Adam Griesemer

The Role of the Thymus in Tolerance

The thymus serves as the central organ of immunologic self-nonself discrimination. Thymocytes undergo both positive and negative selection, resulting in T cells with a broad range of reactivity to foreign antigens but with a lack of reactivity to self-antigens. The thymus is also the source of a subset of regulatory T cells that inhibit autoreactivity of T-cell clones that may escape negative selection. As a result of these functions, the thymus has been shown to be essential for the induction of tolerance in many rodent and large animal models. Proper donor antigen presentation in the thymus after bone marrow, dendritic cell, or solid organ transplantation has been shown to induce tolerance to allografts. The molecular mechanisms of positive and negative selection and regulatory T-cell development must be understood if a tolerance-inducing therapeutic intervention is to be designed effectively. In this brief and selective review, we present some of the known information on T-cell development and on the role of the thymus in experimental models of transplant tolerance. We also cite some clinical attempts to induce tolerance to allografts using pharmacologic or biologic interventions.

Early-life compartmentalization of human T cell differentiation and regulatory function in mucosal and lymphoid tissues

It is unclear how the immune response in early life becomes appropriately stimulated to provide protection while also avoiding excessive activation as a result of diverse new antigens. T cells are integral to adaptive immunity; mouse studies indicate that tissue localization of T cell subsets is important for both protective immunity and immunoregulation. In humans, however, the early development and function of T cells in tissues remain unexplored. We present here an analysis of lymphoid and mucosal tissue T cells derived from pediatric organ donors in the first two years of life, as compared to adult organ donors, revealing early compartmentalization of T cell differentiation and regulation. Whereas adult tissues contain a predominance of memory T cells, in pediatric blood and tissues the main subset consists of naive recent thymic emigrants, with effector memory T cells (TEM) found only in the lungs and small intestine. Additionally, regulatory T (Treg) cells comprise a high proportion (30–40%) of CD4+ T cells in pediatric tissues but are present at much lower frequencies (1–10%) in adult tissues. Pediatric tissue Treg cells suppress endogenous T cell activation, and early T cell functionality is confined to the mucosal sites that have the lowest Treg:TEM cell ratios, which suggests control in situ of immune responses in early life.

Results of Gal-Knockout porcine thymokidney xenografts

Clinical transplantation for the treatment of end‐stage organ disease is limited by a shortage of donor organs. Successful xenotransplantation could immediately overcome this limitation. The development of homozygous α1,3‐galactosyltransferase knockout (GalT‐KO) pigs removed hyperacute rejection as the major immunologic hurdle to xenotransplantation. Nevertheless, GalT‐KO organs stimulate robust immunologic responses that are not prevented by immunosuppressive drugs. Murine studies show that recipient thymopoiesis in thymic xenografts induces xenotolerance. We transplanted life‐supporting composite thymokidneys (composite thymus and kidneys) prepared in GalT‐KO miniature swine to baboons in an attempt to induce tolerance in a preclinical xenotransplant model. Here, we report the results of seven xenogenic thymokidney transplants using a steroid‐free immunosuppressive regimen that eliminated whole‐body irradiation in all but one recipient. The regimen resulted in average recipient survival of over 50 days. This was associated with donor‐specific unresponsiveness in vitro and early baboon thymopoiesis in the porcine thymus tissue of these grafts, suggesting the development of T‐cell tolerance. The kidney grafts had no signs of cellular infiltration or deposition of IgG, and no grafts were lost due to rejection. These results show that xenogeneic thymus transplantation can support early primate thymopoiesis, which in turn may induce T‐cell tolerance to solid organ xenografts.

Standing the test of time: Outcomes of a decade of prioritizing patients with hepatocellular carcinoma, results of the UNOS natural geographic experiment

Priority is given to patients with hepatocellular carcinoma (HCC) to receive liver transplants, potentially causing significant regional disparities in organ access and possibly outcomes in this population. Our aim was to assess these disparities by comparing outcomes in long waiting time regions (LWTR, regions 5 and 9) and short waiting time regions (SWTR regions 3 and 10) by analyzing the United Network for Organ Sharing (UNOS) database. We analyzed 6,160 HCC patients who received exception points in regions 3, 5, 9, and 10 from 2002 to 2012. Data from regions 5 and 9 were combined and compared to data from regions 3 and 10. Survival was studied in three patient cohorts: an intent‐to‐treat cohort, a posttransplant cohort, and a cohort examining overall survival in transplanted patients only (survival from listing to last posttransplant follow‐up). Multivariate analysis and log‐rank testing were used to analyze the data. Median time on the list in the LWTR was 7.6 months compared to 1.6 months for SWTR, with a significantly higher incidence of death on the waiting list in LWTR than in SWTR (8.4% versus 1.6%, P < 0.0001). Patients in the LWTR were more likely to receive loco‐regional therapy, to have T3 tumors at listing, and to receive expanded‐criteria donor (ECD) or donation after cardiac death (DCD) grafts than patients in the SWTR (P < 0.0001 for all). Survival was significantly better in the LWTR compared to the SWTR in all three cohorts (P < 0.0001 for all three survival points). Being listed/transplanted in an SWTR was an independent predictor of poor patient survival on multivariate analysis (P < 0.0001, hazard ratio = 1.545, 95% confidence interval 1.375‐1.736). Conclusion: This study provides evidence that expediting patients with HCC to transplant at too fast a rate may adversely affect patient outcomes. (Hepatology 2014;60:1956–1961)

