Enhancing Kidney Transplantation and the Role of Xenografts: Report of a Scientific Workshop Sponsored by the National Kidney Foundation.

Chronic kidney disease affects an estimated 37 million people in the United States; of these,>800,000 have end-stage renal disease requiring chronic dialysis or a kidney transplant to survive. Despite efforts to increase the donor kidney supply, approximately 100,000 people are registered on the kidney transplant wait-list with no measurable decrease over the past 2 decades. The outcomes of kidney transplantation are significantly better than for chronic dialysis: kidney transplant recipients have lower rates of mortality and cardiovascular events and better quality of life, but wait-list time matters. Time on dialysis waiting for a deceased-donor kidney is a strong independent risk factor for outcomes after a kidney transplant. Deceased-donor recipients with wait-list times on dialysis of<6 months have graft survival rates equivalent to living-donor recipients with waitlist times on dialysis of>2 years. In 2021,>12,000 people had been on the kidney transplant waitlist for ≥5 years. As the gap between the demand for and availability of donor kidneys for allotransplantation continues to widen, alternative strategies are needed to provide a stable, sufficient, and timely supply. A strategy that is gaining momentum toward clinical application is pig-to-human kidney xenotransplantation. This report summarizes the proceedings of a meeting convened on April 11-12, 2022, by the National Kidney Foundation to review and assess the state of pig-to-human kidney xenotransplantation as a potential cure for end-stage renal disease.

Defects in Long-Term APC Repopulation Ability of Adult Human Bone Marrow Hematopoietic Stem Cells (HSCs) Compared with Fetal Liver HSCs.

Immunodeficient mice reconstituted with immune systems from patients, or personalized immune (PI) mice, are powerful tools for understanding human disease. Compared with immunodeficient mice transplanted with human fetal thymus tissue and fetal liver-derived CD34+ cells administered i.v. (Hu/Hu mice), PI mice, which are transplanted with human fetal thymus and adult bone marrow (aBM) CD34+ cells, demonstrate reduced levels of human reconstitution. We characterized APC and APC progenitor repopulation in human immune system mice and detected significant reductions in blood, bone marrow (BM), and splenic APC populations in PI compared with Hu/Hu mice. APC progenitors and hematopoietic stem cells (HSCs) were less abundant in aBM CD34+ cells compared with fetal liver-derived CD34+ cell preparations, and this reduction in APC progenitors was reflected in the BM of PI compared with Hu/Hu mice 14-20 wk posttransplant. The number of HSCs increased in PI mice compared with the originally infused BM cells and maintained functional repopulation potential, because BM from some PI mice 28 wk posttransplant generated human myeloid and lymphoid cells in secondary recipients. Moreover, long-term PI mouse BM contained functional T cell progenitors, evidenced by thymopoiesis in thymic organ cultures. Injection of aBM cells directly into the BM cavity, transgenic expression of hematopoietic cytokines, and coinfusion of human BM-derived mesenchymal stem cells synergized to enhance long-term B cell and monocyte levels in PI mice. These improvements allow a sustained time frame of 18-22 wk where APCs and T cells are present and greater flexibility for modeling immune disease pathogenesis and immunotherapies in PI mice.

Human stem cell-derived thymic epithelial cells enhance human T-cell development in a xenogeneic thymus.

BACKGROUND: Generation of thymic tissue from pluripotent stem cells would provide therapies for acquired and congenital thymic insufficiency states. OBJECTIVES: This study aimed to generate human thymic epithelial progenitors from human embryonic stem cells (hES-TEPs) and to assess their thymopoietic function in vivo. METHODS: This study differentiated hES-TEPs by mimicking developmental queues with FGF8, retinoic acid, SHH, Noggin, and BMP4. Their function was assessed in reaggregate cellular grafts under the kidney capsule and in hybrid thymi by incorporating them into swine thymus (SwTHY) grafts implanted under the kidney capsules of immunodeficient mice that received human hematopoietic stem and progenitor cells (hHSPCs) intravenously. RESULTS: Cultured hES-TEPs expressed FOXN1 and formed colonies expressing EPCAM and both cortical and medullary thymic epithelial cell markers. In thymectomized immunodeficient mice receiving hHSPCs, hES-TEPs mixed with human thymic mesenchymal cells supported human T-cell development. Hypothesizing that support from non-epithelial thymic cells might allow long-term function of hES-TEPs, the investigators injected them into SwTHY tissue, which supports human thymopoiesis in NOD severe combined immunodeficiency IL2Rγnull mice receiving hHSPCs. hES-TEPs integrated into SwTHY grafts, enhanced human thymopoiesis, and increased peripheral CD4+ naive T-cell reconstitution. CONCLUSIONS: This study has developed and demonstrated in vivo thymopoietic function of hES-TEPs generated with a novel differentiation protocol. The SwTHY hybrid thymus model demonstrates beneficial effects on human thymocyte development of hES-TEPs maturing in the context of a supportive thymic structure.

