[Show abstract][Hide abstract] ABSTRACT: The relative contribution of central and peripheral mechanisms to the generation and maintenance of allograft tolerance is of considerable interest. Here, we present new evidence that regulatory T cells (Foxp3+) maintain skin and heart allograft tolerance in mixed hematopoietic chimeric mice. Transient depletion of both donor- and recipient-derived Foxp3+ cells was necessary and sufficient to induce decisive rejection of long-accepted skin and heart allografts. In contrast, stable hematopoietic chimerism remained, and there was no detectable induction of donor-specific reactivity to hematopoietic cells. Foxp3+ cell depletion did not result in the rejection of skin grafts of only MHC-disparate donors (B6.C-H2d/bByJ), indicating that MHC antigens were not the target in the graft. We conclude that two different mechanisms of tolerance are present in mixed chimeras. Hematopoietic chimerism, resistant to Foxp3+ depletion, is probably due to deletional tolerance to MHC antigens, as supported by previous studies. In contrast, regulatory tolerance mechanisms involving Foxp3+ cells are required to control reactivity against non-MHC antigens not present on hematopoietic lineages.
American Journal of Transplantation 08/2014; 14(10). DOI:10.1111/ajt.12851 · 5.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chronic allograft vasculopathy (CAV) in murine heart allografts can be elicited by adoptive transfer of donor specific antibody (DSA) to class I MHC antigens and is independent of complement. Here we address the mechanism by which DSA causes CAV. B6.RAG1(-/-) or B6.RAG1(-/-)C3(-/-) (H-2(b)) mice received B10.BR (H-2(k)) heart allografts and repeated doses of IgG2a, IgG1 or F(ab')(2) fragments of IgG2a DSA (anti-H-2(k)). Intact DSA regularly elicited markedly stenotic CAV in recipients over 28 days. In contrast, depletion of NK cells with anti-NK1.1 reduced significantly DSA-induced CAV, as judged morphometrically. Recipients genetically deficient in mature NK cells (γ-chain knock out) also showed decreased severity of DSA-induced CAV. Direct NK reactivity to the graft was not necessary. F(ab')(2) DSA fragments, even at doses twofold higher than intact DSA, were inactive. Graft microvascular endothelial cells responded to DSA in vivo by increased expression of phospho-extracellular signal-regulated kinase (pERK), a response not elicited by F(ab')(2) DSA. We conclude that antibody mediates CAV through NK cells, by an Fc dependent manner. This new pathway adds to the possible mechanisms of chronic rejection and may relate to the recently described C4d-negative chronic antibody-mediated rejection in humans.
American Journal of Transplantation 11/2011; 12(2):313-21. DOI:10.1111/j.1600-6143.2011.03836.x · 5.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Complement fixation, as evidenced by C4d in the microvasculature, is a widely accepted criterion of antibody-mediated rejection. Complement fixation has been shown to be essential in acute antibody-mediated rejection, but its role in chronic rejection has not been addressed. Previous studies showed that passive transfer of complement fixing monoclonal IgG2a anti-H-2Kk into B6.RAG1-/- KO recipients of B10.BR hearts led to progressive chronic transplant arteriopathy (CTA) over 14-28 days, accompanied by C4d deposition. The present studies were designed to test whether complement was required for these lesions. We report that a noncomplement fixing donor-specific alloantibody (DSA, monoclonal IgG1 anti-H-2Kk) injected into B6.RAG1-/- KO recipients of B10.BR hearts also promotes CTA, without C4d deposition. Furthermore, a passive transfer of DSA (monoclonal IgG2a anti-H-2Kk) initiated endarteritis followed by CTA in B6.RAG1-/- mice genetically deficient in the third component of complement (RAG1-/-C3-/-). These studies indicate that antibody to class I MHC antigens can trigger chronic arterial lesions in vivo without complement participation, in contrast to acute antibody-mediated rejection. This pathway may be relevant to C4d-negative chronic rejection sometimes observed in patients with DSA, and argues that lack of C4d deposition does not exclude antibody-mediated chronic rejection.
