Intrathymic immunomodulation in sensitized rat recipients of cardiac allografts: requirements for allorecognition pathways.

BACKGROUND: Intrathymic injection of alloantigen in the form of donor cells, soluble major histocompatibility complex (MHC) molecules, or MHC allopeptides induces donor-specific tolerance in a variety of acute allograft rejection models. We have previously shown that a single intrathymic injection of donor spleen cells into pre-sensitized rats abrogates accelerated (circa 24-hour) rejection and prolongs the survival of cardiac allografts to about 7 days. The present study was designed to investigate the mechanisms by which intrathymic administration of donor cells modifies the course of accelerated rejection. METHODS: Lewis RT1(1) (LEW) rats sensitized by transplantation with Wistar-Furth RT1(u) (WF) skin grafts received WF cardiac allografts 7 days later-a classic model of accelerated rejection. At the time of skin challenge, however, certain animals received intrathymic cell suspensions (either allogeneic or syngeneic) or donor-derived class I and/or class II MHC peptides. RESULTS: Control animals (sensitized by skin grafts but receiving no other treatment) rejected cardiac allografts within 24 hours. Intrathymic injection of WF splenocytes at the time of skin transplantation abrogated rejection at 24 hours and prolonged cardiac allograft survival to 6.6+/-0.6 days (p<0.001), whereas intrathymic administration of syngeneic (LEW) or allogeneic third party Brown Norway RT1(n) cells was ineffective in this regard. Intrathymic injection of gamma-irradiated donor cells marginally extended cardiac allograft survival to 3.0+/-0.9 days (p< 0.001), but the grafts were still rejected in an accelerated fashion. Intrathymic injection of donor-derived class I and/or class II MHC allopeptides at the same time period also failed to prolong cardiac allograft survival beyond 3 days. In the group receiving unmodified donor cells, elevated immunoglobulin M (IgM) and immunoglobulin G (IgG) allo-antibodies were found at the time of cardiac transplantation; this pattern was not observed with any other treatment. CONCLUSION: The superiority of non-modified donor spleen cells over gamma-irradiated donor cells or donor specific allopeptides in modifying the course of accelerated cardiac rejection suggests that direct allorecognition is the dominant pathway initiating rejection in sensitized transplant recipients. Marked alterations in the antidonor IgM and IgG responses are associated with successful abrogation of accelerated rejection by thymic immunomodulatory mechanisms.     

Cyclosporin A spares selectively lymphocytes with donor-specific suppressor characteristics

The effect of cyclosporin A (Cy A) on the host responses to heart allografts have been examined in rats following administration of the drug for 7 days after grafting. All grafts functioned greater than 100 days without rejection episodes in animals of major histocompatibility differences. Thymic or splenic lymphocytes (1 X 10(8) from LEW recipients of (LEW X BN)F1 hearts were transferred at varying periods into untreated LEW rats transplanted with (LEW X BN)F1 test hearts 24 hr later. Test grafts survived 12 to 16 days significantly (P less than 0.001) longer than in untreated animals (MST +/- SD = 7 +/- 0.3 days). Cells from normal LEW animals, Cy A-treated but ungrafted, and grafted but not treated animals, all failed to prolong test graft survival. Specificity of the effect was tested in vivo, using hearts from donor and third-party rats, and in vitro, using the mixed lymphocyte response (MLR). In vivo, thymocytes from treated LEW recipients of (LEW X WF)F1 grafts failed to prolong (LEW X BN)F1 test grafts; conversely, transferred thymocytes from LEW recipients of LEW X BN)F1 grafts failed to prolong (LEW X WF)F1 grafts. The MLR of lymphocytes from Cy A-treated rats was significantly decreased against donor lymphocytes but not against third-party lymphocytes. Additionally, both cellular and humoral immunity mounted by Cy A-treated recipients was depressed throughout the entire follow-up period. Prolonged heart graft survival after 7 days of Cy A treatment suggests emergence of cells with specific suppressor activity, which in turn may cause profound abrogation of host effector responses against vascularized organ allografts.     