T1D patient-derived hematopoietic stem cells are programmed to generate Tph, Tfh, and autoimmunity-associated B cell subsets in human immune system mice.

Interactions between B cells and CD4+ T cells play a central role in the development of Type 1 Diabetes (T1D). Two helper cell subsets, follicular (Tfh) and peripheral (Tph) helper T cells, are increased in patients with T1D but their role in driving B cell autoimmunity is undefined. We used a personalized immune (PI) mouse model to generate human immune systems de novo from hematopoietic stem cells (HSCs) of patients with T1D or from healthy controls (HCs). Both groups developed Tfh and Tph-like cells, and those with T1D-derived immune systems demonstrated increased numbers of Tph-like and Tfh cells compared to HC-derived PI mice. T1D-derived immune systems included increased proportions of unconventional memory CD27-IgD- B cells and reduced proportions of naïve B cells compared to HC PI mice, resembling changes reported for patients with systemic lupus erythematosus. Our findings suggest that T1D HSCs are genetically programmed to produce increased proportions of T cells that promote the development of unconventional, possibly autoreactive memory B cells. PI mice provide an avenue for further understanding of the immune abnormalities that drive autoantibody pathogenesis and T1D.

Distribution and compartmentalization of human circulating and tissue-resident memory T cell subsets.

Knowledge of human T cells derives chiefly from studies of peripheral blood, whereas their distribution and function in tissues remains largely unknown. Here, we present a unique analysis of human T cells in lymphoid and mucosal tissues obtained from individual organ donors, revealing tissue-intrinsic compartmentalization of naive, effector, and memory subsets conserved between diverse individuals. Effector memory CD4(+) T cells producing IL-2 predominated in mucosal tissues and accumulated as central memory subsets in lymphoid tissue, whereas CD8(+) T cells were maintained as naive subsets in lymphoid tissues and IFN-γ-producing effector memory CD8(+) T cells in mucosal sites. The T cell activation marker CD69 was constitutively expressed by memory T cells in all tissues, distinguishing them from circulating subsets, with mucosal memory T cells exhibiting additional distinct phenotypic and functional properties. Our results provide an assessment of human T cell compartmentalization as a new baseline for understanding human adaptive immunity.

The Fourth International Workshop on Clinical Transplant Tolerance.

The International Workshop on Clinical Transplant Tolerance is a biennial meeting that aims to provide an update on the progress of studies of immunosuppression minimization or withdrawal in solid organ transplantation. The Fourth International Workshop on Clinical Tolerance was held in Pittsburgh, Pennsylvania, September 5-6, 2019. This report is a summary of presentations on the status of clinical trials designed to minimize or withdraw immunosuppressive drugs in kidney, liver, and lung transplantation without subsequent evidence of rejection. All protocols had in common the use of donor or recipient cell therapy combined with organ transplantation. The workshop also included presentations of mechanistic studies designed to improve understanding of the cellular and molecular basis of tolerance and to identify potential predictors/biomarkers of tolerance. Strategies to enhance the safety of hematopoietic cell transplantation and to improve patient selection/risk stratification for clinical trials were also discussed.

T cell repertoire analysis suggests a prominent bystander response in human cardiac allograft vasculopathy.

