Requirement for both MHC and non-MHC antigens residing on the same erythrocyte for donor erythrocyte-mediated prolongation of rat renal allograft survival

Transfusions of highly purified LEW erythrocytes (E) administered to BN recipients prior to insertion of LEW kidneys markedly prolonged the survival of these allografts (greater than 35 days). Administration of E from syngeneic (BN), third-party (PVG), and MHC-congenic LEW.1N or BN.1L rats did not improve LEW kidney graft survival to the same extent (less than 14 days). BN.1L E were shown to carry at least the same quantity of LEW MHC antigens on their surface as LEW E, thus the failure to prolong LEW kidney graft survival is due to the absence of LEW non-MHC antigens from BN.1L E. Attempts to substitute for this deficiency by mixing LEW.1N E to BN.1L E prior to transfusion failed to restore the beneficial effect, demonstrating that donor E-mediated prolonged renal allograft survival requires the presence of both MHC and non-MHC alloantigens on the same E.     

Studies of the induction and maintenance of long-term graft acceptance by treatment with FK506 in heterotopic cardiac allotransplantation in rats

Immunosuppressive activities of the newly discovered FK506, isolated from Streptomyces tsukubaensis, were examined by using cardiac allotransplantation in the rat, and the mechanisms underlying induction and maintenance of FK506-induced long-term allograft survival were studied. Male rats of WKA (RT1k) and F344 (RT1lvl) strains were used as recipients and donors, respectively, and those of BN (RT1n) strain were used as third-party donors. Treatment with FK506, beginning from the day of allografting for 14, 10, or as few as 4 days, prolonged allograft survival significantly across the major histocompatibility barrier. The minimum doses for prolonging graft survival were 0.1 mg/kg/day by intramuscular treatment and 1.0 mg/kg/day by oral treatment. Treatment with FK506 at a dose of 0.32 mg/kg/day from day 4 until day 10 resulted in all the grafts surviving indefinitely and from days 5 to 10, half the grafts survived indefinitely, suggesting that the agent inhibited ongoing rejection. On the other hand, cyclosporine treatment at a dose of 20 mg/kg/day from day 2 did not prolong graft survival time statistically significantly. Induction of prolonged graft survival was not obtained by pretreatment of the prospective donor or recipient; prolonging effects were observed only when the agent was administered after allografting. Thus, the primary effect of the agent is exerted on responder lymphocytes reacting to the donor antigens in the induction phase of long-term graft acceptance. The mechanisms underlying the maintenance of long-term grafts were analyzed by testing the capacity of lymphocytes or serum of long-term graft-bearing rats to inhibit graft rejection in irradiated grafted hosts. Transfer of 2 x 10(8) lymphocytes from FK506-induced long-term F344 graft-bearing WKA rats resulted in indefinite survival of F344 heart allografts, but it did not prolong survival of third-party BN hearts. Transfer of 2.5 ml serum from long-term graft-bearing rats also prolonged graft survival of F344 hearts, but not BN hearts. These results suggest that donor strain-specific suppressor cells and humoral factor(s) are induced by treatment with FK506 in the presence of allografts, and that they play at least partial roles in the maintenance of long-term allograft acceptance.     

Long-term survival of cardiac allografts in lethally irradiated rats repopulated with host-type hemopoietic cells.

