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Reliable in vitro expansion protocols of regulatory T cells (Tregs) are needed for clinical use. We studied the biology of Mauritian Cynomolgus macaque (MCM) Tregs and developed four in vitro Treg expansion protocols for translational studies. Tregs expanded 3000-fold when artificial antigen presenting cells (aAPCs) expressing human CD80, CD58 and CD32 were used throughout the culture. When donor peripheral blood mononuclear cells (PBMCs) were used as the single source of APCs followed by aAPCs, Tregs expanded 2000-fold. Tregs from all protocols suppressed the proliferation of anti-CD2CD3CD28 bead-stimulated autologous PBMCs albeit with different potencies, varying from 1:2-1:4 Treg:PBMC ratios, up to >1:32. Reculture of cryopreserved Tregs permitted reexpansion with improved suppressive activity. Occasionally, CD8 contamination was observed and resolved by resorting. Specificity studies showed greater suppression of stimulation by anti-CD2CD3CD28 beads of PBMCs from the same donor used for stimulation during the Treg cultures and of autologous cells than of third-party PBMC responders. Similar to humans, the Treg-specific demethylated region (TSDR) within the Foxp3 locus correlated with suppressive activity and expression of Foxp3. Contrary to humans, FoxP3 expression did not correlate with CD45RA or CD127 expression. In summary, we have characterized MCM Tregs and developed four Treg expansion protocols that can be used for preclinical applications.

This review focuses on our recent studies involving nonmyeloablative bone marrow transplantation as an approach to inducing organ allograft tolerance across MHC barriers in nonhuman primates and in patients. The clinical studies are focused on mechanisms of tolerance involved in a protocol carried out at Massachusetts General Hospital in HLA-mismatched haploidentical combinations for the induction of renal allograft tolerance. These studies, in which chimerism was only transient and GVHD did not occur, suggest an early role for donor-specific regulatory T cells in tolerance induction, followed by partial and gradual deletion of donor-reactive T cells. We utilized high-throughput sequencing methodologies in a novel way to identify and track large numbers of alloreactive T cell receptors (TCRs). This method has been shown to identify biologically significant alloreactive TCRs in transplant patients and pointed to clonal deletion as a major mechanism of long-term tolerance in these patients. More recently, we adapted this sequencing method to optimally identify the donor-specific regulatory T cell (Treg) repertoire. Interrogation of the early posttransplant repertoire demonstrated expansion of donor-specific Tregs in association with tolerance. Our studies suggest a role for the kidney graft in tolerance by these mechanisms in patients who had only transient chimerism. Nonhuman primate studies indicate that other organs, including the heart, the lungs and the liver, are less readily tolerated following a period of transient mixed chimerism. Our efforts to extend the reach of mixed chimerism for tolerance induction beyond the kidney are therefore focused on the addition of recipient Tregs to the protocol. This approach has the potential to enhance chimerism while further reducing the risk of GVHD.

NOD-SCID IL2rγcnull (NSG) mice are widely used to model human immune cell development because they are more permissive for human hematopoietic cell engraftment and reconstitution than NOD-SCID mice. While increased human reconstitution in the blood of NSG mice has been attributed to the absence of mouse NK cells, deletion of the common gamma chain (γc) limits development of lymphoid tissue inducer cells and precludes development of normal secondary lymphoid structures. The disorganized lymphoid tissue leads to compromised human T cell-B cell interactions and results in variable human immune cell function in human immune system (HIS) NSG compared to NOD-SCID mice. We attempted to remove mouse NK cells from NOD-SCID mice while retaining other γc-dependent cytokine responses by targeted disruption of the mouse genomic IL15RA locus with CRISPR/Cas9. IL15Rα is required for the development, function and survival of NK cells. NOD-SCID IL15Rα-/- mice showed reductions in NK cells and NK cell function. However, NOD-SCID IL15Rα-/- mice demonstrated accelerated thymic lymphomagenesis and showed earlier mortality compared to NOD-SCID mice. This result suggests that mouse NK cells are important to delay lymphoma development in NOD-SCID mice. We transplanted thymectomized NOD-SCID ILl5Rα-/- mice with human fetal liver CD34+ cells and thymus to determine if these mice supported engraftment and development of a transplanted HIS. Surprisingly, we found that peripheral human engraftment was inferior (mean 0.05% of lymphocytes) to that in both NOD-SCID (mean, 10.5% of lymphocytes) and NSG (mean, 54% of lymphocytes) mice. These results indicate that NOD-SCID IL15Rα-/- mice are not permissive for human CD34+ cell engraftment.

