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Introduction: A better understanding of the immune mechanisms involved in allograft rejection after transplantation is urgently needed to improve patient outcomes. As microRNA-155 (miR155) plays a critical role in inflammation, we postulated that a deficiency of miR155 will improve cardiac allograft survival and enhance tolerance induction after heart transplantation., Methods: We developed an acute rejection mouse model through heterotopic BALB/c cardiac transplantation to C57BL/6 (wild-type) and C57BL/6 miR155 knock-out (miR155KO) mice. Further, we induced tolerance in both groups through a costimulatory blockade with CTLA4-Ig (200 μg; post-transplant day 2) and MRI antibodies (250 μg; post-transplant day 0), targeting CD28/B7 and CD40/CD154 signals, respectively. Finally, we examined the effects of injecting 100 μg of small extracellular vesicles (sEVs) isolated from wild-type mice undergoing rejection into tolerant miR155KO mice., Results: Mean survival time (MST) of the cardiac allografts in wild-type and miR155KO mice was 7 and 15 days, respectively (p < 0.0001). Costimulatory blockade increased MST to 65 days and > 100 days in the wild-type and miR155KO recipients, respectively (p < 0.001). Injection of sEVs isolated from wild-type mice undergoing rejection into tolerant miR155KO mice decreased the allograft survival to 9 days, significantly lower than the tolerant miR155KO mice without injection of sEVs (>100 days; p < 0.0001)., Conclusion: miR155KO mice have improved cardiac allograft survival and enhanced induction of tolerance after heterotopic cardiac transplantation. Injection of sEVs from wild-type mice undergoing rejection into the miR155KO mice reversed these benefits., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest. All authors have approved the final manuscript., (Copyright © 2024. Published by Elsevier B.V.)

The aim of this study (CTOTC-09) was to assess the impact of "preformed" (at transplant) donor-specific anti-HLA antibody (DSA) and first year newly detected DSA (ndDSA) on allograft function at 3 years after pediatric heart transplantation (PHTx). We enrolled children listed at 9 North American centers. The primary end point was pulmonary capillary wedge pressure (PCWP) at 3 years posttransplant. Of 407 enrolled subjects, 370 achieved PHTx (mean age, 7.7 years; 57% male). Pre-PHTx sensitization status was nonsensitized (n = 163, 44%), sensitized/no DSA (n = 115, 31%), sensitized/DSA (n = 87, 24%), and insufficient DSA data (n = 5, 1%); 131 (35%) subjects developed ndDSA. Subjects with any DSA had comparable PCWP at 3 years to those with no DSA. There were also no significant differences overall between the 2 groups for other invasive hemodynamic measurements, systolic graft function by echocardiography, and serum brain natriuretic peptide concentration. However, in the multivariable analysis, persistent first-year DSA was a risk factor for 3-year abnormal graft function. Graft and patient survival did not differ between groups. In summary, overall, DSA status was not associated with worse allograft function or inferior patient and graft survival at 3 years, but persistent first-year DSA was a risk factor for late graft dysfunction., Competing Interests: Disclosure The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation., (Copyright © 2023 American Society of Transplantation & American Society of Transplant Surgeons. Published by Elsevier Inc. All rights reserved.)