American Journal of Transplantation 03/2010; 10(3):510-7. DOI:10.1111/j.1600-6143.2009.02958.x · 5.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Viral infections including those due to cytomegalovirus have been associated with accelerated cardiac allograft vasculopathy (CAV) in clinical trials and some animal models. Evidence demonstrating a direct causal relationship between such infections and de novo formation of coronary vascular lesions is lacking. Heterotopic murine cardiac transplants were performed in a parental to F1 combination in animals lacking both T- and B-lymphocytes (RAG(-/-)). Coronary vasculopathy developed almost exclusively in the presence of recipient infection with lymphocytic choriomeningitis virus but not in uninfected controls. This process was also dependent upon the presence of natural killer (NK) cells as depletion of NK cells abrogated the process. These data show that a viral infection in its native host, and not previously implicated in the production of CAV, can contribute to the development of advanced coronary vascular lesions in cardiac allotransplants in mice. These data also suggest that virus-induced CAV can develop via an NK-cell-dependent pathway in the absence of T- and B-lymphocytes.
American Journal of Transplantation 11/2009; 9(11):2479-84. DOI:10.1111/j.1600-6143.2009.02801.x · 5.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cardiac allograft vasculopathy (CAV) is a major source of late posttransplant mortality. Although numerous cell types are implicated in the pathogenesis of CAV, it is unclear which cells actually induce the vascular damage that results in intimal proliferation. Because macrophages are abundant in CAV lesions and are capable of producing growth factors implicated in neointimal proliferation, they are leading end-effector candidates. Macrophages were depleted in a murine heterotopic cardiac transplant system known to develop fulminant CAV lesions. C57BL/6 hearts were transplanted into (C57BL/6 x BALB/c)F(1) recipients, which then received anti-macrophage therapy with intraperitoneal carrageenan or i.v. gadolinium. Intraperitoneal carrageenan treatment depleted macrophages by 30-80% with minimal effects upon T, B or NK cells as confirmed by flow cytometry and NK cytotoxicity assays. Carrageenan treatment led to a 70% reduction in the development of CAV, as compared to mock-treated controls (p = 0.01), which correlated with the degree of macrophage depletion. Inhibition of macrophage phagocytosis alone with gadolinium failed to prevent CAV. Macrophages may represent the end-effector cells in a final common pathway towards CAV independent of T-cell or B-cell alloreactivity and exert their injurious effects through mechanisms related to cytokine/growth factor production rather than phagocytosis.
American Journal of Transplantation 01/2008; 7(12):2675-82. DOI:10.1111/j.1600-6143.2007.01997.x · 5.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Integrin alpha v beta 3 is important for cell survival, signaling and migration, particularly during angiogenesis and tumorigenesis, where it has been proposed as a therapeutic target. alpha v beta 3 is up-regulated following transplantation and beta 3 polymorphisms are associated with increased acute kidney rejection, suggesting that alpha v beta 3 may also play a role in transplant rejection. Here, using a model of allogeneic heart transplantation, we show that allograft survival is prolonged in beta 3 integrin-deficient (beta 3(-/-)) mice. This is associated with Th2-type immune responses and reduced T-cell infiltration into grafts and T cells from beta 3(-/-) mice show impaired adhesion and migration, consistent with a role for alpha v beta 3 in transmigration. These studies provide evidence that targeting beta 3 integrins impairs recruitment of effector cells and alters cytokine production, so prolonging graft survival. We also show that low doses of blocking antibodies against leukocyte function associated antigen-1 (LFA-1)/alpha L beta 2 and very late antigen-4 (VLA-4)/alpha 4 beta 1, when combined with deletion of beta 3, lead to long-term survival of allografts with no evidence of chronic rejection. Hence we provide strong mechanistic evidence supporting previous genetic studies, demonstrate the involvement of beta 3 integrins in both acute and chronic rejection and identify beta 3 as a new target for immunosuppressive therapy.
American Journal of Transplantation 06/2007; 7(5):1080-90. DOI:10.1111/j.1600-6143.2007.01757.x · 5.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Murine heterotopic cardiac allografts were used to reveal some of the fundamental interrelationships between donor-specific alloantibodies (DSA), chronic transplant arteriopathy (CTA) and capillary C4d deposition. B10.BR recipients of B10.A hearts developed transient DSA and C4d deposition that peaked on day 7 and became undetectable at day 56 while CTA developed progressively. Male cardiac grafts in female recipients showed similar degrees of CTA at day 56 but never developed DSA or C4d deposition, indicating that T cell-mediated mechanisms are sufficient to produce CTA. Passive transfer of monoclonal IgG2a anti-H-2K(k) into B6.RAG1 KO recipients of B10.BR hearts over 14-28 days led to progressive CTA. If treatment was stopped on day 14, lesions showed little progression and had no C4d deposition or detectable DSA on day 42. If treatment was stopped on day 28 when the lesions were fully developed, no regression occurred over the next 28 days, even though C4d deposition and circulating antibody became undetectable. Therefore, a minimum threshold of antibody exposure is needed to cause CTA. Once the CTA develops, C4d may become negative after DSA disappears. Thus, serial samples are needed in clinical studies to ascertain the relevance of alloantibody to the lesions of chronic graft rejection.
