Browsing by Author "Kirk, Allan D"
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Item Open Access Age-related effects on thymic output and homeostatic T cell expansion following depletional induction in renal transplant recipients.(American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 2021-09) Xu, He; Lee, Hui-Jie; Schmitz, Robin; Shaw, Brian I; Li, Shu; Kirk, Allan DThymic 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.Item Open Access An age-independent gene signature for monitoring acute rejection in kidney transplantation.(Theranostics, 2020-01) Shaw, Brian I; Cheng, Daniel K; Acharya, Chaitanya R; Ettenger, Robert B; Lyerly, Herbert Kim; Cheng, Qing; Kirk, Allan D; Chambers, Eileen TAcute rejection (AR) remains a significant problem that negatively impacts long-term renal allograft survival. Numerous therapies are used to prevent AR that differ by center and recipient age. This variability confounds diagnostic methods. Methods: To develop an age-independent gene signature for AR effective across a broad array of immunosuppressive regimens, we compiled kidney transplant biopsy (n=1091) and peripheral blood (n=392) gene expression profiles from 12 independent public datasets. After removing genes differentially expressed in pediatric and adult patients, we compared gene expression profiles from biopsy and peripheral blood samples of patients with AR to those who were stable (STA), using Mann-Whitney U Tests with validation in independent testing datasets. We confirmed this signature in pediatric and adult patients (42 AR and 47 STA) from our institutional biorepository. Results: We identified a novel age-independent gene network that identified AR from both kidney and blood samples. We developed a 90-probe set signature targeting 76 genes that differentiated AR from STA and found an 8 gene subset (DIP2C, ENOSF1, FBXO21, KCTD6, PDXDC1, REXO2, HLA-E, and RAB31) that was associated with AR. Conclusion: We used publicly available datasets to create a gene signature of AR that identified AR irrespective of immunosuppression regimen or recipient age. This study highlights a novel model to screen and validate biomarkers across multiple treatment regimens.Item Open Access Association of a Network of Immunologic Response and Clinical Features With the Functional Recovery From Crotalinae Snakebite Envenoming.(Frontiers in immunology, 2021-01) Gerardo, Charles J; Silvius, Elizabeth; Schobel, Seth; Eppensteiner, John C; McGowan, Lauren M; Elster, Eric A; Kirk, Allan D; Limkakeng, Alexander TBackground
The immunologic pathways activated during snakebite envenoming (SBE) are poorly described, and their association with recovery is unclear. The immunologic response in SBE could inform a prognostic model to predict recovery. The purpose of this study was to develop pre- and post-antivenom prognostic models comprised of clinical features and immunologic cytokine data that are associated with recovery from SBE.Materials and methods
We performed a prospective cohort study in an academic medical center emergency department. We enrolled consecutive patients with Crotalinae SBE and obtained serum samples based on previously described criteria for the Surgical Critical Care Initiative (SC2i)(ClinicalTrials.gov Identifier: NCT02182180). We assessed a standard set of clinical variables and measured 35 unique cytokines using Luminex Cytokine 35-Plex Human Panel pre- and post-antivenom administration. The Patient-Specific Functional Scale (PSFS), a well-validated patient-reported outcome of functional recovery, was assessed at 0, 7, 14, 21 and 28 days and the area under the patient curve (PSFS AUPC) determined. We performed Bayesian Belief Network (BBN) modeling to represent relationships with a diagram composed of nodes and arcs. Each node represents a cytokine or clinical feature and each arc represents a joint-probability distribution (JPD).Results
Twenty-eight SBE patients were enrolled. Preliminary results from 24 patients with clinical data, 9 patients with pre-antivenom and 11 patients with post-antivenom cytokine data are presented. The group was mostly female (82%) with a mean age of 38.1 (SD ± 9.8) years. In the pre-antivenom model, the variables most closely associated with the PSFS AUPC are predominantly clinical features. In the post-antivenom model, cytokines are more fully incorporated into the model. The variables most closely associated with the PSFS AUPC are age, antihistamines, white blood cell count (WBC), HGF, CCL5 and VEGF. The most influential variables are age, antihistamines and EGF. Both the pre- and post-antivenom models perform well with AUCs of 0.87 and 0.90 respectively.Discussion
