Rat Heart Allograft Model Research Articles

MHC-derived allopeptide activates TCR-biased CD8+ Tregs and suppresses organ rejection

In a rat heart allograft model, preventing T cell costimulation with CD40Ig leads to indefinite allograft survival, which is mediated by the induction of CD8+CD45RClo regulatory T cells (CD8+CD40Ig Tregs) interacting with plasmacytoid dendritic cells (pDCs). The role of TCR-MHC-peptide interaction in regulating Treg activity remains a topic of debate. Here, we identified a donor MHC class II-derived peptide (Du51) that is recognized by TCR-biased CD8+CD40Ig Tregs and activating CD8+CD40Ig Tregs in both its phenotype and suppression of antidonor alloreactive T cell responses. We generated a labeled tetramer (MHC-I RT1.Aa/Du51) to localize and quantify Du51-specific T cells within rat cardiac allografts and spleen. RT1.Aa/Du51-specific CD8+CD40Ig Tregs were the most suppressive subset of the total Treg population, were essential for in vivo tolerance induction, and expressed a biased, restricted Vβ11-TCR repertoire in the spleen and the graft. Finally, we demonstrated that treatment of transplant recipients with the Du51 peptide resulted in indefinite prolongation of allograft survival. These results show that CD8+CD40Ig Tregs recognize a dominant donor antigen, resulting in TCR repertoire alterations in the graft and periphery. Furthermore, this allopeptide has strong therapeutic activity and highlights the importance of TCR-peptide-MHC interaction for Treg generation and function.

ANTAGONISM OF THE CHEMOKINE RECEPTOR CXCR4 DOES NOT LEAD TO PROLONGATION OF GRAFT SURVIVAL IN THE RAT HETEROTOPIC HEART ALLOGRAFT MODEL

O192 Aims: The importance of chemokine receptors in graft rejection is well documented. CXCR4 is the specific receptor for SDF-1 and is widely expressed on immune cells but also on other cell types. CXCR4 and SDF-1 are implicated in homeostatic as well as pathologic migration of lymphocytes, hematopoietic progenitor cells, neutrophils and tumor cells. CXCR4 and SDF-1 were shown to be upregulated in rejecting kidney and liver grafts, suggesting that they might play a role in the recruitment of immune cells to the graft and thereby contribute to rejection. This hypothesis could not be tested so far due to embryonic lethality of both CXCR4 and SDF-1 deficiency in mice and the lack of appropriate CXCR4 antagonists. We identified a novel, selective and highly potent low molecular weight CXCR4 antagonist, NIBR-1816 and used this compound to explore the contribution of CXCR4 in the rat heart allograft rejection model. Methods: In vitro ligand binding, Ca2+ mobilization and chemotaxis assays were used to characterize the antagonistic activity of NIBR-1816 on CXCR4. The effect of NIBR-1816 in vivo was determined by flow cytometry, measuring the inhibition of SDF-1 mediated actin polymerization on blood cells from rats treated with NIBR-1816. For transplantation experiments the heterotopic rat heart transplant model (strain combination BN to LEW) was used. Recipient rats were treated either with NIBR-1816 (30mg/kg/day s.c.) or a combination of NIBR-1816 plus a subtherapeutic dose of CsA, (0.1mg/kg/day s.c.). Survival of heart allografts was monitored by daily palpation. Haematology was performed at baseline and at rejection using the Technicon/H1 cell counter. Results: NIBR-1816 is a highly potent CXCR4 antagonist with IC50 values of 11-80 nM in a variety of in vitro assays, about equipotent on human and rat CXCR4 and is highly selective over other receptors. Osmotic minipump administration (s.c.) of NIBR-1816 for 5 d led to constant plasma levels of ∼ 1μM. These levels of NIBR-1816 resulted in almost complete suppression of SDF-1 mediated actin polymerization in blood neutrophils and monocytes, indicative of full blockade of CXCR4 in treated animals. In addition, NIBR-1816 treatment increased circulating blood neutrophils, a known consequence of CXCR4 blockade resulting in release of neutrophils from the bone marrow pool. When administered to transplanted animals NIBR-1816 monotherapy did not lead to significant prolongation of graft survival (MST 7 d vs. 9.5 d for controls). Similarly, no significant effect was observed when NIBR-1816 was combined with suboptimal CsA treatment (MST 11.5 d vs. 9 d with CsA alone; p=n.s.). Histology at rejection did not show any difference between the grafts from the different groups. Conclusions: The results obtained in the heterotopic rat heart allograft model provide strong evidence that CXCR4 does not play a major role in the rejection process, since blockade of CXCR4 did not result in a prolongation of graft survival. We conclude that despite its strong upregulation in clinical and experimental graft rejection, CXCR4 is dispensable for the processes leading to graft 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.