To determine the effects and immunological mechanisms of low-dose interleukin-2 (IL-2) in a murine model of chronic cardiac allograft rejection (BALB/c to C57BL/6) after costimulatory blockade consisting of MR1 (250 μg/ip day 0) and CTLA4-Ig (200 μg/ip day 2), we administered low-dose IL-2 (2000 IU/day) starting on posttransplant day 14 for 3 weeks. T regulatory (Treg) cell infiltration of the grafts was determined by immunohistochemistry; circulating exosomes by western blot and aldehyde bead flow cytometry; antibodies to donor MHC by immunofluorescent staining of donor cells; and antibodies to cardiac self-antigens (myosin, vimentin) by ELISA. We demonstrated that costimulation blockade after allogeneic heart transplantation induced circulating exosomes containing cardiac self-antigens and antibodies to both donor MHC and self-antigens, leading to chronic rejection by day 45. Treatment with low-dose IL-2 prolonged allograft survival (>100 days), prevented chronic rejection, and induced splenic and graft-infiltrating CD4+ CD25+ Foxp3 Treg cells by day 45 and circulating exosomes (Foxp3+) with PD-L1 and CD73. MicroRNA 142, associated with the TGFβ pathway, was significantly downregulated in exosomes from IL-2-treated mice. In conclusion, low-dose IL-2 delays rejection in a murine model of chronic cardiac allograft rejection and also induces graft-infiltrating Tregs and circulating exosomes with immunoregulatory molecules., (© 2022 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Antibodies to HLA resulting in positive cytotoxicity crossmatch are generally considered a contraindication for cardiac transplantation. However, cardiac transplantations have been performed in children by reducing the Abs and modifying immunosuppression. To identify mechanisms leading to allograft acceptance in the presence of Abs to donor HLA, we analyzed priming events in endothelial cells (EC) by incubating with sera containing low levels of anti-HLA followed by saturating concentration of anti-HLA. Pre-transplant sera were obtained from children with low levels of Abs to HLA who underwent transplantation. EC were selected for donor HLA and exposed to sera for 72 h (priming), followed by saturating concentrations of anti-HLA (challenge). Priming of EC with sera induced the phosphatidylinositol 3-kinase/Akt mediated by the BMP4/WNT pathway and subsequent challenge with panel reactive antibody sera increased survival genes Bcl2 and Heme oxygenase-1, decreased adhesion molecules, induced complement inhibitory proteins and reduced pro-inflammatory cytokines. In contrast, EC which did not express donor HLA showed decreased anti-apoptotic genes. Primed EC, upon challenge with anti-HLA, results in increased survival genes, decreased adhesion molecules, induction of complement inhibitory proteins, and downregulation of pro-inflammatory cytokines which may result in accommodation of pediatric cardiac allografts despite HLA sensitization., (Copyright © 2019 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Exosomes are extracellular vesicles that express self-antigens (SAgs) and donor human leukocyte antigens. Tissue-specific exosomes can be detected in the circulation following lung, heart, kidney and islet cell transplantations. We collected serum samples from patients who had undergone lung (n = 30), heart (n = 8), or kidney (n = 15) transplantations to isolate circulating exosomes. Exosome purity was analyzed by Western blot, using CD9 exosome-specific markers. Tissue-associated lung SAgs, collagen V (Col-V) and K-alpha 1 tubulin (Kα1T), heart SAgs, myosin and vimentin, and kidney SAgs, fibronectin and collagen IV (Col-IV), were identified using western blot. Lung transplant recipients diagnosed with bronchiolitis obliterans syndrome had exosomes with higher expression of Col-V (4.2-fold) and Kα1T (37.1-fold) than stable. Exosomes isolated from heart transplant recipients diagnosed with coronary artery vasculopathy had a 3.9-fold increase in myosin and a 4.7-fold increase in vimentin compared with stable. Further, Kidney transplant recipients diagnosed with transplant glomerulopathy had circulating exosomes with a 2-fold increased expression of fibronectin and 2.5-fold increase in Col-IV compared with stable. We conclude that circulating exosomes with tissue associated SAgs have the potential to be a noninvasive biomarker for allograft rejection., (Copyright © 2018 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Anti-HLA donor-specific antibodies are associated with worse outcomes after organ transplantation. Among sensitized pediatric heart candidates, requirement for negative donor-specific cytotoxicity crossmatch increases wait times and mortality. However, transplantation with positive crossmatch may increase posttransplantation morbidity and mortality. We address this clinical challenge in a prospective, multicenter, observational cohort study of children listed for heart transplantation (Clinical Trials in Organ Transplantation in Children-04 [CTOTC-04]). Outcomes were compared among sensitized recipients who underwent transplantation with positive crossmatch, nonsensitized recipients, and sensitized recipients without positive crossmatch. Positive crossmatch recipients received antibody removal and augmented immunosuppression, while other recipients received standard immunosuppression with corticosteroid avoidance. This first CTOTC-04 report summarizes study rationale and design and relates pretransplantation sensitization status using solid-phase technology. Risk factors for sensitization were explored. Of 317 screened patients, 290 were enrolled and 240 underwent transplantation. Core laboratory evaluation demonstrated that more than half of patients were anti-HLA sensitized. Greater than 80% of sensitized patients had class I (with or without class II) HLA antibodies, and one-third of sensitized patients had at least 1 HLA antibody with median fluorescence intensity of ≥8000. Logistic regression models demonstrated male sex, weight, congenital heart disease history, prior allograft, and ventricular assist device are independent risk factors for sensitization., (© 2018 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Long-term success of heart transplantation is hindered by humoral and cell-mediated immune responses. We studied preexisting antibodies to cardiac self-antigens, myosin and vimentin, and exosomes induced by antibodies to self-antigens in eliciting immune responses to cardiac grafts. After syngeneic heterotopic murine heart transplantation, rabbit anti-myosin or normal rabbit immunoglobulin was administered at day 0 or 7. Sera were collected after heartbeat cessation, cellular infiltration was analyzed, and exosomes were isolated from sera. Histopathologic examination of the controls' transplanted hearts demonstrated normal architecture, and their sera demonstrated neither antibodies to self-antigens nor exosomes expressing self-antigens. Administration of antibodies to cardiac myosin immediately posttransplantation (day 0) but not on day 7 triggered graft failure on day 7, and histopathologic examination revealed marked cellular infiltration with neutrophils and lymphocytes. Histopathologic examination of rejected hearts also demonstrated myocyte damage as sera had increased antibodies to myosin and vimentin and development of exosomes expressing self-antigens. Administration of exosomes isolated from failed grafts containing self-antigens induced graft dysfunction; exosomes isolated from stable mice did not induce graft failure. Antibodies to self-antigens can induce exosomes containing self-antigens, initiating an immune response and causing graft failure after cardiac transplantation., (© 2018 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Therapeutic plasma exchange (TPE) is an increasingly utilized immunosuppressive adjunct for treatment of antibody-mediated rejection (AMR) following organ transplantation. TPE works through removal of donor-specific HLA antibodies (DSAs) in the recipient's plasma. However, there is no clear laboratory measure evaluating efficacy of removal of DSAs or predicting clinical outcome. We hypothesized that semi-quantitative DSA measurement by multiplex HLA antibody immunoassay may provide qualitative and quantitative data for DSA clearance and predict treatment efficacy. To evaluate this, we retrospectively investigated DSA concentrations and clinical outcome for 21 pediatric patients who received 31 cycles of TPE peri-operatively as an adjunct treatment for transplantation in the setting of a positive cytotoxic crossmatch (CXM) and in recipients with AMR following heart or lung transplantation. Immunoassay measurement of DSAs during 15/20 cycles correlated significantly with clinical outcome in the AMR treatment group (P = 0.02), demonstrating the utility of DSA measurement in predicting clinical outcome. In contrast, immunoassay correlated with clinical outcome in only 7/11 patients treated peri-operatively with TPE for CXM-positive transplantations (P = 0.58). Changes in mean fluorescence intensity (MFI) for the DSAs correlated better with clinical response than surrogate CXM titers in a subset of patients. We conclude that semi-quantitative measurement of DSAs by immunoassay can predict clinical response to TPE for treatment of AMR is more reliable than surrogate CXM titer, and should be used to guide TPE treatment of AMR., (Copyright © 2013 Wiley Periodicals, Inc.)

Chronic rejection in the form of cardiac allograft vasculopathy is one of the major factors that affects long-term graft and patient survival after heart transplantation. Whereas multiple factors contribute to the development of cardiac allograft vasculopathy, immunologic mechanisms play the predominant role in the chronic rejection process, because both alloimmune and autoimmune responses are causal factors. In addition, many nonimmune donor and recipient factors also affect the development of cardiac allograft vasculopathy, including hyperlipidemia, cytomegalovirus infection, baseline coronary artery disease, and the mechanism of brain death in the donor. Modern immunosuppression maintenance therapies have the potential to limit the development of cardiac allograft vasculopathy in the long term. Further research initiatives are needed to identify patient-specific immunosuppressive drug regimens and to elucidate factors that contribute to the chronic rejection of cardiac transplant allografts.

Pathophysiology of chronic rejection strongly supports that inflammation and subsequent tissue remodeling during the post-transplant period cause exposure of cryptic self-antigens (SAgs) or their determinants within the graft, which, along with a subsequent cytokine response, leads to loss of peripheral tolerance. These events lead to the activation of cell-mediated immunity towards development of de novo immune responses to SAgs. There is also evidence for a role for interplay between allo- and autoimmunity in the development of chronic rejection. Experimental results using murine models of Obliterative Airway Disease (OAD) akin to chronic lung allograft rejection have clearly demonstrated that autoimmune responses to Collagen V (ColV) and K-alpha 1 Tubulin (KalT) were induced by administration of antibodies (Abs) against class I major histocompatibility complex antigens. Further, inhibition of interleukin (IL)-17 abrogated the autoimmune response and development of OAD. This shows an important relationship between alloimmunity, autoimmunity to SAgs such as KalT, and a significant role for IL-17 pathway of immune activation. Recent reports demonstrate that in addition to lung transplant recipients, kidney transplant recipients diagnosed with transplant glomerulopathy can develop de novo Abs to Sags, including Col-IV and fibronectin and heart transplant recipients can develop immune responses to cardiac myosin and vimentin. Abs to SAgs were identified frequently with donor specific anti-human leukocyte antigen antibodies, supporting the concept of crosstalk between auto- and alloimmunity. The increased frequency of SAg specific interferon-gamma and IL-17 cells with reduction in IL-10 demonstrates tolerance breakdown to SAgs which may play a significant role in the pathogenesis of chronic rejection.

