Douglas Johnston

The role of regulatory cells in miniature swine rendered tolerant to cardiac allografts by donor kidney cotransplantation.

To determine the mechanism by which cotransplantation of a kidney allograft induces tolerance to a donor heart in miniature swine, we examined the role of CD25+ cells in heart/kidney recipients. Tolerance was induced to class‐I MHC mismatched hearts by cotransplanting a donor‐specific kidney with a 12‐day course of cyclosporine. Peripheral blood leukocytes (PBL) were isolated from tolerant heart/kidney recipients and used in cell‐mediated lympholysis (CML) coculture assays as either unmodified PBL, PBL enriched for CD25+ cells or PBL depleted of CD25+ cells to assess their ability to suppress CML responses of naïve recipient‐matched leukocytes against mismatched target cells. Primed PBL from tolerant heart/kidney recipients completely suppressed lysis by naïve cells. Complete suppression of the response of naïve recipient‐matched leukocytes against donor‐matched target cells was lost following the depletion of CD25+ cells from tolerant heart/kidney animal PBL, but it was reestablished by incubation of naïve cells with small populations of CD25+ cells from tolerant heart/kidney animals. These data suggest that peripheral blood from tolerant heart/kidney recipients contains regulatory cells that, upon priming, can suppress the response of naïve‐matched PBL in coculture CML assays, and that suppression appears to be dependent on cells expressing CD25.

Indirect recognition of MHC class I allopeptides accelerates lung allograft rejection in miniature swine.

The role of indirect allorecognition in graft rejection is examined in two experiments using a swine lung transplantation model. First, two swine received class I mismatched grafts without immunosuppression; another two recipients were treated postoperatively with cyclosporine (CsA). These swine exhibited acute and chronic rejection, respectively. All four recipients developed T‐cell reactivity to donor‐derived class I major histocompatibility complex (MHC) peptides. Second, six swine were immunized with synthetic donor‐derived class I allopeptides prior to transplantation. Control groups consisted of nonimmunized recipients (n = 6) and recipients immunized with an irrelevant peptide (n = 3). These recipients all received a 12‐day course of post‐operative CsA. Swine immunized with allopeptides exhibited accelerated graft rejection, as compared to both control groups (p < 0.01 and p = 0.03, respectively). Within the experimental group, the dominant histologic finding was acute rejection (AR). Obliterative bronchiolitis (OB) was seen in the graft with the longest survival. Both control groups showed a lesser degree of AR, with four out of six nonimmunized swine ultimately developing OB. These studies suggest that indirect allorecognition is operative during lung allograft rejection, and that pre‐transplant sensitization to donor‐derived MHC allopeptides can accelerate graft rejection.

Composite ‘Thymoheart’ Transplantation Improves Cardiac Allograft Survival

We have generated a novel composite organ, the thymoheart, which facilitates the contemporaneous transfer of fully vascularized and functional donor thymic tissue to the host at the time of cardiac transplantation. Composite thymoheart allografts were prepared in MHC‐inbred miniature swine by implanting autologous thymic tissue into donor hearts 60–90 days before organ procurement. Thymoheart allografts and unmanipulated control hearts were then transplanted into three groups, each treated with the same 12 days of cyclosporine. MHC‐matched thymohearts transplanted into euthymic recipients had a minimum survival ranging between 72 and 194 days vs. 42–64 days for unmanipulated control hearts (p = 0.02). MHC class I‐disparate thymohearts transplanted into euthymic recipients had a minimum survival ranging between 64 and 191 days vs. 30–55 days for unmanipulated control hearts (p = 0.01). MHC class I‐disparate thymohearts transplanted into thymectomized recipients survived between 41 and 70 days vs. 8–27 days for unmanipulated control hearts (p = 0.01). Cellular and humoral functional assays, and skin grafting, confirmed the presence of donor‐specific hyporesponsiveness in long‐term thymoheart allografts recipients. The transfer of vascularized, functional donor thymic tissue to the host at the time of cardiac transplantation may provide a novel approach to the induction of tolerance in human heart transplant recipients.

