Advancing mouse models for transplantation research.

Mouse models have been instrumental in understanding mechanisms of transplant rejection and tolerance, but cross-study reproducibility and translation of experimental findings into effective clinical therapies are issues of concern. The Mouse Models in Transplantation symposium gathered scientists and physician-scientists involved in basic and clinical research in transplantation to discuss the strengths and limitations of mouse transplant models and strategies to enhance their utility. Participants recognized that increased procedure standardization, including the use of prespecified, defined endpoints, and statistical power analyses, would benefit the field. They also discussed the generation of new models that incorporate environmental and genetic variables affecting clinical outcomes as potentially important. If implemented, these strategies are expected to improve the reproducibility of mouse studies and increase their translation to clinical trials and, ideally, new Food and Drug Administration-approved drugs.

Single-Cell-Based High-Throughput Ig and TCR Repertoire Sequencing Analysis in Rhesus Macaques.

Recent advancements in microfluidics and high-throughput sequencing technologies have enabled recovery of paired H and L chains of Igs and VDJ and VJ chains of TCRs from thousands of single cells simultaneously in humans and mice. Despite rhesus macaques being one of the most well-studied model organisms for the human adaptive immune response, high-throughput single-cell immune repertoire sequencing assays are not yet available due to the complexity of these polyclonal receptors. We used custom primers that capture all known rhesus macaque Ig and TCR isotypes and chains that are fully compatible with a commercial solution for single-cell immune repertoire profiling. Using these rhesus-specific assays, we sequenced Ig and TCR repertoires in >60,000 cells from cryopreserved rhesus PBMCs, splenocytes, and FACS-sorted B and T cells. We were able to recover every Ig isotype and TCR chain, measure clonal expansion in proliferating T cells, and pair Ig and TCR repertoires with gene expression profiles of the same single cells. Our results establish the ability to perform high-throughput immune repertoire analysis in rhesus macaques at the single-cell level.

Xenorecognition and costimulation of porcine endothelium-derived extracellular vesicles in initiating human porcine-specific T cell immune responses.

Porcine vascular endothelial cells (PECs) form a mechanistic centerpiece of xenograft rejection. Here, we determined that resting PECs release swine leukocyte antigen class I (SLA-I) but not swine leukocyte antigen class-II DR (SLA-DR) expressing extracellular vesicles (EVs) and investigated whether these EVs proficiently initiate xenoreactive T cell responses via direct xenorecognition and costimulation. Human T cells acquired SLA-I+ EVs with or without direct contact to PECs, and these EVs colocalized with T cell receptors. Although interferon gamma-activated PECs released SLA-DR+ EVs, the binding of SLA-DR+ EVs to T cells was sparse. Human T cells demonstrated low levels of proliferation without direct contact to PECs, but marked T cell proliferation was induced following exposure to EVs. EV-induced proliferation proceeded independent of monocytes/macrophages, suggesting that EVs delivered both a T cell receptor signal and costimulation. Costimulation blockade targeting B7, CD40L, or CD11a significantly reduced T cell proliferation to PEC-derived EVs. These findings indicate that endothelial-derived EVs can directly initiate T cell-mediated immune responses, and suggest that inhibiting the release of SLA-I EVs from organ xenografts has the potential to modify the xenograft rejection. We propose a secondary-direct pathway for T cell activation via xenoantigen recognition/costimulation by endothelial-derived EVs.

A comparison of deprivation indices and application to transplant populations.

The choice of deprivation index can influence conclusions drawn regarding the extent of deprivation within a community and the identification of the most deprived communities in the United States. This study aimed to determine the degree of correlation among deprivation indices commonly used to characterize transplant populations. We used a retrospective cohort consisting of adults listed for liver or kidney transplants between 2008 and 2018 to compare 4 deprivation indices: neighborhood deprivation index, social deprivation index (SDI), area deprivation index, and social vulnerability index. Pairwise correlation between deprivation indices by transplant referral regions was measured using Spearman correlations of population-weighted medians and upper quartiles. In total, 52 individual variables were used among the 4 deprivation indices with 25% overlap. For both organs, the correlation between the population-weighted 75th percentile of the deprivation indices by transplant referral region was highest between SDI and social vulnerability index (liver and kidney, 0.93) and lowest between area deprivation index and SDI (liver, 0.19 and kidney, 0.15). The choice of deprivation index affects the applicability of research findings across studies examining the relationship between social risk and clinical outcomes. Appropriate application of these measures to transplant populations requires careful index selection based on the intended use and included variable relevance.

