Donor IL-4-treatment induces alternatively activated liver macrophages and IDO-expressing NK cells and promotes rat liver allograft acceptance

Most approaches to transplant tolerance involve treatment of the recipient to prevent rejection. This study investigates donor treatment with IL-4 for its effect on subsequent rat liver allograft survival. Rat orthotopic liver transplants were performed in rejecting (PVG donor to Lewis recipient) or spontaneously tolerant (PVG to DA) strain combinations. Donors were untreated or injected intraperitoneally with IL-4 (30,000 U/day) for 5 days. Tissue infiltrates and gene expression were examined by immunohistochemistry and real-time quantitative PCR. IL-4 induced a marked leukocyte infiltrate in donor livers prior to transplant. Macrophages comprised the major population, although B cells, T cells and natural killer (NK) cells also increased. IL-4-induced liver macrophages had an alternatively activated phenotype with increased expression of mannose receptor but not inducible nitric oxide synthase (NOS2). IL-4 also induced IDO and IFN-gamma expression by NK cells. Donor IL-4-treatment converted rejection to acceptance in the majority of Lewis recipients (median survival time > 96 days) and did not prevent acceptance in DA recipients. Acceptance in Lewis recipients was associated with increased donor cell migration to recipient spleens and increased splenic IL-2, IFN-gamma and IDO expression 24 h after transplantation. Donor IL-4-treatment increased leukocytes in the donor liver including potentially immunosuppressive populations of alternatively activated macrophages and IDO-expressing NK cells. Donor treatment led to long-term acceptance of most livers in association with early immune activation in recipient lymphoid tissues.     

Induction of antigraft and antirecipient antibody responses after fully allogeneic and semiallogeneic rat small bowel transplantation

BACKGROUND: Given the potential influence of alloantibodies on organ graft outcome, this study investigated the induction of antigraft and antirecipient antibodies after allogeneic and semiallogeneic rat small bowel transplantation. METHODS: Fully allogeneic, unidirectional rejection and unidirectional graft-versus-host disease (GvHD) heterotopic small bowel transplantation was performed using DA, PVG, and (PVGxDA)F1 donor-recipient combinations. Serum was obtained before and at time points after transplantation and incubated with blood from untransplanted DA and PVG rats. Antibody binding to T cells was detected by whole blood flow cytometry using FITC-conjugated anti-rat IgM murine monoclonal antibody. Antibody levels were determined by reference to a standard curve of fluorescent intensity generated using a serum sample with known anti-target cell IgM activity. Data are presented as arbitrary units/ml (AU/ml). RESULTS: In the PVG-->DA combination, five of six DA recipients had detectable anti-graft (PVG) antibodies by day 4 after transplantation (mean 72 AU/ml) and all animals were positive by day 6 (976 AU/ml). Antirecipient (DA) antibodies were also induced, however, they were only apparent after 6 days in five of eight animals (90 AU/ml). Antigraft (DA) antibody responses were also induced in the DA-->PVG combination (day 6-218 AU/ml), however no antirecipient (PVG) response was apparent. Transplantation induced antirecipient (DA) antibodies in the unidirectional GvHD model (day 6-90 AU/ml) and an anti-graft (PVG) response in the unidirectional rejection model (day 6-60 AU/ml). However, the latter was quantitatively lower than that generated in the PVG-->DA combination (day 6-976 AU/ml). CONCLUSIONS: Antigraft and antirecipient antibody responses are simultaneously induced after fully allogeneic small bowel transplantation, despite rejection being the predominant clinical feature. Further studies are required to elucidate their influence on graft outcome.     

