Elimination of donor-specific alloreactivity prevents cytomegalovirus-accelerated chronic rejection in rat small bowel and heart transplants

BACKGROUND: The primary cause for late failure of vascularized allografts is chronic rejection (CR) characterized by transplant vascular sclerosis (TVS). Cytomegalovirus (CMV) infection accelerates TVS and CR by unclear mechanisms involving direct effects of CMV, indirect effects of the recipient's immune response to CMV, or interactions between CMV and the recipient's alloreactivity. This study examined the role of CMV and the alloreactive response in the development of TVS using bone marrow chimerism (BMC) in rat small bowel (SB) and heart transplantation models. METHODS: Fisher 344 (F344) rat heart or SB grafts were transplanted into F344/Lewis bone marrow chimera. F344 heart or SB grafts transplanted into Lewis recipients (low-dose cyclosporine) were positive controls for the development of TVS. Lewis heart or SB grafts transplanted into Lewis recipients (+/-cyclosporine) were transplantation controls. The effect of rat CMV (RCMV) (5x105 plaque-forming units) on TVS (neointimal index, NI) and graft survival was studied in these groups. RCMV infection was assessed by serologic analysis and quantitative polymerase chain reaction techniques (TaqMan). RESULTS: RCMV infection accelerated the time to graft CR (SB 70-38 days; hearts 90-45 days) and increased the severity of TVS in both the SB allografts (day 38, NI=27 vs. 52) and the heart allografts (day 45, NI=43 vs. 83). Grafts from CMV-infected syngeneic recipients failed to develop TVS and CR. Donor-specific tolerance induced by BMC prevented allograft TVS and CR in both transplant models. In contrast to na?ve Lewis recipients, RMCV infection failed to cause allograft TVS and CR in bone marrow (BM) chimeras. CONCLUSIONS: The events in CMV-induced acceleration of TVS involve a crucial interplay between CMV infection and the recipient's alloreactive immune response.     

Combination of donor-specific blood transfusion with anti-CD28 antibody synergizes to prolong graft survival in rat liver transplantation

Donor-specific blood transfusion (DST) has been shown to effectively induce tolerance to certain allografts. In addition, it is well known that blockade of costimulatory signals reduces the ability of T cells to respond to alloantigens, prolonging allograft survival in some transplant models. We assessed the effects of single or multiple DSTs in the absence or presence of anti-CD28 monoclonal antibodies (mAbs) on graft function and host survival in rat liver transplantation (LTx). Fully MHC-mismatched adult male Dark Agouti (DA) and Lewis (LEW) rats were used as donors and recipients, respectively. Heparinized DA blood was administered to na?ve LEW rats 7 days before LTx [DST(-7d)], 14 and 7 days before LTx [DST(1 x 2)], twice a week for 2 weeks prior to LTx [DST(2 x 2)] and once a week for 4 weeks prior to LTx [DST(1 x 4)]. For some experiments, two different monoclonal antibodies (mAb) to rat CD28 (JJ316 and JJ319) were administered in combination with some DST treatments. We found that DST administration induced a time- and dose-dependent increase in host survival. Treatment of LEW rats with JJ316 or JJ319 mAb alone failed to prolong graft survival over untreated rats; however, the combination of DST(1 x 2) with JJ316 or JJ319 mAb induced indefinite survival at 100 days following surgery. We found that this protective effect was associated with increased numbers of splenic CD4+ CD45RC- but not CD4+ CD25+ foxp3+ T-cells in long-term survivors. Our data suggest that the combination of suboptimal DST with CD28 mAb induces donor-specific tolerance that correlates with enhanced numbers of regulatory T-cells.     

15-Deoxyspergualin and cyclophosphamide, but not mycophenolate mofetil, prolong survival and attenuate renal disease in a murine model of ANCA-associated crescentic nephritis.