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.     

Acceptance of cardiac allografts by lethally irradiated rats repopulated with syngeneic bone marrow. Role of class I and class II alloantigens

LEW rats (RT1l) that are lethally irradiated and repopulated with syngenic bone marrow accept WF (RT1u) cardiac allografts. If bone marrow repopulation is delayed for two days after irradiation and operation, grafts containing passenger leukocytes (nonperfused grafts) are generally rejected, but perfused grafts, which have fewer passenger leukocytes, are accepted. If bone marrow is given on the same day as irradiation and surgery, then both perfused and nonperfused grafts are accepted. The difference in acceptance of grafts by recipients that are repopulated on day 0 as opposed to day 2 depends on class II alloantigens, because grafts that are similar to LEW for class II antigens are not rejected by day-2-repopulated recipients. Also, the acceptance of totally major histocompatibility complex (MHC)-mismatched cardiac allografts by day-0-repopulated recipients is influenced by a radiation-resistant host cell. Splenectomy of the irradiated and repopulated recipient prevents tolerance induction unless syngeneic irradiated spleen cells are returned to the recipient. Thus class II alloantigen disparities appear to be a major barrier to tolerance induction in the system of total body irradiation and syngeneic bone marrow reconstitution, although proper timing of bone marrow administration can minimize rejection of completely MHC-mismatched grafts.     

Maturing thymocytes in accelerated rejection of cardiac allografts in presensitized rats.

LBNF1 cardiac allografts are rejected within 36 hr in LEW rats sensitized with BN skin grafts 7 days earlier (acute rejection in unmodified hosts = 8 days). We have studied and compared the function and migration patterns of thymocytes one day after engraftment in sensitized recipients, unmodified hosts, and normal naive rats. Thymocytes from animals experiencing accelerated rejection were more mature and functionally active, as shown by a significant elevation in percentage of OX-44+ (CD37+) cells, increased alloreactivity to BN and WF antigens, and proliferative responses to Con A and exogenous IL-2. However, the cells could neither lyse BN targets in vitro nor trigger rejection of otherwise indefinitely functioning test cardiac allografts in immunologically unresponsive T cell-deficient (B) rats after adoptive transfer. The traffic of 111In-labeled thymocytes was then evaluated. The migration index increased significantly during accelerated graft rejection, with thymocytes preferentially circulating in the blood, penetrating peripheral lymph nodes--and, interestingly, migrating back to the thymus. Thus, immunoresponsive and functionally active thymocytes, which lack the ability to recognize primed specific antigen, appear during accelerated rejection of cardiac allografts in sensitized rats. These cells migrate to the periphery, and then return in large numbers to their site of origin, the thymus. Hence, this study describes a novel behavior of thymocytes in the state of host alloreactivity that is distinct from the physiological one in otherwise normal thymus.     

Induction of indefinite survival of fully mismatched cardiac allografts and generation of regulatory cells by sarpogrelate hydrochloride

BACKGROUND: At initiation of the immunologic response, platelets rapidly release chemical mediators such as serotonin (5-hydroxytryptamine, [5-HT]) and cytokines. Sarpogrelate hydrochloride (SH), a selective 5-HT2-receptor antagonist, is used to treat patients with peripheral arterial disease. We investigated the effect of SH on the alloimmune response in a murine cardiac transplantation model. METHODS: CBA mice underwent transplantation of a C57BL/10 heart and received a short course of SH treatment. Survival of the allograft was recorded. An adoptive transfer study was performed to determine whether regulatory cells were generated. Immunohistochemistry studies of intercellular adhesion molecule 1 (ICAM-1), histological, cell-proliferation, and cytokine assessments were performed. RESULTS: Untreated CBA mice rejected C57BL/10 cardiac grafts acutely (median survival time [MST], 8 days). In mice given 10 mg/kg of SH, all allografts survived indefinitely (MST, >100 days); these mice also had significantly prolonged survival of donor-specific skin grafts but acute rejection of third-party skin grafts. Secondary CBA recipients given not only whole but also CD4 splenocytes from primary SH-treated CBA recipients with C57BL/10 cardiac allograft had indefinite survival of C57BL/10 hearts (MST, >100 days). SH inhibited upregulation of ICAM-1 on endothelial cells in the allografts. Graft acceptance and hyporesponsiveness were confirmed by the histological and cell-proliferation studies, respectively. Production of interleukin-4 and interleukin-10 from splenocytes of SH-treated transplant recipients increased compared to that from splenocytes of untreated recipients. CONCLUSION: SH induced indefinite survival of fully allogeneic cardiac allografts, generated CD4 regulatory cells, inhibited ICAM-1 expression in the allografts, and upregulated IL-4 and IL-10 production.     

