Serology of liver transplantation in the rat. I. Alloantibody responses and evidence for tolerance in a nonrejector combination

The antibody response against class I and class II RT1 antigens has been studied in PVG rats grafted with DA liver. In this nonrejector combination, liver grafts survive permanently in all normal recipients and in about 50% of recipients presensitized by a DA skin graft, with concurrent induction of transplant tolerance for other DA organs and skin. Using a two-stage radioimmunoassay, the anti-class I (RT1Aa) levels in sera of normal PVG recipients of DA liver grafts were found to be low (maximal titer 1:50 serum dilution or less); after peaking at 2 weeks posttransplantation, they diminished to background levels by 6 weeks. The anti-RT1Aa response showed a close parallel to cell-mediated rejection events in the liver graft recipients. In contrast, anti-class II (RT1Ba/Da) responses reached much higher titers (over 1:1000), which were maintained for several weeks before declining after 4 months. Similar observations were made in presensitized recipients. The induction of tolerance in the alloantibody response was indicated by the inability of DA skin grafts to restimulate anti-RT1 antibody in liver recipients. The observations support the picture of "split tolerance" indicated by previous cellular studies in this combination.     

The characterization of reconstituted passenger leukocytes on the induction of tolerance in rat liver transplantation

The tolerance induced by orthotopic liver transplantation [DA (RT1^a) rats to PVG (RT1^c) rats] can be prevented by total body irradiation of the donor rat. Reconstitution of the irradiated donor with DA splenic leukocytes reintroduces this tolerance. To investigate the major histocompatibility complex (MHC) specificity of passenger leukocytes, irradiated DA donors were reconstituted by third-party BN (RT1ⁿ) splenic leukocytes. The reconstitution with BN splenocytes re-established DA-specific tolerance in PVG recipients, as confirmed by subsequent DA cardiac allografting, while BN hearts were rejected with second-set tempo. To determine which cell components play an important role in re-establishing liver graft tolerance, DA splenic leukocytes were further purified into three types: T, B, and adherent cells. Only “T-cell-enriched” preparations restored liver graft tolerance in three out of five PVG recipients. These results suggest that passenger leukocytes of differing MHC types can help to induce liver-specific tolerance and that T cells in the liver graft may be essential to regulate tolerance induction. Received: 31 December 1996 Received after revision: 14 April 1997 Accepted: 15 April 1997     

Telomerase activity in rat liver allografts

BACKGROUND: Telomerase activity in grafts may be involved in the alteration of cellular senescence after transplantation or its relevant immunological events. METHODS: At the age of 20 weeks, donor livers harvested from DA (RT1a) were orthotopically transplanted into PVG (RT1c) or LEW (RT1(1)) rats. Rats having undergone orthotopic liver transplantation (OLT; DA-PVG) naturally overcome rejection, whereas all OLT (DA-LEW) rats die from acute rejection within 14 days. Telomerase activity in liver allografts was measured at various intervals post OLT. RESULTS: At day 7 when the most severe rejection episode was observed in OLT (DA-LEW) and OLT (DA-PVG), the telomerase activity was significantly higher than in syngeneic OLT (DA-DA) rats, in which no rejection occurred. Telomerase activity in tolerogenic OLT (DA-PVG) livers remained elevated for at least 2 months. CONCLUSION: These results suggest that telomerase activity in allogeneic OLT livers may reflect regenerating hepatocytes or activation of lymphocytes and/or hematopoietic stem cells associated with rejection or tolerance.     

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.     

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.     

Immunological tolerance-related genes in a spontaneous tolerant model of rat liver transplantation explored by suppression subtractive hybridization

Natural immunological tolerance can be induced in certain types of allogeneic liver transplantation in rats. To screen for genes associated with the induction of tolerance, suppression subtractive hybridization was performed in the rat liver transplantation model between a DA donor and PVG recipient combination where spontaneous immunological tolerance is known to occur without any immunosuppressive treatment. As a result, 112 genes were cloned from a DA liver graft that survived for 20 days in the fully allogeneic PVG recipient. After confirmation of the expression intensity using an in-house manufactured DNA array with cDNAs from the DA graft, 36 genes were classified in the highly expressed group and 26 moderately expressed group. In the first group, there were 8 immunoglobulin-related genes and 6 MHC class II-related genes, suggesting the existence of an underlying rejection response. Among those genes, an antiapoptotic gene in the p38 MAP kinase pathway, heme oxygenase gene (HO-1), and a ras cascade gene, IQ motif containing GTPase activating protein 1 (Iqgapl), retained biological significance. The results suggested that the molecular response to a liver graft tends to be antiapoptotic and to terminate the rejection response. Unfortunately, there was no gene identified that qualified as a putative immunosuppressive protein, liver suppressor factor-1 (LSF-1). The panel of genes identified in the present work will be a useful panel of candidate genes to investigate the induction of spontaneous tolerance.     