T cells are implicated in the pathogenesis of cardiac allograft vasculopathy (CAV), yet their clonality, specificity, and function are incompletely defined. Here we used T cell receptor ß chain (TCRB) sequencing to study the T cell repertoire in the coronary artery, endomyocardium, and peripheral blood at the time of retransplant in four cases of CAV and compared it to the immunoglobulin heavy chain variable region (IGHV) repertoire from the same samples. High-dimensional flow cytometry coupled with single-cell PCR was also used to define the T cell phenotype. Extensive overlap was observed between intragraft and blood TCRBs in all cases, a finding supported by robust quantitative diversity metrics. In contrast, blood and graft IGHV repertoires from the same samples showed minimal overlap. Coronary infiltrates included CD4+ and CD8+ memory T cells expressing inflammatory (IFNγ, TNFα) and profibrotic (TGFß) cytokines. These were distinguishable from the peripheral blood based on memory, activation, and tissue residency markers (CD45RO, CTLA-4, and CD69). Importantly, high-frequency rearrangements were traced back to endomyocardial biopsies (2-6 years prior). Comparison with four HLA-mismatched blood donors revealed a repertoire of shared TCRBs, including a subset of recently described cross-reactive sequences. These findings provide supportive evidence for an active local intragraft bystander T cell response in late-stage CAV.

Rapid thymectomy of NSG mice to analyze the role of native and grafted thymi in humanized mice.

We developed a rapid method to remove the native mouse thymus from NSG mice, which allowed us to compare the behavior of human immune cells in the presence or absence of human T cells in human immune system mice. Removing the native mouse thymus is critical for studies of human thymopiesis in grafted thymic tissue in humanized mice.

Role of the thymus in spontaneous development of a multi-organ autoimmune disease in human immune system mice.

We evaluated the role of the thymus in development of multi-organ autoimmunity in human immune system (HIS) mice. T cells were essential for disease development and the same T cell clones with varying phenotypes infiltrated multiple tissues. De novo-generated hematopoietic stem cell (HSC)-derived T cells were the major disease drivers, though thymocytes pre-existing in grafted human thymi contributed if not first depleted. HIS mice with a native mouse thymus developed disease earlier than thymectomized mice with a thymocyte-depleted human thymus graft. Defective structure in the native mouse thymus was associated with impaired negative selection of thymocytes expressing a transgenic TCR recognizing a self-antigen. Disease developed without direct recognition of antigens on recipient mouse MHC. While human thymus grafts had normal structure and negative selection, failure to tolerize human T cells recognizing mouse antigens presented on HLA molecules may explain eventual disease development. These new insights have implications for human autoimmunity and suggest methods of avoiding autoimmunity in next-generation HIS mice.

Mixed xenogeneic porcine chimerism tolerizes human anti-pig natural antibody-producing cells in a humanized mouse model.

BACKGROUND: A major obstacle to the success of organ transplantation from pigs to humans, necessitated by the shortage of human organs, is robust humoral immune rejection by pig-reactive human antibodies. Mixed xenogeneic hematopoietic chimerism induces xenoreactive B cell tolerance in rodents, but whether mixed pig/human chimerism could induce tolerance of human B cells to pig xenoantigens is unknown. METHODS: We investigated this question using a humanized mouse model in which durable mixed (pig-human) xenogeneic chimerism can be established. RESULTS: Human natural anti-pig cytotoxic antibodies, predominantly IgM, are detectable in non-chimeric humanized mouse serum, and pig-reactive antibodies were reduced in mixed chimeric versus non-chimeric humanized mice. This difference required persistent mixed chimerism and was not due to the adsorption of antibodies on pig cells in vivo. Furthermore, human B cells from spleens of mixed chimeric mice produced lower levels of anti-pig antibodies when stimulated in vitro compared with those from non-chimeric mice. CONCLUSIONS: Our findings demonstrate that mixed chimerism reduces human natural antibodies to pig xenoantigens, providing the first in vivo evidence of human B cell tolerance induction by mixed xenogeneic chimerism and supporting further evaluation of this approach for inducing human B cell tolerance to xenografts.

Antibody reactivity with new antigens revealed in multi-transgenic triple knockout pigs may cause early loss of pig kidneys in baboons.