BACKGROUND: Here we compare the efficacy of cyclophosphamide (CYC) for treatment of crescentic nephritis (CGN) with the newer immunosuppressants 15-deoxyspergualin (DSG) and mycophenolate mofetil (MMF) in SCG/Kj mice, an inbred mouse strain that spontaneously develops CGN, systemic necrotizing vasculitis and antineutrophil cytoplasmic antibodies (ANCAs). METHODS: Mice were randomly assigned to intraperitoneal treatment with either DSG (2 mg/kg/day), CYC (50 mg/kg/week), MMF (60 or 100 mg/kg/day) or vehicle (VEH, dextrose 5% 0.3 ml/day) beginning at the 10th week of life. ANCA, blood urea nitrogen (BUN) and proteinuria were determined in all animals regularly, and survival was calculated. Renal histology was obtained in the 18th week of life in the MMF- or VEH-treated groups and in the 24th week in DSG- or CYC-treated animals. RESULTS: Mean survival in VEH-treated animals was 123 days. At that point, survival was 100% in the CYC- or DSG-treated animals (P<0.001). Survival in the MMF group (pooled data) was not significantly different from the VEH-treated animals [MMF, 117 days (95% CI 108-127)]. BUN (18th week, CYC 43+/-9 mg/dl and DSG 36+/-6 mg/dl vs VEH 73+/-28 mg/dl, P<0.001, MMF 66+/-26 mg/dl), 24 h proteinuria (18th week, CYC 0.4+/-0.2 mg and DSG 0.7+/-0.6 mg vs VEH 2.7+/-3 mg, P<0.001, MMF 2.2+/-3 mg) crescent formation (18th week, VEH 42+/-9%, MMF 39+/-11%; CYC 5+/-2% and DSG 22+/-7% vs VEH, P<0.05), glomerular immune complex deposition, and ANCA formation were significantly improved in CYC- and DSG- but not in MMF-treated animals when compared with controls. CONCLUSION: DSG and CYC, but not MMF, prolong life, limit renal damage and prevent autoantibody formation in SCG/Kj mice.     

Inability of donor total body irradiation to prolong survival of vascularized bone allografts: experimental study in the rat

At the present time, the toxic side effects of recipient immunosuppression cannot be justified for human non-vital organ transplantation. Total body irradiation has proven effective in ablating various bone-marrow-derived and endothelial immunocompetent cellular populations, which are responsible for immune rejection against donor tissues. Irradiation at a dose of 10 Gy was given to donor rats six days prior to heterotopic transplantation of vascularized bone allografts to host animals. Another group of recipient rats also received a short-term (sixth to fourteenth day after grafting), low dose of cyclosporine. Total body irradiation was able merely to delay rejection of grafts across a strong histocompatibility barrier for one to two weeks, when compared to nonirradiated allografts. The combination of donor irradiation plus cyclosporine did not delay the immune response, and the rejection score was similar to that observed for control allografts. Consequently, allograft viability was quickly impaired, leading to irreversible bone damage. This study suggest that 10 Gy of donor total body irradiation delivered six days prior to grafting cannot circumvent the immune rejection in a vascularized allograft of bone across a strong histocompatibility barrier.     

Isogeneic MSC application in a rat model of acute renal allograft rejection modulates immune response but does not prolong allograft survival

Application of mesenchymal stromal cells (MSCs) has been proposed for solid organ transplantation based on their potent immuno-modulatory effects in vitro and in vivo. We investigated the potential of MSCs to improve acceptance of kidney transplants in an MHC-incompatible rat model including isogeneic kidney transplantation (RTx) as control. MSCs were administered i.v. or i.a. at time of transplantation. No immunosuppression was applied. Renal function was monitored by serum-creatinine, histopathology, immunochemistry for graft infiltrating cells and expressions of inflammatory genes. We demonstrated the short-term beneficial effects of MSC injection. In the long term, however, MSC-related life-threatening/shortening events (thrombotic microangiopathy, infarctions, infections) were evident despite decreased T- and B-cell infiltration, lower interstitial inflammation and downregulated inflammatory genes particularly after i.a. MSC injection. We conclude that i.a. MSC administration provides efficient immunomodulation after allogeneic RTx, although timing and co-treatment strategies need further fine-tuning to develop the full potential of powerful cell therapy in solid organ transplantation.     