To test the hypothesis that hemopoietic cells within a tissue graft are responsible for its immunogenicity, two experimental protocols were followed. LEW hearts were grafted into (LEW X BN)F-1 host rats and LEW or F-1 lymphocytes were injected into the apex of the grafted heart. The LEW but not the F-1 cells induced a local reaction, apparently because the circulating F-1 cells were the necessary immunogens. The second protocol took advantage of the knowledge that lethally irradiated LEW rats were able to reject WF Ag-B-incompatible hemopoietic cells (but not tissue allografts) within a few days. LEW rats were lethally irradiated and grafted with WF hearts on day 0. A mixture of LEW marrow, thymus, spleen and lymph node cells, or marrow cells only were infused either on day 0 or day 2. Cardiac allografts in hosts repopulated with the mixture of lymphoid cells survived a mean of 11.3 days in hosts infused on day 0, but survived indefinitely if the lymphoid cells were infused on day 2. The 2-day interval also prolonged the survival of allografts in rats infused with only marrow cells. The long-term recipients, without any further treatment, rejected WF skin grafts as first-set reactions 1 year later but did not reject second WF cardiac allografts. Lymphoid cells from long-term recipients imparied the rejection of WF cardiac allografts by LEW host rats. The lack of rejection of the original cardiac allograft supported the hypothesis tested. Certain hemopoietic cells responsible for the immunogenicity of cardiac allografts were probably eliminated in the 2-day interval at least in part by host effector cells capable of rejecting allogeneic hemopoietic cells. However, the mechanism of long-term "unresponsiveness" to WF hearts could have been caused by loss of accessory cells during the 2-day interval followed by infusion of immunocompetent cells. Skin rejections in these recipients may have been attributable to reactions against skin differentiation-specific antigens.     

Induction of stable chimerism and transplantation tolerance to rat islet and heart allografts by ultraviolet-B modulation of bone marrow cells.

Ultraviolet-B irradiation (UV-B) (700 J/m2) of BM cells prior to transplantation into lethally gamma-irradiated (1050 rads) allogeneic rats prevents the development of GVHD and results in stable chimerism. This study was developed to determine if UV-B modulation of BMT is useful for preconditioning recipients for the induction of tolerance to donor islets and heart allografts. Lethally irradiated Lewis rats that received UV-B irradiated (700 J/m2) WF BMT (10(8) BM cells) demonstrated stable chimerism without any evidence of GVHD. The stable Lewis chimeras were made diabetic with streptozotocin (STZ) at 28-35 days after BMT and subdivided into 3 experimental groups that received 1000-1200 islets from WF, Lewis, or BN (third-party), respectively. The results showed that group I diabetic Lewis chimeras accepted permanently (greater than 300 days) BM donor WF islets and became normoglycemic. When 3 of 6 Lewis chimeras transplanted with WF islets were rechallenged with WF hearts 60 days after islet grafts, they accepted both islets and cardiac allografts permanently (greater than 240 days). Similarly, the remaining 3 animals accepted Lewis cardiac allografts permanently, thus indicating tolerance to both donor and recipient alloantigens. Group II diabetic chimeras accepted permanently (greater than 300 days) recipient (Lewis) islets. In contrast, group III chimeras rejected acutely (7-8 days) third-party (BN) islets. However, when these animals that rejected BN islets and again became diabetic were retransplanted with BM donor-type (WF) islets, they became permanently normoglycemic (greater than 200 days). This finding emphasizes the specificity of the induction of tolerance in this model and the apparent lack of organ-specific sensitization. To define the underlying mechanism of tolerance, in vivo adoptive transfer of 10(8) spleen cells to naive Lewis or WF recipients, obtained from tolerant Lewis chimeras carrying donor islets and heart allografts, showed no prolongation of cardiac allografts in the unmodified syngeneic hosts, thus questioning the role of suppressor mechanisms in the tolerant rats. Furthermore, cells from the tolerant chimeras that showed no mixed lymphocyte reaction (MLR) response to Lewis or; WF alloantigens failed to suppress anti-Lewis and anti-WF MLR-response in coculture MLR. These results suggest that tolerance to donor alloantigens in the UV-B BMT model is most likely due to selective elimination of anti-BM donor helper or effector cell precursors (clonal deletion) rather than induction of suppressor cell activity. This study demonstrates that this relatively simple and effective approach to modulation of T cells in BM treatment may be potentially useful in the induction of tolerance to donor organs.     

