cells of the transplanted liver are capable of inducing acute GVHD in 1% to 2% of immunosuppressed recipients . Recently, complete hematopoietic chimerism and tolerance of an HLA-mismatched liver allograft from a deceased male donor was reported in a female child, with no evidence of GVHD, 17 months after liver transplantation . Notably, in 1992/1993, Starzl et al.  made the original observation of persistent multilineage hematopoietic cell microchimerism (defined as <1% donor cells) in both lymphoid and nonlymphoid tissues of long-surviving liver or kidney graft recipients, including patients off immunosuppression for many years posttransplantation. These findings provided the basis for the hypothesis that persistent microchimerism might be an important determinant of long-term graft survival and transplantation tolerance. The mechanism envisaged was mutual exhaustion of immune reactivity, with ensuing clonal deletion of alloreactive cell populations. These findings led to extensive investigative work in small animal models to further ascertain the nature of the chimeric cells and their functional significance and the dependence of transplantation tolerance on microchimerism . In addition, clinical studies were undertaken to evaluate the potential benefit of deliberate augmentation of chimerism in conventionally treated liver and other allograft recipients by donor BM cell infusion .
In studies of the function of donor-derived hematopoietic cells in liver transplantation, the DC became the focus of attention because of the ability of this important migratory APC to traffick from the liver to host lymphoid tissue and regulate alloimmune responses. Following liver transplantation in mice, immature donor-derived DC could be propagated from the BM of nonimmunosuppressed graft recipients that became tolerant to the donor . The presence and persistence of these cells in host lymphoid tissues raised key questions regarding their functional significance. Importantly, infusion of immature liver-derived DC into prospective pancreatic islet allograft recipients could prolong transplantation survival . These findings and their implications constituted a paradigm shift from the established perception, at the time (early 1990s), of DC solely as instigators of organ allograft rejection, to cells with ability to regulate (allo) immunity. Moreover, they prompted many subsequent studies that have validated the ability of Tolerogenic DC (tol DC) to promote tolerance in preclinical models . Tol DC therapy is highly effective in the prevention of lethal GVHD and leukemia relapse following experimental allogeneic BM cell transplantation in animals with leukemia . Currently, clinical grade human tol DC are being generated for potential therapeutic use in transplantation and autoimmune disease .
Tol DCs can promote the survival/proliferation of Tregs at the expense of effector T cells. In turn, Tregs can exert effects on DCs that promote their tolerogenic properties . Both tol DCs and Tregs have been implicated in experimental liver transplantation tolerance in the absence of immunosuppressive drug therapy , whereas elevations in Treg have been documented in clinically tolerant human liver allograft recipients [31,32]. Of particular note is the potential of DCs for the generation/expansion of allo-Ag-specific Tregs with ability to suppress allo-Ag-specific effector T cell responses, thus sparing those effector T cells able to combat infection/mediate antitumor immunity. Although initial phase 1 clinical trials of Treg for the prevention of GVHD in recipients of cord blood transplantations have used polyclonal Tregs expanded using anti-CD3/CD28 monoclonal Abs and IL-2 , future studies of Treg cell therapy are also likely to employ allo-Ag-specific Tregs generated using tol DC.
Supported by grants NIH R01HL091749 (PSz), NoNIHR01 AI066219 and the EU-sponsored One Study (WB), and NIH P01 AI81678 (to AWT).
Financial disclosure: The authors have nothing to disclose.