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.     

Treatment of streptozotocin-induced diabetes mellitus in rats by transplantation of islet cells from two major histocompatibility complex disparate rats in combination with intra bone marrow injection of allogeneic bone marrow cells

BACKGROUND: We have established a new method for the transplantation of allogeneic pancreatic islets obtained from two different rat strains in combination with a newly developed bone marrow transplantation (BMT) method in which bone marrow cells (BMCs) are directly injected into the bone marrow cavity (intra bone marrow BMT [IBM-BMT]). METHODS: Streptozotocin-induced diabetic Brown Norway (BN: RT1A(n)) rats were injected with fludarabine, irradiated with 5.0 Gy x 2, and BMCs from two allogeneic rat strains, Fischer 344 (F344: RT1A(1)) and PVG (PVG: RT1A(c)), were then directly injected into the bone marrow cavity (IBM-BMT). Simultaneously, approximately 600 pancreatic islets (PIs) from F344 and PVG rats were mixed and transplanted into the liver by way of the portal vein. RESULTS: All the recipients thus treated showed normoglycemia 30 days after the treatment. Hematolymphoid cells were completely reconstituted with the two donor-type cells, and immunologic tolerance to F344 and PVG major histocompatibility complex (MHC) determinants were induced. CONCLUSIONS: The transplantation of PIs from two MHC-disparate donors was completely achieved in combination with IBM-BMT, resulting in the improvement of blood glucose levels and the amelioration of diabetes mellitus.     

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.     

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.     

The effects of perioperative portal venous inoculation with donor lymphocytes on renal allograft survival in the rat. I. Specific prolongation of donor grafts and suppressor factor in the serum

In order to investigate the in vivo functional role of the liver in the immune responses in organ transplantation, effects of perioperative portal venous p.v. administration of donor lymphocytes on renal allograft survival were tested in the rat kidney transplant model. Donor lymphocytes were prepared from BN (BN, RT-1n) or third-party DA (RT1a) rat spleens and lymph nodes and injected p.v. or intravenously to Lewis (LEW, RT-1l) hosts on the day of transplantation (day 0). Untreated LEW hosts rejected BN renal grafts at 7.8 +/- 0.6 days (n = 10). Intravenous administration of 1 x 10(8) BN cells to LEW hosts on day 0 caused a slight, but not significant, prolongation of renal allograft survival (MST = 9.5 +/- 3.0 days, n = 13, NS), whereas portal venous inoculation of 1 x 10(8) BN cells on day 0 remarkably prolonged renal graft survival to 22.2 +/- 5.3 (n = 10, P less than 0.01). The prolongation of graft survival was antigen-specific; the administration of 1 x 10(8) DA cells p.v. to LEW hosts did not prolong the survival of BN renal grafts (MST = 7.4 +/- 0.8, n = 5). Spleen cells from p.v. treated LEW hosts 10 days after transplantation had no suppressor effect on the one-way MLC reaction of normal LEW responder cells toward donor BN or third-party DA stimulators. On the other hand, when serum from p.v.-treated LEW hosts was added to MLC at a concentration of 3 per cent of total volume, it suppressed the MLC reaction toward donor BN cells by 71.6 per cent, but not toward third-party DA stimulators (-8.5 per cent suppression, NS). Histological examination of p.v.-treated LEW hosts at 10 days after transplantation revealed that the liver had normal lobular architecture without expansion of portal tracts and infiltration of inflammatory cells. On the other hand, the transplanted kidney demonstrated a moderate mononuclear cell infiltration around the artery without an interstitial hemorrhage. Moreover, adoptive transfer of the serum from p.v.-treated LEW rats into the virgin secondary LEW hosts significantly prolonged the graft survival of BN kidneys from 7.8 days to 18.9 +/- 5.5 days (P less than 0.01), but not third-party DA graft survivals (MST = 7.5 +/- 0.6 days), indicating that an antigen-specific tolerogenic factor was released into the circulation through the process of allogeneic cells in the liver.     