In humans receiving intestinal transplantation (ITx), long-term multilineage blood chimerism often develops. Donor T cell macrochimerism (≥4%) frequently occurs without graft-versus-host disease (GVHD) and is associated with reduced rejection. Here we demonstrate that patients with macrochimerism had high graft-versus-host (GvH) to host-versus-graft (HvG) T cell clonal ratios in their allografts. These GvH clones entered the circulation, where their peak levels were associated with declines in HvG clones early after transplant, suggesting that GvH reactions may contribute to chimerism and control HvG responses without causing GVHD. Consistently, donor-derived T cells, including GvH clones, and CD34(+) hematopoietic stem and progenitor cells (HSPCs) were simultaneously detected in the recipients’ BM more than 100 days after transplant. Individual GvH clones appeared in ileal mucosa or PBMCs before detection in recipient BM, consistent with an intestinal mucosal origin, where donor GvH-reactive T cells expanded early upon entry of recipient APCs into the graft. These results, combined with cytotoxic single-cell transcriptional profiles of donor T cells in recipient BM, suggest that tissue-resident GvH-reactive donor T cells migrated into the recipient circulation and BM, where they destroyed recipient hematopoietic cells through cytolytic effector functions and promoted engraftment of graft-derived HSPCs that maintain chimerism. These mechanisms suggest an approach to achieving intestinal allograft tolerance.

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.

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.

Summary The therapeutic applications of regulatory T cells (Tregs) include treating autoimmune diseases, graft-versus-host disease and induction of transplantation tolerance. For ex-vivo expanded Tregs to be used in deceased donor transplantation, they must be able to suppress T cell responses to a broad range of human leukocyte antigen (HLA). Here, we present a novel approach for the expansion of polyspecific Tregs in cynomolgus macaques that was adapted from a good manufacturing practice-compliant protocol. Tregs were isolated by fluorescence-activated cell sorting (FACS) and expanded in the presence of a panel of CD40L-stimulated B cells (CD40L-sBc). Prior to Treg culture, CD40L-sBc were expanded in vitro from multiple major histocompatibility complex (MHC)-disparate macaques. Expanded Tregs expressed high levels of forkhead box protein 3 (FoxP3) and Helios, a high percentage of Treg-specific demethylated region (TSDR) demethylation and strong suppression of naïve T cell responses in vitro. In addition, these Tregs produced low levels of inflammatory cytokines and were able to expand post-cryopreservation. Specificity assays confirmed that these Tregs were suppressive upon activation by any antigen-presenting cells (APCs) whose MHC was shared by CD40L-sBc used during expansion, proving that they are polyspecific. We developed an approach for the expansion of highly suppressive cynomolgus macaque polyspecific Tregs through the use of a combination of CD40L-engineered B cells with the potential to be translated to clinical studies. To our knowledge, this is the first report that uses a pool of MHC-mismatched CD40L-sBc to create polyspecific Tregs suitable for use in deceased-donor transplants.

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 β 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 (TGFβ) 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.

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), β-1,4-N-acetyl-galactosaminyl transferase 2 (B4) KO pigs can cause rejection of xenografts in baboons. METHODS: Five baboons that had 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.

During T cell development in mice, thymic negative selection deletes cells with the potential to recognize and react to self-antigens. In human T cell-dependent autoimmune diseases such as Type 1 diabetes, multiple sclerosis, and rheumatoid arthritis, T cells reactive to autoantigens are thought to escape negative selection, traffic to the periphery and attack self-tissues. However, physiological thymic negative selection of autoreactive human T cells has not been previously studied. We now describe a human T-cell receptor-transgenic humanized mouse model that permits the study of autoreactive T-cell development in a human thymus. Our studies demonstrate that thymocytes expressing the autoreactive Clone 5 TCR, which recognizes insulin B:9–23 presented by HLA-DQ8, are efficiently negatively selected at the double and single positive stage in human immune systems derived from HLA-DQ8+ HSCs. In the absence of hematopoietic expression of the HLA restriction element, negative selection of Clone 5 is less efficient and restricted to the single positive stage. To our knowledge, these data provide the first demonstration of negative selection of human T cells recognizing a naturally-expressed tissue-restricted antigen. Intrathymic antigen presenting cells are required to delete less mature thymocytes, while presentation by medullary thymic epithelial cells may be sufficient to delete more mature single positive cells. These observations set the stage for investigation of putative defects in negative selection in human autoimmune diseases., Highlights • In the presence of the HLA-restriction element on hematopoietic stem cells (HSCs) and thymus.•Thymocytes bearing insulin peptide-reactive TCRs express markers of thymic negative selection.•Insulin peptide-reactive T cells are efficiently negatively selected in the human thymus.•The few transgenic T cells that escape negative selection largely express endogenous TCRs. • HLA-restriction element on HSCs is required for efficient negative selection.