Pre- and post-antivenom networks of cytokines and clinical features were associated with functional recovery measured by the PSFS AUPC over 28 days. With additional data, we can identify prognostic models using immunologic and clinical variables to predict recovery from SBE.Item Open Access Cryopreserved Mesenchymal Stromal Cells Are Susceptible to T-Cell Mediated Apoptosis Which Is Partly Rescued by IFNγ Licensing.(Stem cells (Dayton, Ohio), 2016-09) Chinnadurai, Raghavan; Copland, Ian B; Garcia, Marco A; Petersen, Christopher T; Lewis, Christopher N; Waller, Edmund K; Kirk, Allan D; Galipeau, JacquesWe have previously demonstrated that cryopreservation and thawing lead to altered Mesenchymal stromal cells (MSC) functionalities. Here, we further analyzed MSC's fitness post freeze-thaw. We have observed that thawed MSC can suppress T-cell proliferation when separated from them by transwell membrane and the effect is lost in a MSC:T-cell coculture system. Unlike actively growing MSCs, thawed MSCs were lysed upon coculture with activated autologous Peripheral Blood Mononuclear Cells (PBMCs) and the lysing effect was further enhanced with allogeneic PBMCs. The use of DMSO-free cryoprotectants or substitution of Human Serum Albumin (HSA) with human platelet lysate in freezing media and use of autophagy or caspase inhibitors did not prevent thaw defects. We tested the hypothesis that IFNγ prelicensing before cryobanking can enhance MSC fitness post thaw. Post thawing, IFNγ licensed MSCs inhibit T cell proliferation as well as fresh MSCs and this effect can be blocked by 1-methyl Tryptophan, an Indoleamine 2,3-dioxygenase (IDO) inhibitor. In addition, IFNγ prelicensed thawed MSCs inhibit the degranulation of cytotoxic T cells while IFNγ unlicensed thawed MSCs failed to do so. However, IFNγ prelicensed thawed MSCs do not deploy lung tropism in vivo following intravenous injection as well as fresh MSCs suggesting that IFNγ prelicensing does not fully rescue thaw-induced lung homing defect. We identified reversible and irreversible cryoinjury mechanisms that result in susceptibility to host T-cell cytolysis and affect MSC's cell survival and tissue distribution. The susceptibility of MSC to negative effects of cryopreservation and the potential to mitigate the effects with IFNγ prelicensing may inform strategies to enhance the therapeutic efficacy of MSC in clinical use. Stem Cells 2016;34:2429-2442.Item Open Access Cultured thymus tissue implementation promotes donor-specific tolerance to allogeneic heart transplants.(JCI insight, 2020-04-30) Kwun, Jean; Li, Jie; Rouse, Clay; Park, Jae Berm; Farris, Alton B; Kuchibhatla, Maragatha; Turek, Joseph W; Knechtle, Stuart J; Kirk, Allan D; Markert, M LouiseEighty-six infants born without a thymus have been treated with allogeneic cultured thymus tissue implantation (CTTI). These infants, who lack T cells and are profoundly immunodeficient at birth, after CTTI from an unmatched donor develop genetically-recipient T cells that are tolerant to both their own major histocompatibility antigens and those of the donor. We tested use of CTTI with the goal of inducing tolerance to unmatched heart transplants in immunocompetent rats. We thymectomized and T cell depleted Lewis rats. The rats were then given Lewis x Dark Agouti (LWxDA) CTTI under the kidney capsule and vascularized DA heart transplants in the abdomen. Cyclosporine was administered for 4 months. The control group did not receive CTTI. Recipients with CTTI showed repopulation of naïve and recent thymic emigrant CD4 T cells; controls had none. Recipients of CTTI did not reject DA cardiac allografts. Control animals did not reject DA grafts, due to lack of functional T cells. To confirm donor-specific unresponsiveness, MHC-mismatched Brown Norway (BN) hearts were transplanted 6 months after the initial DA heart transplant. LW rats with (LWxDA) CTTI rejected the third-party BN hearts (mean survival time 10d; n=5). Controls did not (n=5). CTTI recipients produced antibody against third party BN donor but not against the DA thymus donor demonstrating humoral donor-specific tolerance. Taken together, F1(LWxDA) CTTI given to Lewis rats resulted in specific tolerance to the allogeneic DA MHC expressed in the donor thymus with resulting long-term survival of DA heart transplants after withdrawal of all immunosuppression.Item Open Access Damage- and pathogen-associated molecular patterns play differential roles in late mortality after critical illness.