American Journal of Transplantation 02/2007; 7(1):57-65. DOI:10.1111/j.1600-6143.2006.01599.x · 5.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We previously demonstrated that cardiac allografts to fully tolerant chimeric mice developed cardiac allograft vasculopathy (CAV). Here we begin to examine which components of the immune system are responsible for the pathogenesis of CAV in such tolerant recipients. B10.A/B6 mixed chimeric mice were created by receiving injections of bone marrow cells from B10.A (H-2k) mice given to C57BL/6 (B6; H-2b) mice with some preparations. B10.A skin grafts were first placed onto B10.A/B6 mixed chimeric recipients. When the donor strain skin grafts had survived perfectly for at least 56 days, B10.A hearts were transplanted heterotopically into B10.A/B6 mixed chimeric recipients. Hearts were examined for the presence of CAV 56 days later. To determine the effector cells that contribute to the development of CAV, they were treated weekly with a combination of anti-CD4/CD8 monoclonal antibodies (mAbs) or anti-NK1.1 mAb continuing until 56 days. 14 B10.A cardiac transplants of 18 otherwise untreated B10.A/B6 chimeric recipients developed CAV; concurrent B6 isografts were unaffected (0/7). In chimeric recipients treated with anti-CD4/8 mAbs, the prevalence of CAV was greatly reduced (0/6, P < .01 compared to the untreated group). Anti-NK1.1 mAb was not effective in the prevention of CAV (4/5). These data suggest that T cells may contribute in some way to the development of CAV that occurs in those fully tolerant recipients. Host T cells that may still be responsive to non-major histocompatability complex antigens, including tissue-specific antigens presented not on skin but on heart, may also be responsible for the development of CAV in tolerant animals.
[Show abstract][Hide abstract] ABSTRACT: In prior experiments, we found that recipients, even though specifically tolerant of donor antigens, will develop striking cardiac allograft vasculopathy (CAV) in allogeneic mouse heart transplants. This suggested that innate immune responses, in addition to conventional adaptive immunity, may be involved in the development of CAV. We accordingly performed transplants in the parental-to-F1 combination seeking supportive evidence of NK cell activity directed against the vessels of parental donor transplants as a manifestation of "hybrid resistance." When such lesions were indeed found, we investigated their pathogenesis employing immunopathological analysis, in vitro measurements of NK cytotoxicity, and donor-specific T-cell activity in F1 recipients of parental donor hearts. We present evidence that NK cells can promote cardiac allograft vasculopathy. Since NK cell activity is not well targeted by current immunosuppressive therapy, its control may offer a valuable new possibility for improving the long-term outcome of transplanted organs.
[Show abstract][Hide abstract] ABSTRACT: In order for xenotransplantation to become a clinical reality, and fulfill its promise of overcoming shortages of human organs and tissues, rejection mediated by the host's immune system must first be overcome. In primates, preformed natural antibodies that bind the carbohydrate antigen Galalpha1-3Galbeta1-4GIcNAc-R (alphaGal), which is synthesized by UDP galactose:beta-D-galactosyl-1,4-N-acetyl-D-glucosaminide alpha(1-3)galactosyltransferase (E.C. 126.96.36.199) or simply alphaGT, mediate rigorous rejection of transplanted pig organs and tissues. In alphaGT knockout mice (GT0 mice), which like humans contain in their serum antibodies that bind alphaGal, expression of a retrovirally transduced alphaGT in bone marrow-derived cells is sufficient to prevent production of alphaGal-reactive antibodies. Here, we demonstrate that reconstitution of lethally irradiated GT0 mice with alphaGT-transduced bone marrow cells from GT0 littermates prevents antibody-mediated rejection of cardiac transplants from wild-type mice. These data suggest that gene therapy can be used to induce immunological tolerance to defined antigens and thereby overcome transplant rejection.