Specific unresponsiveness to fully allogeneic kidney allografts in rats induced by procarbazine hydrochloride and antilymphocyte serum

A short course of procarbazine hydrochloride (PCH; 50 mg/kg) and antilymphocyte serum (ALS; 5 ml/kg), administered to Lewis (LEW;RT1(1] rats in the first week following transplantation of Brown Norway (BN;RT1n) kidneys, substantially prolonged allograft survival and induced long-term survival in 62% of the grafts. The two agents acted synergistically, in that neither of them administered alone had much effect. Graft recipients did not produce detectable cytotoxic antibodies and antigen-reactive cells injected i.v. were not diverted to the liver, thus showing that neither antibodies nor immune complexes are likely to mediate the unresponsiveness. Spleen cells from graft-bearing recipients failed to cause graft-versus-host responses (GVHR) in both (LEW X BN)F1 and (LEW X DA)F1 hybrids, but they specifically suppressed the GVHR given by normal syngeneic cells to donor strain (BN) antigens. This suppression was specific because the response against third-party antigens (DA; RT1a) was unaffected. Adoptive transfer of spleen and thymus cells from PCH-ALS-treated LEW rats bearing healthy BN kidneys caused a profound prolongation of BN graft survival in sublethally irradiated LEW recipients. This transfer was specific and mediated by W3/13+ (T) lymphocytes. It is concluded that a limited regimen of PCH and ALS given in the first postoperative week incites the generation of specific suppressor T lymphocytes and that this form of immunosuppression, even without preoperative donor antigen, is an effective way of prolonging kidney allograft survival.     

Down-regulation of the immune response to histocompatibility antigens and prevention of sensitization by skin allografts by orally administered alloantigen.

The effects of oral administration of major histocompatibility antigens on the alloimmune response have not been investigated. Lymphocytes from inbred LEW (RT1u) rats that were pre-fed allogeneic WF (RT1l) splenocytes exhibited significant antigen specific reduction of the mixed lymphocyte response in vitro and delayed-type hypersensitivity response in vivo, when compared with unfed controls. In an accelerated allograft rejection model, LEW rats were presensitized with BN (RT1n) skin allografts 7 days before challenging them with (LEW x BN)F1 or BN vascularized cardiac allografts. While sensitized control animals hyperacutely reject their cardiac allografts within 2 days, animals prefed with BN splenocytes maintained cardiac allograft survival to 7 days, a time similar to that observed in unsensitized control recipients. This phenomenon was antigen-specific, as third-party WF grafts were rejected within 2 days. Immunohistologic examination of cardiac allografts harvested on day 2 from the fed animals had markedly reduced deposition of IgG, IgM, C3, and fibrin. In addition, there were significantly fewer cellular infiltrates of total white blood cells, neutrophils, macrophages, T cells, IL-2 receptor-positive T cells, and mononuclear cells with positive staining for the activation cytokines IL-2 and IFN-g. On day 6 posttransplant, the grafts from fed animals showed immunohistologic changes typical of acute cellular rejection usually seen in unsensitized rejecting controls. Feeding allogeneic splenocytes prevents sensitization by skin grafts and transforms accelerated rejection of vascularized cardiac allografts to an acute form typical of unsensitized recipients. Oral administration of alloantigen provides a novel approach to down-regulate the specific systemic alloimmune response against histocompatibility antigens.     

Effects of portal venous administration with allogenic cells on renal allograft survival in the rat