A novel CD154 monoclonal antibody in acute and chronic rat vascularized cardiac allograft rejection

BACKGROUND: The CD40-CD154 interaction is critically important in the cell-mediated immune responses. Blockade of this costimulatory pathway has been shown to prevent acute allograft rejection in murine, as well as nonhuman primate models. However, the role of the CD40-CD154 pathway in the development of chronic rejection and the effects of CD154 targeting on progression of chronic rejection have not been evaluated. METHODS: We examined the effect of AH.F5, a new hamster anti-rat CD154 monoclonal antibody, in a fully allogeneic acute(u) into Lewis [LEW] (RT11) and chronic [WF.1L (RT1l) into LEW (RT1l)] vascularized cardiac allograft rejection model. In the chronic model, the antibody was evaluated for prevention (starting day of transplant) and interruption of progression (starting day 30 or 60 after transplant) of chronic vasculopathy. Graft survival, morphology, and immunohistology were evaluated. RESULTS: In the acute rejection model, anti-CD154 therapy alone prevented acute allograft rejection and resulted in 50% long-term allograft survival (>200 days) and donor-specific tolerance. In recipients treated with anti-CD154 monoclonal antibody in combination with a short course of cyclosporine, 100% of allografts survived long-term and all recipients achieved donor-specific tolerance. In the chronic rejection model, allografts from animals treated with the anti-CD154 antibody had a statistically significant lower score of graft arteriosclerosis and fibrosis in both the prevention and 30-day interruption groups when compared with control allografts. In addition, immunohistochemistry showed a decrease in intragraft mononuclear cell infiltration and activation. CONCLUSION: A new anti-CD154 antibody not only prevents acute allograft rejection, but also inhibits and interrupts the development of chronic rejection. In the acute rejection model cyclosporine acts synergistically with anti-CD154 therapy to prolong allograft survival and induce tolerance. In the chronic rejection model relatively early initiation of therapy is essential to prevent progression of chronic allograft vasculopathy and fibrosis.     

Donor-specific cellular immunity in rejecting and long-term-surviving class I-disparate rat renal allograft recipients

