Small Bowel Allograft Survival Research Articles

Histologic Analysis of Chronic Rejection in Small Bowel Transplantation

Chronic rejection is a significant barrier to small bowel allograft survival. Although chronic rejection primarily involves vessels of the submucosa, serosa, and mesentery, some mucosal alterations have been suggested to be correlative. We retrospectively investigated explanted small bowel allografts for clinical characteristics and histological alterations in the mucosa, submucosa, and serosa. Crypt epithelial mucin loss, submucosal fibrosis, and length of time to explant were all statistically associated with chronic rejection. Medium-sized and large-sized vessels of the serosa and mesentery preferentially demonstrated histologic changes of chronic rejection. These results further define chronic vascular rejection and the relationship between the mucosal changes and chronic rejection.

Prolongation of Rat Intestinal Allograft Survival by Administration of Triptolide-Modified Donor Bone Marrow-Derived Dendritic Cells

Severe graft rejection remains an important obstacle in intestinal transplantation. In this study, dendritic cells (DCs) isolated from rat bone marrow were cultured for 5 days, and triptolide applied for 3 more days. The recipient rats were pretreated with donor triptolide-modified or not modified DC. Small bowel transplantation was performed to observed survival times. We demonstrated that triptolide markedly inhibited both the expression of CD80 and MHCII expression on DCs. Triptolide-modified DCs stimulated lower proliferative responses among allogeneic T cells, prolonging the survival of intestinal allografts in rats. These results suggested that pretreatment with triptolide-modified DC prolonged the survival of rat small bowel allografts after transplantation.

Effect of FTY720 and Ex Vivo Graft Irradiation in Rat Small Bowel Transplantation: Apoptosis of Crypt Cells and Lymphocytes

Objective We investigated the extent of apoptosis in crypt cells and Peyer’s patches (PPs) during small bowel allograft rejection in rats to examine the effect of FTY720 and ex vivo graft irradiation during rejection. Materials and Methods Orthotopic small bowel transplantations (SBT) were performed from Brown Norway (BN) rats to Lewis (LEW) rats. Four groups of SBT animals were studied on days 3, 5, and 7 after operations: untreated allograft, allograft with FTY720, allograft with irradiation, and allograft with FTY720 + irradiation. Cryostat sections were prepared from the grafts, including PPs. An in situ end-labeling (ISEL) technique was used to detect apoptotic cells. Indirect immunoperoxidase staining was also performed using monoclonal antibodies against rat Fas/FasL. Results The graft survival was prolonged in the FTY720-treated groups. In the FTY720-treated group, the number of ISEL-positive enterocytes was significantly down-regulated on days 3, 5, and 7 compared with the untreated allograft group. The number of ISEL-positive mononuclear cells was also significantly down-regulated compared with the untreated allograft group. The FTY720 the radiation and the FTY720 + irradiation treated groups showed significantly down-regulated numbers of Fas/FasL-positive enterocytes on day 7 compared with the untreated allograft group. Fas/FasL-positive mononuclear cells were also significantly down-regulated in the allograft compared with the untreated allograft group. Conclusions FTY720 and ex vivo graft irradiation prevented up-regulation of the number of apoptotic enterocytes, lymphocytes, and Fas/FasL-positive lymphocytes, and also prolonged small bowel allograft survival. Combination FTY720 and ex vivo graft irradiation did not affect graft survival and apoptotic cell expression compared with the FTY720 only group. These findings suggest that FTY720 may prevent both rejection-associated and sepsis-induced apoptosis during the late phase of small bowel graft rejection.

Effect of FTY720 in Rat Small Bowel Transplantation: Apoptosis of Crypt Cells and Lymphocytes in Gut-Associated Lymphoid Tissues