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.

This review focuses on mechanistic studies performed in recipients of non-myeloablative bone marrow transplant regimens developed at Massachusetts General Hospital in HLA-identical and HLA-mismatched haploidentical combinations, initially as a platform for treatment of hematologic malignancies with immunotherapy in the form of donor leukocyte infusions, and later in combination with donor kidney transplantation for the induction of allograft tolerance. These studies point to a prominent early role for regulatory T cells in tolerance induction, followed by partial and gradual deletion of donor-reactive T cells. In patients with permanent mixed chimerism, central deletion may be a major mechanism of long-term tolerance. In patients in whom donor chimerism is only transient, the kidney itself plays a significant role in maintaining long-term tolerance. A high throughput sequencing approach to identifying and tracking a significant portion of the alloreactive T cell receptor repertoire has demonstrated biological significance in transplant patients and has been useful in pointing to clonal deletion as a long-term tolerance mechanism in recipients of HLA-mismatched combined kidney and bone marrow transplants with only transient chimerism.

This review describes recent progress in tolerance-inducing strategies across xenogeneic immunological barriers as well as the potential benefit of a tolerance strategy for islets and kidney xenotransplantation.Using advanced gene editing technologies, xenotransplantation from multitransgenic alpha-1,3-galactosyltransferase knockout pigs has demonstrated marked prolongation of renal xenograft survival, ranging from days to greater than several months for life-supporting kidneys, and more than 2 years in a heterotopic nonlife-supporting cardiac xenograft model. Continuous administration of multiple immunosuppressive drugs has been required and attempts to taper immunosuppression have been unsuccessful. It appears likely that low levels of T cell dependent antibodies and activation of innate responses are responsible for xenograft loss. Mixed chimerism and thymic transplantation approaches have achieved xenogeneic tolerance in pig-to-mouse models and both have recently been extended to pig-to-baboon models. Encouraging results have been reported, including persistence of macrochimerism, prolonged pig skin graft survival, donor-specific unresponsiveness in vitro and detection of recent T cell emigrants in vivo.Although tolerance induction in vivo has not yet been achieved in pig-to-baboon models, recent results are encouraging that this goal will be attainable through genetic engineering of porcine donors.

Little is known about innate lymphoid cell (ILC) populations in the human gut, and the turnover of these cells and their subsets after transplantation has not been described.Intestinal samples were taken from 4 isolated intestine and 3 multivisceral transplant recipients at the time of any operative resection, such as stoma closure or revision. ILCs were isolated and analyzed by flow cytometry. The target population was defined as being negative for lineage markers and double-positive for CD45/CD127. Cells were further stained to define ILC subsets and a donor-specific or recipient-specific HLA marker to analyze chimerism.Donor-derived ILCs were found to persist greater than 8 years after transplantation. Additionally, the percentage of cells thought to be lymphoid tissue inducer cells among donor ILCs was far higher than that among recipient ILCs.Our findings demonstrate that donor-derived ILCs persist long-term after transplantation and support the notion that human lymphoid tissue inducer cells may form in the fetus and persist throughout life, as hypothesized in rodents. Correlation between chimerism and rejection, graft failure, and patient survival requires further study.

There is an urgent and unmet need for humanized in vivo models of type 1 diabetes to study immunopathogenesis and immunotherapy, and in particular antigen-specific therapy. Transfer of patient blood lymphocytes to immunodeficient mice is associated with xenogeneic graft-versus-host reactivity that complicates assessment of autoimmunity. Improved models could identify which human T cells initiate and participate in beta-cell destruction and help define critical target islet autoantigens. We used humanized mice (hu-mice) containing robust human immune repertoires lacking xenogeneic graft-versus-host reactivity to address this question. Hu-mice constructed by transplantation of HLA-DQ8+ human fetal thymus and CD34+ cells into HLA-DQ8-transgenic immunodeficient mice developed hyperglycemia and diabetes after transfer of autologous HLA-DQ8/insulin-B:9-23 (InsB:9-23)-specific T-cell receptor (TCR)-expressing human CD4+ T cells and immunization with InsB:9-23. Survival of the infused human T cells depended on the preexisting autologous human immune system, and pancreatic infiltration by human CD3+ T cells and insulitis were observed in the diabetic hu-mice, provided their islets were stressed by streptozotocin. This study fits Koch's postulate for pathogenicity, demonstrating a pathogenic role of islet autoreactive CD4+ T-cell responses in type 1 diabetes induction in humans, underscores the role of the target beta-cells in their immunological fate, and demonstrates the capacity to initiate disease with T cells, recognizing the InsB:9-23 epitope in the presence of islet inflammation. This preclinical model has the potential to be used in studies of the pathogenesis of type 1 diabetes and for testing of clinically relevant therapeutic interventions.