Application of single-antigen solid-phase immunoassay (SPI) in virtual crossmatch-based organ allocation has been hindered by continued debate over the biologic relevance of detected antibodies and the relationship between cutoff mean fluorescence intensity (MFI) values with crossmatch testing results. To define SPI parameters accurately predicting crossmatch testing, we analyzed a series of anti-HLA antibodies from highly-sensitized patients awaiting lung or heart transplantation. Serial dilution of serum for SPI and cytotoxic crossmatch (CXM) enabled comparison over a wide spectrum of antibody "strengths". Receiver operating characteristic (ROC) analysis identified predictive cutoff values for HLA Class I and DR-specific antibodies. However, antibodies to HLA-DQ antigens demonstrated a significantly different characteristic, highlighting difficulties in interpretation of clinical significance. We also quantitatively characterized two data handling methods, MFI ratio (MR) and relative ratio (RR), to examine their potential impact on identifying unacceptable antigens. In combination with user defined cutoff values, MFI, MR and RR lead to discordant identification of antibodies. Establishment of cutoff values for MR and RR that are comparable to MFI demonstrated increased consistency in antibody identification. This single laboratory experience is an example of establishing statistically robust cutoff values and validation across different data handling methods for use of SPI in virtual crossmatch., (Copyright © 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Introduction: Mizoribine (MZR) is an inosine monophosphate dehydrogenase inhibitor. It has been widely used in Japan in the treatment of autoimmune diseases and is known to inhibit T and B cell proliferation. The aim of this study was to evaluate the efficacy of MZR as an immunosuppressive agent and determine its ability to synergize with a commonly used calcineurin inhibitor Cyclosporine A (CsA) in prolonging survival of murine islet cells and heart transplanted across major histocompatibility barrier., Methods: Murine allogeneic islet cell transplantation between Balb/c donor mice and C57BL/6 recipient mice and heterotopic heart transplantation was done between C3H/He donor mice and Balb/c recipient mice. Recipients were divided into groups based on immunosuppression: Group 1-No immunosuppression, Group 2-MZR alone (20 mg/kg/day), Group 3-CsA alone (20 mg/kg/day), Group 4-MZR+CsA (20 mg/kg/day). Donor specific IFN-γ, IL-10, IL-2, IL-4 secreting cells were enumerated by ELISpot. Serum cytokine and chemokine concentration was measured by Luminex., Results: Islet cell allograft recipients treated with CsA and MZR had prolonged islet function compared to other groups [normoglycemia (blood glucose <200 mg/dL) up to 32±4 days, p<0.05]. Similarly, heart allograft survival was significantly improved in mice treated with CsA and MZR compared to other groups (50% 30-day survival, p=0.04). Donor specific IFN-γ, IL-4, IL-2 secreting cells were significantly decreased in recipients treated with CsA and MZR with marked increase in IL-10 secreting cells (p<0.05). There was also an increase in serum IL-10 with decrease in IFN-γ, IL-4, IL-2, MCP-1, and IL-6 in mice treated with CsA and MZR CONCLUSION: MZR and CsA when used in combination are potent immunosuppressive agents in murine islet cell and heart transplantation models. These agents lead to a decrease in donor specific IFN-γ with increase in IL-10 secreting cells leading to improved allograft survival and function., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: Presence of donor-specific antibodies (Abs) is detrimental to posttransplant allograft function. Some sensitized recipients have successfully undergone transplantation after pretransplant conditioning regimen using plasmapheresis and/or intravenous immunoglobulin therapy, but underlying mechanisms that confer such allograft protection are undefined., Methods: We developed a single human leukocyte antigen (HLA)-mismatched heterotopic murine heart transplant model (HLA-A2 into HLA-A2-sensitized-C57BL/6) to determine whether pretreatment of donors with low concentration of HLA class I (W6/32) or control Ab (C1.18.4) will confer protection. Expression levels of survival genes, Bcl-2 and heme oxygenase-1, were analyzed by gene array analysis and quantitative real-time polymerase chain reaction. Expression levels of cytokine panel were analyzed by Luminex. Role of Bcl-2 in the induction of allograft protection was analyzed by silencing the Bcl-2 expression in the donor hearts using a small hairpin (shRNA) specific for Bcl-2., Results: Control Ab-pretreated hearts were rejected in less than 5 days demonstrating hemorrhage, Ab, and C4 deposition. In contrast, W6/32-pretreated hearts were rejected at 15 days (P<0.05) that was prolonged to 25 days with antilymphocyte serum treatment. W6/32-pretreated hearts on day 5 exhibited increased expression of Bcl-2 (5.5-folds), Bcl-xl (5.5-folds), and heme oxygenase-1 (4.4-folds); decreased expression of ICAM-1, VCAM-1 (3.2-fold), along with reduced levels of cytokines interleukin (IL)-1β (4.4-folds), tumor necrosis factor α (3.7-folds), IL-6 (7.5-folds), IL-12 (2.3-folds) and chemokines monocyte chemotactic protein 1 (4.5-folds), MIG (4.4-folds), MIP-1α (3.4-folds), and IL-8 (3.1-folds). Silencing of Bcl-2 in accommodated hearts before transplant resulted in loss of protection with rejection (9±3 vs. 15±2days, P<0.05)., Conclusion: Pretreatment of hearts with low levels of anti-HLA Abs increases expression of antiapoptotic genes that inhibits caspases, leading to decreased inflammatory cytokines and chemokines, which promote allograft survival.

Background: We determined the role of donor-specific antibodies (DSA) and antibodies (Abs) to self-antigens, collagen-V (Col-V), and K-α1-Tubulin (KAT) in pathogenesis of acute antibody-mediated rejection (AMR) and cardiac allograft vasculopathy (CAV) after human heart transplantation (HTx)., Methods: One hundred thirty-seven HTx recipients, with 60 early period (≤ 12 months) and 77 late period (>12 months), were enrolled in this study. Circulating DSA was determined using LUMINEX. Abs against Col-I, II, IV, V, and KAT were measured using ELISA. Frequency of CD4+T helper cells (CD4+Th) secreting interferon (IFN)-γ, interleukin (IL)-5, -10, or -17 specific to self-antigens were determined using Enzyme Linked Immunosorbent Spot assay., Results: A significant association between AMR and DSA was demonstrated. Development of DSA in AMR patients correlated well with the development of auto-Abs to Col-V (AMR[+]: 383 ± 72 μg/mL, AMR[-]: 172 ± 49 μg/mL, P=0.033) and KAT (AMR[+]: 252 ± 49 μg/mL, AMR[-]: 61 ± 21 μg/mL, P=0.014). Patients who developed AMR demonstrated increased frequencies of CD4+Th secreting IFN-γ and IL-5 with reduction in IL-10 specific for Col-V/KAT. Patients diagnosed with CAV also developed DSA and auto-Abs to Col-V (CAV[+]: 835 ± 142 μg/mL, CAV[-]: 242 ± 68 μg/mL, P=0.025) and KAT (CAV[+]: 768 ± 206 μg/mL, CAV[-]: 196 ± 72 μg/mL, P=0.001) with increased frequencies of CD4+Th secreting IL-17 with reduction in IL-10 specific for Col-V/KAT. CONCLUSIONS.: Development of Abs to human leukocyte antigens and self-antigens are associated with increases in CD4+Th secreting IFN-γ and IL-5 in AMR and IL-17 in CAV, with reduction in CD4+Th secreting IL-10 in both AMR and CAV.