American Society of Transplant Surgeons-American Society of Transplantation report of FDA meeting on regulatory expectations for xenotransplantation products.

In June 2022, the US Food and Drug Administration Center for Biologics Evaluation and Research held the 73rd meeting of the Cellular, Tissue, and Gene Therapies Advisory Committee for public discussion of regulatory expectations for xenotransplantation products. The members of a joint American Society of Transplant Surgeons/American Society of Transplantation committee on xenotransplantation compiled a meeting summary focusing on 7 topics believed to be key by the committee: (1) preclinical evidence supporting progression to a clinical trial, (2) porcine kidney function, (3) ethical aspects, (4) design of initial clinical trials, (5) infectious disease issues, (6) industry perspectives, and (7) regulatory oversight.

Development, Deployment, and Implementation of a Machine Learning Surgical Case Length Prediction Model and Prospective Evaluation.

OBJECTIVE: To implement a machine learning model using only the restricted data available at case creation time to predict surgical case length for multiple services at different locations. BACKGROUND: The operating room is one of the most expensive resources in a health system, estimated to cost $22 to $133 per minute and generate about 40% of hospital revenue. Accurate prediction of surgical case length is necessary for efficient scheduling and cost-effective utilization of the operating room and other resources. METHODS: We introduced a similarity cascade to capture the complexity of cases and surgeon influence on the case length and incorporated that into a gradient-boosting machine learning model. The model loss function was customized to improve the balance between over- and under-prediction of the case length. A production pipeline was created to seamlessly deploy and implement the model across our institution. RESULTS: The prospective analysis showed that the model output was gradually adopted by the schedulers and outperformed the scheduler-predicted case length from August to December 2022. In 33,815 surgical cases across outpatient and inpatient platforms, the operational implementation predicted 11.2% fewer underpredicted cases and 5.9% more cases within 20% of the actual case length compared with the schedulers and only overpredicted 5.3% more. The model assisted schedulers to predict 3.4% more cases within 20% of the actual case length and 4.3% fewer underpredicted cases. CONCLUSIONS: We created a unique framework that is being leveraged every day to predict surgical case length more accurately at case posting time and could be potentially utilized to deploy future machine learning models.

Immune Phenotype and Postoperative Complications After Elective Surgery.

OBJECTIVES: To characterize and quantify accumulating immunologic alterations, pre and postoperatively in patients undergoing elective surgical procedures. BACKGROUND: Elective surgery is an anticipatable, controlled human injury. Although the human response to injury is generally stereotyped, individual variability exists. This makes surgical outcomes less predictable, even after standardized procedures, and may provoke complications in patients unable to compensate for their injury. One potential source of variation is found in immune cell maturation, with phenotypic changes dependent on an individual's unique, lifelong response to environmental antigens. METHODS: We enrolled 248 patients in a prospective trial facilitating comprehensive biospecimen and clinical data collection in patients scheduled to undergo elective surgery. Peripheral blood was collected preoperatively, and immediately on return to the postanesthesia care unit. Postoperative complications that occurred within 30 days after surgery were captured. RESULTS: As this was an elective surgical cohort, outcomes were generally favorable. With a median follow-up of 6 months, the overall survival at 30 days was 100%. However, 20.5% of the cohort experienced a postoperative complication (infection, readmission, or system dysfunction). We identified substantial heterogeneity of immune senescence and terminal differentiation phenotypes in surgical patients. More importantly, phenotypes indicating increased T-cell maturation and senescence were associated with postoperative complications and were evident preoperatively. CONCLUSIONS: The baseline immune repertoire may define an immune signature of resilience to surgical injury and help predict risk for surgical complications.

Functional Characteristics and Phenotypic Plasticity of CD57+PD1- CD4 T Cells and Their Relationship with Transplant Immunosuppression.

Costimulation blockade (CoB)-based immunosuppression offers the promise of improved transplantation outcomes with reduced drug toxicity. However, it is hampered by early acute rejections, mediated at least in part by differentiated, CoB-resistant T cells, such as CD57+PD1- CD4 T cells. In this study, we characterize these cells pretransplant, determine their fate posttransplant, and examine their proliferative capacity in vitro in humans. Our studies show that CD57+PD1- CD4 T cells are correlated with increasing age and CMV infection pretransplant, and persist for up to 1 y posttransplant. These cells are replication incompetent alone but proliferated in the presence of unsorted PBMCs in a contact-independent manner. When stimulated, cells sorted by CD57/PD1 status upregulate markers of activation with proliferation. Up to 85% of CD57+PD1- cells change expression of CD57/PD1 with stimulation, typically, upregulating PD1 and downregulating CD57. PD1 upregulation is accentuated in the presence of rapamycin but prevented by tacrolimus. These data support a general theory of CoB-resistant cells as Ag-experienced, costimulation-independent cells and suggest a mechanism for the synergy of belatacept and rapamycin, with increased expression of the activation marker PD1 potentiating exhaustion of CoB-resistant cells.