Manipulation of CD45 antigen in transplantation tolerance.

CD45 is known to have tyrosine phosphatase activity for signal transduction of T cells. Immunomodulation of CD45 has been tried to prevent T cell-mediated graft rejection in organ transplantation. In vitro study showed that blockade of CD45RB, an alternative splicing isoform of CD45, inhibited proliferative response of T cells after allogeneic stimulation. Treatment with a monoclonal antibody (mAb) against CD45RB induced long-term allograft acceptance in some mouse organ transplantation models. In a rat heart allograft model, a single injection of anti-rat CD45 (RT7) mAb which bound to allomorphic region of RT7 also induced allograft acceptance. CD45/RT7 is also a useful tool of targeting hematopoietic cells, because of the selective expression on all hematopoietic cells. There are two allomorphic forms of CD45 (RT7a and RT7b) in the rat. Using RT7 system, a rat heart allograft model from RT7a donors to RT7b recipients was designed to test functional relevance of graft-associated hematopoietic cells (microchimerism) to allograft acceptance. Then donor-derived hematopoietic cells were selectively depleted using anti-RT7a mAb in vivo. Depletion on day 0 prevented allograft acceptance and was associated with severe acute or chronic graft rejection, while depletion on day 18 after transplantation showed no effect. This experimental study showed a crucial role of microchimerism in induction phase of allograft acceptance. In conclusion, the CD45/RT7 system is not only a target molecule for tolerance induction, but also an useful tool for experimental models in transplantation immunology. In this review, we introduce basic properties of CD45 and recent results with manipulation of CD45.

New method for detection of heart allograft rejection: validation of sensitivity and reliability in a rat heterotopic allograft model.

Patients with inflammatory heart muscle diseases would benefit from a safe, convenient, rapidly performed diagnostic technique with real-time results not involving tissue removal. We have performed a detailed evaluation of detection of heart allograft rejection by autofluorescence in a heterotopic abdominal rat heart allograft model ex vivo. Recipient rats with allograft (Lewis to Fisher 344; n=71) and isograft (Lewis to Lewis; n=33) hearts, treated with cyclosporine or untreated, were killed at days 2, 4, 7, 14, 21, 28, and 56 after transplant. Nontransplant controls with (n=24) or without (n=24) immunosuppressive therapy were also studied. When the rats were killed, autofluorescence spectra were acquired under blue-light excitation from midtransverse ventricular sections of native and transplanted hearts. Corresponding sections were then evaluated pathologically by a modified International Society for Heart and Lung Transplantation (ISHLT) grading schema. The spectral differences between rejecting and nonrejecting hearts were quantified by linear discriminant functions, producing scores that decreased progressively with increasing severity of tissue rejection. Mean+/-SD discriminant function scores were 2.9+/-1.6, 1.8+/-2.2, -0.1+/-2.8, -1.2+/-2.3, and -2.3+/-3.0 for isografts and allograft ISHLT grades 0, I, II, and III, respectively (Spearman rank-order correlation -0.6; P<0.001, test for trend). Cyclosporine had no detectable effect on the spectra. The correlation between changes in autofluorescence spectra and ISHLT rejection grade strongly supports the possibility of catheter-based, fluorescence-guided surveillance of rejection.