Xenotransplantation: Immunological hurdles and progress toward tolerance

The discrepancy between organ need and organ availability represents one of the major limitations in the field of transplantation. One possible solution to this problem is xenotransplantation. Research in this field has identified several obstacles that have so far prevented the successful development of clinical xenotransplantation protocols. The main immunologic barriers include strong T‐cell and B‐cell responses to solid organ and cellular xenografts. In addition, components of the innate immune system can mediate xenograft rejection. Here, we review these immunologic and physiologic barriers and describe some of the strategies that we and others have developed to overcome them. We also describe the development of two strategies to induce tolerance across the xenogeneic barrier, namely thymus transplantation and mixed chimerism, from their inception in rodent models through their current progress in preclinical large animal models. We believe that the addition of further beneficial transgenes to Gal knockout swine, combined with new therapies such as Treg administration, will allow for successful clinical application of xenotransplantation.

Allosensitization does not increase the risk of xenoreactivity to α1,3-galactosyltransferase gene-knockout miniature swine in patients on transplantation waiting lists

Background. The recent availability of α1,3-galactosyltransferase knockout (GalT-KO) miniature swine has eliminated anti-Gal antibodies as the major barrier to xenotransplantation, potentially bringing this modality closer to clinical application. Highly-allosensitized patients, who have poor prospects of receiving a suitable cross-match negative human organ, might be the first patients to benefit from xenotransplantation of porcine organs. However, concerns exist regarding cross-reactivity of alloreactive anti-human leukocyte antigen (HLA) antibodies against xenogeneic swine leukocyte antigen (SLA) antigens. We have investigated this question using sera from such patients on GalT-KO target cells. Methods. Using flow cytometry and complement-dependent cytotoxicity (CDC) assays, we have tested a panel of 88 human serum samples from patients awaiting cadaveric renal allotransplantation for reactivity against: 1) human; 2) standard miniature swine; and 3) GalT-KO peripheral blood lymphocytes (PBL) and cultured endothelial cells. Results. Anti-swine IgM and IgG antibody binding, as well as CDC, were significantly attenuated on GalT-KO versus standard swine. No correlation was found between the degree of anti-human panel reactive antibodies (PRA) and xenoreactivity against either standard or GalT-KO miniature swine. Treatment of sera with dithiothreitol (DTT) showed that the majority of remaining lymphocytotoxicity against GalT-KO swine was mediated by preformed IgM antibodies. Patients with high alloreactivity but low anti-GalT-KO xenoreactivity were readily identified. Conclusions. Highly allosensitized patients awaiting renal transplants appear to be at no increased risk of xenosensitization over their non-sensitized cohorts, and could therefore be candidates for xenotransplantation using GalT-KO swine donors.

Renal and Cardiac Endothelial Heterogeneity Impact Acute Vascular Rejection in Pig-to-Baboon Xenotransplantation

Xenograft outcomes are dictated by xenoantigen expression, for example, Gal α1, 3Gal (Gal), but might also depend on differing vascular responses. We investigated whether differential vascular gene expression in kidney and cardiac xenografts correlate with development of thrombotic microangiopathy (TM) and consumptive coagulation (CC). Immunosuppressed baboons underwent miniswine or hDAF pig kidney (n = 6) or heart (n = 7), or Gal‐transferase gene‐knockout (GalT‐KO) (thymo)kidney transplantation (n = 14). Porcine cDNA miniarrays determined donor proinflammatory, apoptosis‐related and vascular coagulant/fibrinolytic gene expression at defined time points; validated by mRNA, protein levels and immunopathology. hDAF‐transgenic and GalT‐KO xenografts, (particularly thymokidneys) exhibited prolonged survival. CC was seen with Gal‐expressing porcine kidneys (3 of 6), only 1 of 7 baboons postcardiac xenotransplantation and was infrequent following GalT‐KO grafts (1 of 14). Protective‐type genes (heme oxygenase‐I, superoxide dismutases and CD39) together with von Willebrand factor and P‐selectin were upregulated in all renal grafts. Transcriptional responses in Gal‐expressing xenografts were comparable to those seen in the infrequent GalT‐KO rejection. In cardiac xenografts, fibrin deposition was associated with increased plasminogen activator inhibitor‐1 expression establishing that gene expression profiles in renal and cardiac xenografts differ in a quantitative manner. These findings suggest that therapeutic targets may differ for renal and cardiac xenotransplants.