The effect of donor-specific transfusion and cyclosporin A on small bowel transplantation in the rat

Pre-transplant blood transfusions are given as a means of desensitization to reduce the required dose of cyclosporin A (CsA). In this study, the effect of pretransplant blood transfusion on host survival and T-cell function against alloantigen were investigated. Male Lewis rats (RT1¹) were used as the recipients in all experiments, and male DA rats (RT1^a) were used as the blood and small bowel donors, and as a source of allogeneic stimulator cells. Male BUF rats (RT1^b) were used as donors of third party blood, and of allo-stimulator cells in a delayed-type hypersensitivity (DTH) response. In our experimental design, Lewis rats were divided into the following groups according to the type of administration: (1) a donor-specific blood transfusion (DST) 8 days preoperatively and a concurrent 5-day course of CsA at 10 mg/kg per day; (2) a nonspecific third party blood transfusion (NST) and CsA at 10 mg/kg per day from day 8 to day 4 preoperatively; (3) CsA alone from day 8 to day 4 preoperatively; (4) DST alone 8 days preoperatively; or (5) no treatment, being the control group. Postoperative treatment consisted of CsA at 2.5 mg/kg per day for 30 days. Rats conditioned with NST plus CsA, CsA alone, DST alone, and the untreated control rats survived for 7.2 ±1.2, 9.0 ± 2.2, 6.8 ± 0.4, and 7.4 ± 1.6 days, respectively. In contrast, the five rats conditioned with DST plus CsA survived for 100 days or more. This study demonstrates that long-term survival of a small bowel allograft can be achieved by host-conditioning with a combined treatment of DST and low-dose CsA.This paper was presented at the 10th Congress of the Asian Association of Pediatric Surgeons held in Seoul, Korea from March 26–29, 1990, and at the 90th Annual Meeting of the Japanese Surgical Society held in Sapporo, Japan in 1990.     

Evidence that indefinite survival of small bowel allografts achieved by a brief course of cyclosporine or FK506 is not due to systemic hyporesponsiveness.

The immunological status of Lewis (LEW) recipients of indefinitely surviving (greater than 400 days) orthotopic Brown-Norway (BN) small bowel allografts was investigated 1 to 1 1/2 years after cessation of immunosuppressive therapy with either cyclosporine or FK506 and compared with recipients of syngeneic grafts. A normal proliferative response (as measured by a mixed lymphocyte culture) of recipient peripheral lymph node lymphocytes in response to the donor-specific (BN) and the third-party (ACI) antigen, was observed in all experimental groups. Cytolytic T cell generation (as measured by a standard 51Cr-release cytotoxicity assay) in response to the donor-specific (BN) and the third-party (ACI) antigen was observed also in all groups. A FACS analysis of allograft-recipient splenocytes showed no evidence for systemic lymphoid chimerism. BN or ACI skin grafts transplanted onto recipients of allogeneic and syngeneic small bowel grafts were rejected completely in 12-17 days, while the intestinal grafts remained functional. Immunohistologic evaluation of the allografts, using anti-BN class I and anti-Lewis class II monoclonal antibodies showed anti-BN staining on the epithelial and endothelial structures, whereas the mononuclear cells in the lamina propria stained positively with the anti-LEW monoclonal antibody. However, lymphoid depletion and scarring of Peyer's patches and mesenteric lymph nodes as well as focal obliterative mesenteric arteriopathy, indicative of an indolent chronic rejection, were observed. These data demonstrate that recipients of indefinitely surviving small bowel allografts remain immune competent and do not retain the intestinal graft on the basis of specific hyporesponsiveness to the donor antigens.     