Alloimmune T cells are central mediators of rejection and graft-versus-host disease in both solid organ and hematopoietic stem cell transplantation. Unique among immune responses in terms of its strength and diversity, the T cell alloresponse reflects extensive genetic polymorphisms between allogeneic donors and recipients, most prominently within the major histocompatibility complex (MHC), which encodes human leukocyte antigens (HLAs) in humans. The repertoire of alloreactive T cell clones is distinct for every donor-recipient pair and includes potentially thousands of unique HLA/peptide specificities. The extraordinary magnitude of the primary alloresponse and diversity of the T cell population mediating it have presented technical challenges to its study in humans. High-throughput T cell receptor sequencing approaches have opened up new possibilities for tackling many fundamental questions about this important immunologic phenomenon.

The success of organ transplantation is limited by the complications of immunosuppression, by chronic rejection, and by the insufficient organ supply, and thousands of patients die every year while waiting for a transplant. With recent progress in xenotransplantation permitting porcine organ graft survival of months or even years in nonhuman primates, there is renewed interest in its potential to alleviate the organ shortage. Many of these advances are the result of our heightened capacity to modify pigs genetically, particularly with the development of CRISPR-Cas9-based gene editing methodologies. Although this approach allows the engineering of pig organs that are less prone to rejection, the clinical application of xenotransplantation will require the ability to avoid the ravages of a multifaceted attack on the immune system while preserving the capacity to protect both the recipient and the graft from infectious microorganisms. In this review, we will discuss the potential and limitations of these modifications and how the engineering of the graft can be leveraged to alter the host immune response so that all types of immune attack are avoided.

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.

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.

We studied human T cell repertoire formation using high-throughput T cell receptor β (TCRβ) complementarity-determining region 3 (CDR3) sequencing in immunodeficient mice receiving human hematopoietic stem cells (HSCs) and human thymus grafts. Replicate humanized mice generated diverse and highly divergent repertoires. We observed repertoire narrowing and increased CDR3β sharing during thymocyte selection. Whereas hydrophobicity analysis implicated self-peptides in positive selection of the overall repertoire, positive selection favored shorter shared sequences that had reduced hydrophobicity at positions 6 and 7 of CDR3βs, suggesting weaker interactions with self-peptides than were observed with unshared sequences, possibly allowing escape from negative selection. Sharing was similar between autologous and allogeneic thymi and occurred between different cell subsets. Shared sequences were enriched for allo–cross-reactive CDR3βs and for type 1 diabetes–associated autoreactive CDR3βs. Single-cell TCR sequencing showed increased sharing of CDR3αs compared with CDR3βs between mice. Our data collectively implicate preferential positive selection for shared human CDR3βs that are highly cross-reactive. Although previous studies suggested a role for recombination bias in producing “public” sequences in mice, our study is the first to our knowledge to demonstrate a role for thymic selection. Our results implicate positive selection for promiscuous TCRβ sequences that probably evade negative selection, given their low affinity for self-ligands, in the abundance of “public” human TCRβ sequences.

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.

Background We have achieved greater than a 6-month survival of a life-supporting kidney co-transplanted with a vascularized thymic graft into non-human primates (NHPs). Although we have achieved pig-specific unresponsiveness in vitro, immunosuppression was not able to be fully weaned. Studies in mice and humanized mice suggest that a hybrid pig thymus (Hyb-thy)-containing host thymic epithelial cells (TECs) can optimize intra-thymic selection, achieving xenograft tolerance with improved reconstitution of T-cell function. Methods We have tested the feasibility of the preparation of a Hyb-thy that contains NHP TECs in the donor thymic grafts. We first prepared the Hyb-thy in the donor pigs 2-3 weeks before xeno-Tx. We performed six cases of Hyb-thy preparation in six juvenile miniature swine. Two pigs received non-manipulated cynomolgus monkey thymic cells that were isolated from an excised atrophic thymus via injection into their thymic lobes (Group 1). The remaining four received thymic cells that were isolated from non-atrophic thymic glands (Groups 2 and 3). Pigs in Group 2 received unmanipulated thymic cells in one thymic lobe, as well as CD2-positive cell-depleted TEC-enriched cells in the contralateral lobe. Pigs in Group 3 received TEC-enriched cells alone. Results All thymus-injected pigs received tacrolimus and rapamycin until endpoint (POD16). We detected cynomolgus monkey TEC networks in pig thymus from Groups 1 and 3, while pigs in Group 2 rejected the thymic cells. We demonstrated the preparation of Hyb-thy in pigs using tacrolimus plus rapamycin therapy. Conclusions Our results suggest that the enrichment of TEC from the excised NHP thymus facilitated NHP TEC engraftment in pig thymus.