Thymic rejuvenation and the induction of tolerance by adult thymic grafts

The thymus, the site of origin of T cell immunity, shapes the repertoire of T cell reactivity through positive selection of developing T cells and prevents autoimmunity through negative selection of autoreactive T cells. Previous studies have demonstrated an important role for the thymus not only in central deletional tolerance, but also in the induction of peripheral tolerance by vascularized renal allografts in juvenile miniature swine recipients. The same protocol did not induce tolerance in thymectomized recipients nor in recipients beyond the age of thymic involution. We subsequently reported that vascularized thymic lobe grafts from juvenile donors were capable of inducing tolerance in thymectomized juvenile hosts. However, the important question remained whether aged, involuted thymus could also induce tolerance if transplanted into thymectomized hosts, which, if true, would imply that thymic involution is not an intrinsic property of thymic tissue but is rather determined by host factors extrinsic to the thymus. We report here that aged, involuted thymus transplanted as a vascularized graft into juvenile recipients leads to rejuvenation of both thymic structure and function, suggesting that factors extrinsic to the thymus are capable of restoring juvenile thymic function to aged recipients. We show furthermore that rejuvenated aged thymus has the ability to induce transplant tolerance across class I MHC barriers. These findings indicate that it may be possible to manipulate thymic function in adults to induce transplantation tolerance after the age of thymic involution.

Laparoscopic living donor left lateral sectionectomy: a new standard practice for donor hepatectomy

OBJECTIVE The aim of the study was to compare the short-term donor outcomes of laparoscopic left lateral sectionectomy (LLLS) for adult to child living donor liver transplantation (A-C LDLT) and laparoscopic donor nephrectomy (LDN). BACKGROUND Although laparoscopy has become the standard approach in kidney donors, its use remains limited and controversial in LLS for A-C LDLT due to the lack of conclusive assessment of procedure-related morbidity. METHODS From 2001 to 2014, 124 healthy donors undergoing laparoscopic LLLS for A-C LDLT at 5 tertiary referral centers in Europe, North America, and Asia, and 300 healthy donors undergoing LDN at 2 tertiary centers in Europe were retrospectively analyzed. The outcomes of LLLS were compared with those of LDN including the use of the comprehensive complication index (CCI). RESULTS Although liver donors experienced significantly less overall (16.9% vs 31.7%, P = 0.002) and grade 1 to 2 (12.1% vs 24.7%, P = 0.004) complications than kidney donors, the rates of major complication (≥ grade 3) were similar between the 2 groups. In both groups, donors experiencing postoperative complications had similar CCI (19.3 vs 21.9 for liver and kidney donors, respectively, P = 0.29). After propensity score analysis allowing for matching donors on age, sex, and body mass index, the postoperative outcomes remained comparable between the 2 groups. CONCLUSION Laparoscopic LLS for A-C LDLT yields at least similar short-term donor outcomes as LDN. These results provide the first validation for a laparoscopic donor hepatectomy and suggest that the laparoscopic approach should be considered a new standard practice for retrieval of left lateral section liver grafts as it is for kidney donation.

Absence of Replication of Porcine Endogenous Retrovirus and Porcine Lymphotropic Herpesvirus Type 1 with Prolonged Pig Cell Microchimerism after Pig-to-Baboon Xenotransplantation

ABSTRACT Porcine endogenous retrovirus (PERV), porcine cytomegalovirus (PCMV), and porcine lymphotropic herpesvirus (PLHV) are common porcine viruses that may be activated with immunosuppression for xenotransplantation. Studies of viral replication or transmission are possible due to prolonged survival of xenografts in baboon recipients from human decay-accelerating factor transgenic or α-1,3-galactosyltransferase gene knockout miniature swine. Ten baboons underwent xenotransplantation with transgenic pig organs. Graft survival was 32 to 179 days. Recipient serial samples of peripheral blood mononuclear cells (PBMC) and plasma were analyzed for PCMV, PERV, and PLHV-1 nucleic acids and viral replication using quantitative PCR assays. The PBMC contained PERV proviral DNA in 10 animals, PLHV-1 DNA in 6, and PCMV in 2. PERV RNA was not detected in any PBMC or serum samples. Plasma PLHV-1 DNA was detected in one animal. Pig cell microchimerism (pig major histocompatibility complex class I and pig mitochondrial cytochrome c oxidase subunit II sequences) was present in all recipients with detectable PERV or PLHV-1 (85.5%). Productive infection of PERV or PLHV-1 could not be demonstrated. The PLHV-1 viral load did not increase in serum over time, despite prolonged graft survival and pig cell microchimerism. There was no association of viral loads with the nature of exogenous immune suppression. In conclusion, PERV provirus and PLHV-1 DNA were detected in baboons following porcine xenotransplantation. Viral detection appeared to be due to persistent pig cell microchimerism. There was no evidence of productive infection in recipient baboons for up to 6 months of xenograft function.