BACKGROUND: Recent advances in gene editing technology have enabled the production of multi-knockout (KO) and transgenic pigs in order to overcome immunologic barriers in xenotransplantation (XTx). However, the genetic manipulations required to produce these changes may have the unintended consequence of producing or revealing neoantigens reactive with natural antibodies present in baboons. In this study, we examined whether the neoantigens that develop in multi-transgenic (mTg) GalT, Cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH), ß-1,4-N-acetyl-galactosaminyl transferase 2 (B4) KO pigs can cause rejection of xenografts in baboons. METHODS: Five baboons that had <35% cytotoxicity against GalT-KO peripheral blood mononuclear cells (PBMCs) in a pre-screening assay received pig kidneys and vascularized thymic grafts (VT + K) from multi-transgenic hCD47, human thrombomodulin (hTBM), human endothelial protein C receptor (EPCR) with/without hCD46 and hCD55 with GalT-KO/NeuGC-KO/B4-KO (mTg Tri-KO) swine. In order to further examine the effects of anti-donor non-Gal natural antibody (nAb), anti-pig preformed IgM and IgG nAb binding against the GalT-KO PBMCs was compared with the donor-type PBMCs using donor pretransplant sera as well as 5 additional naïve baboon sera by flow cytometric analysis. RESULTS: Five baboons that received VT + K grafts had stable renal function in the first 11 days (serum creatinine < 1.5 mg/dL). Two of the five baboons had higher binding of preformed IgG to mTg Tri-KO PBMCs than to GalT-KO PBMCs (mTg Tri-KO > GalT-KO), and they rejected their grafts at POD 20. In contrast, the other three baboons demonstrated either mTg Tri-KO = GalT-KO or mTg Tri-KO < GalT-KO, and they maintained renal function 43, 52, and 154 days without rejection. Among 10 baboon sera, two had less antibody binding against PBMCs that were syngeneic to the mTg Tri-KO than against GalT-KO PBMCs (mTg Tri-KO < GalT-KO); three had similar binding to mTg Tri-KO and GalT-KO PBMCs (mTg Tri-KO = GalT-KO); and five had higher binding to m Tg Tri-KO than to GalT-KO PBMCs (mTg Tri-KO > GalT-KO). CONCLUSIONS: These data suggest that neoantigens associated with mTg Tri-KO promote acute xenograft rejection in a pig-to-baboon VT + K XTx model. The screening assays may be useful to select "safe" recipients to receive mTg Tri-KO kidneys.

Expression of human CD47 in pig glomeruli prevents proteinuria and prolongs graft survival following pig-to-baboon xenotransplantation.

BACKGROUND: Nephrotic syndrome is a common complication of pig-to-baboon kidney xenotransplantation (KXTx) that adversely affects outcomes. We have reported that upregulation of CD80 and down-regulation of SMPDL-3b in glomeruli have an important role in the development of proteinuria following pig-to-baboon KXTx. Recently we found induced expression of human CD47 (hCD47) on endothelial cells and podocytes isolated from hCD47 transgenic (Tg) swine markedly reduced phagocytosis by baboon and human macrophages. These observations led us to hypothesize that transplanting hCD47 Tg porcine kidneys could overcome the incompatibility of the porcine CD47-baboon SIRPα interspecies ligand-receptor interaction and prevent the development of proteinuria following KXTx. METHODS: Ten baboons received pig kidneys with vascularized thymic grafts (n = 8) or intra-bone bone marrow transplants (n = 2). Baboons were divided into three groups (A, B, and C) based on the transgenic expression of hCD47 in GalT-KO pigs. Baboons in Group A received kidney grafts with expression of hCD47 restricted to glomerular cells (n = 2). Baboons in Group B received kidney grafts with high expression of hCD47 on both glomerular and tubular cells of the kidneys (n = 4). Baboons in Group C received kidney grafts with low/no glomerular expression of hCD47, and high expression of hCD47 on renal tubular cells (n = 4). RESULTS: Consistent with this hypothesis, GalT-KO/hCD47 kidney grafts with high expression of hCD47 on glomerular cells developed minimal proteinuria. However, high hCD47 expression in all renal cells including renal tubular cells induced an apparent destructive inflammatory response associated with upregulated thrombospondin-1. This response could be avoided by a short course of weekly anti-IL6R antibody administration, resulting in prolonged survival without proteinuria (mean 170.5 days from 47.8 days). CONCLUSION: Data showed that transgenic expression of hCD47 on glomerular cells in the GalT-KO donor kidneys can prevent xenograft nephropathy, a significant barrier for therapeutic applications of xenotransplantation. The ability to prevent nephrotic syndrome following KXTx overcomes a critical barrier for future clinical applications of KXTx.