SPLENIC-INJECTION OF AUTOLOGOUS HEPATOCYTES GENETICALLY MODIFIED TO EXPRESS SOLUBLE DONOR-MHC CLASS I ANTIGEN PROLONGS HEART ALLOGRAFT SURVIVAL

881 Previous studies suggest that liver allografts can provide protection for other transplanted organs. In this respect heart allograft rejection can be prevented, and even reversed, with a liver transplant from the same donor. One possible explanation for this phenomenon is that hepatocytes produce relatively large amounts of potentially immunosuppressive soluble MHC class I antigen (Ag). In support of this theory we have recently shown that hepatocyte-secreted, soluble, allogeneic-MHC class I Ag downregulates both cytotoxic and helper T lymphocyte responses in vivo. In the present study we examined the potential immunosuppressive effects of hepatocyte-secreted donor-MHC Ag in a rat heart allograft model. Hepatocytes were obtained by collagenase digestion of a Lewis (RT11) liver. Cells were cultured and subsequently transfected by lipofection to express the soluble donor-MHC class I Ag, RT1.Aa (plasmid: pcRQ.B3). One day prior to ACI (RT1a) heterotopic heart allografting, Lewis recipients were injected with 1 × 107 pcRQ.B3-transfected hepatocytes via the spleen. Controls did not receive hepatocytes or were injected with hepatocytes transfected with a gene encoding firefly-luciferase (pCMVLux). All animals received a short-course of cyclosporin A (10 mg/kg/day, i.p.) on days 0 to +7, relative to heart transplantation. Graft survival results showed that recipients injected with hepatocytes expressing soluble donor-MHC class I Ag experienced a prolongation of ACI heart allograft survival, when compared to control. (Table)TableThese results suggest that the immunosuppressive effects of a single mismatched soluble donor-MHC class I Ag are sufficiently potent to promote heart allograft survival in a fully MHC-disparate, high-responder, rat-strain combination. This is one of the first steps in the development of a gene transfer-based system to deliver soluble donor-MHC class I molecules to organ transplant recipients. We are currently refining this gene transfer system to allow for more prolonged soluble donor-MHC expression in vivo, which we predict is a critical factor to provide for longer immunologic protection of heart allografts. Also, it is likely that delivery of a single type of mismatched donor-MHC Ag will be more effective between strain combinations where the MHC is not completely different. In summary, these results provide incentive to determine the full potential of soluble donor-MHC class I gene therapy in organ transplantation.

Factors relevant for the induction of rejection by indirect recognition in a rat heart allograft model: effect of CTLA4Ig treatment on indirect alloactivation induced by immunization with donor MHC I peptides.

We have defined factors relevant for the induction of rejection by indirect recognition in a rat heart allograft model and analyzed the influence of CTLA4Ig treatment on indirect alloactivation induced by donor MHC I peptides in a DA-->LEW heart allograft model. Indirect allorecognition of MHC I led to accelerated graft rejection and was accompanied by the induction of anti-peptide antibodies and donor peptide-activated T cells. In an attempt to block the B7-induced costimulatory signal of T cell activation, CTLA4Ig was administered to graft recipients in addition to MHC I peptide treatment. CTLA4Ig therapy, however, was not effective in preventing the humoral or cellular anti-donor immune response, nor did it prevent accelerated graft rejection.

Factors relevant for the induction of rejection by indirect recognition in a rat heart allograft model: effect of CTLA4lg treatment on indirect alloactivation induced by immunization with donor MHC I peptides

Abstract We have defined factors relevant for the induction of rejection by indirect recognition in a rat heart allograft model and analyzed the influence of CTLA4Ig treatment on indirect alloactivation induced by donor MHC I peptides in a DA → LEW heart allograft model. Indirect allorecognition of MHC I led to accelerated graft rejection and was accompanied by the induction of anti-peptide antibodies and donor pep-tide-activated T cells. In an attempt to block the B7-induced costimulatory signal of T cell activation, CTLA4Ig was administered to graft recipients in addition to MHC I peptide treatment. CTLA4Ig therapy, however, was not effective in preventing the humoral or cellular anti-donor immune response, nor did it prevent accelerated graft rejection.