Combination treatment with donor-specific transfusions and cyclosporine a induces long-term survival of cardiac allografts in miniature swine

Background. To evaluate whether pretransplant donor-specific transfusions (DST) can induce tolerance to cardiac allografts in large animals, heterotopic cardiac transplants were performed across a class I MHC barrier in inbred miniature swine. Methods. Experimental animals received two DSTs, each containing 1.4x108 viable peripheral blood mononuclear cells, 14 and 7 days prior to transplantation together with a 12-day course of cyclosporine (CyA) (13 mg/kg IV) starting on postoperative day (POD) 0. Results. Untreated (n=2) and DST-only (n=2) treated control animals rejected between POD 6 and 8. Animals treated with CyA alone (n=3) exhibited graft survival to 53, 52 and 59 days. In contrast, the combination of DST and CyA (n=3) led to stable graft function for >200 days. Long-term survivors maintained peripheral CML response against donor antigen. Following DSTs, the donor-specific proliferative response of CD8+ recipient T cells was significantly increased (P=0.011), and a significant number of CD8+ T cells underwent apoptosis (10.1% on POD 0; 5.2% on POD –14; P=0.04). None of the DST-treated animals developed donor-specific antibodies. Conclusions. These results are the first to demonstrate the ability of DST to induce operational tolerance to cardiac allografts in large animals, and they suggest that peripheral mechanisms of tolerance mediate this effect.

Recipients of MHC class I disparate lung grafts develop T cell reactivity to donor MHC class I allopeptides in an orthotopic swine model

logical reactivity to donor antigens, followed by vascularized heterotopic heart transplantation to determine if CAV still develops in the tolerant recipient. Procedures: In mice, specific immunological nonreactivity was achieved in recipients by (1) neonatal administration of allogeneic spleen cells from F1 donors between class I mismatched donor and recipient strains, (2) acheivement of mixed chimerism by bone marrow infusion of irradiated adult recipients, (3) use of genetically immunodeficient recipients (RAG1-/or SCID). In swine, reduced reactivity was achieved through the induction of mixed chimerism. After nonreactivity was induced and verified through extended persistence of skin grafts, hearts from donors were heterotopically transplanted into the recipients. Results: Most hearts transplanted into tolerant or immunodeficeint mice developed striking vasculopathy in their coronary arteries (neonatal tolerance: 12 of 15, mixed chimeras: 15 of 23, immunodeficient recipients: 16 of 31). In contrast, isotransplants were virtually free of CAV. Three of the four transplanted swine hearts showed neointimal proliferation characteristic of CAV in 1%-6% of donor coronary arteries. Control hearts transplanted into untreated recipients across similar minor antigen barriers were acutely rejected within 44 days whereas control isografts survived indefinitely without evidence of CAV. Conclusions: Elimination of T and B cell immune responses was not sufficient to prevent the development of CAV. Other immune effectors, perhaps NK cells, could play a role in the development of CAV in this immunodeficient state. Additional results on these alternate effectors will be presented.

Disparity in spatial distribution of pericardial calcifications in constrictive pericarditis

Background Pericardial calcification is seen among patients with constrictive pericarditis (CP). However, the pattern of pericardial calcium distribution and the association with clinical outcomes and imaging data are not well described. Methods This was a retrospective study from 2007 to 2013 to evaluate the pattern of pericardial calcium distribution by CT in CP using a semiquantitative calcium scoring system to calculate total pericardial calcium burden and distribution. Calcium localisation was allocated to 20 regions named after the corresponding heart structure. Baseline clinical data, imaging data and clinical outcomes were collected and compared between the calcified pericardium and non-calcified pericardium groups. We assessed the effect of pericardial calcium on clinical outcomes and echocardiographic data between the two groups. Results Of the 123 consecutive patients with CP (93 male; mean age 61±13 years) between 2007 and 2013, 49 had calcified pericardium and 74 had non-calcified pericardium. Distribution of calcium on the left ventricle (LV) basal anterior, mid-anterior and apical segments in addition to right ventricle (RV) apical segment was involved in <30% of the cases with the remaining segments involved in >35% of cases. A potential protective role of RV calcium on regional myocardial mechanics was noted. Conclusion Preferential distribution of calcium in CP in a partial band-like pattern (from basal anterolateral LV going inferiorly and then encircling the heart to reach the RV outflow tract) with extension into the mitral and tricuspid annuli was noted. Pericardial calcium was not significantly associated to clinical outcomes.