The effects of pre- and posttransplant immunization on graft survival, infiltrate intensity, and host in situ/systemic cellular immune responsiveness were examined for class I MHC-disparate rat renal allograft recipients. Naive, unsensitized PVG (RT1c) recipients of class I MHC disparate PVG.R1 (RT1.Aa on PVG background) orthotopic kidney transplants displayed long-term (greater than 50 days) survival (LTS) in a majority (41/52) of cases. Pretransplant immunization of recipients with a PVG.R1 skin graft most often resulted in rejection (mean survival greater than 21.3 days) with 8/15 rats surviving less than or equal to 2 weeks and only 3/15 with LTS. Pretransplant immunization with a skin graft from a fully MHC-disparate PVG.1A (RT1a on PVG background) donor resulted in acute rejection (mean = 6.1 days) with 0/8 rats surviving greater than or equal to 2 weeks. Donor-specific class I and II disparate (PVG.1A), and third-party (LEW) skin transplants applied on LTS (greater than 50 days) PVG.R1 kidney graft recipients showed typical 1st- and accelerated 2nd-set skin graft rejection, but had no effect on kidney graft survival. In contrast, 6/7 LTS PVG.R1 kidney graft recipients accepted PVG.R1 skin grafts indefinitely following their transient partial rejection. Histologic analysis of kidney allografts revealed the highest degree of mononuclear cell infiltrates in animals specifically sensitized by PVG.1A skin grafts prior to transplant. Donor class I-specific cytotoxic T lymphocyte precursor (pCTL) frequencies, as determined by limiting dilution assays, were increased and equivalent at 1 week posttransplant in kidney allograft cell eluates from nonrejecting naive recipients (1/127-1/2209) and rejecting presensitized animals (1/470-1/7848). LTS animals had decreased intragraft pCTL at greater than 50 days (1/2969-1/61875), as did LTS at greater than 50 days that received PVG.R1, PVG.1A, or LEW skin grafts posttransplant. In all groups, splenocyte pCTL frequencies were significantly lower than the corresponding values within the allograft. By comparison, no significant differences in intragraft or splenic proliferative T lymphocyte (pPTL) precursor frequencies were observed between any groups. These results indicate that unsensitized recipients of class I-disparate renal cells grafts are capable of maintaining graft survival in the early posttransplant period, despite the presence of significant in situ antidonor class I MHC-specific cellular immune responsiveness. These findings also indicate that long-surviving PVG recipients of class I-disparate renal allografts develop specific functional tolerance to donor class I alloantigens, that may be associated with a diminished frequency of anti-class I cytotoxic (but not proliferative) T cell precursors.     

Further study of anti-ICOS immunotherapy for rat cardiac allograft rejection

PURPOSE: To study the effect of B7-CD28 costimulatory signal blockade by adenovirus-mediated cytotoxic T-lymphocyte-associated antigen 4 immunoglobulin (AdCTLA-4Ig) on cardiac allograft survival in DA (RT1(a)) to LEW (Lewis RT1(l)) rat combinations. METHODS: We evaluated the effect of combined AdCTLA-4Ig and anti-inducible costimulator (ICOS) antibody immunotherapy on rat cardiac allograft acceptance. RESULTS: Unlike AdCTLA-4Ig alone, anti-ICOS immunotherapy combined with AdCTLA-4Ig induced stable tolerance without causing chronic rejection. The combined immunotherapy also prevented the accelerated cardiac rejection caused by donor-type test skin grafting. Immunohistochemical analyses revealed remarkable inflammatory mononuclear cell infiltration with typical vasculopathy, especially ICOS-positive cells in the grafts, in recipients treated with AdCTLA-4Ig alone. In contrast, anti-ICOS therapy combined with AdCTLA-4Ig reduced the ICOS-positive inflammatory cell infiltration of the graft significantly. The most important finding is that possible cardiac arrest caused by secondary donor-type skin graft was prevented by combined immunotherapy of AdCTLA-4Ig and anti-ICOS antibody, despite skin graft rejection. CONCLUSIONS: Our results identified a major role played by the ICOS-ICOSL pathway in chronic and accelerated cardiac allograft rejection, providing a novel approach to preventing the chronic rejection of vascularized organ allografts.     

The role of class I major histocompatibility complex antigens in prolonging the survival of hepatic allografts in the rat

In an attempt to study the role of class I major histocompatibility complex antigens in inducing immunological unresponsiveness, the survival rates of hepatic allografts were compared in rats pretreated with blood taken from various rat strains. A single intravenous injection of 1 ml fresh heparinized whole blood seven days before transplantation significantly prolonged the survival of subsequent donor-specific hepatic allografts in the fully allogeneic ACI(RT1a)-to-LEW(RT1l) rat combination. However, pretreatment with blood taken from the third-party strain BN(RT1n) did not produce suppression of rejection, attesting to the specificity of the pretransplant transfusion effect. Interestingly, pretransplant transfusion of PVG.r1 blood, sharing only the RT1.A MHC region with ACI, significantly prolonged the survival of ACI-to-LEW hepatic allografts. In addition, no lymphocytotoxic antibodies could be detected at 30 or 100 days after transplantation in animals with long-surviving hepatic allografts pretreated with either PVG.r1 or ACI whole blood. On the other hand, pretreatment with PVG(RT1c) blood increased the survival of ACI-to-LEW hepatic allografts only moderately compared with controls. This finding may be consistent with a partial effect of some third-party blood transfusion. The experimental data suggest that the class I MHC antigens can be immunosuppressive in rat hepatic allografts. Adoptive transfer of 5 x 10(7) splenocytes taken from long-term-surviving hepatic allografts pretreated with donor ACI whole blood or PVG.r1 blood into irradiated (750 rads) LEW rats prolonged the survival of donor-type skin grafts, whereas third-party strain (BN) grafts were rejected. This finding suggests the presence of donor-specific suppressor cells.     