Aim We investigated the extent of apoptosis in crypt cells and Peyer’s patches (PPs) during small bowel allograft rejection in rats to examine the effect of FTY720 during rejection. Methods Orthotopic small bowel transplantations (SBTs) were performed from BN to LEW rats. Isografted animals served as controls. Three groups of SBT animals were studied on days 3, 5, and 7 after operation: isograft, untreated allograft, allograft with FTY720. FTY720 was orally administered by gavage (1 mg/kg/d) to allograft recipients on 7 consecutive days. Cryostat sections were prepared from grafts, including PPs. An in situ end-labeling (ISEL) technique was used to detect apoptotic cells. Indirect immunoperoxidase staining was also performed using monoclonal antibodies against rat Fas/Fas-L. Results Graft survival was prolonged in the FTY720-treated group. The number of ISEL-positive enterocytes in the allografts increased significantly on days 3, 5, and 7 compared with the isograft group. In the FTY720-treated group, the number of ISEL-positive enterocytes in the allografts was down-regulated significantly on days 3, 5, and 7 compared with untreated allograft group. In the PPs, the number of ISEL-positive mononuclear cells increased significantly in the allografts compared with the isograft group. In the FTY720-treated groups, the number of ISEL-positive mononuclear cells were down-regulated significantly in the allografts compared with the untreated allograft group. The number of Fas/FasL-positive enterocytes were increased significantly in allografts compared with isograft group. In FTY720-treated groups, the number of Fas/FasL-positive enterocytes were down-regulated significantly on day 7 compared with the untreated allograft group. In the PPs, Fas/FasL-positive mononuclear cells also increased significantly on day 7 in the allografts compared with isografts. In the FTY720-treated groups, Fas/FasL-positive mononuclear cells were down-regulated significantly in the allografts compared with the untreated allograft group. Conclusions The number of apoptotic enterocytes, lymphocytes, and Fas/FasL-positive lymphocytes increased during small bowel graft rejection. FTY720 prevented up-regulation of the number of apoptotic enterocytes, lymphocytes, and Fas/FasL-positive lymphocytes while also prolonging small bowel allograft survival.

Effect of FTY720 in Rat Small Bowel Transplantation: Expression of Mucosal Addressin Cell Adhesion Molecule-1

Aim Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) mediates the homing of lymphocytes to gut-associated tissues (GALT). We performed a semiquantitative analysis of MAdCAM-1 expression during small bowel graft rejection in rat treated with FTY720. Methods Orthotopic small bowel transplantations (SBT) were performed from BN rats to LEW rats. Isografted animals served as controls. Three groups of SBT animals were studied on days 3, 5, 7 after operations (Isograft, untreated allograft, allograft with FTY720). FTY720 was orally administered by gavage (1 mg/kg/d) to allograft models on 7 consecutive days. Cryostat sections were prepared from grafts, including Peyer’s patches (PPs). Indirect immunoperoxidase staining was performed using mAbs against MAdCAM-1. The degree of vascular endothelial staining on high endothelial venules (HEV) in the PPs was graded from 1 (low levels) to 5 (high levels), and in the vessels of the lamina propia from 1 (faint), to 2 (low at the base of villi), 3 (low to the middle of villi), 4 (high to the middle of villi), to 5 (high to villi tip). Results The graft survival was prolonged in the FTY720-treated group. MAdCAM-1 expression on HEVs in PPs was down-regulated during rejection. In contrast its expression on endothelial cells of vessels in the lamina propria was up-regulated during rejection. In the FTY720-treated groups, MAdCAM-1 expression on HEVs in PPs was up-regulated and its expression on endothelial cells of vessels in the lamina propria was down-regulated compared with untreated allograft group. Conclusions Alteration in MAdCAM-1 expression may be associated with the development of SB graft rejection. The vessels at the base of villi, which are associated with lymphocyte recruitment, may become sites of intestine immune reactivity during the early phase of small bowel allograft rejection. FTY720 was found to prevent the down-regulation of MAdCAM-1 expression on HEVs in PPs and the up-regulation of its expression on endothelial cells of vessels in the lamina propria while also prolonging small bowel allograft survival.

Chimerism induction by nonmyeloablactive preconditioning and bone marrow infusion in rat small bowel transplantation