Spontaneous acceptance of mouse kidney allografts is associated with increased Foxp3 expression and differences in the B and T cell compartments

Spontaneous acceptance of organ allografts can identify novel mechanisms of drug-free transplantation tolerance. Spontaneous acceptance occurs in both mouse kidney transplants and rat liver transplants however the early immune processes of mouse kidney acceptance have not been studied. Acceptance of C57BL/6 strain kidney allografts in fully MHC-incompatible B10.BR recipients was compared with rejection (REJ) of heart allografts in the same strain combination. Graft infiltrate and antibody deposition were examined by immunohistochemical staining. Expression of mRNA was measured by quantitative real-time PCR. Apoptosis was examined by TUNEL staining. The majority of kidney allografts were accepted long-term and induced tolerance (TOL) of donor-strain skin grafts, showing that acceptance was not due to immune ignorance. There was an extensive infiltrate of T cells in the TOL kidney that exceeded the level in REJ hearts but subsequently declined. The main differences were deposition of IgG2a antibody in REJ that was absent in TOL, more B cells infiltrating TOL kidneys and a progressive increase in the ratio of CD8:CD4 cells during rejection. There was also significantly greater Foxp3 mRNA expression in TOL. Kidneys from RAG-/- donors were accepted, showing that donor lymphocytes were not necessary for acceptance. Neutralising antibodies to TGF-β administered from day 0 to day 6 did not prevent TOL. On the basis of cytokine expression and apoptosis there was no evidence for immune deviation or deletion as mechanisms of acceptance. In accord with the findings of spontaneous acceptance of liver allografts in rats, the main difference between mouse kidney TOL and heart REJ was in the B cell compartment. The major difference to rat liver allograft acceptance was that apoptosis of infiltrate did not appear to play a role. Instead, increased Foxp3 expression in TOL kidneys implies that regulatory T cells might be important.     

A short course of mycophenolate immunosuppression inhibits rejection, but not tolerance, of rat liver allografts in association with inhibition of interleukin-4 and alloantibody responses

BACKGROUND: Some immunosuppressive drug therapies inhibit transplant tolerance in animal models, and we have shown that treatment of recipients with methylprednisolone, but not cyclosporine, inhibits spontaneous acceptance of liver transplants. This study investigates the effects of mycophenolate mofetil (MMF) on liver acceptance and rejection. METHODS: Piebald Virol Glaxo rat livers were transplanted into Dark Agouti recipients, which spontaneously tolerate (TOL) the liver, or into Lewis recipients, which reject (REJ) the liver. MMF (40 mg/kg/day subcutaneously) was given for 5 days from days 0 to 4 (early) or from days 3 to 7 (late). In separate experiments, liver grafts were collected for assessment of infiltrate and of interleukin (IL)-2, IL-4, IL-10, and interferon-gamma mRNA expression. RESULTS: TOL liver transplants had a median survival time (MST) of more than 100 days (n=6), and neither early nor late MMF treatment of TOL transplants reduced survival (MST 85 days, P=0.19 and 78 days, P=0.08, respectively). Liver failure in most of these animals was the result of biliary problems, not rejection. There were few consistent differences between treated and untreated TOL animals in infiltrate or liver cytokine expression, although there was a moderate reduction in T-cell infiltrate in MMF-treated TOL animals (P=0.003 on day 5 TOL). In contrast, REJ transplants had an MST of 13 days (n=10), and early MMF treatment led to five of six animals surviving more than 100 days (P=0.0002), whereas late treatment was much less effective, with one of six animals surviving more than 100 days. REJ livers had significantly more IL-4 mRNA expression and immunoglobulin G1 deposition in the graft than TOL livers, and this was inhibited by early, but not late, MMF treatment. CONCLUSIONS: MMF treatment inhibited rejection but not acceptance of liver allografts. Early administration was more effective in preventing rejection and demonstrated a more marked effect on IL-4 expression and alloantibody deposition than on graft T-cell infiltrate and expression of other cytokines.     

The role of passenger leukocyte genotype in rejection and acceptance of rat liver allografts