[Show abstract][Hide abstract] ABSTRACT: Much evidence supports the conclusion that immunological responses to donor-specific incompatibilities are a major factor in producing "chronic" transplant rejection, including the arteriopathy (atherosclerosis) commonly present. Our experiments explored the effects of altered immunological responsiveness to these Ags on the formation of arteriopathy in transplanted mouse hearts. Specific immunological nonreactivity, or tolerance, was induced either by neonatal administration of allogeneic spleen cells (from F(1) donors between class I-mismatched donor and recipient strains), resulting in "classical" immunological tolerance, or by bone marrow infusion to suitably prepared adult recipients, either fully MHC mismatched or class I mismatched, yielding "mixed chimerism." Both approaches obviated systemic graft-versus-host effects. In both groups, donor-specific skin grafts survived perfectly and donor cell chimerism persisted. Specific Abs were undetectable in all recipients. Most transplants to either group of tolerant recipients developed striking vasculopathy in their coronary arteries (12 of 15 in neonatal tolerance and 15 of 23 in mixed chimeras). Neointimal infiltrates included CD4 and CD8 T cells and macrophages. Only 2 of 29 contemporary isotransplants showed any evidence of vasculopathy. Recipients essentially incapable of T and B cell responses (C.B-17/SCID and RAG1(-/-)) were also used. Transplants into these animals developed vasculopathy in 16 of 31 instances. Accordingly, in this setting, vasculopathy develops in the presence of H-2 gene-determined incompatibility even with minimal conventional immune reactivity. Perhaps innate responsiveness, that could include NK cell activity, can create such arteriopathic lesions. More evidence is being sought regarding this process.
The Journal of Immunology 12/2001; 167(10):5731-40. DOI:10.4049/jimmunol.167.10.5731 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Arteriopathy, sometimes termed accelerated atherosclerosis, often impairs transplants. We employed apolipoprotein-E-deficient, hypercholesterolemic mice to determine how the hyperlipidemic environment affected transplanted hearts. Strain 129 hearts transplanted to C57BL/6 normal or C57BL/6 apolipoprotein-E-deficient recipients were evaluated by immunochemical and histological techniques. Analyses were possible both of differences in the coronary lesions that developed in a normolipidemic as compared with a hyperlipidemic environment and of the coronary atherosclerotic process in transplanted hearts compared with native hearts in the same hyperlipidemic environment. Aortas and coronary arteries of transplanted aortas in both recipient groups developed florid intimal thickening by 4 to 10 weeks, with marked lipid deposition, foamy macrophages, and infiltration of smooth muscle alpha-actin-positive cells in apolipoprotein-E-deficient mice. Lipid was layered against the internal elastic lamina as in human transplants. VCAM-1 was increased in various sites in both groups. Allotransplants to apolipoprotein-E-deficient recipients had more severe aortic and coronary lesions with characteristic T cell infiltration than native hearts. In this sense, transplants suffered from accelerated atherosclerosis. The character of coronary vascular changes in transplanted hearts was distinctly affected by their lipid environment, but their severity, in terms of luminal encroachment, was not markedly different.
American Journal Of Pathology 08/1996; 149(1):91-9. · 4.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Atherosclerotic lesions in the coronary arteries of transplanted mouse hearts manifest high expression of ICAM-1 (CD54), especially on endothelial surfaces, and of LFA-1 (CD11a) on migratory mononuclear cells. The possible participation of cellular adhesion systems in the evolution of these complex lesions was suggested by the increased expression of intercellular adhesion molecule-1 (ICAM-1) and leukocyte function-associated antigen-1 (LFA-1) and also by our previous studies with this experimental system. In our studies, we have found that administration of a monoclonal antibody (mAb) to gamma-interferon will greatly suppress coronary changes, and gamma-interferon is known to stimulate the formation of these adhesion molecules. The present experiments were to evaluate how administration to murine heart transplant recipients of mAbs against ICAM-1, LFA-1, or both affected the development of coronary atherosclerosis. It was found that treatment with either mAb alone did not alter the severity of coronary atherosclerosis, but that both mAbs given together can significantly suppress lesion formation at 30 days compared with controls (P < 0.044). Continuing treatment was even more effective when extended to 60 days (P < 0.003). The mAbs to ICAM-1 and LFA-1 bound their targets in vivo (primarily endothelium and mononuclear cells, respectively), but complete, long-term saturation of combining sites was not attained, even with very high doses. No appreciable reduction in arterial endothelial ICAM-1 expression was evident. It is concluded that the ICAM-1/LFA-1 system is of central importance in the evolution of accelerated coronary atherosclerosis.