Tissue-resident memory T cells (TRM) maintain immunity in diverse sites as determined in mouse models, whereas their establishment and role in human tissues have been difficult to assess. Here, we investigated human lung TRM generation, maintenance, and function in airway samples obtained longitudinally from human leukocyte antigen (HLA)-disparate lung transplant recipients, where donor and recipient T cells could be localized and tracked over time. Donor T cells persist specifically in the lungs (and not blood) of transplant recipients and express high levels of TRM signature markers including CD69, CD103, and CD49a, whereas lung-infiltrating recipient T cells gradually acquire TRM phenotypes over months in vivo. Single-cell transcriptome profiling of airway T cells reveals that donor T cells comprise two TRM-like subsets with varying levels of expression of TRM-associated genes, whereas recipient T cells comprised non-TRM and similar TRM-like subpopulations, suggesting de novo TRM generation. Transplant recipients exhibiting higher frequencies of persisting donor TRM experienced fewer adverse clinical events such as primary graft dysfunction and acute cellular rejection compared with recipients with low donor TRM persistence, suggesting that monitoring TRM dynamics could be clinically informative. Together, our results provide spatial and temporal insights into how human TRM develop, function, persist, and affect tissue integrity within the complexities of lung transplantation.

BACKGROUND: 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 non-human primates and humans. Herein, we report the safety profile and efficacy of two modifications of the same anti-CD2 monoclonal antibody in cynomolgus macaques. METHODS: 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 MLRs was determined. RESULTS: 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. CONCLUSIONS: 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.

Specific immune tolerance of transplanted organs in association with either transient or sustained lymphohematopoietic chimerism has been demonstrated in several preclinical animal models and clinically in patients who are full donor chimeras after hematopoietic stem cell transplantation and subsequently received kidney transplants from the same donor. Most recently, tolerance induction has been extended to patients in whom chimerism was intentionally induced at the time of kidney transplantation.Twenty years ago, we reported the first successful histocompatibility leukocyte antigen-matched sibling donor bone marrow and kidney transplant following nonmyeloablative conditioning in a patient with multiple myeloma and end-stage renal disease (ESRD). After 2 decades, she has normal renal function in the absence of ongoing systemic immunosuppressive therapy. Nine patients have subsequently undergone similar treatment for multiple myeloma with ESRD.In the initial patient, hematopoietic chimerism was detectable for only 105 days after the transplant. In subsequent patients, chimerism detection ranged from 49 days to14 years. Nevertheless, a long remission of the myeloma and long-term immunosuppression-free survival of the kidney allograft were achieved in 7 of the 10 patients, 5 of whom currently survive.This initial patient demonstrated the feasibility of performing combined histocompatibility leukocyte antigen-matched, sibling donor bone marrow and kidney transplantation for ESRD due to multiple myeloma. This experience paved the way for extending the initial trial to 9 additional patients with multiple myeloma and ESRD and, more recently, to tolerance induction strategies involving combined bone marrow and kidney transplantation for patients with and without an underlying malignancy.

The currently available human tumor xenograft models permit modeling of human cancers in vivo, but in immunocompromised hosts. Here we report a humanized mouse (hu-mouse) model made by transplantation of human fetal thymic tissue plus hematopoietic stem cells transduced with a leukemia-associated fusion gene MLL-AF9. In addition to normal human lymphohematopoietic reconstitution as seen in non-leukemic hu-mice, these hu-mice showed spontaneous development of B-cell acute lymphoblastic leukemia (B-ALL), which was transplantable to secondary recipients with an autologous human immune system. Using this model, we show that lymphopenia markedly improves the antitumor efficacy of recipient leukocyte infusion (RLI), a GVHD-free immunotherapy that induces antitumor responses in association with rejection of donor chimerism in mixed allogeneic chimeras. Our data demonstrate the potential of this leukemic hu-mouse model in modeling leukemia immunotherapy, and suggest that RLI may offer a safe treatment option for leukemia patients with severe lymphopenia., Highlights • NSG mice grafted with thymus/oncogenic HSC develop human immune system and leukemia. • Leukemia transfer to mice with autologous immunity suffices to model immunotherapy. • Lymphopenia enhances RLI-mediated HVGR and anti-leukemia activity in mixed chimeras. This study establishes a humanized mouse model with human immunity and autologous leukemia. Using this model, the authors demonstrate that lymphopenia promotes the rejection of donor hematopoietic chimerism and the associated anti-leukemia response by recipient leukocyte infusion in mixed allogeneic chimeras.