Humoral immune responses to mismatched donor human leukocyte antigen (HLA) and major histocompatibility complex (MHC) class I-related chain A (MICA) have been reported to contribute to immunopathogenesis of antibody-mediated rejection (AMR) in the early period and cardiac allograft vasculopathy (CAV) in the late period after cardiac transplantation (HTx). The goal of this study is to define the roles of donor-specific antibodies (DSA) and anti-MICA in AMR and CAV. A total of 95 post-HTx recipients were enrolled; 43 patients in the early period (≤ 12 months post-HTx) and 52 patients in the late period (>12 months post-HTx). Development of DSA and anti-MICA were serially monitored using Luminex. Development of DSA (AMR+: n = 6/8.75%, AMR-: n = 4/35.11%, p = 0.009) and anti-MICA (AMR+: n = 5/8.63%, AMR-: n = 4/35.11%, p = 0.002) was significantly associated with AMR. AMR+DSA+ patients demonstrated increased anti-MICA levels compared with AMR+DSA- patients (p=0.01). Serial monitoring revealed DSA (2.7 ± 1.4 months) preceded development of anti-MICA (6.5 ± 2.1 months) in recipients diagnosed with AMR at 8.3 ± 2.5 months post-HTx. Development of DSA (CAV+: n = 8/12.67%, CAV-: n = 5/40.13%, p = 0.004) and anti-MICA (CAV+: n = 9/12.75%, CAV-: n = 5/40.13%, p = 0.001) was significantly associated with CAV. CAV+DSA+ patients demonstrated increased anti-MICA levels compared with CAV+DSA- patients (p = 0.01). Antibodies to HLA are associated with and precede development of anti-MICA in AMR and CAV. Therefore, DSA and anti-MICA can be used as noninvasive markers for monitoring AMR and CAV., (Copyright © 2010 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Background: Herein we study the role of donor-specific antibodies (DSA) to mismatched human leukocyte antigen (HLA) and antibodies (Abs) to the cardiac self-antigens myosin (MYO) and vimentin (VIM) in the pathogenesis of acute antibody-mediated rejection (AMR) in the early post-transplant period (EP, <12 months) and cardiac allograft vasculopathy (CAV) in the late post-transplant period (LP, >12 months) after heart transplantation (HTx)., Methods: One hundred forty-eight HTx recipients (65 in EP, 83 in LP) were enrolled in the study. Development of DSA was determined by Luminex. Circulating Abs against MYO and VIM in sera were measured using enzyme-linked immunoassay (ELISA). Frequency of CD4+ T-helper cells (CD4+ Th) secreting interferon (IFN)-γ, interleukin (IL)-17, IL-10 or IL-5 specific to either MYO or VIM were analyzed in vitro using ELISpot assays., Results: AMR patients were more likely DSA positive (AMR-: 15%; AMR+: 70%; p = 0.03) and demonstrated increased Abs to MYO (AMR-: 144 ± 115 μg/ml; AMR+: 285 ± 70 μg/ml; p = 0.033) and VIM (AMR-: 37 ± 19 μg/ml; AMR+: 103 ± 43 μg/ml; p = 0.014). AMR patients demonstrated increased IL-5 CD4+ Th cells specific to MYO (5.2 ± 0.9 fold, p = 0.003) and VIM (7.3 ± 2.9-fold, p = 0.004) and decreased IL-10 CD4+ Th cells specific to MYO (2.2 ± 0.4-fold, p = 0.009) and VIM (1.7 ± 0.2-fold, p = 0.03). CAV patients were more likely DSA positive (CAV-): 25%; CAV+: 79%; p = 0.03) and demonstrated increased Abs to MYO (CAV-: 191 ± 120 μg/ml; CAV+: 550 ± 98 μg/ml; p = 0.025) and VIM (CAV-: 55 ± 25 μg/ml; CAV+: 255 ± 49 μg/ml; p = 0.001). CAV patients demonstrated increased IL-17 CD4+ Th cells specific to MYO (10.5 ± 7.3-fold, p = 0.002) and VIM (7.0 ± 3.9-fold, p = 0.003)., Conclusions: The presence of DSA in AMR and CAV is significantly associated with development of Abs to MYO and VIM in post-HTx patients. Induction of high CD4+ Th cells specific to cardiac self-antigens that secrete predominantly IL-5 and IL-17 plays a significant role in the development of Abs to self-antigens leading to AMR and CAV, respectively., (Copyright © 2010 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

Background: Pediatric heart transplant recipients with a positive complement-dependent cytotoxic (CDC) donor-recipient crossmatch are at high risk for rejection. We sought to correlate the pattern of C3d and C4d myocardial capillary deposition and pericapillary macrophage infiltration, possible markers of antibody-mediated rejection, to clinical evidence of rejection in these patients., Methods: Were studied 15 pre-sensitized pediatric patients who had 21 rejection episodes, as defined by International Society for Heart and Lung Transplantation (ISHLT) biopsy Grade >or=2R and/or the development of abnormal left ventricular (LV) function at >1 week after transplant. Archived paraffin-embedded endomyocardial biopsies (n = 74) from these patients were subjected to immunoperoxidase staining for C3d, C4d and CD68 in duplicate. Positive and negative controls were included for each assay., Results: C3d deposition was present in 74 of 74 (100%) specimens. C4d deposition was present in 6 of 74 (8%) biopsies from 4 patients. Biopsies with C4d deposition had ISHLT Grade >or=2R cellular infiltration in 5 of 6 specimens. Pericapillary macrophage infiltration, defined as positive staining for CD68, was found in 34 of 74 (46%) biopsies, and was associated with ISHLT Grade >or=2R in 10 of 34 (29%)., Conclusions: C3d deposition was universally present after heart transplantation with a CDC(+) donor/recipient crossmatch. C4d deposition and pericapillary macrophage infiltration were found with some, but not all, episodes of rejection. Further study is needed to understand the significance of the presence of complement fragments and pericapillary macrophage infiltration in these endomyocardial biopsies.