BACKGROUND: Although graft-resident passenger leukocytes are known to mediate acute rejection by triggering direct allorecognition, they may also act in an immunomodulatory fashion and play an important role in tolerance induction. Our purpose in the current study was to utilize rat bone marrow chimeras to evaluate the role of the genotype of passenger leukocytes in both acute rejection and tolerance of liver allografts. METHODS: The fate of livers bearing donor-type, recipient-type, and third-party passenger leukocytes was evaluated in the MHC class I and II mismatched rejector combination ACI-->LEW and the acceptor combination PVG-->DA. RESULTS: We report that although treatment of ACI liver donors with lethal irradiation does not lead to prolongation of graft survival in the ACI-->LEW strain combination, ACI livers bearing recipient-type (LEW) or third-party passenger leukocytes (BN) are rejected at a significantly slower rate. We confirm that lethal irradiation of PVG donor animals leads to abrogation of tolerance induction with acute rejection of their livers by DA recipients. However, the majority of PVG livers carrying donor-type (PVG), recipient-type (DA), or third-party (LEW) passenger leukocytes are accepted for >100 days. These DA recipients develop immune tolerance to the donor parenchyma (PVG). CONCLUSIONS: Our findings demonstrate that long-term acceptance of liver allografts and tolerance induction is not dependent on the presence of donor-type passenger leukocytes and can be achieved with organs carrying donor-type, recipient-type, or third-party passenger leukocytes. The importance of the MHC framework on the surface of passenger leukocytes as a critical regulator of the immune response after transplantation of chimeric organs is substantiated by the delayed tempo of rejection of ACI livers bearing recipient-type or third-party passenger leukocytes in the ACI-->LEW strain combination.     

A short course of methylprednisolone immunosuppression inhibits both rejection and spontaneous acceptance of rat liver allografts

BACKGROUND: The effects of immunosuppressive drugs on transplant tolerance have not been extensively studied, although their effect on rejection is well established. METHODS: We examined the effects of a short course of treatment with the immunosuppressive drug methylprednisolone (MP) on the survival of PVG liver allografts in Dark Agouti (DA) recipients that accepted the livers and in Lewis recipients that rejected the livers. Infiltration of liver allografts was examined by immunohistochemical staining of liver sections, and apoptosis was measured by terminal deoxynucleotide transferase-mediated dUTP nick end labeling. RESULTS: A 5-day course of MP (days 0 to 4) led to rejection of four of six livers (mean survival time [MST] 99 days) in DA recipients compared with long-term survival (MST >100 days) in untreated animals. Delayed administration of MP (days 3 to 7) exacerbated rejection in DA recipients, and all eight animals rejected the graft (MST 68.5 days). Treatment of Lewis recipients with MP did not significantly prolong survival when administered from days 0 to 4 (MST 13 days), although delay of administration improved the outcome. Treatment from days 3 to 7 resulted in an MST of 21 days, whereas treatment from days 7 to 11 resulted in an MST of 41.5 days. MP treatment from day 3 to day 7 reduced T cells and interleukin 2 receptor-expressing cells but increased the numbers of apoptotic cells infiltrating both DA and Lewis strain allografts. CONCLUSIONS: These results show that immunosuppression with MP inhibits both spontaneous tolerance and rejection of liver allografts in a rat model and question the efficacy of administering MP to all liver allograft recipients from the time of transplantation.     

Antimurine immunoglobulin antibody responses after the administration of murine monoclonal antibodies to rats are altered by small bowel allograft rejection

BACKGROUND: This study monitored the induction of antimurine immunoglobulin antibody responses after the administration of anti-CD4 (OX38) and anti-LFA-1 (WT.1) monoclonal antibodies to DA rats. METHODS: Monoclonal antibody was administered i.v. on 3 consecutive days to untransplanted DA rats, and DA recipients of PVG small bowel allografts. Control animals received no monoclonal antibody. Antimurine immunoglobulin antibody levels in serum samples were determined by enzyme immunoassay. RESULTS: No antimurine immunoglobulin antibody was detected in untransplanted animals receiving OX38 alone. Reactivity was apparent in WT.1-treated animals, but this response was totally abrogated by the co-administration of OX38. A combination of OX38 and WT.1 had no effect on allograft recipient survival and antimurine immunoglobulin antibody responses were detected in all allograft recipients, irrespective of the treatment regimen. CONCLUSIONS: Although OX38 inhibited the antibody response both to itself and to WT.1 in untransplanted animals, the immune reaction induced by small bowel allograft rejection overcame this inhibitory capacity.     