Deletion of donor-reactive T cell clones after human liver transplant.

We recently developed a high throughput T cell receptor ß chain (TCRß) sequencing-based approach to identifying and tracking donor-reactive T cells. To address the role of clonal deletion in liver allograft tolerance, we applied this method in samples from a recent randomized study, ITN030ST, in which immunosuppression withdrawal was attempted within 2 years of liver transplantation. We identified donor-reactive T cell clones via TCRß sequencing following a pre-transplant mixed lymphocyte reaction and tracked these clones in the circulation following transplantation in 3 tolerant and 5 non-tolerant subjects. All subjects showed a downward trend and significant reductions in donor-reactive TCRß sequences were detected post-transplant in 6 of 8 subjects, including 2 tolerant and 4 non-tolerant recipients. Reductions in donor-reactive TCRß sequences were greater than those of all other TCRß sequences, including 3rd party-reactive sequences, in all 8 subjects, demonstrating an impact of the liver allograft after accounting for repertoire turnover. Although limited by patient number and heterogeneity, our results suggest that partial deletion of donor-reactive T cell clones may be a consequence of liver transplantation and does not correlate with success or failure of early immunosuppression withdrawal. These observations underscore the organ- and/or protocol-specific nature of tolerance mechanisms in humans.

Siplizumab selectively depletes effector memory T cells and promotes a relative expansion of alloreactive regulatory T cells in vitro.

Siplizumab, a humanized anti-CD2 monoclonal antibody, has been used in conditioning regimens for hematopoietic cell transplantation and tolerance induction with combined kidney-bone marrow transplantation. Siplizumab-based tolerance induction regimens deplete T cells globally while enriching regulatory T cells (Tregs) early posttransplantation. Siplizumab inhibits allogeneic mixed-lymphocyte reactions (MLRs) in vitro. We compared the impact of siplizumab on Tregs versus other T cell subsets in HLA-mismatched allogeneic MLRs using PBMCs. Siplizumab predominantly reduced the percentage of CD4+ and CD8+ effector memory T cells, which express higher CD2 levels than naïve T cells or resting Tregs. Conversely, siplizumab enriched proliferating CD45RA- FoxP3HI cells in MLRs. FoxP3 expression was stable over time in siplizumab-containing cultures, consistent with enrichment for bona fide Tregs. Consistently, high-throughput TCRß CDR3 sequencing of sorted unstimulated and proliferating T cells in MLRs revealed selective expansion of donor-reactive Tregs along with depletion of donor-reactive CD4+ effector/memory T cells in siplizumab-containing MLRs. These results indicate that siplizumab may have immunomodulatory functions that may contribute to its success in tolerance-inducing regimens. Our studies also confirm that naïve in addition to effector/memory T cells contribute to the allogeneic MLR and mandate further investigation of the impact of siplizumab on alloreactive naïve T cells.

Pharmacokinetic and pharmacodynamic study of a clinically effective anti-CD2 monoclonal antibody.

The humanized IgG1κ monoclonal antibody siplizumab and its rat parent monoclonal IgG2b antibody BTI-322 are directed against the CD2 antigen. Siplizumab is species-specific, reacting with human and chimpanzee cells but not with cells from any other species, including other non-human primates. Because siplizumab treatment has recently shown great potential in clinical transplantation, we now present the results of our previous pharmacokinetic, pharmacodynamic and safety studies of both antibodies. Fourteen chimpanzees received 1-3 doses of 0.143 to 5.0 mg/kg iv The effects were followed with flow cytometry on peripheral lymphocytes and staining of lymph nodes. Side effects were recorded. Serum antibody concentrations were followed. Across the doses, a rapid, transient depletion of CD2, CD3, CD4 and CD8 lymphocytes and NK cells was observed for both antibodies. Immune reconstitution was more rapid for BTI-322 compared to siplizumab. Paracortical lymph node T cell depletion was moderate, estimated at 45% with doses of >0.6 mg/kg. Restoration of lymph node architecture was seen after two weeks to two months for all animals. All four subjects receiving BTI-322 experienced AEs on the first dosing day, while the eight subjects dosed with siplizumab experienced few mild, transient AEs. Infusion with siplizumab and BTI-322 resulted in rapid depletion of CD2+ cells in circulation and tissue. Siplizumab had a longer t1/2 and fewer AEs compared to BTI-322.