Cytomegalovirus infection, viral DNA, and immediate early-1 gene expression in rejecting rat liver allografts

BACKGROUND: Cytomegalovirus (CMV) infection has been linked to acute and chronic rejection. We have previously shown that concomitant rat cytomegalovirus (RCMV) infection increases portal inflammation and bile duct destruction in rejecting rat liver allografts. Many of the pro-inflammatory effects of CMV have been attributed to the immediate early (IE) proteins of CMV. We wanted to investigate whether RCMV and IE-1 gene expression persist in the liver graft in our model. METHODS: Liver transplantations were performed from PVG (RT1c) into BN (RT1n) rats. One day after transplantation, the rats were infected with RCMV. No immunosuppression was given. The graft infection was studied by viral culture, immunofluorescence, DNA in situ hybridization and RT-PCR for the detection of IE-1 mRNA at various time points. RESULTS: RCMV caused an active infection from 5 days to 2 weeks after transplantation, during which infectious virus was found in the graft. Thereafter the cultures were negative. RCMV antigens and DNA were found in hepatocytes, endothelial, inflammatory, and bile duct cells during the active infection. At 4 weeks, RCMV DNA positive hepatocytes, endothelial, inflamma tory, and bile duct cells could still be found, but in much smaller quantities. IE-1 mRNA expression was, however, only detected during the active infection, not at 4 weeks postinfection. CONCLUSIONS: RCMV IE-1 expression does not persist in the graft after the active infection, although some viral DNA can be detected in the graft up to 4 weeks. In our model, the CMV-induced increase in graft damage does not seem to require the continued expression of IE-1.     

No association between tumor necrosis factor-alpha production and gene polymorphisms among inbred rat strains

Differences in spontaneous allograft acceptance after liver transplantation among inbred rat strains might be explained by variation in the local production of TNF-alpha as a potent mediator of the inflammatory response. In this study, we hypothesize that nucleotide differences in the rat Tnf gene influence TNF-alpha protein expression. As such, polymorphisms in the Tnf gene may also provide a possible explanation for differences in survival of allogeneic liver grafts among inbred rat strains. We therefore investigated the capacity of mononuclear cells to produce TNF-alpha in response to a mitogenic stimulus and the Tnf locus was sequenced in six different inbred rat strains. Among the six strains (AUG, BN, DA, LEW, PVG and WF), 44 nucleotide differences including 36 single nucleotide polymorphisms (SNPs), five simple sequence length polymorphisms, two deletions and one insertion, were found in the Tnf gene. Although, the inbred rat strains differed significantly in mean levels of maximum TNF-alpha production (P = 0.001), no associations were found with nucleotide differences within the Tnf gene. In conclusion, our results indicate that differential in vitro TNF-alpha responses among inbred rat strains are not associated with nucleotide differences within non-coding regulatory regions of the rat TNF-alpha gene. Without an established relationship between polymorphisms and expression of the TNF-alpha gene, it is preliminary to address a possible association of Tnf gene polymorphisms with rat liver allograft survival.     

Restoration of tolerance to rat hepatic allografts by spleen-derived passenger leukocytes

The tolerance induced by orthotopic liver transplantation (OLT) in certain combinations of rat strains can be prevented by total body irradiation (TBI) of the donor. We demonstrate here that the intravenous inoculation of splenic leukocytes into irradiated donors before OLT could re-establish tolerance in association with a state of microchimerism detected in the recipients. When donor DA (RT1^a) strain rats were irradiated with 1000 rad 24 h before liver harvesting and subsequent liver implantation into PVG recipients, five out of six rats died from rejection in this normally tolerogenic OLT (DA-PVG) combination. Injection of 1.5x10⁸ splenic leukocytes from naive DA rats into the irradiated DA donor rats 24 h before OLT restored the tolerogenic potential of the liver allografts. Immunofluorescence assay revealed an increased number of donor (DA) type cells in the PVG recipient bearing a repopulated DA liver, compared to the PVG recipient of an irradiated liver. These results suggest that passenger leukocytes reconstituted by splenic leukocytes have the capacity to protect liver allografts.     