Introducing thymus for promoting transplantation tolerance.

Establishing tolerance remains a central, if elusive, goal of transplantation. In solid-organ transplantation, one strategy for inducing tolerance has been cotransplantation of various forms of thymic tissue along with another organ. As one of the biological foundations of central tolerance, thymic tissue carries with it the ability to induce tolerance to any other organ or tissue from the same donor (or another donor tissue-matched to the thymic tissue) if successfully transplanted. In this review, we outline the history of this approach as well as work to date on its application in organ transplantation, concluding with future directions. We also review our experience with allogeneic processed thymus tissue for the treatment of congenital athymia, encompassing complete DiGeorge syndrome and other rare genetic disorders, and consider whether allogeneic processed thymic tissue implantation may offer a novel method for future experimentation with tolerance induction in organ transplantation.

IFI16-STING-NF-κB signaling controls exogenous mitochondrion-induced endothelial activation.

Mitochondria released from injured cells activate endothelial cells (ECs), fostering inflammatory processes, including allograft rejection. The stimulator of interferon genes (STING) senses endogenous mitochondrial DNA, triggering innate immune activation via NF-κB signaling. Here, we show that exogenous mitochondria exposure induces EC STING-NF-κB activation, promoting EC/effector memory T cell adhesion, which is abrogated by NF-κB and STING inhibitors. STING activation in mitochondrion-activated ECs is independent of canonical cGMP-AMP synthetase sensing/signaling, but rather is mediated by interferon gamma-inducible factor 16 (IFI16) and can be inhibited by IFI16 inhibition. Internalized mitochondria undergo mitofusion and STING-dependent mitophagy, leading to selective sequestration of internalized mitochondria. The exposure of donor hearts to exogenous mitochondria activates murine heart ECs in vivo. Collectively, our results suggest that IFI16-STING-NF-κB signaling regulates exogenous mitochondrion-induced EC activation and mitophagy, and exogenous mitochondria foster T cell-mediated CoBRR. These data suggest a novel, donor-directed, therapeutic approach toward mitigating perioperative allograft immunogenicity.

Social determinants of health data in solid organ transplantation: National data sources and future directions.

Health equity research in transplantation has largely relied on national data sources, yet the availability of social determinants of health (SDOH) data varies widely among these sources. We sought to characterize the extent to which national data sources contain SDOH data applicable to end-stage organ disease (ESOD) and transplant patients. We reviewed 10 active national data sources based in the United States. For each data source, we examined patient inclusion criteria and explored strengths and limitations regarding SDOH data, using the National Institutes of Health PhenX toolkit of SDOH as a data collection instrument. Of the 28 SDOH variables reviewed, eight-core demographic variables were included in ≥80% of the data sources, and seven variables that described elements of social status ranged between 30 and 60% inclusion. Variables regarding identity, healthcare access, and social need were poorly represented (≤20%) across the data sources, and five of these variables were included in none of the data sources. The results of our review highlight the need for improved SDOH data collection systems in ESOD and transplant patients via: enhanced inter-registry collaboration, incorporation of standardized SDOH variables into existing data sources, and transplant center and consortium-based investigation and innovation.

A Multidimensional Bioinformatic Platform for the Study of Human Response to Surgery.