Mechanisms of Mixed Chimerism-Based Transplant Tolerance.

Immune responses to allografts represent a major barrier in organ transplantation. Immune tolerance to avoid chronic immunosuppression is a critical goal in the field, recently achieved in the clinic by combining bone marrow transplantation (BMT) with kidney transplantation following non-myeloablative conditioning. At high levels of chimerism such protocols can permit central deletional tolerance, but with a significant risk of graft-versus-host (GVH) disease (GVHD). By contrast, transient chimerism-based tolerance is devoid of GVHD risk and appears to initially depend on regulatory T cells (Tregs) followed by gradual, presumably peripheral, clonal deletion of donor-reactive T cells. Here we review recent mechanistic insights into tolerance and the development of more robust and safer protocols for tolerance induction that will be guided by innovative immune monitoring tools.

Reduced positive selection of a human TCR in a swine thymus using a humanized mouse model for xenotolerance induction.

BACKGROUND: Tolerance-inducing approaches to xenotransplantation would be optimal and may be necessary for long-term survival of transplanted pig organs in human patients. The ideal approach would generate donor-specific unresponsiveness to the pig organ without suppressing the patient's normal immune function. Porcine thymus transplantation has shown efficacy in promoting xenotolerance in humanized mice and large animal models. However, murine studies demonstrate that T cells selected in a swine thymus are positively selected only by swine thymic epithelial cells, and therefore, cells expressing human HLA-restricted TCRs may not be selected efficiently in a transplanted pig thymus. This may lead to suboptimal patient immune function. METHODS: To assess human thymocyte selection in a pig thymus, we used a TCR transgenic humanized mouse model to study positive selection of cells expressing the MART1 TCR, a well-characterized human HLA-A2-restricted TCR, in a grafted pig thymus. RESULTS: Positive selection of T cells expressing the MART1 TCR was inefficient in both a non-selecting human HLA-A2- or swine thymus compared with an HLA-A2+ thymus. Additionally, CD8 MART1 TCRbright T cells were detected in the spleens of mice transplanted with HLA-A2+ thymi but were significantly reduced in the spleens of mice transplanted with swine or HLA-A2- thymi. [Correction added on October 15, 2019, after first online publication: The missing superscript values +, -, and bright have been included in the Results section.] CONCLUSIONS: Positive selection of cells expressing a human-restricted TCR in a transplanted pig thymus is inefficient, suggesting that modifications to improve positive selection of cells expressing human-restricted TCRs in a pig thymus may be necessary to support development of a protective human T-cell pool in future patients.

Transgenic expression of human CD47 reduces phagocytosis of porcine endothelial cells and podocytes by baboon and human macrophages.

BACKGROUND: Our initial studies utilizing a galactosyl-α1-3-galactosyltransferase gene knockout (GalTKO) pig-to-baboon renal transplant model demonstrated that the early development of nephrotic syndrome has been a significant obstacle to the long-term survival of baboon recipients. We have recently documented that sphingomyelin phosphodiesterase-3 (SMPDL3b) and CD80 expressed on podocytes in porcine kidney grafts contribute to this complication. We have hypothesized that one regulator of immune function is CD47 and that incompatibilities in CD47 between pig and baboon could potentially affect macrophage function, increasing the susceptibility of the kidney grafts to immunologically induced injury. METHODS: In order to address this hypothesis in vitro, we isolated and cultured porcine podocytes and ECs from GalTKO alone, human CD47 (hCD47)/hCD55 expressing transgenic (Tg) GalTKO swine, and GalTKO hCD46/hCD55 Tg swine along with baboon or human macrophages. RESULTS: We found that baboon macrophages phagocytosed porcine ECs in a similar manner to human macrophages, and this response was significantly reduced when porcine ECs and podocytes expressed hCD47/hCD55 but not hCD46/hCD55 without hCD47. Furthermore, masking hCD47 by anti-hCD47 antibody on hCD47/hCD55Tg ECs restored phagocytosis. These results are consistent with the hypothesis that CD47 incompatibility plays an important role in promoting macrophage phagocytosis of endogenous cells from the transplanted kidney. CONCLUSIONS: The similar levels of phagocytosis of porcine cells by baboon and human macrophages suggest that the expression of hCD47Tg on glomerular cells in donor porcine kidneys may prove to be a key strategy for preventing proteinuria following kidney xenotransplantation in a pig-to-human as well as a pig-to-baboon model.