A rat model of pregnancy-induced sensitization to transplants

Previous pregnancy is a known risk factor for alloantibody production and graft rejection in clinical transplantation. However, in previous rat models, immune responses to RT1.A antigens induced by allogeneic pregnancy resulted in prolonged survival of subsequent allografts. This study was designed to investigate the effects of a previous pregnancy on alloantibody response, complement activation, and allograft survival in a highly immunogenic rat strain combination. C6-sufficient and -deficient female PVG.1U (RT1.A(u)B(u)) rats were mated with allogeneic PVG.R8 (RT1.A(a)B(u)) males or control isogeneic PVG.1U (RT1.A(u)B(u)) males. Three weeks after parturition, experimental and control females received cardiac allografts from female PVG.R8 donors. A low dose of cyclosporine (CsA, 5 mg/kg on alternate days) was used for immunosuppression after transplantation. Allogeneic, but not control isogeneic, pregnancy elicited a weak, transient IgG alloantibody response that declined before transplantation. Experimental female recipients produced a rapid, vigorous IgM and IgG alloantibody response to the transplant despite CsA treatment. C6-sufficient recipients rejected their transplants at an accelerated rate (5 days, n = 6) compared with control animals (7 days, n = 5). In contrast, allografts to C6-deficient recipients functioned until sacrifice at 90 days in both the experimental group (n = 7) and control group (n = 4). Most experimental C6-deficient recipients continued to produce strong IgG alloantibodies for 90 days. Complement activation resulting from the alloantibody response was evidenced by the diffuse deposition of C3d on the vascular endothelium of the grafts. In summary, previous pregnancy leads to memory alloantibody responses that accelerate allograft rejection even with immunosuppression. Membrane attack complex is required for accelerated rejection induced by previous pregnancy.     

C4d deposition and cellular infiltrates as markers of acute rejection in rat models of orthotopic lung transplantation

BACKGROUND: C4d is a useful marker of antibody-mediated rejection in cardiac and renal transplants, but clinical studies examining correlations between circulating alloantibodies, C4d deposition, and rejection in lung transplants have yielded conflicting results. METHODS: We studied circulating alloantibody levels and C4d deposition in two rat models of lung transplantation: Brown Norway (BN) to Wistar-Kyoto (WKY) and PVG.R8 to PVG.1U lung allografts. The availability of C6 deficient (C6-) and C6 sufficient (C6+) PVG 1U rats allowed evaluation of the effects of the terminal complement components on graft injury and C4d deposition. RESULTS: The lung allografts had histologic features resembling human posttransplant capillaritis, characterized by neutrophilic infiltration of alveoli, edema, and hemorrhage. Immunoperoxidase stains on cross sections of allografts showed intense, diffuse, C4d deposition in a continuous linear pattern on the vascular endothelium. C4d deposits were found in both BN to WKY and PVG R8 to 1U allografts, whereas no staining was detectable in WKY to WKY isografts or native lungs. Complement deposition was associated with vascular disruption in C6+, but not in C6- recipients. The presence of circulating donor-specific alloantibodies was verified by flow cytometry. Cell-specific staining revealed perivascular accumulation of macrophages and T lymphocytes whereas neutrophils were sequestered in the intravascular and alveolar capillary compartments. CONCLUSIONS: The deposition of C4d on vascular endothelium as well as the coincident presence of alloantibodies is consistent with previous findings in antibody-mediated rejection of renal and cardiac transplants. Furthermore, the histological features of our allografts support the concept that posttransplant capillaritis is a form of humoral rejection.     

Stimulation of CD4+ T lymphocytes by allogeneic MHC peptides presented on autologous antigen-presenting cells. Evidence of the indirect pathway of allorecognition in some strain combinations.

A preliminary analysis of the alloantibody response to free, unconjugated class I and class II MHC peptides in several rat and mouse strains was performed, to screen for an effective interaction between the allogeneic MHC peptides and recipient MHC molecules. The PVG rat strain was noted to produce very strong, MHC-restricted, primary and secondary responses to a synthetic peptide derived from the alpha helical region of the alpha 2 domain of an RT1.C/E class I MHC molecule of the DA strain. In vitro proliferation studies demonstrated that CD4+ but not CD8+ T cells of the PVG strain responded in a recipient APC-dependent manner to the peptide, whereas the BN strain (which showed no antibody response to this peptide) gave no T cell proliferation. Immunization of PVG rats with the peptide did not influence the rejection of DA skin allografts. The relevance of these studies to the possible mechanisms of allograft rejection by an indirect pathway are discussed.     

