Chronic Heart Allograft Rejection Research Articles

T-regulatory cell treatment prevents chronic rejection of heart allografts in a murine mixed chimerism model

BackgroundThe mixed chimerism approach induces donor-specific tolerance in both pre-clinical models and clinical pilot trials. However, chronic rejection of heart allografts and acute rejection of skin allografts were observed in some chimeric animals despite persistent hematopoietic chimerism and tolerance toward donor antigens in vitro. We tested whether additional cell therapy with regulatory T cells (Tregs) is able to induce full immunologic tolerance and prevent chronic rejection.MethodsWe recently developed a murine “Treg bone marrow (BM) transplantation (BMT) protocol” that is devoid of cytoreductive recipient pre-treatment. The protocol consists of a moderate dose of fully mismatched allogeneic donor BM under costimulation blockade, together with polyclonal recipient Tregs and rapamycin. Control groups received BMT under non-myeloablative irradiation and costimulation blockade without Treg therapy. Multilineage chimerism was followed by flow cytometry, and tolerance was assessed by donor-specific skin and heart allografts.ResultsDurable multilineage chimerism and long-term donor skin and heart allograft survival were successfully achieved with both protocols. Notably, histologic examination of heart allografts at the end of follow-up revealed that chronic rejection is prevented only in chimeras induced with the Treg protocol.ConclusionsIn a mouse model of mixed chimerism, additional Treg treatment at the time of BMT prevents chronic rejection of heart allografts. As the Treg-chimerism protocol also obviates the need for cytoreductive recipient treatment it improves both efficacy and safety over previous non-myeloablative mixed chimerism regimens. These results may significantly impact the development of protocols for tolerance induction in cardiac transplantation.

Allochimeric molecules and mechanisms in abrogation of cardiac allograft rejection

Dendritic cells are professional antigen presenting cells that perform antigen processing and antigen presentation functions and rely on the proper functioning and distribution of the endoplasmic reticulum (ER) and Golgi apparatus and of vesicular trafficking pathways. We previously developed a model system to study the mechanisms governing inhibition of chronic rejection of heart allografts. Heterotopic cardiac transplants were placed intra-abdominally and the major histocompatibility class (MHC) class I allochimeric molecule, [α1h1/u]-RT1.Aa, which contains donor-type (Wistar Furth, WF; RT1u) immunogenic epitopes displayed on recipient-type (ACI, RT1a) sequences, was delivered by portal vein to the recipients of heterotopic hearts. Dendritic cells were isolated from the recipient bone marrow at 1 and 3 days after transplantation and were immunostained or processed for Western blotting with anti-RhoB, translationally controlled tumor protein (TCTP), Sprouty-related (Spred1) protein, ER, and Golgi antibodies. Western blotting analyses showed the downregulation of RhoB GTPase, TCTP, and Spred1 in dendritic cells isolated from allochimeric molecule-treated rats. Immunostaining showed that in these cells, Spred 1 was shifted to the base of cellular processes, Rho B formed nonvesicular band in the cell equator, and TCTP was highly enriched in the cell nucleus. The Golgi apparatus was drastically reduced in size and formed a tiny nonvesicular aggregate, and the ER partially lost vesicular appearance. The function of allochimeric molecule in the abrogation of heart allograft rejection may rely on the downregulation of RhoB pathway components that regulate the structure and function of the ER/Golgi/vesicular trafficking pathways involved in antigen processing and presentation by dendritic cells.