Praeoperative Donor-spezifische Transfusionen kombiniert mit Cyclosporin induzieren Toleranz für MHC-l-inkompatible Herztransplantate im Miniatur Schwein Modell

Purpose: To evaluate the effect of pre-transplant donor-specific transfusion on sensitization and tolerance induction to cardiac allografts in a clinically relevant large-animal model. Methods: Heterotopic cardiac transplants were performed across a major histocompatibility complex (MHC) class I barrier in inbred miniature swine. Experimental animals were treated with two donor-specific transfusions (DSTs), each containing 1.4×l08 viable peripheral blood mononuclear cells, 14 and 7 days prior to heart transplantation. All animals received a 12-day course of cyclosporine (CyA) (13 mg/kg IV) starting on post-operative day (POD) 0. Control groups received CyA alone, DST alone or no treatment. Cell mediated lympholysis (CML) assays were performed at regular basis. T cell priming and activation induced cell death (AICD) were assessed by carboxyfluoroscein succinimidyl ester (CFSE) proliferation assays and Annexin V staining. Rejection was monitored by serial biopsies. Results: Untreated (n = 2) and DST-only (n = 2) treated control animals rejected between POD 6 and 8. Animals treated with CyA alone (n = 3) exhibited graft survival to 53, 52 and 59 days. In contrast, the combination of DST-preteatment and CyA (n = 3) led to stable graft function for > 200, > 200, and > 200 days. Grafts in both the control and experimental groups showed equally severe cellular infiltrates on POD 28 and 50 (ISHLT lb to 3b/4). However, in the control groups, rejection continued to worsen over time, whereas in the experimental group, the cellular infiltrate diminished over time (ISHLT 0/4 on POD 100 and 200). Like the controls, experimental animals maintained peripheral CML response against donor and third party antigen. Following two DSTs, CD4+and CD8+recipient T cells demonstrated an increased donor-specific proliferative response on CFSE assay (CD4 +: 29.9% anti-donor, 16.4% anti-third-party; CD8 +: 45.9% anti-donor, 31.5% anti-third-party; n = 6). There was no difference in the rate of dividing T cells undergoing AICD in response to donor versus third-party antigen stimulation by Annexin V staining. None of the animals developed anti-donor antibodies following DST treatment. Conclusion: The combination of pre-transplant DST and a short course of CyA appears to facilitate tolerance to MHC class I disparate cardiac allografts in a preclinical miniature swine model. The sustained peripheral anti-donor CML response and the fact that alloreactive T cells were primed by the DST prior to transplantation suggest an active regulatory mechanism rather than peripheral deletion.

Regulatory cells in the maintenance phase of tolerance in miniature swine: coculture cell mediated lympholysis assay as approach for a tolerance assay

that CM/IFA-injected animals grafted with MHC class II KO hearts experienced significant prolongation of heart survival as compared to untreated mice (11 1 d vs 24 2 d). We conclude that modulation of CM response can on its own achieve graft prolongation when CD4 alloresponse is oligoclonal, i.e. mediated via indirect allorecognition. Our findings have important implications for the design of future therapies in heart transplantation.

Intracoronary interferon-γ accellerates cardiac allograft rejection in miniature swine

Purpose: The effect of interferon(IFN) on allograft rejection remains controversial. Some have proposed that IFNhas a protective effect on the microcirculation of allografts, while others have shown IFNto be deleterious. We investigated the effects of intracoronary IFNon cardiac rejection in a preclinical large animal model. Methods: MHC class I disparate hearts were heterotopically transplanted into swine treated with a 12-day course of cyclosporine (10-14mg/kg/day) (n 3). An Alzet osmotic mini-pump delivered IFNinto the coronary vasculature at a rate of 100ng/day. Control recipients were treated with a similar course of cyclosporine but without IFN(n 3). Results: Swine treated with IFNdeveloped an earlier onset of high grade acute rejection as compared to controls, and rejected their allografts in 19 days vs. 46 days for controls (p 0.02). Elastin staining of the cytokine treated allografts showed severe endothelialitis and intimal thickening as early as POD 13. Conclusion: The delivery of IFNinto the coronary circulation of cardiac allografts appears to accelerate both acute and chronic rejection. Targeting IFNin antirejection regimens may ameliorate both of these processes.