Synergistic interaction between castanospermine and tacrolimus in a rat heart allograft model

Synergistic interaction between castanospermine and tacrolimus in a rat heart allograft model

Castanospermine, an oligosaccharide processing inhibitor, reduces membrane expression of adhesion molecules and prolongs heart allograft survival in rats

The inhibition of intracellular oligosaccharide processing is a new approach to immunosuppression in allotransplantation. The net effect of such inhibition is reduction in the membrane expression of certain glycoproteins. Hence cellcell interaction in allorejection may be impaired in the presence of glycoprotein processing inhibitors because the expression of key ligand-receptor pairs of N-linked glycoproteins including adhesion molecules is inhibited. The aims of this study were to measure the immunosuppressive ability of castanospermine (CAST) in a rat heart allograft model, to measure its effect on membrane expression of adhesion molecules (LFA-1α, LFA-1β, ICAM-1), class I and class II MHC antigens and on other T cell associated molecules (CD4, CD8, CD39, CD45, W 3 13 ), to test its tolerogenic potential and its toxicity. Membrane expression of these molecules was measured by flow cytometry for single cells and by immunoperoxidase staining for the allograft. In grafted rats CAST significantly reduced the expression of LFA-1α on lymphoid cells in the thymus, lymph node, spleen and heart allografts, ICAM-1 expression on endothelial cells of the allograft vasculature, class I and class II MHC expression on lymphoid cells in the thymus, class II MHC expression on lymphoid cells in the allograft; and CD4, CD8, CD45 and W 3 13 expression on lymphoid cells in some organs. By contrast, in non-grafted rats CAST significantly upregulated expression of class I MHC and CD45 in the thymus, lymph node and spleen, ICAM-1 and CD4 on lymphoid cells in the spleen, but reduced expression of LFA-1α on lymphoid cells in the thymus. It also prolonged rat heart allograft survival in a dose-dependent manner and with limited testing was relatively non-toxic. In conclusion, CAST is an immunosuppressive molecules which may work by downregulation of the ligand-receptor adhesion molecule pair, LFA-1α-ICAM-1 although subtle downregulation of class I and II MHC, CD4 and CD8 molecules could also contribute to its immunosuppressive activity. Hence, both lymphocyte-endothelial cell binding and lymphocyte activation may be inhibited by CAST. This work suggests that CAST may hold significant potential as a transplant immunosuppressant probably as an adjuvant agent to inhibitors of interleukin 2 secretion.

The effects of blood transfusion on cytokine production by TH1 and TH2 lymphocytes in the mouse.

The effects of blood transfusion on cytokine production by TH1 and TH2 lymphocytes in the mouse.

Low-dose combination therapy of DUP-785 and RS-61443 prolongs cardiac allograft survival in rats.

The introduction of cyclosporine into the immunosuppressive armamentarium has revolutionized transplant surgery with significant improvements in graft survival. The apparent lack of effect of cyclosporine on humoral rejection mechanisms makes the search for other immunosuppressive agents desirable. Two anti-metabolites affecting nucleotide synthesis via different pathways have recently been evaluated for their immunosuppressive potential. DUP-785 (DUP), also known as brequinar sodium, reversibly inhibits de novo pyrimidine synthesis by blocking dihydro-orotate dehydrogenase, thus resulting in the deletion of critical precursors for RNA and DNA synthesis. RS-61443, a morpholinoethyl ester of mycophenolic acid, reversibly and non-competitively blocks inosin monophosphate dehydrogenase, the key enzyme in purine de novo synthesis. A possible additive effect of both drugs was investigated in the rat heart allograft model.