Increased Connective Tissue Growth Factor Expression in a Rat Model of Chronic Heart Allograft Rejection

Chronic rejection limits the long-term success of cardiac transplantation and the underlying cause of the disease is unknown. Connective tissue growth factor (CTGF) is considered as a mitogenic and chemotactic factor for fibroblasts, and is associated with cell proliferation and collagen synthesis. We evaluated the expression of CTGF in a rat model of heart allograft chronic rejection. Intra-abdominal heterotopic heart transplantation was performed from 20 Wistar rats to 20 Sprague-Dawley (SD) rats that received cyclosporine, mycophenolate mofetil and methylprednisolone as immunosuppression. Ten heart allografts were explanted at 2 and 8 weeks postoperatively for analysis of morphologic changes. The hearts from 10 normal Wistar rats served as a control group. Coronary artery density, luminal loss of myocardial coronary arteries, and myocardial fibrosis were measured. The expression of CTGF was studied by immunohistochemistry. Correlation between CTGF expression and development of cardiac allograft vasculopathy (CAV) or fibrosis was studied. Allografts harvested at 8 weeks postoperatively showed more coronary intimal proliferation, fibrosis and CTGF expression compared with the 2-week allografts (p < 0.05) and the controls (p < 0.01), but the coronary artery density was lower than in the control group (p < 0.05). However, the control group showed negligible CTGF expression. There were strong negative correlations between the gray value of CTGF protein expression and cardiac fibrosis and coronary intimal occlusion (r = -0.734, -0.713; p < 0.01), which demonstrated that CTGF protein expression was positively correlated with cardiac fibrosis and coronary intimal occlusion. CTGF is expressed in cardiomyocytes in CAV. Increased expression of CTGF in cardiac allografts is associated with development of CAV and fibrosis formation.

Sanglifehrin A Blocks Key Dendritic Cell Functions In Vivo and Promotes Long-Term Allograft Survival Together with Low-Dose CsA

Sanglifehrin A (SFA) is a novel compound with unsurpassed cyclophilin-binding affinity, but relatively low direct antilymphocytic activity. Here, we report the capacity of SFA to selectively inhibit key dendritic cell (DC) functions in vivo and to suppress acute and chronic heart allograft rejection. We show that in vivo, SFA profoundly decreases DC receptor-mediated endocytosis and macropinocytosis and DC-T-cell allostimulatory activity. Furthermore, SFA abrogates >90% of IL-12p70 production in vivo while having no significant effect on IL-10 and TNF-alpha production. In a rat vascularized heart transplant model, SFA alone did not prevent graft rejection and rejection occurred within 23 days after low-dose CsA, whereas addition of SFA to low-dose CsA promoted long-term graft survival (median survival time >100 days; p = 0.0007). With respect to chronic rejection, SFA + CsA almost completely prevented graft arteriosclerosis compared to animals treated with CsA alone and controls. We propose that SFA represents a novel class of immunophilin-binding immunosuppressants with high activity against DCs and the development of graft vasculopathy in CsA-treated recipients.

Tissue inhibitor of metalloproteinases 4 (TIMP4) is involved in inflammatory processes of human cardiovascular pathology

Tissue inhibitors of matrix metalloproteinases (TIMPs) comprise a family of four members, of which TIMP4 is characterized by being primarily restricted to cardiovascular structures. We demonstrate with immunohistochemical analysis of healthy human tissue that TIMP4 is present in medial smooth muscle cells and adventitial capillaries of arteries as well as in cardiomyocytes. Animal studies have suggested a role for TIMP4 in several inflammatory diseases and cardiovascular pathologies. We therefore examined whether TIMP4 is involved in human inflammatory cardiovascular disorders, specifically atherosclerosis, giant cell arteritis and chronic rejection of heart allografts. TIMP4 was most clearly visible in cardiovascular tissue areas populated by abundant inflammatory cells, mainly macrophages and CD3+ T cells. Using western blotting and immunocytochemistry, human blood derived lymphocytes, monocytes/macrophages and mast cells were shown to produce TIMP4. In advanced atherosclerotic lesions, TIMP4 was detected around necrotic lipid cores, whereas TIMP3 and caspase 3 resided within and around the core regions, indicating different roles for TIMP3 and TIMP4 in inflammation-induced apoptosis and in matrix turnover. In conclusion, the data demonstrate upregulation of TIMP4 in human cardiovascular disorders exhibiting inflammation, suggesting its future use as a novel systemic marker for vascular inflammation.