Intra-bone bone marrow transplantation from hCD47 transgenic pigs to baboons prolongs chimerism to >60 days and promotes increased porcine lung transplant survival.

BACKGROUND: We have recently demonstrated that human-CD47 (hCD47) expressed on endothelial cells of porcine lung xenografts extended median graft survival from 3.5 days to 8.7 days in baboons. Intra-bone bone marrow transplantation (IBBMTx) in a pig-to-baboon model was previously shown to markedly prolong the duration of macrochimerism up to 21 days from 1 to 4 days by intravenous BMTx. We now examined whether the use of hCD47 transgenic (Tg) BM further prolonged the duration of chimerism following IBBMTx. We then tested if lung xenograft survival was prolonged following IBBMTx. METHODS: Baboons received GalTKO-hCD47/hCD55Tg (n = 5) or -hCD55Tg (n = 1) or -hCD46/HLA-E Tg (n = 1) pig IBBMTx. Macrochimerism, anti-pig T cells and antibody responses were assessed. Animals received lung xenografts from either hCD47+ or hCD47- porcine lungs 1-3 months later. RESULTS: All baboons that received hCD47Tg porcine IBBM maintained durable macrochimerism >30 days, and two maintained chimerism for >8 weeks. Notably, anti-pig antibody levels decreased over time and anti-pig cellular unresponsiveness developed following IBBMTx. Lungs from hCD47Tg IBBMTx matched pigs were transplanted at day 33 or day 49 after IBBMTx. These animals showed extended survival up to 13 and 14 days, while animals that received lungs from hCD47 negative pigs displayed no prolonged survival (1-4 days). CONCLUSION: This is the first report demonstrating durable macrochimerism beyond 8 weeks, as well as evidence for B cell tolerance in large animal xenotransplantation. Using hCD47Tg pigs as both IBBMTx and lung donors prolongs lung xenograft survival. However, additional strategies are required to control the acute loss of lung xenografts.

Safety and pharmacodynamics of anti-CD2 monoclonal antibody treatment in cynomolgus macaques - an experimental study.

Anti-CD2 treatment provides targeted immunomodulatory properties that have demonstrated clinical usefulness to condition the immune system and to treat transplant rejection. The treatment is species-specific due to structural CD2 antigen differences between nonhuman primates and humans. Herein, we report the safety profile and efficacy of two modifications of the same anti-CD2 monoclonal antibody in cynomolgus macaques. Twelve subjects received one i.v. anti-CD2 (of rat or rhesus type) dose each, range 1-4 mg/kg, and were followed for 1-7 days. Treatment effects were evaluated with flow cytometry on peripheral blood and histopathological evaluation of secondary lymphoid organs. In vitro inhibitory activity on primary MHC disparate mixed lymphocyte reactions (MLRs) was determined. Upon anti-CD2 treatment, CD4+ , CD8+ memory subsets were substantially depleted. Naïve T cells and Tregs were relatively spared and exhibited lower CD2 expression than memory T cells. Early immune reconstitution was noted for naïve cells, while memory counts had not recovered after one week. Both antibodies displayed a concentration-dependent MLR inhibition. Lymph node examination revealed no significant lymphocyte depletion. None of the animals experienced any significant study drug-related adverse events. This study outlines the safety and pharmacodynamic profile of primate-specific anti-CD2 treatment, relevant for translation of anti-CD2-based animal models into clinical trials.