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.     

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.     

Experimental vascularized bone allografting

Presented here is a compendium of studies investigating the fate of vascularized bone allografts. The first set of experiments employ the posterior rib graft in two canine models. The rib-to-mandible model was used to evaluate the rejection phenomena of vascularized bone allografts in an outbred dog model. This ascertained the time course of rejection and histological characteristics of the grafts. Immunosuppression of the graft recipients was attempted with azathioprine and cyclosporine. The results demonstrated that azathioprine was not an effective immunosuppressant, whereas cyclosporine resulted in survival of cortical osteons. The use of the vascularized rib allograft, with and without azathioprine, to bridge the defect in the dog femur was met with failure. Further studies employed a genetically defined rat model to determine the effect of different histocompatibilities on the survival of vascularized knee allografts. Grafts were transplanted from Lewis rats to syngeneic Lewis rats as isografts and to Fischer-344 rats (F-344) and Brown-Norway rats (BN) as allografts. Grafts across a major histocompatibility barrier to BN were rejected by 7 days, whereas grafts across a weak histocompatibility barrier to F-344 were rejected more slowly. The use of cyclosporine in this model abrogated the rejection response when administered to both groups continuously. However, a short course of cyclosporine was effective in preventing rejection in the F-344 animals. Efforts to induce tolerance by blood transfusions, from the donor strain or from a third-party donor, were not effective in preventing rejection.     

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)     

Enhanced growth of tumor isografts in rats after skin allografting.

Full-thickness skin grafts from either BN (Ag-B3) or WF (Ag-B2) rats were transplanted to WF recipients of the same sex. Six to seven days after grafting, recipients were challenged with isografts of a chemically induced rat colon carcinoma, NG-W1. In three of four experiments, mean challenge tumor volumes were greater after allografting than after skin isografting. Tumor incidences, however, were no different in rats after skin allograft or isograft placement. When isografts of a polyoma virus-induced sarcoma, P-W13, were used to challenge WF rats after skin grafting, tumor incidence was significantly greater in animals which has received allografts, whether or not they also had been immunized to P-W13 before challenge. Thus, in otherwise untreated inbred rats, grafting of full-thickness skin from donors differing at a major histocompatibility locus led to facilitated growth of solid tumor isografts in animals undergoing allograft rejection.     

Low molecular weight fucan prevents transplant coronaropathy in rat cardiac allograft model