Inhibition of Chronic Rejection and Development of Tolerogenic T Cells after ICOS-ICOSL and CD40-CD40L Co-stimulation Blockade

: Blockade of the CD40-CD40L pathway results in long-term allograft survival but does not prevent chronic rejection. ICOS-ICOSL are members of the CD28-B7 family that play an important role in T-cell activation. : The authors analyzed the effect of single or combined treatment with an anti-ICOS monoclonal antibody and CD40 immunoglobulin (Ig) on acute and chronic rejection of heart allografts in rats. : Treatment with anti-ICOS resulted in a modest but significant prolongation of allograft survival. Treatment with CD40Ig resulted in long-term graft survival but the cardiac grafts developed chronic rejection lesions. Combined CD40Ig+anti-ICOS treatment led to indefinite graft survival in all recipients and a significant decrease of chronic rejection lesions compared with CD40Ig alone. Importantly, four of the seven CD40Ig+anti-ICOS-treated recipients showed a complete absence of chronic rejection lesions, whereas all of the CD40Ig-treated recipients showed chronic rejection. The CD40Ig+anti-ICOS group also showed significant decreased graft infiltration, decreased antidonor cytotoxic T-lymphocyte activity, and decreased alloantibodies compared with the CD40Ig-treated group. Adoptive transfer of splenocytes indefinitely prolonged allograft survival, whereas those depleted of T cells did not, suggesting the development of T-regulatory mechanisms. : These data indicate that the chronic rejection mechanisms that are CD40-CD40L independent are ICOS-ICOSL dependent. These results were obtained with conservation of cognate immune responses and development of tolerogenic T cells.

Combinations of Anti-LFA-1, Everolimus, Anti-CD40 Ligand, and Allogeneic Bone Marrow Induce Central Transplantation Tolerance through Hemopoietic Chimerism, Including Protection from Chronic Heart Allograft Rejection

Central transplantation tolerance through hemopoietic chimerism initially requires inhibition of allogeneic stem cell or bone marrow (BM) rejection, as previously achieved in murine models by combinations of T cell costimulation blockade. We have evaluated LFA-1 blockade as part of regimens to support mixed hemopoietic chimerism development upon fully allogeneic BALB/c BM transfer to nonirradiated busulfan-treated B6 recipient mice. Combining anti-LFA-1 with anti-CD40 ligand (CD40L) induced high incidences and levels of stable multilineage hemopoietic chimerism comparable to chimerism achieved with anti-CD40L and everolimus (40-O-(2-hydroxyethyl)-rapamycin) under conditions where neither Ab alone was effective. The combination of anti-LFA-1 with everolimus also resulted in high levels of chimerism, albeit with a lower incidence of stability. Inhibition of acute allograft rejection critically depended on chimerism stability, even if maintained at very low levels around 1%, as was the case for some recipients without busulfan conditioning. Chimerism stability correlated with a significant donor BM-dependent loss of host-derived Vbeta11(+) T cells 3 mo after BM transplantation (Tx). Combinations of anti-CD40L with anti-LFA-1 or everolimus also prevented acute rejection of skin allografts transplanted before established chimerism, albeit not independently of allospecific BMTx. All skin and heart allografts transplanted to stable chimeras 3 and 5 mo after BMTx, respectively, were protected from acute rejection. Moreover, this included prevention of heart allograft vascular intimal thickening ("chronic rejection").

Chronic heart allograft rejection in rats demonstrates a dynamic interplay between IFN-γ and IL-10 producing T cells