SEB combined with IL-1ra could prolong the survival of the rat allografts in high-risk corneal transplantation

PURPOSE: To determine whether the superantigen Staphylococcal enterotoxin B (SEB) combined with interleukin-1 receptor antagonist (IL-1ra) prolong allograft survival better than individual agents in high-risk corneal transplantation in a rat model. METHODS: Fisher 344 donor corneas were transplanted into Lewis recipients. High-risk transplantation meant that the transplants were sutured into the recipient beds with corneal neovascularization induced by placing three interrupted sutures in the host cornea. All of the recipients were divided in blinded fashion into four groups. Group I was injected with saline buffer. Group II was injected intraperitoneally with 0.2 mL SEB (75 microg/kg) at 4-day intervals on three occasions before transplantation. Group III was injected with 0.1 mL IL-1ra (1 mg/mL) subconjunctivally from the first day after transplantation for 2 weeks. Group IV received both SEB and IL-1ra. All transplants were evaluated for signs of rejection for 4 weeks after surgery. Ten days after transplantation, two recipients in each group were sacrificed for histopathological and immunological evaluation. RESULTS: The mean survival time of the allografts in the control group was 5.89 +/- 0.79 days; in SEB group, 10.70 +/- 2.52 days; in IL-1ra group, 8.25 +/- 0.71 days; in the SEB and IL-1ra group, 17.36 +/- 2.39 days. CD4+ and CD8+ lymphocyte infiltration into the allografts and the percentage of the lymphocytes in the spleen and mandibular lymphatic nodes was significantly decreased among the treated groups with dampened lymphocyte reactivity. The SEB plus IL-1ra combination group showed the strongest inhibition. CONCLUSION: SEB and IL-1ra are most effective in combination to treat high-risk corneal transplants.     

Use of allochimeric proteins to mitigate graft-versus-host and host-versus-graft immune responses to rat small bowel allografts

BACKGROUND: We aimed to identify the polymorphic epitopes that mitigate graft-versus-host disease (GvHD) and host-versus-graft response (HvGR) toward rat small bowel allografts in rats. METHODS: We tailored class I major histocompatibility complex (MHC) allochimeric antigens encoding 10 al-helical (alpha(1h)l58-80-RT1.Aa) or 4 (alpha(1h)l/u62-69-RT1.Aa) polymorphic amino acids. In the GvHD model, ACI (RT1a) donors were pretreated (day -14) with an intrathymic injection of alpha(1h)l58-80-RT1.Aa, alpha(1h)l/u62-69-RT1.Aa, or RT1.Al protein, with or without simultaneous intravenous injection of anti-T-cell receptor R73 monoclonal antibodies. Wistar-Furth (WF; RT1u) donors were tested with a similar protocol. In the HvGR model, ACI recipients were treated with a protocol designed to induce transplantation tolerance toward WF heart allografts: a portal vein injection of alpha(1h)l/u62-69-RT1.Aa protein and cyclosporine (4 mg/kg, intramuscular; days 0-6). RESULTS: GvHD was prevented in all (ACI x LEW) F1 recipients (RT1a/l) by pretreating ACI donors with R73 monoclonal antibody and recipient RT1.Al or alpha(1h)l58-80-RT1.Aa protein. Similarly, pretreatment of WF donors with RT1.Aa protein also prevented GvHD in (ACI x WF) F1 recipients. However, in a combined GvHD/HvGR model, ACI recipient perioperative treatment designed to prevent HvGR only modestly prolonged WF small bowel allograft survival (27.7+/-5.3 days compared to 17.4+/-4.6 days in the cyclosporine-alone group). In contrast, application of the two protocols significantly prolonged WF allograft survival (55.6+/-34.6 days), with two of seven recipients surviving more than 100 days. CONCLUSION: Simultaneous inhibition of GvHD and HvGR significantly prolongs small bowel allograft survival.     