New Immunosuppressive Proteins (KX-2, -4, and -5) Induced by Liver Transplantation

Abstract: The presence in rat serum of three novel immunosuppressive proteins is associated with induction of tolerance after liver grafting. One of these, KX-5 (40 kilodal-ton molecular weight) is present 40–100 days after grafting of DA liver into PVG, a combination in which liver grafts are not rejected; two others, KX-2 (87 kilodaltons) and KX-4 (10 kilodaltons), are present in PVG sera in a retransplantation model. N-terminal amino acid sequencing indicated that none of the proteins had been described previously. Induction of KX-5 was inhibited by cyclosporin A. All three proteins inhibited mixed lymphocyte responses nonspecifically. Administration of a single dose of 300 ^g purified KX-5 to PVG rats receiving heterotopic heart allografts led to prolonged graft acceptance. The results raise the possibility of clinical application of the immunosuppressive properties of liver grafts through administration of soluble proteins.     

T-cell anergy: a consequence of interaction between T cells and allogeneic rat renal epithelial cells

Abstract In order to study the immunogenicity of parenchymal cells within an allograft, renal tubular cells were propagated from both PVG and DA strain rats. These cells were induced to express class II major histocompatibility (MHC) antigens by stimulation for 4 days with interferon-gamma (IFN- y ). It was found that resting lymphoid cells derived from Lewis rats responded vigorously after stimulation with irradiated splenic cells from PVG rats. However, stimulation with renal cells from PVG rats did not result in interleukin (IL-2) production or lymphoprolife-ration. Furthermore, lymphocytes from this mixture failed to respond to secondary stimulation by PVG splenic cells; lymphocytes primed by mixture with DA renal cells responded normally to secondary stimulation by PVG splenic cells. These results indicate that renal epithelial cells can specifically anergise allogeneic lymphocytes.     

Detection of chimerism following vascularized bone allotransplantation by polymerase chain reaction using a Y-chromosome specific primer.

Chimerism following allogeneic organ transplantation is a phenomenon known to occur and be associated with development of immunologic tolerance in allotransplantation. However, little is known about graft cell migration following vascularized bone allografting. In this study, chimerism was assessed following vascularized tibia transplantation from male DA or PVG donors to female PVG rat recipients using a semi-quantitative polymerase chain reaction for the Y-chromosome. FK-506 (Tacrolimus) was administered after transplantation for immunosuppression. All immunosuppresssed PVG rat recipients of PVG bone grafts showed a high level of chimerism (1%) in the thymus, spleen, liver and cervical lymph nodes at 18 weeks post-transplant. Donor cells were also detected in the contralateral tibia and humerus. In non-immunosuppressed PVG rat recipients of DA bone grafts, donor cells were detected in the spleen in three of five rats within 2 weeks post-transplant. In these animals the bone grafts were severely rejected. In immunosuppressed PVG rat recipients of DA bone grafts, two of five, four of eight and eight of 10 rats showed low level chimerism (0.1%) in peripheral blood at 1, 12, and 18 weeks post-transplant. Six rats showed a high level of chimerism in the spleen and thymus. Histological studies revealed no rejection findings through 18 weeks post-transplant. Our results indicate that chimerism, or the presence of graft cells in host tissue, may occur in the face of acute rejection and be demonstrable following vascularized isograft and allograft living bone transplantation when chronic immunosuppression is maintained. Graft vascular patency during the short-term likely allows cellular migration, even in the face of acute rejection. Long-term survival and proliferation of graft marrow elements in host tissue may be possible with adequate immunosuppression.     

Specific suppression of allograft rejection by soluble class I antigen and complexes with monoclonal antibody

In this experiment, we investigated the effect of daily injection or continuous slow infusion of either DA (MHC haplotype, RT1a) serum or soluble DA class 1 MHC antigen or its complexes with monoclonal antibody on rejection of heterotopic heart allograft in the combination of PVG.RT1a (RT1a) donor into PVG (RT1c) recipient. DA serum delayed significantly both the early and late rejection of PVG.RT1a heart grafts in PVG recipients. Removal of soluble class I MHC antigen from DA serum by affinity chromatography on a monoclonal anti-class I antibody column completely abolished the immunosuppressive effect. Continuous infusion of purified soluble class I antigen from DA rat liver, even from day 4 after heart grafting, induced a significant prolongation of graft survival. This effectiveness was donor-specific and amplified by the mixture of monoclonal anti-class 1 (RT1a) antibody with DA serum--this being induced only by using continuous infusion but not by daily injection. The results indicate that soluble class I antigen can act as a specific immunosuppressive agent in allograft rejection and that its effect is amplified by monoclonal anti-class 1 antibody.     

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.