Effect of liver transplantation on islet allografts: up-regulation of Fas ligand and apoptosis of T lymphocytes are associated with islet graft tolerance

BACKGROUND: Liver allografts in spontaneously tolerant strain combinations can protect other organs of the same donor origin from rejection and reverse ongoing rejection in previously placed grafts. The aims of this study were to examine whether liver allografts have the same protective effect on islet allografts and to investigate the underlying mechanisms. METHODS: PVG islets were transplanted beneath the kidney capsule of streptozotocin-induced diabetic DA rats with or without liver allografting. The cellular infiltrate, and the extent of apoptosis and of Fas ligand (FasL) expression in the islet grafts were evaluated on days 2, 4, and 7 after transplantation by means of immunostaining and the in situ terminal deoxynucleotide transferase-mediated dUTP nick end labeling assay. Donor and recipient mixed lymphocyte reactions (MLR) were determined at 7 days or 100 days after islet transplantation. RESULTS: Islet allografts transplanted alone were rapidly rejected within 5-8 days. Rejection was delayed, but not prevented, when islets were transplanted simultaneously with the liver. Liver transplantation 1 month before islet transplantation resulted in long-term survival (>100 days) of islet grafts in three of seven animals, whereas the other four died of liver rejection with functional islet grafts. Liver transplantation on day 4 after islet grafting reversed ongoing islet rejection and led to indefinite islet graft survival in three of seven cases. There was a progressive increase of cellular infiltration in all of the islet allografts, but the intensity of the infiltrate did not correlate with the outcome of the islet allografts. Islet rejection was characterized by an early dominance of monocytes/macrophages and CD25+ T cells in the infiltrates, a high incidence of apoptotic beta cells in grafts, and a sensitized status in the MLR. Tolerance of islet allografts was associated with increased numbers of dendritic cells in the graft infiltrates, up-regulation of FasL, and prominent apoptosis of alloreactive leukocytes in the islet grafts, as well as donor-specific MLR suppression in long-term survivors. CONCLUSIONS: These results demonstrate that the extent of the protective effect of liver transplantation on islet allografts varies with the time of liver grafting, ranging from delay in islet rejection to complete islet acceptance. Islet graft tolerance induced by liver transplantation is the result of an immune process that involves up-regulation of Fas ligand expression on, and apoptosis of, islet graft infiltrating lymphocytes.     

Studies on the participation of different T cell subsets in rat liver allograft rejection

In this study, we investigated which subsets of rat T cells (CD8 + vs. CD4 + ) are involved in the rejection of liver allografts by the in vivo administration of monoclonal antibody (OX-8 or OX-38, and W3/25 MAb) into thymectomized recipient Lewis (RTI¹) rats prior to DA (RTI^a) liver transplantation. We also compared the results of allograft survival of liver and heart transplants under the same experimental conditions. In order to deplete either CD8 + T cells or CD4 + T cells from recipient animals, 0.4 ml of OX-8 (ascitic form) or a 0.8 ml cocktail of MAb W3/25 and OX-38 (0.4 ml each) was injected into thymectomized recipient rats, respectively. Untreated Lewis rats consistently rejected donor DA liver grafts between 9 and 11 days (n = 7, 9.8 days ± 1.1 days). In contrast, anti-CD8 MAb pretreatment extended the survival times of DA liver grafts for up to 40 days (n = 5, 26.8 days ± 8.4 days). Furthermore, survival of DA liver grafts was significantly prolonged in Lewis rats that had been pretreated with anti-CD4 MAb (n = 7,35.6 days ± 17.9 days). Two out of seven recipient animals survived for more than 60 days. For heart transplantation, untreated Lewis rats rejected DA heart grafts between 6 and 8 days after operation (n = 6, 6.5 days ± 1.2 days). Anti-CD4 MAb treatment prolonged heart graft survival for more than 60 days in all cases (n = 3, > 60 days). However, there was virtually no effect of anti-CD8 MAb treatment on heart graft survival (n = 4, 7.0 days ± 0.9 days). These results suggested that when whole MHC disparity prevailed between donor and recipient, both subsets of T cells were required for the rejection of liver allografts and that class II reactive T cells predominantly mediated liver graft rejection. Furthermore, CD8 + T cells played a differential role in the rejection of rat liver and heart allograft.     