Low-dose combination therapy of DUP-785 and RS-61443 prolongs cardiac allograft survival in rats

The introduction of cyclosporine into the immunosuppressive armamentarium has revolutionized transplant surgery with significant improvements in graft survival. The apparent lack of effect of cyclosporine on humoral rejection mechanisms makes the search for other immuno-suppressive agents desirable. Two anti-metabolites affecting nucleotide synthesis via different pathways have recently been evaluated for their immunosuppressive potential. DUP-785 (DUP), also known as brequinar sodium, reversibly inhibits de novo pyrimidine synthesis by blocking dihydro-orotate dehydrogenase, thus resulting in the delretion of critical precursors for RNA and DNA synthesis [2]. RS-61443, a morpholinoethyl ester of mycophenolic acid, reversibly and non-competitively blocks inosin monophosphate dehydrogenase, the key enzyme in purine de novo synthesis [10]. A possible additive effect of both drugs was investigated in the rat heart allograft model.

Examination of the clonal deletion hypothesis following transfusions in rat cardiac transplants.

To test the clonal deletion hypothesis, different levels of immunization were carried out by 0, 1, 2, or 3 transfusions preoperatively, together with 10 methods of immunosuppression. In this way, it was hoped that we could determine the optimal way to immunize and the optimal immunosuppressant treatment to deactivate the responding cells. With the Buffalo-to-Lewis rat heart allograft model, the control mean survival rate was 7 days. Rats with 2 or 3 transfusions had 5- and 12-day survival rates, respectively. If azathioprine was given in addition before grafting, 19- and 18-day survival rates were seen, and if azathioprine was given after grafting, extended survivals of 33 and 34 days were achieved. The longest survival rate of greater than 52 days was obtained by a single transfusion and 25 mg/kg/day of cyclophosphamide given before transplantation. Splenectomy following 2 or 3 transfusions prolonged survival rates to 16-18 days, suggesting that mechanical removal of immunized cells is somewhat effective. Most of the antibody produced by one transfusion was IgM, as were antibodies that resulted from transfusions followed by azathioprine. It is possible that reduction of IgG-producing cells is important. These results are consistent with the clonal deletion theory, although they do not necessarily provide final proof. The experiments suggest that the optimal transfusion effect may be obtained with minimal immunization (1 transfusion) and proper immunosuppression before transplantation.

The effect of donor-specific transfusions on rat heart allograft survival.

A rat heart allograft model employing donor-specific transfusions (DSTs) was used to investigate several questions relevant to their clinical usage. The effects of varying blood storage duration (stored vs. fresh), number and timing of DSTs as well as use of concomitant azathioprine and cyclosporine (CsA) were assessed in terms of allograft survival and recipient sensitization. A comparison of stored and fresh-blood DSTs revealed that blood stored for up to 5 weeks was as effective as fresh blood and that a 2-week storage was optimal. Increased storage appeared to be associated with decreased sensitization. Multiple DSTs were more effective than a single DST and the peak effect appeared after six. Transfusions given at the time of transplantation were ineffective. The addition of concomitant (preoperative) azathioprine or CsA resulted in a further decrease in sensitization but also resulted in a dose-dependent diminution of the transfusion effect.

Donor-specific blood transfusions with stored and fresh blood in a rat heart allograft model

Donor-specific transfusions (DSTs) using stored and fresh blood were investigated in a rat heart allograft model. Lewis rats were recipients of preoperative DSTs and cardiac allografts from ACI donors. Blood was stored for 0, 1, 2, 3, 4, and 5 weeks prior to transfusion. Allograft survival and recipient sensitization were recorded. In all DST groups, allograft survival was superior to nontransfused controls ( P < 0.05). Stored blood was equal or better than fresh blood, and a 2-week storage was optimal ( P < 0.05). Storage of 2 weeks or greater resulted in decreased sensitization compared to storage for 1 week or less ( P < 0.05). This study supports the use of stored blood to enhance allograft survival and reduce recipient sensitization.

Suppressor cells in antithymocyte globulin-induced transplantation tolerance in the adult rat.