(JCI insight, 2019-08) Eppensteiner, John; Kwun, Jean; Scheuermann, Uwe; Barbas, Andrew; Limkakeng, Alexander T; Kuchibhatla, Maggie; Elster, Eric A; Kirk, Allan D; Lee, JaewooMultiple organ failure (MOF) is the leading cause of late mortality and morbidity in patients who are admitted to intensive care units (ICUs). However, there is an epidemiologic discrepancy in the mechanism of underlying immunologic derangement dependent on etiology between sepsis and trauma patients in MOF. We hypothesized that damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs), while both involved in the development of MOF, contribute differently to the systemic innate immune derangement and coagulopathic changes. We found that DAMPs not only produce weaker innate immune activation than counterpart PAMPs, but also induce less TLR signal desensitization, contribute to less innate immune cell death, and propagate more robust systemic coagulopathic effects than PAMPs. This differential contribution to MOF provides further insight into the contributing factors to late mortality in critically ill trauma and sepsis patients. These findings will help to better prognosticate patients at risk of MOF and may provide future therapeutic molecular targets in this disease process.Item Open Access Defining the Need for Causal Inference to Understand the Impact of Social Determinants of Health: A Primer on Behalf of the Consortium for the Holistic Assessment of Risk in Transplantation (CHART).(Annals of surgery open : perspectives of surgical history, education, and clinical approaches, 2023-12) Bhavsar, Nrupen A; Patzer, Rachel E; Taber, David J; Ross-Driscoll, Katie; Deierhoi Reed, Rhiannon; Caicedo-Ramirez, Juan C; Gordon, Elisa J; Matsouaka, Roland A; Rogers, Ursula; Webster, Wendy; Adams, Andrew; Kirk, Allan D; McElroy, Lisa MObjective
This study aims to introduce key concepts and methods that inform the design of studies that seek to quantify the causal effect of social determinants of health (SDOH) on access to and outcomes following organ transplant.Background
The causal pathways between SDOH and transplant outcomes are poorly understood. This is partially due to the unstandardized and incomplete capture of the complex interactions between patients, their neighborhood environments, the tertiary care system, and structural factors that impact access and outcomes. Designing studies to quantify the causal impact of these factors on transplant access and outcomes requires an understanding of the fundamental concepts of causal inference.Methods
We present an overview of fundamental concepts in causal inference, including the potential outcomes framework and direct acyclic graphs. We discuss how to conceptualize SDOH in a causal framework and provide applied examples to illustrate how bias is introduced.Results
There is a need for direct measures of SDOH, increased measurement of latent and mediating variables, and multi-level frameworks for research that examine health inequities across multiple health systems to generalize results. We illustrate that biases can arise due to socioeconomic status, race/ethnicity, and incongruencies in language between the patient and clinician.Conclusions
Progress towards an equitable transplant system requires establishing causal pathways between psychosocial risk factors, access, and outcomes. This is predicated on accurate and precise quantification of social risk, best facilitated by improved organization of health system data and multicenter efforts to collect and learn from it in ways relevant to specialties and service lines.Item Open Access Development, Deployment, and Implementation of a Machine Learning Surgical Case Length Prediction Model and Prospective Evaluation.(Annals of surgery, 2023-06) Zaribafzadeh, Hamed; Webster, Wendy L; Vail, Christopher J; Daigle, Thomas; Kirk, Allan D; Allen, Peter J; Henao, Ricardo; Buckland, Daniel MObjective
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 (OR) is one of the most expensive resources in a health system, estimated to cost $22-133 per minute and generate about 40% of the hospital revenue. Accurate prediction of surgical case length is necessary for efficient scheduling and cost-effective utilization of the OR 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 in Aug-Dec 2022. In 33,815 surgical cases across outpatient and inpatient platforms, the operational implementation predicted 11.2% fewer under-predicted cases and 5.9% more cases within 20% of the actual case length compared with the schedulers and only over-predicted 5.3% more. The model assisted schedulers to predict 3.4% more cases within 20% of the actual case length and 4.3% fewer under-predicted 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.Item Open Access IFI16-STING-NF-κB signaling controls exogenous mitochondrion-induced endothelial activation.