IL-13 prolongs allograft survival: association with inhibition of macrophage cytokine activation

Th2 cytokines, especially IL-4 and IL-10, may facilitate transplant tolerance induction but the role of IL-13, another Th2 cytokine, is not known. This study examined the effects of rat recombinant IL-13 (rIL-13) on alloimmune responses. In vitro effects of rIL-13 were compared in mixed lymphocyte cultures (MLC) on rat lymphocytes cultured with PVG stimulator cells. DA rats grafted with fully allogeneic PVG neonatal heart grafts were treated with 40,000 units of rIL-13 for 10 days and graft survival monitored by ECG. Cytokine mRNA expression in the graft and lymphoid tissues was studied by RT-PCR and alloantibody levels assayed. rIL-13 had no effect on MLC, unlike rIL-4 which enhanced proliferation and induced Th2 and inhibited Th1 cytokines in MLC. rIL-13 inhibited IL-12p35, IL-12p40 and TNF-alpha mRNA induction in dendritic cell cultures. Treatment with rIL-13 prolonged fully allogeneic PVG neonatal heart graft survival to 18-21 (13-27) days (median (range)); compared to 12 (9-15) days in untreated normal rejection (p<0.05) and 14 (10-24) days in sham treated controls (p<0.05). RT-PCR studies on graft tissue identified reduced mRNA expression for the dendritic cell/macrophage molecules iNOS, TNF-alpha and IL-12 compared to normal rejection. rIL-13 treatment did not increase Th2 cytokines as compared to normal rejection, or the Th2 dependent IgG1 alloantibody response, while IL-4 did. These studies demonstrated that rIL-13 can prolong allograft survival associated with inhibition of IL-12, TNF-alpha and iNOS mRNA induction, and suggest IL-13 could modify graft rejection by inhibition of dendritic cell and/or macrophage function.     

Suppression of kidney allograft rejection across full MHC barriers by recipient-specific antibodies to class II MHC antigens.

The aim of these studies was to see if recipient-specific antibodies to class II MHC antigens might be effective in suppressing kidney graft rejection in rats. For these experiments, the polymorphic BMAC-4 mouse IgG1 monoclonal antibody to RT1-D class II MHC antigens was raised. This antibody reacts with the DA, LEW, PVG, and SHR strains, but not the BN or WAG strains, and is therefore recipient-specific in the WAG to PVG combination. Initial in vivo titrations demonstrated that 1 ml doses of the BMAC-4 and also of the MRC OX6 (monomorphic mouse IgG1 anti-RT1-B class II) antibody resulted in the maintenance of free antibody levels in blood for greater than 24 hr. Treatment of PVG recipients of WAG kidney allografts with the BMAC-4 antibody, but not the MRC OX6 antibody, resulted in greatly prolonged graft survival. To examine possible mechanisms, several experiments were performed. After intravenous injection, the antibody was found to have ready access to the connective tissues of nonlymphoid organs, to the red and white pulp of the spleen, and to the medulla of lymph nodes. However, there was poor early access to the cortex and paracortex of lymph nodes. Both MRC OX6 and BMAC-4 could completely suppress PVG anti-WAG and WAG anti-PVG mixed lymphocyte culture reactions. Both antibodies were also equally effective for opsonisation of class II-positive cells from the blood circulation. However, only the recipient-specific, anti-RT1-D BMAC-4 antibody suppressed graft rejection. Thus, while the BMAC-4 antibody is likely to have had a variety of different effects on RT1-D positive recipient cells, the locus specificity of the immunosuppression is consistent with an important component of those effects being the blocking of presentation of WAG donor alloantigens by PVG RT1-D class II antigens on PVG antigen-presenting cells.     