Background: Cardiac transplant recipients frequently receive a large number of transfusions. The objective of this study was to determine whether there is an association between total number of blood transfusions and cardiac allograft rejection., Methods: A retrospective analysis of all cardiac transplants between October 1, 1997, and December 31, 2001, was performed. Total number of transfusions, total number of rejection episodes Grade 3A or more, rejection-free survival, and overall survival were analyzed. Comparisons between patients bridged to transplantation with a Novacor left ventricular assist device (LVAD) and the primary transplant group were also made., Results: Eighty-two patients were transplanted. Fifteen were bridged to transplantation, and 67 underwent primary heart transplantation. Age and sex were similar for the LVAD group and the primary transplant group (45 +/- 11 vs 47 +/- 15 years and 67% vs 58% male sex, respectively). Mean follow-up was 658 +/- 486 days for the LVAD group and 708 +/- 548 days for the primary transplant group. Transfusions received were 50 +/- 34 U of packed red blood cells for the LVAD group and 7 +/- 12 for the primary transplant group (p < 0.001). There were no differences in donor characteristics between the 2 groups. The incidence of acute rejection within 1 year was 27% for the LVAD group and 39% for the primary transplant group (p = .28). Freedom from rejection was 71% at 1 year in the LVAD group compared with 59% for the primary transplant group (p = 0.39). In all 82 patients, the total number of transfusions was inversely correlated with the development of acute rejection (p = 0.011). Survival was 80% and 62% for the LVAD group at 1 and 3 years after transplantation and 88% and 85%, respectively, for the primary transplant group (p = 0.045)., Conclusions: The number of blood transfusions received by heart transplant recipients is inversely related with the number of acute rejection episodes.

Background: Minor histocompatibility antigen (mHag) discordances have been shown to play a critical role in graft-versus-host disease after bone marrow transplantation. However, the role of mHag in rejection of solid-organ allografts remains unknown. Therefore, the goal of this study was to define the role of a single mHag discordance derived from the polymorphic H13 allele in the development of cardiac allograft rejection in mice. The H13a and H13b alleles encode for the SSVVGVWYL (SVL9) and SSVIGVWYL (SIL9) mHag bound to the H2Db molecule, respectively., Methods: C56BL/10SnJ (H13a) cardiac allografts were transplanted into congenic B10.CE-H13b Aw(30NX)/Sn (H13b) mice. Allograft function was monitored daily and rejection was defined by cessation of heart beat. Rejection was confirmed histologically. The phenotypic and functional characteristics of the graft-infiltrating cells were analyzed by in situ and in vitro staining with H13a-specific tetramers and by chromium-51 (51Cr)-release assay, respectively., Results: Sixty-five percent of H13-incompatible allografts were rejected in 37.0+/-14.5 days. Sixty-eight percent of the H13a allografts transplanted into H13a-sensitized mice were rejected earlier, in 27.6+/-15.9 days. Rejected allografts showed histopathologic signs of chronic rejection with diffuse mononuclear cell infiltration, concentric intimal hyperplasia, and fibrosis. Both CD8+ (87%) and CD4+ (13%) T cells were observed in rejected allografts. In addition, 60% of the graft-infiltrating CD8+ T cells recognized a H2Db/SVL9 tetramer. Graft-infiltrating CD8+ T cells showed a significant H2Db-restricted, SVL9-specific cytotoxic activity., Conclusions: A single mHag discordance, as demonstrated with H13 disparity, results in the pathogenesis of chronic rejection of major histocompatibility complex-matched vascularized solid-organ allograft.

Background: Long-term follow-up of heart, liver, and lung transplantation has led to an increased recognition of secondary end-stage renal failure (ESRF) in transplant recipients. This study examines our center's experience with renal transplantation following previous solid organ transplantation., Methods: From January 1, 1992, to September 30, 1999, our center performed 18 renal transplants in previous solid organ recipients. During the same period, 815 total renal transplants were performed. One- and 3-year graft and patient survival, recipient demographics, donor type, and reason for transplantation were compared between these groups., Results: Of the 18 recipients, 7 had prior heart transplants, 4 had prior liver transplants, and 7 had prior lung transplants. Cyclosporine toxicity contributed to renal failure in 17 (94.4%) of the patients-either as a sole factor (11 patients) or in combination with hypertension, renal artery stenosis, or tacrolimus toxicity (6 patients). Kaplan-Meier 1- and 3-year patient survival was 82.9% and 73.7%, compared with 95.5% and 90.7% in all renal transplant recipients. No surviving patient has suffered renal allograft loss. Mean current creatinine level is 1.4 mg/dL., Conclusions: Renal transplantation is an excellent therapy for ESRF following prior solid organ transplantation. One and 3-year patient and graft survival demonstrate the utility of renal transplantation in this patient population.

To further define the role of indirect allorecognition, cardiac allografts from HLA-A2-transgenic (HLA-A2+) C57BL/6 mice were heterotopically transplanted into normal C57BL/6, CD4 T cell-knockout (KO) C57BL/6 mice, CD8 T cell-KO C57BL/6 mice, fully MHC-discordant BALB/c mice (allogeneic control), and HLA-A2+ C57BL/6 mice (syngeneic control). HLA-A2+ grafts were acutely rejected when transplanted into BALB/c mice (mean survival time: 10+/-0.8 days), normal C57BL/6 mice (mean survival time: 16.5+/-2.1 days) as well as CD8-KO mice (mean survival time: 12.8+/-1.3 days). Histopathological analysis revealed classical acute cellular rejection with moderate to severe diffuse interstitial CD4+ and CD8+ cellular infiltrates and significant intra-graft deposition of IgG and complement. In contrast, HLA-A2+ grafts were not rejected when transplanted into CD4-KO mice or HLA-A2+ mice. CD8-KO recipients treated with an anti-CD4 monoclonal antibody, but not with an anti-NK monoclonal antibody, failed to reject their allografts with prolonged administration of antibody (30 days). Spleen cells from mice rejecting HLA-A2+ allografts failed to lyse HLA-A2+ target cells indicating a lack of involvement of CD8+ T cells in the rejection process. In contrast, spleen cells from rejecting animals proliferated significantly to both HLA-A2+ cells and to a peptide derived from the HLA-A2 molecule. Development of anti-HLA-A2 antibodies was observed in all animals rejecting HLA-A2+ allografts. These results suggest that indirect allorecognition of donor MHC class I molecules leads to rejection of cardiac allografts and development of alloantibodies in this unique transplant model in which there is a single MHC discordance between donor and recipient.