The effects of administration of donor lymphocytes via portal vein (PV) on capacity of alloreactivity and renal allograft survival were investigated in comparison with those of intra-venous (IV) administration in the rats. Orthotopic renal transplantations were performed from Brown-Norway (BN, RT-In) to Lewis (LEW, RT-11) male rats. Donor lymphocytes were prepared from BN or third party DA(RT-1a) rat spleens and lymph nodes and injected via PV or IV to LEW rats on the day of transplantation (day 0). Untreated LEW hosts rejected BN grafts at 7.8 +/- 0.6 days (n = 10). IV administration of 1 x 10(8) BN cells to LEW rats caused a slight prolongation of BN graft survival to 10.4 +/- 3.1 days (n = 9, p less than 0.05), whereas PV inoculation of the same number of BN cells further prolonged graft survival to 28.9 +/- 9.2 days (n = 9, p less than 0.01). This effect was antigen specific; the administration of 1 x 10(8) third party DA cells via PV to LEW rats did not prolong survival of BN graft (MST = 7.4 +/- 0.8, n = 6). Serum from tolerant recipients had significant antigen specific suppressor effect (70.6%) on the MLR proliferative reaction of LEW responder cells toward donor BN cells, but not third party DA cells. Spleen cells from these recipients did not show any suppressive effect. These results demonstrate that PV administration of donor lymphoid cells to recipients results in rapidly inducible and long-lasting immunologic tolerance specific to donor alloantigen, and that this tolerance is mediated by serum factor induced in hosts, but not by suppressor cells.     

Suppression of antibody response and prolongation of skin graft survival by multiple blood transfusions in the rat

The effect of blood transfusions (BT) on antibody response and skin graft survival was studied in the strongly MHC-incompatible BN and LEW combination. One-to-three BT induced high titer antibodies. Additional BT, however, led to a decrease of antibody titers. After 15 BT the recipients either had no detectable antibodies, or they had very low antibody titers. This suppression of response was shown to be distinct from a simple loss of antibody activity caused by lack of further antigenic challenge. In multiple transfused rats, humoral nonreactivity persisted in spite of rechallenge with antigen; in animals that lost their antibodies as a result of lack of further stimulation, an additional BT boosted strong antibody production. In LEW recipients of multiple BN transfusions, not only the specific anti-BN response but also reactivity to third-party BUF blood was suppressed. However, whereas the donor-specific response (anti-BN) was largely inhibited after a ten-week interval, the response to third-party BUF blood recovered. The state of humoral nonreactivity could be transferred by spleen cells to nontransfused syngeneic animals. In LEW rats that received three injections of 5 X 10(7) "suppressor" spleen cells, the antibody response to BN blood was strongly impaired as compared with animals that received normal spleen cells. BN or (BN X LEW)F1 skin grafts survived significantly better in multiple transfused LEW rats than in nontransfused controls. This was even more pronounced when ALS was given additionally. Third-party grafts (BUF) survived only slightly better than controls. It is concluded that multiple BT (1) result in humoral anti-donor nonreactivity secondary to an initial antibody response, (2) induce strong specific and weak nonspecific suppressor cell activity, and (3) increase skin graft survival.     

Efficacy of Perioperative Portal Venous Inoculation with Donor Lymphocytes on Skin Graft Survivals in the Rat