Blocking of mononuclear cell accumulation, cytokine production, and endothelial activation within rat cardiac allografts by CD4 monoclonal antibody therapy.

CD4 monoclonal antibody therapy prolongs allograft survival in a variety of experimental models and is currently undergoing clinical trials, though surprisingly little is known about the effects of CD4 mAb therapy on intragraft effector mechanisms that mediate rejection. We previously reported the significantly improved survival of (LEWxBN)F1 cardiac allografts in LEW rats treated for 10 days with the new CD4 mAb, BWH-4, at a dose of 700 micrograms/day, i.v., starting at the time of engraftment. Thus, CD4-treated rats showed prolongation of allograft survival to a median of 37 days (range 22 to greater than 100 days) post-Tx, compared with rejection at 7 days in untreated controls. We now report the results of detailed immunohistologic studies of allografts collected from these rats. Comparison of acutely rejecting allografts in untreated rats with well-functioning allografts collected at day 7 post-Tx from CD4-treated rats showed that CD4 mAb: (1) significantly reduced mononuclear cell infiltration, interstitial edema, hemorrhage formation and vascular and extravascular thrombosis; (2) inhibited mononuclear cell induction of receptors for IL-2 and transferrin, and upregulation of class II antigens and ICAM-1 on leukocytes and endothelial cells; (3) suppressed intragraft mononuclear cell and/or endothelial production of the cytokines IL-1, IL-2, IL-6, IFN-gamma, and TNF; and (4) blocked upregulation of endothelial tissue factor and downregulation of thrombomodulin, and consequently inhibited fibrin deposition. Studies of allografts from CD4-treated rats collected at day 30 post-Tx, prior to clinical rejection, showed a resurgence of CD4+ cells within allografts and a dense cellular immune response. We conclude that short-term CD4 mAb therapy has potent and extensive inhibitory effects on cytokine-related mononuclear cell and endothelial activation in vivo, blocking multiple afferent and efferent steps of the alloresponse.     

Allograft acceptance and rejection, mediated by a liver suppressor factor, LSF-1, purified from serum of liver transplanted rats

In certain rat strain combinations liver allografts are spontaneously accepted without immunosuppression and induce donor-specific tolerance to further skin and heart grafts in the recipient. Such an effect is also transferrable using serum from orthotopically liver transplanted rats (OLT serum). In the OLT serum of one such combination. DA (RT1^a) donor into PVG (RT1^c) recipient, a 40 kDa protein (liver suppressor factor, LSF-1) has been identified and shown to be immunosupressive in vitro. The aim of the present study is to investigate the immunological effect of LSF-1 and a polyclonal antibody (anti-LSF-1) against this molecule, in a rat heterotopic heart transplant (HHT) model and OLT model, respectively. Intramuscular injection of 300 μg of LSF-1, 1 h postoperatively, into a PVG recipient of either a DA or BN (RT1ⁿ) cardiac allograft caused significant prolongation of graft survival. Intravenous injection of polyclonal rabbit sera raised against an N-terminal peptide of LSF-1 (anti-LSF-1), within 1 h postoperatively, had variable effects on the survival of DA liver grafts in PVG recipients. In cases injection of between 1 and 2 ml of anti-LSF-1 resulted in death of the recipient. Histological examination of the liver showed severe rejection with lymphoid cell infiltration of the portal tract and sinusoids and extensive damage to the parenchyma. All control rats survived for more than 60 days without any signs of rejection. The anti-LSF-1 polyclonal antibody prevented the induction of tolerance in the normally tolerogenic model (DA into PVG). This, together with the in vivo results, suggests a role for LSF-1 in the induction of tolerance.     