OBJECTIVE: To design and establish a prospective biospecimen repository that integrates multi-omics assays with clinical data to study mechanisms of controlled injury and healing. BACKGROUND: Elective surgery is an opportunity to understand both the systemic and focal responses accompanying controlled and well-characterized injury to the human body. The overarching goal of this ongoing project is to define stereotypical responses to surgical injury, with the translational purpose of identifying targetable pathways involved in healing and resilience, and variations indicative of aberrant peri-operative outcomes. METHODS: Clinical data from the electronic medical record combined with large-scale biological data sets derived from blood, urine, fecal matter, and tissue samples are collected prospectively through the peri-operative period on patients undergoing 14 surgeries chosen to represent a range of injury locations and intensities. Specimens are subjected to genomic, transcriptomic, proteomic, and metabolomic assays to describe their genetic, metabolic, immunologic, and microbiome profiles, providing a multidimensional landscape of the human response to injury. RESULTS: The highly multiplexed data generated includes changes in over 28,000 mRNA transcripts, 100 plasma metabolites, 200 urine metabolites, and 400 proteins over the longitudinal course of surgery and recovery. In our initial pilot dataset, we demonstrate the feasibility of collecting high quality multi-omic data at pre- and postoperative time points and are already seeing evidence of physiologic perturbation between timepoints. CONCLUSIONS: This repository allows for longitudinal, state-of-the-art geno-mic, transcriptomic, proteomic, metabolomic, immunologic, and clinical data collection and provides a rich and stable infrastructure on which to fuel further biomedical discovery.

Viral Micro-RNAs Are Detected in the Early Systemic Response to Injury and Are Associated With Outcomes in Polytrauma Patients.

OBJECTIVES: To evaluate early activation of latent viruses in polytrauma patients and consider prognostic value of viral micro-RNAs in these patients. DESIGN: This was a subset analysis from a prospectively collected multicenter trauma database. Blood samples were obtained upon admission to the trauma bay (T0), and trauma metrics and recovery data were collected. SETTING: Two civilian Level 1 Trauma Centers and one Military Treatment Facility. PATIENTS: Adult polytrauma patients with Injury Severity Scores greater than or equal to 16 and available T0 plasma samples were included in this study. Patients with ICU admission greater than 14 days, mechanical ventilation greater than 7 days, or mortality within 28 days were considered to have a complicated recovery. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Polytrauma patients (n = 180) were identified, and complicated recovery was noted in 33%. Plasma samples from T0 underwent reverse transcriptase-quantitative polymerase chain reaction analysis for Kaposi's sarcoma-associated herpesvirus micro-RNAs (miR-K12_10b and miRK-12-12) and Epstein-Barr virus-associated micro-RNA (miR-BHRF-1), as well as Luminex multiplex array analysis for established mediators of inflammation. Ninety-eight percent of polytrauma patients were found to have detectable Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus micro-RNAs at T0, whereas healthy controls demonstrated 0% and 100% detection rate for Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus, respectively. Univariate analysis revealed associations between viral micro-RNAs and polytrauma patients' age, race, and postinjury complications. Multivariate least absolute shrinkage and selection operator analysis of clinical variables and systemic biomarkers at T0 revealed that interleukin-10 was the strongest predictor of all viral micro-RNAs. Multivariate least absolute shrinkage and selection operator analysis of systemic biomarkers as predictors of complicated recovery at T0 demonstrated that miR-BHRF-1, miR-K12-12, monocyte chemoattractant protein-1, and hepatocyte growth factor were independent predictors of complicated recovery with a model complicated recovery prediction area under the curve of 0.81. CONCLUSIONS: Viral micro-RNAs were detected within hours of injury and correlated with poor outcomes in polytrauma patients. Our findings suggest that transcription of viral micro-RNAs occurs early in the response to trauma and may be associated with the biological processes involved in polytrauma-induced complicated recovery.

Malnutrition and immune cell subsets in children undergoing kidney transplantation.

BACKGROUND: Malnutrition, including obesity and undernutrition, among children is increasing in prevalence and is common among children on renal replacement therapy. The effect of malnutrition on the pre-transplant immune system and how the pediatric immune system responds to the insult of both immunosuppression and allotransplantation is unknown. We examined the relationship of nutritional status with post-transplant outcomes and characterized the peripheral immune cell phenotypes of children from the Immune Development of Pediatric Transplant (IMPACT) study. METHODS: Ninety-eight patients from the IMPACT study were classified as having obesity, undernutrition, or normal nutrition-based pre-transplant measurements. Incidence of infectious and alloimmune outcomes at 1-year post-transplantation was compared between nutritional groups using Gray's test and Fine-Gray subdistribution hazards model. Event-free survival was estimated by Kaplan-Meier method and compared between groups. Differences in immune cell subsets between nutritional groups over time were determined using generalized estimating equations accounting for the correlation between repeated measurements. RESULTS: We did not observe that nutritional status was associated with infectious or alloimmune events or event-free survival post-transplant. We demonstrated that children with obesity had distinct T-and B-cell signatures relative to those with undernutrition and normal nutrition, even when controlling for immunosuppression. Children with obesity had a lower frequency of CD8 Tnaive cells 9-month post-transplant (p < .001), a higher frequency of CD4 CD57 + PD1- T cells, and lower frequencies of CD57-PD1+ CD8 and CD57-PD1- CD8 T cells at 12-month transplant (p < .05 for all). CONCLUSIONS: Children with obesity have distinct immunophenotypes that may influence the tailoring of immunosuppression.