In our previous work we demonstrated that the use of donor specific bone marrow infusions ( DSBMI ) after small bowel transplantation did not improve the graft survival after a short course of immunossupression. PURPOSE: In the current study, we evaluated whether recipient preconditioning with different regimens of radiation combined with DSBMI may enhance small bowel allograft survival with minimum recipient morbidity. METHODS: Heterotopic small bowel transplantation (SBTx) was performed with Lewis rats as recipients and DA rats as donors, which were immunossupressed with a short course of tacrolimus (FK 506 ) at 1mg/Kg/day for 5 days and distributed in 4 groups: group 1 (n= 4) without both irradiation and DSBMI; Groups 2 (n= 6), 3 (n= 9) and 4 (n= 6) received 100 x 10(6) DSBM cells at the time of the transplant. Groups 3 and 4 were irradiated with 250 and 400 rd respectively. Animals were examined daily for clinical signs of rejection or GVHD. Blood samples were taken weekly for chimeric studies by FC and intestinal biopsies were performed every 2 weeks. RESULTS: Animals in G1 and G2 had minimal rejection at day 15 after SBTx while GVHD was clinically and histologically characterized in G 3 and G 4. Total chimerism and T-cell chimerism was higher in irradiated groups when compared to non-irradiated groups. With exception of G1 and 2 where rejection was the cause of death, all animals in G3 and 4 died of GVHD. CONCLUSION:We concluded that low cytoreductive of irradiation can successfully decrease the graft rejection but not prevent the occurrence of GVHD.

Prolongation of small bowel allograft survival with a sequential therapy consisting of a synthetic MHC class II peptide and temporarily low-dose cyclosporine A

It has been extensively documented the role of the indirect pathway of allorecognition in allograft rejection. However, recent data demonstrate that the manipulation of this pathway could be also sufficient to promote prolongation of allograft survival. In the present study we evaluated the effect of preoperative immunization with the WF-specific MHC class II peptides RT1.D2 and RT1.B2 in combination with low-dose CsA from days 0 to 7 (5 mg/kg/day) and from days 8 to 30 (1 mg/kg/day) after WF small bowel transplantation. Seven days before and on the day of transplantation, LEW recipients were immunized with the two WF MHC class II peptides RT1.B2 and RT1.D2. The CsA monotherapy induced an allograft survival of 49.3 ± 6.1 days. MHC class II peptide immunization had a limited effect on allograft survival for RT1.D2 (47.1 ± 3.8 days) and induced prolongation of allograft survival for RT1.B2 (73.6 ± 34.6 days). This effect seems to be based on the absence or silence of RT1.B2-reactive T cells and rejection seems to be correlated with the presence of RT1.B2-specific T cells in the late phase. Therefore, the combination of RT1.B2 with low-dose CsA shifts the immunological response and protects small bowel allograft rejection.

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

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. 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). 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. Simultaneous inhibition of GvHD and HvGR significantly prolongs small bowel allograft survival.

Effect of anti-CD4 monoclonal antibody administration on rat small bowel allograft survival and circulating leukocyte populations.

This study assessed the effect of an anti-rat CD4 monoclonal antibody (OX38) on heterotopic small bowel allograft rejection. Fully allogeneic small bowel transplants were performed in the PVG-to-DA-rat strain combination. Animals received either i) short course (days -1, 0 and 1) of 1 mg/kg per day OX38, ii) short course of 5 mg/kg per day or iii) extended course (days -2, -1, 0, 1, 2 and twice weekly thereafter) of 1 mg/kg per day. Both the high dose (13 days) and extended low-dose (12 days) courses prolonged graft survival compared to untreated control animals (7 days). The low-dose, short-course treatment had no effect. Similar regimens were given to animals that did not receive transplants and in which peripheral blood CD4+ cell counts fell to between 20 and 55 % of pretreatment levels and 20-30% of binding sites were blocked. In summary, anti-CD4 monoclonal antibody therapy delayed rejection of rat small bowel allografts; however, long-term survival was not achieved.

Extracellular matrix proteins in organ transplantation.

Extracellular matrix proteins in organ transplantation.

Effect of anti-CD4 monoclonal antibody administration on rat small bowel allograft survival and circulating leukocyte populations

Abstract This study assessed the effect of an anti-rat CD4 monoclonal antibody (OX38) on heterotopic small bowel allograft rejection. Fully allogeneic small bowel transplants were performed in the PVG-to-DA-rat strain combination. Animals received either i) short course (days -1, 0 and 1) of 1 mg/kg per day OX38, ii) short course of 5 mg/kg per day or iii) extended course (days -2, -1, 0, 1, 2 and twice weekly thereafter) of 1 mg/kg per day. Both the high dose (13 days) and extended low-dose (12 days) courses prolonged graft survival compared to untreated control animals (7 days). The low-dose, short-course treatment had no effect. Similar regimens were given to animals that did not receive transplants and in which peripheral blood CD4+ cell counts fell to between 20 and 55% of pretreatment levels and 20–30% of binding sites were blocked. In summary, anti-CD4 monoclonal antibody therapy delayed rejection of rat small bowel allografts; however, long-term survival was not achieved.