Although the administration of donor lymphocytes via portal vein (PV) on the day of transplantation significantly prolongs rat renal allograft survivals and the unresponsive state is mediated by an antigen-specific suppressor factor in the serum, significant variations exist among rodent models in terms of immunogenecity and mechanism of antigen presentation. The present studies sought to assess the effect of perioperative PV inoculation with donor lymphocytes on skin allograft survivals. Donor lymphocytes were prepared from Brown-Norway (BN, RT-1^a) or third-party DA (RT-1^a) rat spleens and lymph nodes and injected via PV or intravenously to Lewis (LEW, RT-1¹) hosts on the day of skin grafting. Untreated LEW hosts rejected BN skin grafts at 9.0 ± 1.4 days (n= 10). Intravenous administration of 1 ± 10⁸ BN cells into LEW hosts on day 0 did not prolong the skin graft survivals (MST= 8.6 ± 1.2 days, n= 7, NS), whereas PV inoculation of 1 × 10⁸ BN cells prolonged skin graft survival to 13.4 ± 3.9 (n= 8, P <. 01). PV administration of 1 × 10⁸ DA cells to LEW hosts did not prolong the survival of BN skin grafts (MST= 8.6 ± 1.5 days, n= 6). PV inoculation with BN cells inhibited the generation of anti-BN delayed-type hypersensitivity (DTH) response in the hosts, whereas untreated control hosts or hosts inoculated with third-party DA cells could not inhibit the anti-BN DTH response. When serum harvested from PV-treated LEW hosts on day 7 was added to MLC, it suppressed the reaction toward donor BN cells by 61%, but not toward third-party DA stimulators (4.8% suppression, NS). Moreover, adoptive transfer of the serum from PV-treated LEW rats into the virgin secondary LEW hosts significantly prolonged the graft survivals of BN kidney from 7.8 to 16.3 ±1.1 days (n= 6, P ≤. 01), but not third-party DA renal graft (MST= 7.7 ± 0.5, n= 8, NS) or BN skin graft (MST= 9.8 ± 1.2 days, n= 5, NS). These studies suggest that the suppressor mechanism demonstrated in the PV-treated renal transplant model might be similarly active in the hosts who received skin grafts simultaneously with the PV inoculation of donor cells.     

Assessment of peripheral tolerance in anti-CD4 treated C57BL/6 mouse heart transplants recipients.

The study was designed to compare second heart and skin grafts and in vitro assays as a means of assessing peripheral tolerance in C57BL/6 mice. Vascularized heterotopic BALB/c hearts were placed in C57BL/6 recipients treated with anti-CD4, GK1.5 (1 mg total per 20 g mouse i.p. on days 0, 1, 2, 3). Those mice in which hearts survived for >60 days were challenged with donor and third-party (CBA) skin grafts or with second heart grafts, of donor or third-party origin, attached to the carotid artery and jugular vein. In vitro alloreactivity was assessed by mixed lymphocyte reactions (MLR) and cell mediated lympholysis (CML) using recipient spleen cells. Parenchymal damage, cellular infiltration and vascular disease were assessed from the histology of long-term allografts and isografts. Allografts in untreated recipients were rapidly rejected while isografts survived > 100 days. Primary allografts in anti-CD4 treated recipients also survived > 100 days, as did donor strain secondary heart transplants given at >60 days after the first graft. Third-party hearts were rapidly rejected, as were donor and third-party skin grafts placed on recipients with long-term allografts. These recipients showed low MLR response to both donor and third-party stimulators and donor-specific suppression of CML at 60 days post graft. Long-surviving heart allografts all showed evidence of parenchymal damage and vascular intimal thickening. Thus in the BALB/c to C57BL/6 donor-recipient strain combination, hearts, but not skin grafts, could be used to demonstrate peripheral tolerance, which seemed to be both organ and major histocompatibility complex (MHC) specific. Despite long survival, BALB/c hearts all showed evidence of parenchymal damage and vascular intimal thickening, a sign of chronic rejection.     

Development of donor-specific immunoregulatory T-cells after local CTLA4Ig gene transfer to pancreatic allograft

BACKGROUND: CTLA4Ig gene transfer directly to graft tissue might have the potential to avoid the need for systemic immunosuppression. In our previous studies of bio-breeding (BB) rats, local adenovirus-mediated CTLA4Ig gene transfer protected the pancreas from autoimmune and alloimmune responses. This study investigated the potency of local CD28/B7 costimulatory blockade for induction of donor-specific tolerance and further examined the existing mechanisms. METHODS: Brown Norway (BN; RT1)-pancreaticoduodenal grafts transfected with Ad.CTLA4Ig via intraarterial ex vivo perfusion were transplanted into streptozotocin-induced diabetic Lewis (LEW; RT1) rats. RESULTS: Ad.CTLA4Ig transduced grafts combined with a short course of FK506 resulted in indefinitely prolonged survival (>156 days vs. 19.5 days with FK506 alone). CTLA4Ig was predominantly expressed in grafts on day 4. The expression was gradually diminished and was only slightly detectable at day >100. The proliferative responses against BN antigen were remarkably enhanced among recipients with rejected grafts, but the T-cells from tolerant recipients (>100 days) showed poor cytotoxic responses. On adoptive transfer assay, the splenic T-cells of tolerant recipients were able to suppress the rejection of BN, but not third-party Wistar Furth (WF; RT1) hearts in irradiated (480 cGy) LEW recipients. The percentage of CD4CD25 splenic T-cells was significantly increased in tolerant recipients (13.53 +/- 4.06% vs. 6.06 +/- 0.56% in naive rats). CONCLUSION: CTLA4Ig gene transfer to the pancreaticoduodenal allograft combined with a short course of FK506 induces donor-specific tolerance. The mechanism of maintaining tolerance could be explained by development of splenic T suppressor cells.     

