Catharine M. Chase

Alloantibody- and T cell-mediated immunity in the pathogenesis of transplant arteriosclerosis : lack of progression to sclerotic lesions in B cell-deficient mice

BACKGROUND The relative roles of humoral and cell-mediated immunity in generating chronic allograft arteriopathy have been considered for several years. We have sought definitive evidence regarding these questions using heart transplants between mouse strains selected to isolate the effects of each form of immune responsiveness. METHODS B10.BR hearts were transplanted to B cell-deficient recipients that are devoid of immunoglobulins (muMT). Their vessels were compared with those of transplants to fully reactive recipients of the same genetic background (C57BL/6). Additional evidence came from comparisons in other strain combinations. RESULTS Transplants to B cell-deficient and normal recipients developed cellular coronary endothelialitis, with destruction of the arterial media, accompanied by the adherence of T lymphocytes and macrophages to endothelial surfaces. In B cell-deficient recipients, there was no centripetal migration of smooth muscle, alpha-actin-positive myointimal cells and little deposition of collagen or ground substance, compared with lesions in fully reactive C57BL/6 recipients in which these changes are prominent. In two other donor-recipient combinations in which anti-donor antibodies are generally undetectable (B10.BR-->B10.A and 129-->C57BL/6), intimal fibrosis was uncommon. However, B10.A recipients became capable of producing fibrous lesions in B10.BR hearts when given anti-donor, class I antibody by passive transfer, as we have observed previously in scid recipients. CONCLUSIONS Taken together, these findings indicate that endothelialitis is antibody-independent, whereas antibodies potentiate and can be sufficient for fully developed, fibrous, chronic allograft vasculopathy. Therapeutic strategies for controlling chronic lesions must consider inhibition of the humoral response.

A novel pathway of chronic allograft rejection mediated by NK cells and alloantibody.

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‐2b) mice received B10.BR (H‐2k) heart allografts and repeated doses of IgG2a, IgG1 or F(ab’)2 fragments of IgG2a DSA (anti‐H‐2k). 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.

NK Cells Can Trigger Allograft Vasculopathy: The Role of Hybrid Resistance in Solid Organ Allografts

Progressive arterial stenosis (cardiac allograft vasculopathy (CAV)) is a leading cause of long-term failure of organ transplants. CAV remains intractable, in part because its mechanisms are insufficiently understood. A central proposition is that MHC-driven alloimmune processes play a necessary role in CAV, as shown by the absolute requirement for histoincompatibility between donor and recipient for its production. Two immunological pathways have been implicated involving reactivity to donor MHC Ags by either T or B cells. In this study, we use a novel system of semiallogeneic cardiac transplants between parental donors and F1 hybrid recipients to provide evidence that NK cells, members of the innate immune system, also contribute to the generation of CAV in mice. This finding marks the first demonstration that the hybrid resistance phenomenon occurs in solid organ allografts. Extension of these experiments to recipients deficient in T cells demonstrates that this third pathway of CAV, the NK cell-triggered pathway, involves the recruitment of T cells not responsive to donor alloantigens. Finally, transplants performed with donors or recipients deficient in IFN-γ revealed that recipient-derived IFN-γ is necessary for CAV formation in parental to F1 transplants, suggesting a possible effector mechanism by which NK cells can promote CAV. Together, these results define a previously unknown pathway toward CAV and assign a novel role to NK cells in organ allograft rejection.

Chronic cardiac transplant arteriopathy in mice: relationship of alloantibody, C4d deposition and neointimal fibrosis.

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‐2Kk 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.

Tolerance, Mixed Chimerism, and Chronic Transplant Arteriopathy

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 F1 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.

Coronary Atherosclerosis In Transplanted Mouse Hearts: Iii. Effects of Recipient Treatment with a Monoclonal Antibody to Interferon-γ

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.

Complement Independent Antibody‐Mediated Endarteritis and Transplant Arteriopathy in Mice

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.

Macrophage Depletion Suppresses Cardiac Allograft Vasculopathy in Mice

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 × BALB/c)F1 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.

Early Acceptance of Renal Allografts in Mice Is Dependent on Foxp3+ Cells

Mouse renal allografts have a remarkable ability to promote acceptance across full major histocompatibility complex incompatibilities in certain strain combinations without immunosuppression. The mechanism is unknown but is believed to involve immunoregulation. This study tests whether Foxp3(+) T-regulatory cells are responsible in the early phase of graft acceptance, using B6.Foxp3(DTR) mice that express diphtheria toxin receptor (DTR) in Foxp3(+) cells. The administration of DT to B6.Foxp3(DTR) recipients with accepted DBA/2 kidneys, 3 weeks to 3 months after transplantation, caused a marked depletion of Foxp3 cells and triggered acute cellular rejection, manifested by a sudden increase in blood urea nitrogen within a week. None of the controls showed an increase in blood urea nitrogen, including DT-treated B6 wild-type recipients of DBA/2 kidneys or B6.Foxp3(DTR) recipients of isografts. Accepted DBA/2 allografts showed prominent lymphoid sheaths around arteries containing numerous CD3(+)Foxp3(+) cells, CD4(+) cells, dedritic cells, and B cells, which was independent of CCR4. The lymphoid sheaths disintegrate after Foxp3 depletion, accompanied by widespread CD8 interstitial mononuclear inflammation, tubulitis, and endarteritis. The Foxp3 depletion caused an increased frequency of donor-reactive cells in the spleen by interferon (IFN) γ enzyme-linked immunosorbent spot (ELISPOT) assays and increased expression of the maturation markers, CD86 and IA(b), on dendritic cells in the spleen and kidney. We conclude that Foxp3(+) cells are needed to maintain acceptance of major histocompatibility complex-incompatible renal allografts in the first 3 months after transplantation and may act by inhibiting DC maturation.

Coronary atherosclerosis in transplanted mouse hearts. IV: Effects of treatment with monoclonal antibodies to intercellular adhesion molecule-1 and leukocyte-function-associated antigen-1

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.