Effect of anti-LFA-1 monoclonal antibody on rat small bowel allograft survival and circulating leukocyte populations

Anti-LFA-1 monoclonal antibodies (mAb) prolong graft survival in several animal models. This study assessed the effect of an anti-LFA-1 mAb (WT.1) on small bowel allograft rejection, circulating leukocyte subsets and in vivo target cell antigen blockade. Heterotopic small bowel transplantation was performed between PVG donor and DA recipient rats. Transplanted animals received 1 mg/kg per day WT.1 on days −1, 0 (day of transplantation) and 1. Three doses of WT.1 were also administered to a group of untransplanted animals to monitor circulating leukocyte populations and in vivo binding. WT.1 prolonged recipient survival from 7 to 14 days. Peripheral leukocyte counts increased more than twofold, primarily due to marked increases in both CD4 + and CD8 + lymphocytes. Approximately 85% of WT.1 binding sites on lymphocytes and monocytes were blocked/modulated after the course of therapy. WT.1 has marked effects on circulating leukocytes and target cell binding capacities and can affect the survival of rat small bowel transplant recipients.

15AU81, a prostacyclin analog, enhances donor-specific hepatocytes to prolong the survival of rat heart but not small bowel allografts.

Prostacyclin analogs have previously been shown to have not only cytoprotective but also independent immunosuppressive effects. The effect of one such analog, 15AU81, to enhance the immunosuppressive effects of liver was investigated. We have previously demonstrated that cyclosporine (CsA) in conjunction with rapamycin (RAPA) potentiates class I+, class II- donor-specific hepatocytes to prolong rat cardiac and small bowel allograft survival. Brown Norway (BN; RT1n) hepatocytes alone (5 x 10(7)/kg, administered intrasplenically) failed to prolong the survival of BN heart allografts in Wistar Furth (WFu; RT1u) recipients, beyond that of untreated controls (MST = 7.2 +/- 0.8 days). Survival of BN hearts was increased to 11.4 +/- 1.7 days in WFu recipients treated with BN hepatocytes and 50 microg/kg/day 15AU81 administered by continuous s.c. infusion for 14 days using osmotic pumps (p < 0.05). The further addition of RAPA 0.0075 mg/kg/day and CsA 0.375 mg/kg/day delivered for 14 days by continuous i.v. infusion (CIVI) using osmotic pumps (a combination that alone prolonged BN heart allografts in WFu hosts to 18.4 +/- 1.3 days and in conjunction with BN hepatocytes prolonged survival to 27.2 +/- 1.9 days) prolonged allograft survival to 35.2 +/- 5.2 days. In contrast, the survival of small bowel allografts was not enhanced by 15AU81 administration. Survival of BN small bowel transplants in LEW recipients treated with hepatocytes alone (MST = 11.6 +/- 1.5 days) or hepatocytes plus 15AU81 (MST = 10.0 +/- 1.0 days) was similar to controls (MST = 10.2 +/- 1.9 days). Treatment with hepatocytes and RAPA/CsA increased survival to 21.2 +/- 1.5 days. The further addition of 15AU81 failed to augment this (MST = 17.0 +/- 1.9 days). In vitro WFu lymphocyte proliferative responses from animals pretreated with BN hepatocytes, 15AU81, or both treatments, for 2 weeks prior to harvesting, exhibited a reduction of at least 50%, compared to untreated controls upon allostimulation with irradiated BN or ACI spleen cells. These findings demonstrate that 15AU81 interacts favorably with hepatocytes either alone or in conjunction with RAPA and CsA to enhance their immunosuppressive effects on rat heart allograft survival. The failure to enhance small bowel allograft survival may be explained by the inability at this low dosage of 15AU81 to influence the intense graft versus host reaction elicited by small bowel transplants.

Correlation between allograft survival and chimeric state after bone marrow infusion in rat small bowel transplantation.