Alloreactive T lymphocytes are the primary mediators of allograft rejection. The size and diversity of the HLA-alloreactive T cell repertoire has thus far precluded the ability to follow these T cells and thereby to understand their fate in human transplant recipients. This review summarizes the history, challenges, and recent advances in the study of alloreactive T cells. We highlight the historical development of assays to measure alloreactivity and discuss how high-throughput T cell receptor (TCR) sequencing-based assays can provide a new window into the fate of alloreactive T cells in human transplant recipients. A specific approach combining a classical in vitro assay, the mixed lymphocyte reaction, with deep T cell receptor sequencing is described as a tool to track the donor-reactive T cell repertoire for any specific HLA-mismatched donor-recipient pair. This assay can provide mechanistic insights and has potential as a non-invasive, highly specific biomarker for rejection and tolerance.

Biologic or biomechanical therapy capable of replacing the β cell mass has the potential to positively impact the health and well being of millions of people with insulindependent diabetes. Research in this area stands at a pivotal moment at which a number of viable strategies exist or are under development. Broad application depends on achieving both technical and financial feasibility. The ultimate goal of a "true cure," in which diabetic individuals achieve euglycemia with a single procedure associated with minimal risk, without long-termtoxic drugs, and unfettered by external devices and/or frequent monitoring, appears to still be some years away. However, dramatic progress has been achieved toward themore proximate objectives of improved glycemic control and elimination of hypoglycemia and long-term vascular complications. Long-term whole organ pancreas and isolated islet results have improved significantly over the last decade,with the latter now approaching the success of the former in insulin independence rates at the 5-year mark. It seems likely that allogeneic pancreas and islet transplantation will remain a treatment of choice for the foreseeable future in kidney recipients already obligated to lifelong immunosuppression until a more complete and permanent restoration of euglycemia is available. Nascent tolerance promoting protocols could aid in improving the risk-to-benefit balance for both islets and whole organ pancreas. With the present supply of transplantable pancreases used optimally, nomore than 13% of the annual incident cases of T1D can be cured. In practical terms though, today, fewer than 5% of the annual incident cases are transplanted. The reality of the limited supply of deceased donor organs ultimately constrains the impact of islet and pancreas transplantation and compels researchers to press forward to develop broader strategies such as the AP, xenogeneic islets, and stem cell-derived β cells for which the supply will be limitless; in these areas, recent progress has been most impressive. The AP continues to be refined withmore sophisticated delivery algorithms, improved sensors and exploration of mobile device control. For xenogeneic islets, dramatic progress is evident in the long-term survival of porcine islets in primates using genetically modified donors and/or improved biologic immunosuppressants. Microencapsulation and macroencapsulation devices that exclude direct immunity by physical means may further aid in fostering xenogeneic islet graft survival but will likely find their primary place in the containment and protection of early versions of stem cell-derived allogeneic β cells. Deriving functional β cells from stem cells has experienced the most celebrated recent advances. Improved differentiation protocols that permit large scale/ unlimited production of IPCs are nowavailable, and although "normal" β-cell function has not yet been achieved, the ever quickening pace of progress suggests they are not far off. Importantly, this therapeutic modality will ultimately need to confront the likely requirement for a containment device and the need to be retransplanted periodically. These blemishes notwithstanding, the tremendous perceived potential of the approach for clinical application is evident in the huge venture capital investment that was rapidly garnered after the report of the most recent advance in embryonic stem cell differentiation into proper β cells. Consistent with the informal survey we conducted, iPS-derived β cells, which currently suffer from regulatory hurdles and the lack of a viable business model, and the seemingly more remote regeneration of native β cells,may offer the ultimate chance for a personalized true cure of insulin-dependent diabetes by avoidance of barrier devices and toxic immunosuppressive drugs. The research agenda we have detailed is designed to facilitate full exploration of the potential of each proposed β-cell replacement solution so the optimal therapy is advanced as quickly as possible. Success in this endeavor will require broad and deep financial support from philanthropic (JDRF, Diabetes Research and Wellness Foundation, ADA, and so on) and public funding agencies worldwide; the investment needed is large but the potential reward will be profound. It is imperative that high impact, scientifically sound approaches are not overwhelmed by industry, private, or venture capital-supported priorities just because they hold a more lucrative near-term business model; scientific merit should dictate the course. The adherence of the historical funding agencies to traditional peer-reviewed methodology will be the incubator of novel approaches. This is a rapidly evolving landscape, and new data and novel ideas may radically divert the path forward. However, the diverse recent progress is tangible and undeniable, and the next decade is bound to witness a fascinating unfolding of competing solutions to cure insulin-dependent diabetes. Our assessment of the data presented creates the opportunity for IPITA/TTS to endorse the following broad agenda for specific support by the peer-reviewed agencies.