Background: Intrathymic (IT) alloantigen combined with administration of rabbit anti-rat anti-lymphocyte serum (ALS) intraperitoneally induces donor-specific tolerance to rat cardiac transplants. The purpose of this study was to examine the effect of a brief course (4 days) of cyclosporine (CsA) on the development of IT tolerance., Methods: Buffalo (BUF) (RT1b) rats were given 25x10(6) fully MHC-mismatched Lewis (LEW) (RT1l) splenocytes by IT injection plus 1.0 ml of ALS intraperitoneally. Twenty-one days later, IT donor-specific LEW (group 1) or third-party (ACI, RT1a) (group 2) hearts were heterotopically transplanted to the abdominal aorta A third group of BUF (group 3) were given daily CsA (10 mg/kg) by oral gavage for 4 days before administration of IT LEW cells and ALS. Rejection as defined by the cessation of a palpable heartbeat was confirmed by histology. Cytokine profiles of allografts from all groups were then analyzed using a multi-probe RNase protection assay., Results: Sixty-seven percent of IT/ALS-treated BUF recipients not pretreated with CsA accepted LEW heart grafts for greater than 90 days. However, 86% of animals treated with CsA for 4 days before IT injection and ALS rejected allografts at 10.7+/-3.2 days. Third-party allografts (ACI) were uniformly rejected (7.0+/-0.0 days). Histology confirmed cellular rejection in CsA-treated allografts and cytokine analysis detected increased interleukin (IL)-3, IL-5, and tumor necrosis factor-alpha when compared to increased IL-2 and interferon-gamma in rejecting untreated controls., Conclusions: CsA can prevent the induction of intrathymic alloantigen tolerance. These results support the development of a CsA-sensitive, but IL-2-independent, active regulatory mechanism after intrathymic exposure to donor-specific alloantigen and depletion of mature peripheral T cells.

We have recently found that donor-specific tolerance to a cardiac allograft can be achieved after the intrathymic (i.t.) injection of donor splenocytes and a single intraperitoneal injection of rabbit antirat lymphocyte serum. The present study evaluated whether the tolerance induced by splenocytes injected i.t. could also prevent the rejection of kidney and skin allografts. Male Buffalo (RT1b) rats were given 25 x 10(6) fully MHC-mismatched unfractionated Lewis (RT1l) splenocytes by i.t. injection plus 1 ml of ALS i.p. and 21 days later underwent a Lewis heterotopic cardiac, orthotopic renal, or skin transplant. Lewis i.t. injection induced a donor-specific tolerance with indefinite cardiac allograft survival (> 153.1 days) in 88% of the recipients without the need for further immunosuppression, while renal and skin allograft survival was prolonged (kidney 14.8 days vs. control 7.8 days; skin 11.6 days vs. control 9.2 days) but were still rejected. Buffalo recipients with a long-surviving Lewis cardiac allograft after Lewis i.t. injection were still able to reject a third-party heterotopic ACI (RT1a) cardiac allograft in normal time (7.0 days), but did not reject a second Lewis cardiac allograft (> 100.0 days). In contrast, however, Buffalo recipients with long-surviving Lewis cardiac allografts did reject a Lewis skin allograft in normal time (10.0 days) and a Lewis renal allograft in a prolonged manner (17.6 days) without causing the rejection of the Lewis cardiac allografts. These data support the important role tissue-specific non-MHC antigens may play in the rejection of kidney and skin allografts.

Cyclosporine has gained acceptance as the immunosuppressive agent of choice in cardiac transplantation, but the validity of this assumption has yet to be established. Since January 1983, 25 patients have been randomly assigned to receive either conventional immunosuppression (azathioprine/antithymocyte globulin/prednisone) and pretransplant transfusion (PAAP, n = 11) or cyclosporine immunosuppression (cyclosporine and prednisone [CyA], n = 14). There was no difference in the age distribution (41 +/- 9 vs 38 +/- 11 years), indications for transplantation, preoperative serum creatinine level (1.2 +/- 0.2 vs 1.4 +/- 0.3 mg/dl), or postoperative follow-up time (13.5 +/- 5.4 vs 13.5 +/- 5.2 months). Mortality was not different (PAAP = 2, CyA = 3) and there was no difference in rejection episodes per patient (PAAP = 1.8, CyA = 1.9). Patients in the PAAP group had more serious infections (PAAP = 8, CyA = 3; P less than .02), but those in the CyA group developed a greater incidence of systemic hypertension (PAAP = 1, CyA = 10; p less than .02), pericardial effusion (PAAP = 0, CyA = 6; p = .05), and impaired renal function (creatinine 1.5 mg/dl, PAAP = 2, CyA = 11; p less than .02). Thus it appears that in this small series, cyclosporine is not associated with a significant increase in early survival. It does appear that patients on PAAP immunosuppression develop a greater number of serious infections, but the incidence of rejection episodes appears to be the same. Renal dysfunction and hypertension in patients receiving cyclosporine continue to be long-term concerns and may add to the morbidity and mortality of patients treated with this immunosuppressive regimen.

Cyclosporine is a new immunosuppressive drug that acts early in the exposure of a host to allogeneic stimulation. It is a peptide of fungal origin. It has selective action on T cells, leaving the other cells of the immune system intact. It acts by preventing the function of the early activation signals of T cells, such as the acquisition of receptors for Il 2 and Il 1. It is lipophilic, moderately well absorbed by the gut, and metabolized by the liver. Factors affecting absorption or hepatic metabolism alter the amount of cyclosporine available in the circulation. Circulating levels can be measured by radioimmunoassay or HPLC. Doses should be tailored to trough levels taken approximately 12 hours after an oral or intravenous dose or to individual pharmacokinetic curves. The drug is nephrotoxic, hepatotoxic, and neurotoxic. In addition, cyclosporine has been associated with hypertension, hemolytic-uremic syndrome, increased incidence of intravascular thrombotic events, hypertrichosis, gum hyperplasia, pericardial effusion, and lymphoproliferative disorders. Despite these complications, cyclosporine usage seems to have improved short-term cardiac allograft survival and to have reduced the complications associated with side effects of steroids. As a result, cyclosporine has spawned a resurgence of interest in cardiac transplantation, which will be of great benefit in prolonging the lives of patients with end-stage cardiac disease.

Since the introduction of cyclosporine in heart transplantation, the search for the ideal combination of immunosuppressive agents continues. Between January 1983 and February 1985, 32 patients have been randomized prospectively to either one of two immunosuppressive regimens: one includes pretransplant transfusion, prednisone, azathioprine and rabbit anti-thymocyte globulin [Group I, n = 14], the other includes cyclosporine and prednisone [Group II, n = 18]. There were no differences between Group I and II in relation to age distribution, indications for transplantation, preoperative serum creatinine, length of follow-up, mortality or number of rejection episodes per patient. However, there was a statistically significant increase in the incidence of serious infections in Group I compared to Group II patients, and also in Group II of the incidence of systemic hypertension (p less than 0.001), of symptomatic pericardial effusion (p less than 0.05) and impaired renal function (p less than 0.02). Adding cyclosporine to azathioprine immunosuppression is effective in treating ongoing rejection in patients not previously treated with cyclosporine. In conclusion, patients treated with azathioprine and prednisone (Group I) develop a greater number of serious infections, but both groups had a similar incidence of rejection. The development of renal dysfunction and hypertension in patients treated with cyclosporine continues to be of concern and may preclude its use as an effective long-term immunosuppressive agent in heart transplant recipients.