Methods to enhance natural microchimerism, which occurs after any successful organ transplant, are currently explored using unmodified donor bone marrow both in experimental and in clinical trials. Because of the potential immunomodulatory effects of donor bone marrow cells, we performed this study to evaluate the effect of single and multiple donor-specific bone marrow infusions (DSBMI) on chimerism and small bowel allograft survival in a fully histoincompatible rat model. Forty-five male DA rats and 45 female Lewis rats were used as donors and recipients, respectively, for a heterotopic small bowel transplant. Animals were separated into 10 groups according to the number of bone marrow infusions and immunosuppressive protocol used. Control groups (groups 1 and 2) did not receive any bone marrow infusion, groups 3 and 4 received one infusion at day 0 (150 x 10(6) cells), groups 5 and 6 received two infusions at days 0 and 4 (75 x 10(6) cells each), groups 7 and 8 received two infusions at days 4 and 10 (75 x 10(6) cells each), and groups 9 and 10 received five infusions at days 4, 10, 15, 20 and 25 (30 x 10(6) cells each). Animals in groups 1, 3, 5, 7 and 9 were immunosuppressed with 0.5 mg/kg FK506 while the remaining groups were immunosuppressed with 1 mg/kg FK506, from day 0 to 4 after transplant. Every 15 days, the chimeric state was determined by flow cytometry in order to detect cells expressing DA rat class I antigen, and small bowel biopsies were obtained from ileostomies. Animals in all groups showed minimal to moderate acute rejection at day 15 after transplant, however, vascular rejection (vasculitis, arteritis) was observed in only bone marrow groups (100% in 0.5 mg/kg and 42.1% in 1 mg/kg FK506 groups). On day 30, 58.3% of bone-marrow-infused animals and 66.6% of controls showed severe acute and early chronic rejection. The chimeric levels varied from 0 to 12% after transplant and were significantly higher in bone-marrow-infused groups compared with controls (p < 0.05). We conclude that modulation of immune response with short-course immunosuppression and a single or multiple DSBMI did not improve allograft or recipient survival. The inability to achieve a stable chimeric state did not allow us to determine the effect of chimerism on graft and recipient survival after small bowel transplantation.

Prolonged small bowel allograft survival following repeated intrathymic injections of donor splenocytes and peripheral anti-lymphocyte treatment

Prolonged small bowel allograft survival following repeated intrathymic injections of donor splenocytes and peripheral anti-lymphocyte treatment

A second intrathymic injection of donor splenocytes improves survival of small bowel allografts

A second intrathymic injection of donor splenocytes improves survival of small bowel allografts

Graft survival and MLC response in LEW-ACI small bowel transplants treated with a novel MHC peptide, BC-1nl

SMALL synthetic peptides corresponding to polymorphic regions of the human HLA-B7-01 molecule have non-MHC-restricted immunomodulatory effects both in vitro and in vitro. In vitro, ALLOTRAP inhibits differentiation of CTL and inhibits CTL and natural killer (NK) lysis. In vivo, ALLOTRAP prolongs LEW.1W . LEW.1A heart allograft survival, prolongs LEW . ACI heart allograft survival when used in combination with cyclosporine, and prolongs the survival of C57BL/6 . CBA murine skin allografts. We have observed that ALLOTRAP administered together with anti-lymphocyte serum significantly prolonged porcine islet xenograft survival in streptozocintreated or NOD mice (unpublished observations). We therefore have asked whether there would be any immunomodulatory effects of BC-1nl, a synthetic peptide of the ALLOTRAP family, on survival of LEW small bowel allografts in ACI rats.

Prolongation of rat small bowel allograft survival by CTLA-4 IG

Prolongation of rat small bowel allograft survival by CTLA-4 IG

Anti-CD4 therapy in combined heart-kidney, heart-liver, and heart-small bowel allotransplants in high-responder rats.