Alloreactive T lymphocytes are the primary mediators of immune responses in transplantation, both in the graft-versus-host and host-versus-graft directions. While essentially all clones comprising the human T cell repertoire have been selected on self-peptide presented by self–human leukocyte antigens (self-HLAs), much remains to be understood about the nature of clones capable of responding to allo-HLA molecules. Quantitative tools to study these cells are critical to understand fundamental features of this important response; however, the large size and diversity of the alloreactive T cell repertoire in humans presents a great technical challenge. We have developed a high-throughput T cell receptor (TCR) sequencing approach to characterize the human alloresponse. We present a statistical method to model T cell clonal frequency distribution and quantify repertoire diversity. Using these approaches, we measured the diversity and frequency of distinct alloreactive CD4+ and CD8+ T cell populations in HLA-mismatched responder-stimulator pairs. Our findings indicate that the alloimmune repertoire is highly specific for a given pair of individuals, that most alloreactive clones circulate at low frequencies, and that a high proportion of TCRs is likely able to recognize alloantigens.

Summary The specific cellular physiology of hematopoietic stem cells (HSCs) is underexplored, and their maintenance in vitro remains challenging. We discovered that culture of HSCs in low calcium increased their maintenance as determined by phenotype, function, and single-cell expression signature. HSCs are endowed with low intracellular calcium conveyed by elevated activity of glycolysis-fueled plasma membrane calcium efflux pumps and a low-bone-marrow interstitial fluid calcium concentration. Low-calcium conditions inhibited calpain proteases, which target ten-eleven translocated (TET) enzymes, of which TET2 was required for the effect of low calcium conditions on HSC maintenance in vitro. These observations reveal a physiological feature of HSCs that can be harnessed to improve their maintenance in vitro.

Emerging data suggest that hypercholesterolemia has stimulatory effects on adaptive immunity and that these effects can promote atherosclerosis and perhaps other inflammatory diseases. However, research in this area has relied primarily on inbred strains of mice whose adaptive immune system can differ substantially from that of humans. Moreover, the genetically induced hypercholesterolemia in these models typically results in plasma cholesterol levels that are much higher than those in most humans. To overcome these obstacles, we studied human immune system-reconstituted mice (hu-mice) rendered hypercholesterolemic by treatment with adeno-associated virus 8-proprotein convertase subtilisin/kexin type 9 (AAV8-PCSK9) and a high-fat/high-cholesterol Western-type diet (WD). These mice had a high percentage of human T cells and moderate hypercholesterolemia. Compared with hu-mice that had lower plasma cholesterol, the PCSK9-WD mice developed a T cell-mediated inflammatory response in the lung and liver. Human CD4+ and CD8+ T cells bearing an effector memory phenotype were significantly elevated in the blood, spleen, and lungs of PCSK9-WD hu-mice, whereas splenic and circulating regulatory T cells were reduced. These data show that moderately high plasma cholesterol can disrupt human T cell homeostasis in vivo. This process may not only exacerbate atherosclerosis, but also contribute to T cell-mediated inflammatory diseases in the hypercholesterolemia setting.

The specific cellular physiology of hematopoietic stem cells (HSCs) is underexplored and their maintenance in vitro remains challenging. We discovered that culture of HSC in low calcium increased their maintenance as determined by phenotype, function and single cell expression signature. HSCs are endowed with low intracellular calcium conveyed by elevated activity of glycolysis-fueled plasma membrane calcium efflux pumps and a low bone marrow interstitial fluid calcium concentration. HSC maintenance in low calcium conditions was mediated by inhibition of calpain proteases. Consistent with the fact that TET enzymes are targets of calpains, low calcium increased TET protein expression and 5-hydroxymethyl cytosine deposition and maintained TET2-asssociated single cell gene signature. Deletion of the TET homologue required for normal HSC function and identity, TET2, rendered HSC impervious to the effect low calcium conditions. These observations reveal a physiological feature of HSCs than can be harnessed to improve their maintenance in vitro.