Long-term survival of orthotopic bowel allografts in the rat treated with short-term low-dose cyclosporine

Orthotopic bowel transplantation continues to be a "difficult" procedure, both experimentally and clinically, despite the advent of cyclosporine. Even with high-dosage CsA administration, long-term survival has been sporadic and short-term mortality high. In the present study, a low-dosage CsA regimen (5 mg/kg/day for 2 weeks postoperatively) produced prolonged survival in BN-Lewis donor/recipient combinations utilizing an orthotopic bowel transplantation model with portal venous anastomosis. The transplanted bowel continued to survive despite the lack of maintenance dosage after initial induction. The recipient animals evinced donor-specific hyporesponsiveness as donor skin grafts were rejected in a slow, indolent fashion. Third-party skin grafts acutely rejected in normal fashion. It was remarkable that the bowel grafts continued to survive in good condition despite the rejection of donor-specific and nonspecific challenging skin grafts placed on the animals. The precise mechanism of this donor-specific hyporesponsiveness is not known.     

Ability of 3M KC1-extracted histocompatibility antigen to potentiate the immunosuppressive effect of cyclosporine to prolong the survival of heterotopic rat cardiac allografts

A soluble histocompatibility antigen preparation (HAg) derived by 3M KCl extraction of donor spleen cells has been shown to prolong rat renal allografts in CsA-treated hosts. In the present study, the effect of combined soluble antigen-CsA treatment on cardiac allograft survival was studied in WFu hosts grafted with BUF hearts. Cardiac allograft survival was prolonged in WFu recipients treated with both BUF HAg and CsA compared with survival time in untreated controls or controls treated with HAg or CsA alone. In addition, experiments were performed to test the antigenicity of the HAg extract. BUF extract given sc to WFu hosts before grafting specifically sensitized the hosts to BUF grafts, as shown by the accelerated rejection of BUF grafts but not third-party grafts. Assays to determine the major histocompatibility antigenic determinants present in the extract showed that class I and class II determinants were present.     

Portal versus systemic drainage of small bowel allografts: comparative assessment of survival,function, rejection,and bacterial translocation

BACKGROUND: Portal venous drainage of small bowel grafts is theoretically more physiologic than systemic drainage, but is technically more demanding. Comparisons in animal models have not demonstrated a clear advantage of one technique over the other, but clinical data are lacking. STUDY DESIGN: Clinical records of 36 patients who underwent 37 small bowel transplantation procedures from January 1995 to August 2001 were reviewed. Portal drainage was performed in 19 patients (PD group). Systemic drainage was performed in 18 patients (SD group). Median followup was 531 days. RESULTS: PD and SD patients had similar ICU stays (median 7 versus 9 days) and endotracheal intubation durations (median 3 versus 5 days). All current survivors, with the exception of one patient in each group, are independent from parenteral nutrition. Liver function tests were similar in both groups. There was a twofold increase in tacrolimus dosage in the PD group to achieve similar trough levels indicating a "first-pass" hepatic clearance effect. Cumulative incidence of acute rejection episodes and OKT3-requiring rejection episodes were similar in both groups. To the contrary, a lower incidence of gram-negative rods of Enterococcus sp. in blood or bronchoalveolar lavage suggested that the clearance of translocated intestinal bacteria was more efficient in the PD group. Graft and patient survival rates were similar in both groups. CONCLUSIONS: Systemic venous drainage of small bowel transplants is a dependable technique, associated with similar results as portal venous drainage, in terms of overall mortality, morbidity, rejection, function, and patient and graft survival. But attention should be paid to an impaired clearance of intestinal bacterial translocation after systemic drainage.     