[Show abstract][Hide abstract] ABSTRACT: Obstructive lesions in arterial vessels of transplanted organs constitute an important factor in the late failure of these organs, especially for the heart. The absence of obstructive lesions in syngeneic donor-recipient combinations, suggests that they depend upon recipient immune responsiveness. It is controversial whether humoral or cellular aspects of the immune response predominate in the process. The present experiments employed hearts transplanted between inbred mice. After brief preoperative immunosuppression of recipients with mAbs to CD4 and CD8 determinants, hearts transplanted between mice incompatible for histocompatibility Ags survived for prolonged periods and most developed typical, obstructive coronary lesions. Our quantitative scoring of vascular changes on tissue sections of excised hearts, grades both their severity and prevalence. Transplants between strains that produced Abs to donor cells (B10.A to B10.BR), developed coronary lesions exceeding those in the reverse combination in which no detectable Ab was formed (B10.BR to B10.A; p < 0.00001), even though their histoincompatibility was similar. Treatment of B10.A recipients of B10.BR hearts with an antiserum against the donor significantly increased coronary lesions (p < 0.0003) in a dose-dependent fashion. Coronary arteries of B10.BR hearts transplanted to C.B-17 SCID mice remained largely free of lesions, whereas transplants to SCID recipients that received continuing injections of an antiserum directed to Ags of the donor developed striking, obstructive coronary lesions. We conclude that humoral immunity can be the prime instigator of atheromatous changes that occur in transplanted hearts.
The Journal of Immunology 06/1994; 152(10):5135-41. · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Obstructive coronary arterial lesions in the vessels of transplanted hearts result from a complex process in which the immune response of the recipient plays a pivotal role. We have devised an experimental system in which mouse hearts, transplanted after brief treatment with mAbs to CD4 and CD8, survive and contract for many weeks. A high percentage of such hearts develop advanced, obstructive coronary lesions by 4 weeks. Migratory cells of recipient origin localize in the linings of affected vessels, and mediators of inflammation, including adhesion molecules, are present in increased amounts in characteristic locations. Histocompatibility antigen expression is also increased, and these substances may promote the formation of vascular lesions by acting as targets for immune responses. IFN-gamma synthesis has been demonstrated in grafts where it is postulated to be important in the expression of MHC molecules and macrophage activation. Here we report that continuing treatment with R4-6A2, an mAb to IFN-gamma, strikingly inhibits the formation of obstructive vascular lesions in mouse hearts transplanted to recipients incompatible for either class I or class II antigens (P < 0.0001 for the former and P < 0.03 for the latter). Immunohistologic studies showed reduction of the class II-positive mononuclear infiltrate, but focally enhanced endothelial class I expression remained. The mechanism for this effect of anti-IFN-gamma probably extends beyond the influence of anti-IFN-gamma on increased expression of histocompatibility antigens.
[Show abstract][Hide abstract] ABSTRACT: An experimental system is described in which coronary arteries of mouse hearts transplanted heterotopically develop obstructive lesions by 4 weeks. Transient immunosuppression permits graft survival. Donor/recipient antigenic differences may be either class I or class II major histocompatibility antigens (H-2) or non-H-2 antigens. An infiltrate including CD4+ and CD8+ T lymphocytes and macrophages concentrates early in the intima and adventitia of larger coronary arteries, with little in the myocardium. Subsequently, the intima expands with cells of donor origin and the infiltrate invades the media. Endothelial and intimal cells express ICAM-1, leukocytes LFA-1: Endothelium expresses class I, but not class II, antigens. As class II disparity alone suffices, the endothelium can apparently be an indirect target of immune injury. We propose that graft atherosclerosis is T cell initiated and elicited by heterogeneous antigens in the endothelium or media. It is separable from rejection of the myocardium.
American Journal Of Pathology 03/1994; 144(2):260-74. · 4.59 Impact Factor