Biomarkers of transplant tolerance would enhance the safety and feasibility of clinical tolerance trials and potentially facilitate management of patients receiving immunosuppression. To this end, we examined blood from spontaneously tolerant renal transplant recipients and patients enrolled in two interventional tolerance trials using flow cytometry and gene expression profiling. Using a previously reported tolerant cohort as well as newly identified tolerant patients, we confirmed our previous finding that tolerance was associated with increased expression of B cell-associated genes relative to immunosuppressed patients. This was not accounted for merely by an increase in total B cell numbers, but was associated with the increased frequencies of transitional and naïve B cells. Moreover, serial measurements of gene expression demonstrated that this pattern persisted over several years, although patients receiving immunosuppression also displayed an increase in the two most dominant tolerance-related B cell genes, IGKV1D-13 and IGLL-1, over time. Importantly, patients rendered tolerant via induction of transient mixed chimerism, and those weaned to minimal immunosuppression, showed similar increases in IGKV1D-13 as did spontaneously tolerant individuals. Collectively, these findings support the notion that alterations in B cells may be a common theme for tolerant kidney transplant recipients, and that it is a useful monitoring tool in prospective trials.

Background Despite recent progress in survival times of xenografts in non-human primates, there are no reports of survival beyond 5 days of histologically well-aerated porcine lung grafts in baboons. Here, we report our initial results of pig-to-baboon xeno-lung transplantation (XLTx). Methods Eleven baboons received genetically modified porcine left lungs from either GalT-KO alone (n = 3), GalT-KO/humanCD47(hCD47)/hCD55 (n = 3), GalT-KO/hD47/hCD46 (n = 4), or GalT-KO/hCD39/hCD46/hCD55/TBM/EPCR (n = 1) swine. The first 2 XLTx procedures were performed under a non-survival protocol that allowed a 72-hour follow-up of the recipients with general anesthesia, while the remaining 9 underwent a survival protocol with the intention of weaning from ventilation. Results Lung graft survivals in the 2 non-survival animals were 48 and >72 hours, while survivals in the other 9 were 25 and 28 hours, at 5, 5, 6, 7, >7, 9, and 10 days. One baboon with graft survival >7 days, whose entire lung graft remained well aerated, was euthanized on POD 7 due to malfunction of femoral catheters. hCD47 expression of donor lungs was detected in both alveoli and vessels only in the 3 grafts surviving >7, 9, and 10 days. All other grafts lacked hCD47 expression in endothelial cells and were completely rejected with diffuse hemorrhagic changes and antibody/complement deposition detected in association with early graft loss. Conclusions To our knowledge, this is the first evidence of histologically viable porcine lung grafts beyond 7 days in baboons. Our results indicate that GalT-KO pig lungs are highly susceptible to acute humoral rejection and that this may be mitigated by transgenic expression of hCD47.

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.

Background Previous studies identified B cell gene signatures and predominance of specific B cell subsets as a marker of operational tolerance after kidney transplantation. These findings suggested a role for B cells in the establishment or maintenance of tolerance. Here we analyzed B cell recovery in 4 subjects, 3 of whom achieved tolerance after combined kidney/bone marrow transplantation. Methods Peripheral B cell subsets were examined longitudinally by flow cytometry. Immunoglobulin heavy chain repertoire analysis was performed using next-generation sequencing. Lastly, the patients' serum reactivity to HLA was assessed by Luminex. Results B cell counts recovered approximately 1 year posttransplant except for 1 subject who experienced delayed reconstitution. This subject resumed immunosuppression for acute rejection at 10 months posttransplant and underwent preemptive retransplantation at 3 years for chronic rejection. B cell recovery was accompanied by a high frequency of CD20 + CD24CD38 transitional B cells and a diversified clonal repertoire. However, all 4 subjects showed prevalence of CD20 + CD27+ memory B cells around 6 months posttransplant when B cell counts were still low and the clonal B cell repertoire very limited. The predominance of memory B cells was also associated with high levels of somatically mutated immunoglobulin heavy chain variable sequences and transient serum reactivity to HLA. Conclusions Our observations reveal the presence of memory B cells early posttransplant that likely escaped the preparative regimen at a time consistent with the establishment of tolerance. Further studies are warranted to characterize the functional properties of these persisting memory cells and evaluate their potential contribution to tolerance induction.