To assess the usefulness of circulating T-lymphocyte analysis in cardiac transplantation, T helper (Th) and T suppressor-cytotoxic (Ts-c) subsets were serially monitored in 33 cardiac allograft recipients treated with cyclosporine. The short-term prognosis of their 47 treated rejection episodes were retrospectively correlated with the changes in T-cell subpopulations. The data indicate three main findings. Reversed Th to Ts-c ratio (less than 1) was associated with a reduced incidence of rejection onset and a benign clinical course after treatment for rejection. Reversed Th:Ts-c ratio caused by antirejection therapy was associated with less chance of recurrence during the rest of hospitalization, regardless of the mode of therapy and irrespective of whether the rejection was primary or recurrent. These changes were mainly mediated by a reduction in T helper cells rather than changes in the T suppressor-cytotoxic subset or total T cells. Titration of antirejection therapy based on these T-cell dynamics may reduce either overtreatment or undertreatment. A prospective randomized study seems warranted to evaluate this approach as an alternative to a predetermined antirejection protocol.

Seventy fresh frozen biopsies of 22 human heart allografts were stained with mouse antihuman monoclonal antibodies (OKT-4, OKT-8, and OKM-1) using the immunoperoxidase method. The numbers of infiltrating cell phenotypes were correlated with patients' clinical status and histopathological diagnoses of the biopsies. At the clinically stable stage the number of OKT-4-positive cells (T-4 cells, helper/inducer), OKT-8-positive cells (T-8 cells, suppressor/cytotoxic), OKM-1-positive cells (M-1 cells, monocyte/macrophage) and T4/T8 ratio were lowest. During the early stage of rejection T-4 cells increased to the highest values. T-8 cells also increased significantly and T4/T8 ratio increased to the peak level as well. During the later stage of rejection, T-8 and M-1 cells increased to the highest values and T-4 cells and T4/T8 ratio decreased. After the treatment of rejection T-4 cells continued to decrease and T-8 cells and M-1 cells decreased to intermediate levels, but T4/T8 ratio still remained level. The numbers of T-4 cells, T-8, cells and M-1 cells were closely associated with the histopathologic severity of rejection. These results were also correlated with the allografts' prognoses. Interestingly, high T4/8 ratios with high number of T-4 cells in biopsies during the quiescent period were often followed by rejection episodes within 7 days, even though the pathological diagnoses were mild rejection. Another important finding was that after the treatment of rejection, persistent M-1 cells and low T4/T8 ratios in situ were frequently accompanied by recurrent rejections. Thus, monitoring of infiltrating cell phenotypes may be beneficial in the management of clinical cardiac transplantations.

Of 57 patients referred to the McGuire Veterans Administration Medical Center, Richmond, Va, for evaluation for cardiac transplantation, 18 received allografts. The overall graft survival rate was 67% from two to 24 months after transplantation. In 15 recipients receiving immunosuppression with rabbit anti-human thymocyte globulin, prednisone, and azathioprine, previous transfusion and HLA-DR matching resulted in improved graft survival. In three patients receiving cyclosporin and prednisone, hypertension, nephrotoxicity, and hepatotoxicity were seen. All surviving recipients were in functional New York Heart Association class 1 or 2. The cost to the Medical Center was $23,275 per transplant. The results achieved at the center suggested that a regionalized cardiac transplant program can be established within the VA health care system with acceptable clinical results and significant financial savings.

The role of pretransplant transfusion in cardiac allograft recipients was determined retrospectively in 68 patients. Three groups were studied: group 1 (n = 29) received no pretransplant transfusion, group 2 (n = 15) received transfusion over one year prior to transplantation, and Group 3 (n = 24) received 5 or 10 50-100 ml units of random donor red blood cells or buffy coat 2-4 weeks prior to transplantation. Data were analyzed for survival, number of rejection episodes, and number of infections. Immunosuppression included azathioprine, prednisone, and antithymocyte globulin. Survival in transfused patients (groups 2 and 3) was 68% and 51% at 1 and 5 years, respectively, while in the nontransfused population (group 1) it was 35% and 16%. The incidence of rejection episodes per year of survival was similar in the three groups (group 1: 1.3, group 2: 1.1, group 3: 1.3; P greater than 0.05). The number of infections per year of survival were greater in the transfused patients but this did not achieve statistical significance (group 1: 1.0, group 2: 1.2, group 3: 1.7; P greater than 0.05). Thus, we conclude that cardiac transplant recipients who have received blood transfusions prior to transplantation may have enhanced survival over patients who have not received preoperative transfusions.

The prophylactic use of rabbit antithymocyte globulin (RATG) was evaluated in 13 cardiac allograft recipients who received a low-dose of RATG (175 +/- 32 mg) after transplantation (group 1). The patients were retrospectively compared with 13 parallel cases receiving the same treatment except for the initial RATG (group 2). There were no differences in the patient composition and the level of the basic immunosuppression therapy with cyclosporine. The patients treated with RATG (group 1) showed substantially greater suppression of T helper and total T cells up to 10 and 20 days after surgery, respectively. The incidence of rejection episodes during the initial admission seemed lower in patients receiving RATG, with a borderline significance (1.4 versus 2.4, p = 0.06). This accounts for the reduced requirement of methylprednisolone in treating rejection in patients in group 1 (1.3 gm versus 4.0 gm, p less than 0.02). Moreover, the patients without initial doses of RATG (group 2) more frequently experienced recurrent rejection (five of 12 versus nine of 10, p less than 0.05), which necessitated rescue RATG; the initial difference in the RATG usage disappeared by the time of discharge. Serious infection occurring after antirejection therapy was common in the group 2 patients without initial doses of RATG, although the overall incidence of infection was not statistically different. Prophylactic use of RATG at the dosage used appeared to reduce the incidence of rejection and the requirement for intravenous steroids and other immunosuppressants. It seems warranted to test this approach in a prospective randomized manner.

Forty-one heart transplant recipients were monitored serially for the expression of transferrin receptors and T-helper/T-suppressor cytotoxic ratios on circulating lymphocytes during the hospitalization periods after heart transplantations (60.5 +/- 18.9 days). These values were retrospectively correlated with the patients' clinical status with respect to rejection and infection. During clinically stable periods the average values of percentage of transferrin receptor-positive lymphocytes and T-helper/T-suppressor cytotoxic ratios were 5.9 +/- 4.3 and 1.5 +/- 1.0, respectively. The percentage of transferrin receptor-positive lymphocytes increased to a level of 12.0 +/- 5.4 (p less than 0.001) during the early prerejection phase and remained at this level throughout the rejection period. T-helper/T-suppressor cytotoxic ratios increased to 1.96 +/- 0.92 during the early prerejection phase (p less than 0.05), peaked at 2.30 +/- 1.21 during the late prerejection phase (p less than 0.01), but began to decline by the rejection period. After rejection treatment percentage of transferrin receptor-positive lymphocytes decreased to 8.4 +/- 5.3 (p less than 0.05), and T-helper/T-suppressor cytotoxic ratios decreased to normal levels. In contrast, in patients with infectious complications, a remarkably elevated percentage of transferrin receptor-positive lymphocytes (20.7 +/- 11.7) and relatively low T-helper/T-suppressor cytotoxic ratios (1.3 +/- 0.5) were noted. The data show an association between the clinical status, such as rejection and infection, and these immunologic measurements as transferrin receptor-positive lymphocytes and T-helper/T-suppressor cytotoxic ratios in heart transplant recipients.(ABSTRACT TRUNCATED AT 250 WORDS)