Detection of serum-blocking factors by inhibition of allorosette formation in rats with long-surviving renal allografts following short-term postoperative ALS treatment.

Thirty-nine (LEW x BN)F1 kidneys were transplanted to LEW rats. Twenty-four untreated recipients survived for a mean time of 16.1 +/- 1.7 days (group 1). Fifteen recipients received 4 ml of antilymphocytic serum per rat (group 3). In the last group 10 recipients survived for more than 4 months. The spleen cells of these permanently surviving 10 rats were obtained by splenectomy and used in a graft-versus-host assay, and this assay showed that the reactivity of these cells was normal. Following splenectomy the animals were given an (LEW x BN)F1 skin allograft, followed 18 days by a second. After another 18 days (LEW x Buf)F1 "third party" skin allografts were transplanted to the same animals. Animals of group 2 rejected their first grafts with a mean survival time of 12.2 +/- 1.2 days, whereas the second grafts were rejected normally as were the third party grafts. Attempts were made to detect lymphocytotoxic antibodies and haemagglutinins before and after the transplantation of skin grafts and none could be found up to day 53. The sera of group 2 inhibited allorosette formation by 38%. This serum-blocking factor was donor specific. It is probable that the survival of the kidney transplants following antilymphocytic serum treatment was brought about by the development of blocking antibodies.     

Genetics of the blood transfusion effect on heart allografts in rats

Working with the recently available recombinant haplotypes of the rat major histocompatibility complex (MHC)--RT1, we investigated the effect of various types of blood transfusion (BT) on allograft prolongation, including blood identical for the whole RT1 haplotype with that of the donor or for only a part of it. One or two milliliters of donor blood significantly prolonged graft survival in the (LEW X BN)F1----LEW or the LEW X 1W----LEW X 1A combination. The optimal regimen consisted of two BTs given 15 and 7 days prior to grafting; BTs given at day -30 were ineffective. A BT given on the day of the operation was effective, but sequential BTs after grafting did not further increase graft survival. In the (LEW X BN)F1----LEW combination, blood from congenic LEW X 1N rats significantly prolonged graft survival, but third-party BTs were ineffective or had only a borderline effect when transfused (1 ml, 8 times) within the three months before transplantation. This showed the major role of the RT1 system as well as the specificity of the model. Although the survival of LEW X 1A heart grafts transplanted into LEW X 1W recipients could not be significantly prolonged by donor blood, with the reverse--and "weaker"--combination (LEW X 1W----LEW X 1A), 2 ml of donor blood led, in all cases, to greater than 100 days graft survival. In this last combination, third-party BT (LEW X 1N) was again totally ineffective. Blood from RT1-recombinant rats was used to test the role of the respective RT1.A, B, and C regions, in the enhancing effect. BTs from LEW X 1AR2 or LEW X 1WR2 recombinants--sharing, respectively, RT1.C and RT1.A with the graft donor--were only moderately effective, as compared with BTs from the graft donor. On the other hand, LEW X 1WR1 BTs--sharing the RT1.A and RT1.B regions with the graft donor--had a much more powerful effect on heart survival. The results strongly suggest that the RT1.B region (coding for Ia-like antigens) must be shared by the graft and blood donor in order to mediate a significant graft prolongation.     