In these experiments, we studied the role of anti-CD4 (Ox38) monoclonal antibody in the induction of allograft unresponsiveness in high-responder Lewis rats in the single liver, kidney, small bowel, and heart versus the combined heart-kidney, heart-liver, and heart-small bowel transplantation models. ACI heart, kidney, liver, and small bowel allografts were transplanted into untreated and anti-CD4 treated Lewis rats. In selected animals bearing long-surviving ACI liver or kidney allografts for over 3 months, donor-matched second heart or third-party (Brown Norway) heart allografts were transplanted. Simultaneously, heart-liver, heart-kidney, and heart-small bowel transplants were performed on the day of operation. Rejected allografts were verified by autopsy and pathology. ACI liver allografts were permanently accepted by Lewis recipients treated with either regular-dose (5 mg/kg for 4 days) or low-dose (5 mg/kg for 2 days) of anti-CD4 monoclonal antibody. Pretransplant anti-CD4 therapy (5 mg/kg for 4 days but not 5 mg/kg for 2 days) resulted in a long-term survival of kidney allografts (mean survival time [MST] > 100.0 days, n=5). Pretransplant anti-CD4 treatment (5 mg/kg for 4 days) could not induce tolerance when single ACI hearts were transplanted; however, long-term survival of ACI heart allografts could be induced when heart transplants were combined with liver (n=7) or kidney (n=8) transplants. The survival of both ACI heart allografts (MST=25.0 days, n=4) and small bowel allografts (MST=28.0 days, n=4) was also prolonged when simultaneous heart and small bowel transplantation was performed in anti-CD4-treated recipients. The second ACI heart allograft was permanently accepted by tolerant Lewis recipients of ACI liver or kidney allografts induced by anti-CD4 treatment, and third-party heart grafts were acutely rejected without affecting survival of the primary allografts. Our current results show that: (1) there is a vigorous rejection of heart > or = small bowel > kidney > liver in high-responder Lewis rats after pretransplant anti-CD4 therapy; and (2) simultaneous or metachronous combined liver-heart and kidney-heart transplants may protect heart allografts from rejection.

Intrasplenic Liver Parenchymal Cells in Conjunction with Low-Dose Rapamycin and Cyclosporine Induce a Unique and Specific Prolongation of Rat Cardiac and Small Bowel Allograft Survival

These experiments investigated the immunosuppressive properties of liver tissue. Brown Norway (BN; RT1 n) rat heart allografts survived in untreated control Wistar Furth (WFu; RTl u) rat recipients for 6.2 ± 1.5 days, while allografts in animals that received rapamycin (RAPA) 0.0075 mg/kg/day and cyclosporine (CsA) 0.375 mg/kg/day delivered for 14 days by continuous intravenous infusion (civi) using osmotic pumps in conjunction with intrasplenic (i.s.) saline survived to 18.4 ± 1.3 days. i.s. addition of 3 M-KCl extracted BN hepatic antigen or unpurified BN hepatocytes (liver parenchymal cells—5 × 10 7/kg), which exhibited a 4.8% class II antigen expression, and which alone failed to prolong allograft survival (MST = 6.0 ± 1.4 days), increased heart allograft survival to 25.3 ± 2.3 and 27.2 ± 1.9 days, respectively ( p < 0.01). Hepatocyte purification using Dynabeads and Percoll reduced class II expression to 0.9% and increased allograft survival to 32.8 ± 1.6 days ( p < 0.01). In contrast, the effect of 5 × 10 8/kg BN erythrocytes, exhibiting only 0.1% class II expression, was much less (23.8 ± 1.9 days). Administration i.s. of BN splenocytes or nonparenchymal liver cells, demonstrated by flow cytometry to exhibit a 47.3 or 55.1% expression of class II antigen, respectively, failed to induce any significant increase in allograft survival (18.4 ± 4.6 and 19.4 ± 0.5 days, respectively). Survival of BN rat small bowel allografts was increased in Lewis (LEW; RTl 1) rat recipients treated with RAPA, CsA, and unfractionated BN hepatocytes from 10.2 ± 1.9 to 21.2 ± 1.5 days. Pretreatment with i.s. BN hepatocytes, 14 days prior to harvesting, reduced WFu lymphocyte responses to allogeneic stimulation with BN or ACI spleen cells by 75 and 70%, respectively. Addition of 1 × 10 5 unpurified donor-specific BN or third-party Buffalo (BUF; RTl b) hepatocytes, but not supernatant, to the responder wells of MLCs resulted in a 61 and 40% suppression, respectively, of the WFu lymphocyte response induced by BN allogeneic stimulation. These findings suggest that while class I MHC expression has a significant role to play in exerting the immunosuppressive effects of hepatocytes, other influences more specific to liver may also prevail.