(American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 2022-06) Li, Shu; Xu, He; Song, Mingqing; Shaw, Brian I; Li, Qi-Jing; Kirk, Allan DMitochondria 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.Item Open Access Immune response profiling in patients with traumatic injuries associated with alcohol ingestion.(Clinical and translational science, 2021-09) Breslin, Adam W; Limkakeng, Alexander T; Silvius, Elizabeth; Staton, Catherine A; Almond, Chandra; Joshi, Mary-Beth; Adams, Bartley; Johnston, Bria; McGowan, Lauren; Kirk, Allan D; Elster, EricTraumatic injuries afflict more than 5 million people globally every year. Current and past animal research has demonstrated association among alcohol, trauma, and impaired immune function, whereas human registries have shown association between alcohol and morbidity as well as mortality. The purpose of this study is to elucidate the immune interactions with alcohol in traumatically injured patients. We prospectively enrolled 379 patients after trauma at three medical centers in the Surgical Critical Care Initiative. Plasma was analyzed using Luminex for up to 35 different cytokines. Collected samples were grouped by patients with detectable plasma alcohol levels versus those without. Univariate testing determined differences in analytes between groups. We built Bayesian belief networks with multiple minimum descriptive lengths to compare the two groups. All 379 patient samples were analyzed. Two hundred eighty-two (74.4%) patients were men, and 143 (37.7%) were White. Patients had a median intensive care unit length of stay (LOS) of 5.8 days and hospital LOS of 12 days. Using single variate analyses, eight different cytokines were differentially associated with alcohol. Cytokines IL-12 and IL-6 were important nodes in both models and IL-10 was a prominent node in the nonalcohol model. This study found select immune function differed between traumatically injured patients with measurable serum alcohol levels as compared with those without. Traumatically injured patients with positive blood alcohol content appear less able to inhibit inflammatory stress. Alcohol appears to suppress pro-inflammatory IL-12 and IL-6, whereas patients without alcohol have greater levels of anti-inflammatory IL-10 expressed at injury and may better regulate anti-inflammatory pathways. Future studies should determine the relationship with these markers with clinically oriented outcomes.Item Open Access Modulation of Xenogeneic T-cell Proliferation by B7 and mTOR Blockade of T Cells and Porcine Endothelial Cells.(Transplantation, 2022-05) Li, Shu; Xu, He; Kirk, Allan DBackground
Activation of porcine endothelial cells (PECs) is the mechanistic centerpiece of xenograft rejection. This study sought to characterize the immuno-phenotype of human T cells in response to PECs and to explore the immuno-modulation of B7 and mammalian target of rapamycin blockade of T cells and/or PECs during xeno-responses.Methods
Rapid memory T-cell (TM) responses to PECs were assessed by an intracellular cytokine staining. T-cell proliferation to PEC with or without belatacept or rapamycin was evaluated by a mixed lymphocyte-endothelial cell reaction (MLER). Additionally, rapamycin-pretreated PECs were used in MLER. Cell phenotypes were analyzed by flow cytometry.Results
Tumor necrosis factor-α/interferon-γ producers were detected in CD8+ cells stimulated by human endothelium but not PECs. MLER showed proliferation of CD4+ and CD8+ cells with predominantly memory subsets. Purified memory and naive cells proliferated following PEC stimulation with an increased frequency of TM in PEC-stimulated naive cells. Proliferating cells upregulated programmed cell death-1 (PD-1) and CD2 expression. Belatacept partially inhibited T-cell proliferation with reduced CD2 expression and frequency of the CD8+CD2highCD28- subset. Rapamycin dramatically inhibited PEC-induced T-cell proliferation, and rapamycin-preconditioned PECs failed to induce T-cell proliferation. PD-1 blockade did not restore T-cell proliferation to rapamycin-preconditioned PECs.Conclusions