IgG alloantibody responses to donor-specific blood transfusion in different rat strain combinations as a predictor of renal allograft survival.

Donor-specific blood transfusion prolongs the survival of fully allogeneic ACI (RT1a) renal allografts in PVG (RT1c) recipients from 7-10 days to greater than 100 days. We have observed significant differences in the alloantibody (Ab1) responses to ACI renal allografts in control and DSBT-treated PVG recipients: DSBT is associated with decreased IgG and IgM alloantibody circulating in serum, deposited in the allograft, and produced in culture by splenocytes. In the present studies the effects of DSBT on alloantibody production and renal allograft survival were extended to examine other recipient strains: F344 (RT1lv1), BN (RT1n), W/F (RT1u) and LEW (RT1l). Animals of each recipient strain were injected i.v. with 0.5 ml of ACI blood alone or followed by a renal allograft. Studies on the kinetics of IgM and IgG alloantibody responses were performed by flow cytometry on lymphocytes from donor ACI, PVG, and PVG.R1 (RT1.Aa class I MHC antigen on PVG background) rats. In F344 and PVG rats, DSBT from ACI rats elicited a transient IgM response that peaked at day 7 and was not followed by a switch to IgG. In control PBS transfused F344 recipients, an ACI renal allograft stimulated both IgM and IgG alloantibody production. DSBT pretreatment significantly decreased circulating IgG alloantibody following ACI renal transplantation and prolonged graft survival in F344 recipients. In DSBT-treated F344 recipients that rejected ACI renal allografts acutely, small amounts of IgG (5-12 mode channel shift) were detected in sera harvested 7 days after transplantation, whereas almost no IgG was detected in the sera from DSBT treated F344 rats that accepted their renal allografts indefinitely. In contrast, DSBT alone from ACI to BN, W/F, or LEW strains elicited a transient IgM response that peaked at day 7 and was followed by a strong IgG response that peaked on days 10-14 and remained high through day 21. DSBT failed to prolong ACI renal allograft survival in any of these strains (survival less than 11 days in control and DSBT rats). The alloantibody response to DSBT in all five recipient strains examined was directed primarily to RT1.Aa class I MHC antigens, as determined by binding studies on lymphocytes from ACI, PVG and PVG.R1 rats and alloantibody blocking studies using biotinylated rat monoclonal antibodies to distinct epitopes of the RT1.Aa antigen. The relative magnitude of blocking of R2/10P and R2/15S binding by sera from BN, W/F, and LEW rats was: control allograft recipients greater than DSBT pretreated allograft recipients greater than DSBT alone.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

328. Mechanismen der langzeitakzeptanz allogener rattenlebertransplantate: makrophagenaustausch und suppressorzellaktivierung

Summary After orthotopic rat liver transplantation in the fully allogeneic BN (RT-1n) to LEW (RT-11) combination, the phenomenon of spontaneous tolerance of donor antigen occurs. We demonstrate two different immune mechanisms that may account for this process. Using adoptive transfer assays we show the presence of donor-specific T-suppressor lymphocytes in the spleens of long-term surviving liver graft, recipients. These cells prolong - adoptively transferred into irradiated syngeneic hosts — the survival of donor-specific (BN) but not third-party (DA) renal allografts (I00 days vs 1I days in control groups). Secondly, we demonstrate the replacement of Kupffer cells in the graft by recipient macrophages using polymorphic monoclonal antibodies in an immunoperoxidase technique. This may contribute to graft adaptation and thus to long-term graft acceptance.     

FK506 suppression of heart and liver allograft rejection. II: The induction of graft acceptance in rats.