Non-depleting anti-CD4, but not anti-CD8, antibody induces long-term survival of xenogeneic and allogeneic hearts in alpha1,3-galactosyltransferase knockout (GT-Ko) mice

The anti-galactose-alpha1,3-galactose (Gal) antibody (Ab) response following pig-to-human transplantation is vigorous and largely resistant to currently available immunosuppression. The recent generation of GT-Ko mice provides a unique opportunity to study the immunological basis of xenograft-elicited anti-Gal Ab response in vivo, and to test the efficacy of various strategies at controlling this Ab response [1]. In this study, we compared the ability of non-depleting anti-CD4 and anti-CD8 to control rejection and antibody production in GT-Ko mice following xenograft and allograft transplantation. Hearts from baby Lewis rat or C3H mice were transplanted heterotopically into GT-Ko. Non-depleting anti-CD4 (YTS177) and anti-CD8 (YTS105) Abs were used at 1 mg/mouse, and given as four doses daily from day -2 to 1 then q.o.d. till day 21. Xenograft rejection occurred at 3 to 5 days post-transplantation in untreated GT-Ko recipients, and was histologically characterized as vascular rejection. Anti-CD4, but not anti-CD8, Ab treatment prolonged xenograft survival to 68 to 74 days and inhibited anti-Gal Ab as well as xeno-Ab production. In four of the five hearts from anti-CD4 mAbs-treated GT-Ko mice, we observed classic signs of chronic rejection, namely, thickened intima in the lumen of vessels, significant IgM deposition, fibrosis and modest mononuclear cell infiltrate of Mac-1+ macrophages and scattered T cells (CD8>CD4). Xenograft rejection in untreated, as well as anti-CD4- and anti-CD8-treated, recipients was associated with increased intragraft IL-6, IFN-gamma and IL-10 mRNA. C3H allografts were rejected in 7 to 9 days by untreated GT-Ko mice and were histologically characterized as cellular rejection. Treatment with anti-CD4 and anti-CD8 mAb resulted in graft survivals of >94.8 and 11.8 days, respectively. Anti-CD4 mAb treatment resulted in a transient inhibition of alloreactive and anti-Gal Ab production. The presence of circulating alloreactive and anti-Gal Abs at >50 days post-transplant was associated with significant IgM and IgG deposition in the graft. Yet, in the anti-CD4 mAb-treated group, the allografts showed no signs of rejection at the time of sacrifice (>100 days post-transplantation). All rejected allografts had elevated levels of intragraft IL-6, IFN-gamma and IL-10 mRNA, while the long-surviving anti-CD4-treated allografts had reduced mRNA levels of these cytokines. Collectively, our studies suggest that the elicited xeno-antibody production and anti-Gal Ab production in GT-Ko mice are CD4+ T-cell dependent. The majority of xenografts succumbed to chronic rejection, while allografts survived with minimal histological change, despite elevated levels of circulating alloAbs. Thus, immunosuppression with anti-CD4 mAb therapy induces long-term survival of allografts more effectively than to xenografts.     

Antibody and complement mediated injury in transplants following sensitization by allogeneic blood transfusion

BACKGROUND: Many patients on the waiting list for transplants are sensitized from previous blood transfusions, pregnancy, or transplants. We investigated the role of complement in acute and chronic pathology in hearts transplanted to sensitized rats. METHODS: Blood was transfused from allogeneic PVG.R8 rats or control isogeneic PVG.1U rats to C6-sufficient and -deficient PVG.1U rats. Three weeks later hearts were transplanted from PVG.R8 donors and low-dose cyclosporin A was initiated. RESULTS: Allogeneic but not isogeneic blood transfusion elicited strong immunoglobulin (Ig) M, IgG1 and IgG2b alloantibody responses. Sensitization caused accelerated acute rejection of cardiac allografts by C6-sufficient recipients (4 days). In contrast, allografts functioned over 40 days in all C6-deficient recipients, but sensitization caused increased interstitial fibrosis and chronic vasculopathy. Circulating alloantibodies were associated with deposits of C4d on the vascular endothelium together with pericapillary accumulation of neutrophils and macrophages in the grafts. In contrast, T cells accumulated in periarterial lymphatics that did not have C4d deposits. CONCLUSIONS: Presensitization by allogeneic blood transfusion causes accelerated acute graft rejection in the presence of the complete complement cascade. In the absence of C6, macrophages colocalized with deposits of C4d and T cells accumulated in the periarterial lymphatics.