Eudaimonia: An Aristotelian approach to transplantation.

Despite extraordinary achievements in over the past 20 years, the field of transplantation remains hindered by relatively narrow metrics for success. Eudaimonia is an Aristotelian concept that refers to flourishing, or achieving the best conditions possible, in every sense. The vast amounts of patient data that are collected throughout the transplant care continuum, ranging from social determinants of health to genomic profiles and patient-reported outcomes, afford us unprecedented opportunity to enhance our definition of success for our transplant patients. We must engage the technologies available for data integration and analysis and apply them in an insightful way, such that our clinical practice evolves beyond patient and graft survival and toward a more comprehensive state of wellness.

Age-related effects on thymic output and homeostatic T cell expansion following depletional induction in renal transplant recipients.

Thymic output and homeostatic mature cell proliferation both influence T cell repopulation following depletional induction, though the relative contribution of each and their association with recipient age have not been well studied. We investigated the repopulating T cell kinetics in kidney transplant recipients who underwent alemtuzumab induction followed by belatacept/rapamycin-based immunosuppression over 36-month posttransplantation. We focused specifically on the correlation between repopulating T cell subsets and the age of patients. Substantial homeostatic Ki67-expressing T cell proliferation was seen posttransplantation. A repertoire enriched for naïve T (TNaïve ) cells emerged posttransplantation. Analysis by generalized estimating equation linear models revealed a strong negative linear association between reconstituting TNaïve cells and advancing age. A relationship between age and persistence of effector memory cells was shown. We assessed thymic output and found an increase in the frequency of recent thymic emigrants (RTEs, CD4+ CD31+ ) at 12-month posttransplantation. Patients under 30 years of age showed significantly higher levels of CD4+ CD31+ cells than patients over 55 years of age pre- and posttransplantation. IL-7 and autologous mature dendritic cells (mDCs) induced CD57- cell proliferation. In contrast, mDCs, but not IL-7, induced CD57+ cell proliferation. This study establishes the relationship between age and thymic output during T cell homeostatic repopulation after alemtuzumab induction. Trial Registration: ClinicalTrials.gov - NCT00565773.

Optimization of de novo belatacept-based immunosuppression administered to renal transplant recipients.

Kidney transplant recipients administered belatacept-based maintenance immunosuppression present with a more favorable metabolic profile, reduced incidence of de novo donor-specific antibodies (DSAs), and improved renal function and long-term patient/graft survival relative to individuals receiving calcineurin inhibitor (CNI)-based immunosuppression. However, the rates and severity of acute rejection (AR) are greater with the approved belatacept-based regimen than with CNI-based immunosuppression. Although these early co-stimulation blockade-resistant rejections are typically steroid sensitive, the higher rate of cellular AR has led many transplant centers to adopt immunosuppressive regimens that differ from the approved label. This article summarizes the available data on these alternative de novo belatacept-based maintenance regimens. Steroid-sparing, belatacept-based immunosuppression (following T cell-depleting induction therapy) has been shown to yield AR rates comparable to those seen with CNI-based regimens. Concomitant treatment with belatacept plus a mammalian target of rapamycin inhibitor (mTORi; sirolimus or everolimus) has yielded AR rates ranging from 0 to 4%. Because the optimal induction agent and number of induction doses; blood levels of mTORi; and dose, duration, and use of corticosteroids have yet to be determined, larger prospective clinical trials are needed to establish the optimal alternative belatacept-based regimen for minimizing early cellular AR occurrence.

Relationship between antithymocyte globulin, T cell phenotypes, and clinical outcomes in pediatric kidney transplantation.