Rapamycin, a potent immunosuppressive drug for vascularized heart, kidney, and small bowel transplantation in the rat

The effectiveness of rapamycin (RAPA) was examined for heart, kidney, and small bowel allografts in rats. Untreated or vehicle only-infused Wistar Furth (RT1u) recipients rejected Buffalo (RT1b) heart allografts within a mean survival time (MST) of 6.5 +/- 0.5 and 6.3 +/- 0.5 days, respectively. In contrast, a 14-day continuous intravenous (i.v.) infusion by an osmotic pump of 0.08 mg/kg/day RAPA to WFu recipients prolonged BUF heart allograft survival to an MST of 34.4 +/- 12.1 days (P = 0.0001). There was a graded dose-response to 0.16 mg/kg (39.0 +/- 8.7 days; P = 0.0001), 0.32 mg/kg (55.7 +/- 3.3 days; P = 0.0001) and 0.8 mg/kg (48.0 +/- 3.6; P = 0.0001). Furthermore, intraarterial/intragraft but not i.v. infusion of 0.02 mg/kg/day prolonged BUF heart allografts--namely, an MST of 14.6 +/- 1.4 days versus 8.6 +/- 2.6 days (P = 0.0001), respectively. Local delivery doses of RAPA were about as effective as the same dose delivered i.v.: 0.08 mg/kg MST 37.0 +/- 18.3 days (P = 0.0001); 0.32 mg/kg, 40.0 +/- 3.9 days (P = 0.0001); and 0.8 mg/kg, 54.8 +/- 8.2 days (P = 0.0001). Systemic i.v. RAPA therapy with 0.08 or 0.8 mg/kg/day prolonged the survival of BUF kidney grafts in WFu recipients--namely, an MST of 52.7 +/- 42.7 (NS) and 90.2 +/- 62.4 (P = 0.001) days, respectively, versus an MST of 11.6 +/- 1.5 days in control WFu recipients only infused with vehicle. While normal WFu rats reject heterotopic BUF small bowel allografts within an MST of 10.0 days, a 14-day course of i.v. RAPA treatment significantly (P = 0.0001) prolonged small bowel allograft survival to an MST of 26.8 +/- 3.7 days.     

Co-transplantation of donor-derived hepatocytes induces long-term tolerance to cardiac allografts in a rat model

BACKGROUND: Liver allografts transplanted between MHC-disparate mice, rats, and swine are spontaneously accepted in most strain combinations without requirement for immunosuppression. The underlying mechanism has, however, remained elusive. Here, we demonstrate that co-transplantation of donor-derived hepatocytes protect Lewis (RT1.A1) cardiac allografts from acute and chronic rejection in DA (RT1.Aa) recipients indefinitely. METHODS: Livers of donor Lewis rats were harvested and the hepatocytes separated from hepatic leukocytes by collagenase digestion and gradient separation. DA recipient animals were transplanted Lewis cardiac allografts and simultaneously intraportally infused either Lewis-derived hepatocytes or hepatic leukocytes. Recipient animals were either not further treated or received a single dose of 15 mg/kg cyclosporine. RESULTS: Donor hepatocytes alone significantly protected syngeneic cardiac allografts from rejection, whereas hepatic leukocytes failed to influence graft survival. In combination with cyclosporine, recipient cardiac allografts were indefinitely protected from rejection. Graft-infiltrating cells in tolerant animals presented as clusters of CD4+ T cells and stained mostly positive for interleukin-4, whereas graft-infiltrating cells in rejected allografts were predominantly positive for interferon-gamma. Adoptive transfer of splenocytes derived from tolerant animals protected Lewis cardiac allografts from rejection in DA recipients without immunosuppression. In contrast, hepatic leukocytes protected only 50% of the allografts from rejection. CONCLUSION: We propose that donor hepatocytes induce permanent engraftment of syngeneic allografts by establishing a Th2 type alloresponse that is transferable to new graft recipients. The results of this study demonstrate that liver parenchymal cells significantly mediate spontaneously liver-induced tolerance.