A rat heart allograft model, for induction of transplantation tolerance by a single dose of antithymocyte globulin, was studied. Experiments were made to transfer the tolerance to syngeneic recipients, either by spleen cells or sera obtained from tolerant rats. Different doses of tolerant spleen cells caused long-term surviving (LTS) allografts when transferred to sublethally irradiated recipients. Spleen cell transfer to untreated recipients resulted in prolonged allograft survival but no LTS grafts. The results indicate the presence of a cell-mediated suppressor mechanism in the tolerant animals.

Treatment of rat heart allograft recipients with postoperative total lymphoid irradiation (TLI)

Pretransplant total lymphoid irradiation (TLI) plus donor bone marrow can result in donor-specific tolerance, but graft-versus-host disease is not consistently avoided. TLI may have greater applicability as an adjuct to generalized immunosuppressive treatment. In a clinical trial, preoperative TLI posed problems for patients with a high percentage of cytotoxic antibodies who sometimes had a long wait for a transplant after completion of TLI. It would be logistically advantageous if TLI could be given in the perioperative period. We investigated the feasibility of this approach in an ACI (RT-1 a) to Lewis (RT-1 l) rat heart allograft model. Untreated recipients rejected the grafts at a mean of 6.1 days. TLI 400 or 600 rads administered as one dose 1 day preoperative did not prolong graft survival (rejected a mean of 5.6 and 6.3 days). A dose fraction schedule previously found to be effective when given preoperatively, 200 rads × 5 consecutive days, did not prolong graft survival when administered beginning the first day postoperatively (mean rejection at 6.3 days). Higher dose fractions were effective: 300 rads × 3, 300 rads × 5, 400 rads × 3, and 400 rads × 4 gave mean rejection times of 11.8, 16.8, 11.5, and 13.6 days, respectively. Combining 600 rads preoperative with 200 rads × 5 days postoperative did not prolong graft survival. Six hundred rads preoperative plus 300 rads × 3 days postoperative prolonged graft survival, but was no more effective than 300 rads × 3 days postoperative alone. The most effective schedule for postoperative TLI was 300 rads × 3, 0 rads × 2, and 200 rads × 5 days; this treatment delayed rejection to 25 days. In conclusion, one dose preoperative TLI has no effect on heart allograft survival, but postoperative TLI in dose fractions of 300 rads is effective in delaying rejection. Postoperative TLI may be an alternative treatment for patients who must wait for suitable crossmatch negative cadaver kidneys.

Total lymphoid irradiation: Critical timing and combination with cyclosporin A for immunosuppression in a rat heart allograft model

Total lymphoid irradiation (TLI), a standard radiotherapeutic technique for the treatment of Hodgkin's disease, is immunosuppressive. TLI has been used for clinical renal transplantation in conjunction with pharmacological immunosuppression. The optimal recipient treatment protocol is not known. Using ACI (RT-1 a) rat heart allograft survival in Lewis (RT-1 1) rat recipients as a measure of treatment efficacy, we have determined the importance of radiation fractionation, the optimum dose per radiation fraction, the effect of delaying transplantation after completion of TLI, the effect of pretransplant versus post-transplant TLI, and the effect of combining TLI with a pharmacological immunosuppressant, cyclosporin A (CsA). Median heart allograft survival in untreated rats, in rats given 1000 rad (single dose), and in rats given 1000 rad in five fractions pretransplant were 6, 9, and 26 days, respectively. Median allograft survival in rats given 1000 rad in <5 or >5 fractions was 10–14 days. A dose fraction of between 125 and 250 rad was optimal. Heart allografts transplanted immediately after 2600 rad TLI (13 × 200 rad) survived 57 days; survival was shortened to 20 days if transplantation was delayed 1 month. Postoperative TLI alone did not prolong allograft survival. CsA (1.25 mg/kg/day) alone, in combination with preoperative TLI (5 × 200 rad), and in combination with postoperative TLI (9 × 200 rad) resulted in median survival times of 15, 96, and 60 days. Both the sequential and concurrent combination of TLI and CsA have synergistic effects on heart allograft survival. For optimal results, TLI should be fractionated preoperatively and transplantation should be performed soon after completion of radiotherapy. Post-transplant TLI is effective only if used in combination with pharmacological immunosuppression.