Humans lack rapid TM-mediated responses to PECs but induce T-cell proliferative responses characterized largely as TM with increasing CD2 and PD-1 expression. B7-CD28 and mammalian target of rapamycin blockade of T cells exhibit dramatic inhibitory effects in altering xeno-proliferating cells. Rapamycin alters PEC xeno-immunogenicity leading to inhibition of xeno-specific T-cell proliferation independent of PD-1-PD ligand interaction.Item Open Access Rapamycin Prevents Expansion of Costimulation Blockade-resistant CD8+ Alloreactive Memory Cells following Depletional Induction in Renal Transplant Recipients.(Journal of immunology (Baltimore, Md. : 1950), 2024-09) Li, Shu; Gao, Qimeng; Xu, He; Kirk, Allan DAlemtuzumab induction with belatacept/rapamycin-based maintenance immunotherapy (ABR) prevents kidney allograft rejection and specifically limits early costimulation blockade-resistant rejection (CoBRR). To evaluate the mechanisms by which this regimen alters CoBRR, we characterized the phenotype and functional response of preexisting memory cells to allogeneic endothelial cells using intracellular cytokine staining and flow cytometry. IL-7-induced lymphocyte proliferation in the presence or absence of rapamycin was assessed to characterize the phenotype of proliferating cells. Lymphocytes from 40 recipients who underwent transplant using the ABR regimen were studied longitudinally. The rapid immunoresponses of preexisting alloreactive cells to allogeneic endothelial cells were predominantly CD8+TNF-α+/IFN-γ+ cells. These cells were effector memory (TEM) and terminally differentiated effector memory cells lacking CD28 expression, and most were CD57+PD1-. Neither rapamycin nor belatacept directly inhibited these cells. IL-7, a cytokine induced during lymphopenia postdepletion, provoked dramatic CD8+ TEM cell proliferation and a low level of CD8+CD57+PD1- cell expansion in vitro. The IL-7 stimulation induced CD8+ cell mTOR phosphorylation, and rapamycin treatment markedly inhibited IL-7-induced TEM and CD57+PD1- cell expansion. This effect was evident in patients receiving the ABR in that the repopulation of CD8+CD57+PD1- TEM cells was substantially suppressed for at least 36 mo after transplant. These findings help define one mechanism by which a costimulation blockade/rapamycin-based therapy following alemtuzumab induction minimizes CoBRR, namely that in the presence of rapamycin, costimulation-resistant alloreactive cells are disproportionately ineffective at repopulating following post-transplant T cell depletion.Item Open Access Toll-like receptor activation as a biomarker in traumatically injured patients(Journal of Surgical Research, 2018-11-01) Darrabie, Marcus D; Cheeseman, Jennifer; Limkakeng, Alexander T; Borawski, Joseph; Sullenger, Bruce A; Elster, Eric A; Kirk, Allan D; Lee, Jaewoo© 2018 Elsevier Inc. Background: Surgical insult and trauma have been shown to cause dysregulation of the immune and inflammatory responses. Interaction of damage-associated molecular patterns (DAMPs) with toll-like receptors (TLRs) initiates innate immune response and systemic inflammatory responses. Given that surgical patients produce high levels of circulating damage-associated molecular patterns, we hypothesized that plasma-activated TLR activity would be correlated to injury status and could be used to predict pathological conditions involving tissue injury. Methods: An observational study was performed using samples from a single-institution prospective tissue and data repository from a Level-1 trauma center. In vitro TLR 2, 3, 4, and 9 activation was determined in a TLR reporter assay after isolation of plasma from peripheral blood. We determined correlations between plasma-activated TLR activity and clinical course measures of severity. Results: Eighteen patients were enrolled (median Injury Severity Score 15 [interquartile range 10, 23.5]). Trauma resulted in significant elevation in circulation high mobility group box 1 as well as increase of plasma-activated TLR activation (2.8-5.4-fold) compared to healthy controls. There was no correlation between circulating high mobility group box 1 and trauma morbidity; however, the plasma-activated TLR activity was correlated with acute physiology and chronic health evaluation II scores (R square = 0.24-0.38, P < 0.05). Patients who received blood products demonstrated significant increases in the levels of plasma-activated TLRs 2, 3, 4, and 9 and had a trend toward developing systemic inflammatory response syndrome. Conclusions: Further studies examining TLR modulation and signaling in surgical patients may assist in predictive risk modeling and reduction in morbidity and mortality.Item Open Access Xenorecognition and costimulation of porcine endothelium-derived extracellular vesicles in initiating human porcine-specific T cell immune responses.(American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 2023-07) Li, Shu; Anwar, Imran J; Canning, Aidan J; Vo-Dinh, Tuan; Kirk, Allan D; Xu, HePorcine 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.