Depletional induction using antithymocyte globulin (ATG) reduces rates of acute rejection in adult kidney transplant recipients, yet little is known about its effects in children. Using a longitudinal cohort of 103 patients in the Immune Development in Pediatric Transplant (IMPACT) study, we compared T cell phenotypes after ATG or non-ATG induction. We examined the effects of ATG on the early clinical outcomes of alloimmune events (development of de novo donor specific antibody and/or biopsy proven rejection) and infection events (viremia/viral infections). Long-term patient and graft outcomes were examined using the Scientific Registry of Transplant Recipients. After ATG induction, although absolute counts of CD4 and CD8 T cells were lower, patients had higher percentages of CD4 and CD8 memory T cells with a concomitant decrease in frequency of naïve T cells compared to non-ATG induction. In adjusted and unadjusted models, ATG induction was associated with increased early event-free survival, with no difference in long-term patient or allograft survival. Decreased CD4+ naïve and increased CD4+ effector memory T cell frequencies were associated with improved clinical outcomes. Though immunologic parameters are drastically altered with ATG induction, long-term clinical benefits remain unclear in pediatric patients.

Immunosuppression Withdrawal in Liver Transplant Recipients on Sirolimus.

BACKGROUND AND AIMS: As conversion from calcineurin inhibitor to sirolimus (SRL), a mechanistic target of rapamycin inhibitor (mTOR-I), has been shown to enhance immunoregulatory profiles in liver transplant (LT) recipients (LTRs), mTOR-I therapy might allow for increased success of immunosuppression (IS) withdrawal. Our aim was to determine if operational tolerance could be observed in LTRs withdrawn from SRL and if blood/graft tolerance biomarkers were predictive of successful withdrawal. APPROACH AND RESULTS: We performed a prospective trial of SRL monotherapy withdrawal in nonimmune, nonviremic LTRs > 3 years post-LT. SRL was weaned over ~6 months, and biopsies were performed 12 months postweaning or at concern for acute rejection. Twenty-one LTRs consented; 6 were excluded due to subclinical acute rejection on baseline biopsy or other reasons, and 15 underwent weaning (age 61.3 ± 8.8 years; LT to SRL weaning 6.7 ± 3 years). Eight (53%) achieved operational tolerance (TOL). Of the 7 who were nontolerant (non-TOL), 6 had mild acute rejection on biopsy near the end of weaning or at study end; 1 was removed from the trial due to liver cancer recurrence. At baseline preweaning, there were statistically increased blood tolerogenic dendritic cells and cell phenotypes correlating with chronic antigen presentation in the TOL versus non-TOL groups. A previously identified biopsy gene signature accurately predicted TOL versus non-TOL in 12/14 LTRs before weaning. At study end, biopsy staining revealed statistically significant increases in antigen-presenting cell:leukocyte pairings, FOXP3+ /CD4+ T cells, Tbet+ /CD8+ T cells, and lobular dendritic cells in the non-TOL group. CONCLUSIONS: This study evaluated IS withdrawal directly from mTOR-I therapy in LTRs and achieved > 50% operational tolerance. Preweaning gene expression and peripheral blood mononuclear cell profiling may be useful as predictors of successful mTOR-I therapy withdrawal. NCT02062944.

Coagulation, inflammation, and CD46 transgene expression in neonatal porcine islet xenotransplantation.

BACKGROUND: Thrombosis is a known consequence of intraportal islet transplantation, particularly for xenogeneic islets. To define the origins of thrombosis after islet xenotransplantation and relate it to early inflammation, we examined porcine islets transplanted into non-human primates using a dual-transplant model to directly compare islet characteristics. METHODS: α1,3-Galactosyltransferase gene-knockout (GTKO) islets with and without expression of the human complement regulatory transgene CD46 (hCD46) were studied. Biologically inert polyethylene microspheres were used to examine the generic pro-thrombotic effects of particle embolization. Immunohistochemistry was performed 1 and 24 hours after transplantation. RESULTS: Xeno-islet transplantation activated both extrinsic and intrinsic coagulation pathways. The intrinsic pathway was also initiated by microsphere embolization, while extrinsic pathway tissue factor (TF) and platelet aggregation were more specific to engrafted islets. hCD46 expression significantly reduced TF, platelet, fibrin, and factor XIIIa accumulation in and around islets but did not alter intrinsic factor activation. Layers of TF+ cells emerged around islets within 24 hours, particularly co-localized with vimentin, and identified as CD3+ and CD68+ cells inflammatory cells. CONCLUSIONS: These findings detail the origins of thrombosis following islet xenotransplantation, relate it to early immune activation, and suggest a role for transgenic hCD46 expression in its mitigation. Layers of TF-positive inflammatory cells and fibroblasts around islets at 24 hours may have important roles in the progressive events of thrombosis, inflammatory cell recruitment, rejection, and the ultimate outcome of transplanted grafts. These suggest that the strategies targeting these elements could yield more progress toward successful xenogeneic islet engraftment and survival.