Aim of the study is to determine the effect of low dose interleukin-2 (IL-2) therapy on the kinetics of induction of circulating exosomes and their immunological properties resulting in prevention of chronic rejection following allogeneic murine cardiac transplantation (HTx). BALB/c (H2d) to C57BL/6 (H2b) HTx was performed heterotopically in the abdominal cavity. Costimulatory blockade consisting of MR1 (250 μg/ip day 0) and CTLA4-Ig (200 μg/ip day 2) was administered to induce tolerance. IL-2 (2000IU/day) was administered from day 14 post-transplant for 3 weeks. Sera were collected on days 7-100 for analysis of exosome induction and characterization of cardiac self-antigens (SAgs) (Myosin, Vimentin) by western blot. Foxp3, PDL1, CD73 in exosomes were detected using aldehyde bead - based flow cytometry. Antibodies (Abs) to donor MHC and SAgs were detected using immunofluorescence and ELISA respectively. BALB/c to C57BL/6 HTx acutely rejected (AR) with median survival time of 8 days. Costimulatory blockade prevented AR but developed chronic rejection with mean of 42 days circulating exosomes contained significantly higher levels of Myosin and Vimentin in the exosomes day 15 (p<0.05) and day 30 (p<0.01). Abs to MHC (32±13.8% vs 2±1.1; p<0.001) was detected on day 30, whereas Abs to Myosin (1114.21±443.4 ng/ml; p<0.0001) and Vimentin (519±241 ng/ml; p<0.0001) were detected on day45 during chronic rejection with fibrosis (57.1±20.7 vs 2.3±1.7%). Low dose IL-2 significantly prolonged the survival, 115 vs 42 (p<0.001) and did not induce exosomes with cardiac SAgs or developed Abs to MHC or SAgs. Low dose IL-2 not only abrogated chronic rejection but also induced splenic CD4+CD25+ Foxp3 T regulatory (Treg) cells in the allograft by day 45. Flow cytometry of circulatory exosomes isolated in day 45 also demonstrated increased Foxp3+ PDL1+ (3.3 fold) and FoxP3+ CD73+ (3 fold) compared to exosomes isolated on day 45 from chronic rejecting animals. Our results demonstrate that low dose IL-2 abrogated the development of chronic rejection following HTx and induced circulating Treg cells derived exosomes with PDL1 and CD73 along with splenic Tregs and graft infiltrating FoxP3 cells. [ABSTRACT FROM AUTHOR]

Aim: The study investigated results of Luminex Single Antigen (SA) testing of sera following different pre-treatments and C1q binding assay (C1q) to identify the method that best correlates with cell based crossmatch. Methods: SA assays (One Lambda) were performed on sera from sensitized pts awaiting heart transplantation. Sera were analyzed untreated, treated with 0.05M DTT, heat treated at 63°C, treated with Adsorb Out beads, diluted at 1:25, or using C1q (One Lambda). Complement dependent cytotoxicity (CDC) and flow (FCXM) crossmatches were performed using cells from surrogate donors. Results: Luminex SA results of the serum pre-treatments and C1q showed considerable variation within each pt, especially between untreated and C1q. SA results of DSA specificities obtained using the various protocols are presented in , and their correlation with crossmatching is presented in . Conclusions: Luminex SA and crossmatch results suggest that high MFI DSA that are not identified on untreated sera, but are identified following DTT, heat treatment, 1:25 dilution, and by C1q, correlate with positive CDC and FCXM. DSA with high MFI on untreated, but not identified by C1q, correlate with positive CDC. These results suggest that high MFI Abs detected by either SA or C1q assay may represent an immunologic barrier. Further testing is needed to determine the most effective means of using these assays to distinguish antibodies that will result in positive crossmatches. [Copyright &y& Elsevier]

Aim of the study is to determine the effect of low dose interleukin-2 (IL-2) therapy on the kinetics of induction of circulating exosomes with tolerance markers (PDL1, CD73) and their role in abrogating chronic rejection following murine heterotopic cardiac transplant (HTx). Allogenic (BALB/c to C57BL/6) HTx was performed heterotopically in abdominal cavity. Costimulatory blockade consisting of MR1 (250 μg/ip day 0) and CTLA4-Ig (200 μg/ip day 2) was performed. Low dose IL-2 (2000IU/day) was administered from day 15 post-transplant for 3 weeks. Sera was collected on days 7-100 for kinetic analysis of exosome induction and characterization of PDL1, CD73, cardiac self-antigens (SAgs) (Myosin, Vimentin), class II MHC trans-activator (CIITA), and 20S proteasome by western blot using specific antibodies (Abs) and Abs to SAgs. BALB/c to C57BL/6 HTx rejected transplanted heart with median survival time of 8 (7-9) days. HTx done following costimulatory blockade developed chronic rejection with mean survival time 26 (7-47) days. Exosome and Ab development demonstrated induction of exosomes with Vimentin (2.2± 1.0 vs 0.6±0.1; p<0.05) and 20S proteasome levels (0.6±0.2 vs 0.2±0.02; p<0.03) on day 15, whereas Myosin (1±0.3 vs 0.1±0.05; p<0.05), CIITA (1.3±0.7 vs 0.2±0.3; p<0.05) on day 45 and no tolerance markers (PDL1, CD73) were detected. Development of Abs to Myosin (1289±409 vs 187±50) and Vimentin (503±218 vs 86±3) significantly increased during chronic rejection of HTx. Low dose IL-2 significantly prolonged the survival, 107 vs 26 (p<0.001). IL-2 therapy induced exosomes with PDL1 (1.4±0.8 vs 0.09±0.14; p<0.01) CD73 (2.3±1.2 vs 0.4±0.3; p<0.01) on day 45, and decreased exosome induction with SAgs (Myosin 0.4±0.2; Vimentin 0.7±0.4) and Abs to SAgs (Myosin 540±283, Vimentin 295±47). Circulating exosomes containing cardiac SAgs and development of Abs to SAgs lead to chronic rejection in a murine model occurring after costimulation blockade following allogenic HTx. Low dose IL-2 abrogated chronic rejection and induced circulating exosome with tolerance markers. This result supports an important role for exosomes, inducing tolerance and significantly delaying development of chronic rejection in murine model of chronic cardiac allograft rejection. [ABSTRACT FROM AUTHOR]