The relative roles of different types of T cells and their modes of alloantigen recognition and cytokine production in chronic rejection have been controversial. This may be due to the use of models involving various immune interventions. We recently reported a rat heart allograft model (PVG.1U-to-PVG.R8) that consistently develops chronic rejection without any immune manipulations. Using this model, we investigated the frequency of indirectly activated alloreactive CD4+ T cells and directly/indirectly induced CD8+ T cells and characterized their cytokine profiles at various times posttransplantation. In vitro quantitative–proliferative and intracellular cytokine assays were performed using recipient lymphocytes as responders against donor and third party stimulator cells. Intragraft transcript levels for IFN-γ were measured using competitive RT-PCR. We observed a steady increase in the frequency of donor reactive CD4+ T cells, peaking on day 18–22 posttransplantation, followed by a decline to background levels on day 60. These cells produced high levels of IL-10 that steadily increased throughout the 100-day experimental period. In contrast, the frequency of donor reactive CD8+ T cells was similar to that of naı̈ve cells and remained unchanged throughout the study. CD8+ T cells produced high amounts of IFN-γ only on days 39–45 posttransplantation when chronic rejection was apparent. There was a sharp increase in intragraft IFN-γ transcripts on days 8–12 that peaked on days 39–45 followed by a steady decrease thereafter. Our results demonstrate that chronic rejection in this model involves a dynamic interplay between alloreactive CD8+IFN-γ+ and CD4+IL-10+ T cells, both of which may have differential effects on the pathogenesis of the disease.

Indirect allorecognition of donor HLA-DR peptides in chronic rejection of heart allografts

Indirect allorecognition of donor HLA-DR peptides in chronic rejection of heart allografts

Cytomegalovirus infection-associated generalized immune activation in heart allograft recipients: a study of cellular events in peripheral blood and endomyocardial biopsy specimens.

An association between cytomegalovirus (CMV) infection, heart allograft rejection, and arteriosclerosis has been reported. To investigate the mechanisms of this association, the cellular immune response in peripheral blood and the inflammation in heart allografts during antigenemia were studied. CMV antigenemia occurred in 13 recipients. In recipients with severe CMV infection, a significantly weaker immune response was recorded in peripheral blood: fewer lymphoid blast cells (max. 2.4% +/- 0.4%) and large granular lymphocytes (LGL; max. 9.3% +/- 1.4%) were seen than in patients with mild or asymptomatic CMV infection (lymphoid blast cells max. 6.5% +/- 0.8% P < 0.01 and LGLs max. 20% +/- 2.3%, P < 0.05). Thus, a strong immune response with lymphoid activation was associated with clinically good outcome of CMV infection. In heart allograft histology, subendothelial inflammation of small intramyocardial vessels was a characteristic finding during CMV antigenemia compared to CMV-free recipients (at the peak P < 0.01). However, no difference in this mild and short-lived inflammatory response was observed between clinically mild or severe CMV infection. The CMV-linked generalized immune activation and inflammation of the vascular structures might contribute to the initiation of allograft vasculopathy and to the pathogenesis of chronic heart allograft rejection.

Invited Commentary: “Immunocytochemical Features in Rejection Monitoring”

During the last few years, a variety of new immunosuppressive regimes have been used in cardiac transplantation. The mostly used was the so called triple drug regimen consisting of steroids, azathioprine and cyclosporin A. These regimens have led to an increase in cardiac transplant survival and have produced a different spectrum of pathologic manifestations and complications (8). Patients treated with these new regimens show a delayed onset of rejection episodes and a possible decrease in the number of such episodes, as well as a dramatic decrease in the number of infections complications. However, the long-term outcome of cardiac transplantation continues to be plagued by the problem of chronic vascular rejection manifested as coronary artery disease of myocardial ischemia (4). The incidence of these complications is reported to be anywhere between 11% and 44% of all patients undergoing cardiac allograft transplantation as of 5 years posttransplant (5). This incidence has not been significantly altered by the use of cyclosporin A or other, newer immunosuppressive regimens and remains the major problem in cardiac transplantation. Endomyocardial biopsy remains to be the golden standard of rejection diagnosis in acute as well as chronic heart allograft rejection (1). Because it is an invasive method and because its sensitivity in chronic rejection is not satisfactory, there still is the need for an easy, sensitive and not invasive method. Therefore a lot of studies are done to find an adequate cytologic immunologic monitoring (3) for a closely meshed diagnostic monitoring in the postoperative phase of cardiac transplantation. In contrast to re-