Lewis recipients of orthotopic ACI livers had permanent graft acceptance induced with 3 doses of i.m. FK506 in the early postoperative period. They were studied 100 and 300 days posttransplantation. The recipients rejected ACI as well as Brown Norway (BN) (third-party) skin grafts, and had lymphocytes with substantial reactivity by mixed lymphocyte culture testing against ACI and third-party (BN) alloantigens. Lymphocyte subset redistribution had not occurred in the peripheral blood or spleens of these animals, and there was no evidence of suppressor cell activation by in vitro and in vivo tests. Graft-versus-host reactivity in splenic lymphoid tissues of these recipients was demonstrated with the popliteal lymph node assay. Attempts at adaptive transfer with recipient lymphocytes were unsuccessful. Heart graft acceptance was far more difficult to accomplish than liver graft acceptance, and probably was never permanent. ACI heart graft prolongation in LEW recipients after a brief induction with FK506 lasted for no more than 3 months in most animals. The temporary heart graft acceptance was specific for hearts of the original ACI donor strain but not for ACI skin. Results of studies of lymphocyte subsets and suppressor cell activity were similar to those in the liver recipients. These studies illustrate how poorly graft acceptance is understood and how badly further work is needed to clarify its mechanism.     

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.     

Cyclosporine and skin allografts for the treatment of thermal injury. I. Extensive graft survival with low-level long-term administration and prolongation in a rat burn model

The hypothesis tested in the present and accompanying study is that an effective treatment for severe burns involves early excision of necrotic tissue followed by skin allografting and cyclosporine (CsA) immunosuppressive therapy. LEW (RT1) rats served as recipients of thermal injury and/or skin allografts. BN x LEW F1 (LBN, RT1(l+n)) rats served as skin donors. LEW burn recipients received a hot water (90 degrees C for 10 sec) 30% body surface area (BSA) full-thickness burn. As expected, LEW recipients treated with CsA (25 mg/kg/day for 20 days) demonstrated significant graft prolongation compared with controls (P less than 0.005). Skin graft survival was similarly prolonged in LEW recipients undergoing burn injury, primary wound excision, and CsA administration compared with burn-skin allograft controls (P less than 0.001). Mortality was not increased in the thermal injury-CsA-treated recipients compared with burn controls. A final experiment was initiated to investigate how low-level long-term (greater than 100 days) maintenance CsA treatment influenced skin allograft survival for possible future consideration in burn trauma. Recipients receiving skin allografts plus CsA (20 days, 8mg/kg/day, followed by every other day thereafter) did not reject their grafts. However, a possible early sign of rejection (a single small ulcerative lesion) was noted in five of these long-term CsA-treated animals at a mean of 34 +/- 11 (SD) days. The lesion in these animals did not progress any further during CsA administration. Histopathologic study of selected animals removed from the CsA maintenance regimen for greater than 50 days following long-term administration revealed a number of interesting chronic lesions similar to those previously reported in the skin component of composite tissue (limb) allografts following long-term low-level CsA intervention. In conclusion, CsA was very successful in preventing rejection of skin allografts in a rat burn model without apparent adverse effects.     

The role of T suppressor cells in the maintenance of spontaneously accepted orthotopic rat liver allografts.

Orthotopic liver allografts from BN donors to LEW recipients are spontaneously accepted, and the recipients develop donor-specific immunological unresponsiveness. This unresponsiveness may be mediated by suppressor T cells. Immunomagnetically purified splenic T cells from LEW rats bearing BN liver grafts were shown to adoptively transfer suppression of skin, heart, and kidney graft rejection in a donor-specific manner, prolonging the survival of BN but not third-party DA grafts. However, the suppressor T cells were sessile, being resident in the spleen but not present in thoracic duct lymph. The presence of a nonrecirculating suppressor T cell in rats spontaneously accepting liver transplants is strongly suggestive of an important function in the maintenance of donor-specific unresponsiveness, although the contribution of other possible mechanisms of unresponsiveness has not been investigated.