INTRODUCTION: Transplant arteriosclerosis is the main cause of long-term failure after cardiac transplantation. Vascular rejection is thought to be due to intimal proliferation occurring in response to arterial wall immune-mediated injury. A low molecular weight fucan (LMWF) compound, a sulfated polysaccharide, has been demonstrated to increase plasma levels of stromal cell-derived factor 1 (SDF-1) and consequently to mobilize bone marrow-derived vascular progenitor cells (BMVPC). The aim of this study was to evaluate the capacity of LMWF to prevent coronary intimal proliferation in a rat cardiac allograft model. METHODS: Heterotopic abdominal cardiac graftings were performed in Brown Norway (BN) and Lewis (LEW) rats. Animals were divided into 4 groups of 10 rats. Two groups were treated intramuscularly with LMWF (5 mg/kg/day) (one BN to BN isograft group, and one BN to LEW allograft group); and two control groups were LMWF-untreated (one BN to BN isograft group and one BN to LEW allograft group). All animals were treated by cyclosporin (15 mg/kg/day) sub-cutaneously and sacrificed at day 30. The cardiac grafts were assessed by morphometry of structural parameters and by histological and immunohistochemical analyses. RESULTS: All cardiac isografts were devoid of any coronary and parenchymal lesions. In contrast, the majority of untreated allografts developed coronary intimal proliferation in close association with intimal and adventitial inflammatory CD68(+) cell infiltration. Further, the parenchyma exhibited large areas of actin(+) cells (myofibroblasts) of recipient origin colocalized with the CD68(+) infiltrating cells. Interestingly, all LMWF-treated allografts were well protected against coronary and parenchymal lesions and the coronary arteries exhibited an intimal monolayer of flat cells, which however were CD34 negative. CONCLUSION: treatment with LMWF appeared very effective in this rat cardiac allograft model to prevent arterial and parenchymal lesions occurring in response to alloimmune injury. However this protective effect does not appear to depend on mobilization of bone marrow-derived cells.     

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.     

Role of antibody in rat renal allograft rejection. II. Failure to enhance renal allografts by sensitization to donor class I antigens presented by donor strain erythrocytes

BN rats were immunized with one or three doses of 1 X 10(8) highly purified LEW erythrocytes (LEW-E) yielding IgM antibody (IgM-BN rats) and IgG antibody (IgG-BN rats) to LEW class I antigens, respectively. LEW kidneys transplanted into IgM-BN rats elicited cytotoxic T cell responses and lymphocytotoxic antibody responses comparable to those elicited by LEW renal grafts in unmodified BN rats. However, LEW kidneys were rejected by IgM-BN hosts in a slightly delayed fashion compared with controls (mean rejection times (MRTs), 9.4 versus 7.1 days); delayed rejection was associated with the absence of anti-LEW IgG hemagglutinins from the recipients' blood and the absence of vasculonecrotic lesions from rejected renal grafts. LEW kidneys inserted into IgG-BN rats were rejected in a slightly accelerated fashion compared with controls (MRT, 6.6 days). Lymphocytotoxins developed in IgG-BN recipients of LEW kidneys in a fashion similar to that of controls, but cytotoxic T cell responses were delayed up to the 6th day after transplantation. These observations confirm our previous finding that cytotoxic T cells do not play a decisive role in acute rejection in this model. The association observed between delayed or accelerated rejection of LEW kidneys by BN rats sensitized with LEW-E and the absence or presence of anti-donor IgG hemagglutinins in the blood of these recipients after transplantation suggests an important role for IgG anti-donor class I antibodies in the rejection of LEW renal allografts by BN rats.     

Mucosal glutamine utilization after small-bowel transplantation: an electrophysiologic study.

A short course of FK 506 after small bowel transplantation averts rejection in the rat and achieves indefinite survival of the recipient whose nutritional status is dependent on the function of the intestinal graft. Ex vivo electrophysiologic studies using the Ussing Cell were conducted to delineate functional competence of the graft by evaluating mucosal ion transport and glutamine utilization. Orthotopic small-bowel transplantation was performed in Lewis (LEW) rats as recipients of either Brown-Norway (BN) allografts or LEW syngeneic grafts. Allograft recipients received FK 506 either as a short course (2 mg/kg on Day 0-4 after transplantation) or continuously (2 mg/kg Day 0-4, then 0.5 mg/kg weekly). Ileal mucosa was harvested from small bowel grafts 9 and 60 days after transplantation and mounted in the Ussing Cell containing Hanks' balanced salt solution with/without L-glutamine (20 mM). Transmembrane potential difference (PD), which represents mucosal active ion transport, and mucosal resistance, an index of membrane integrity, were recorded. Nine days after transplantation, mucosal PD was the same in the ileum from syngeneic grafts, allografts treated with FK 506 and normal LEW and BN rats, and the addition of glutamine increased PD equally in all groups. In comparison, PD was markedly decreased in allografts undergoing rejection, and the glutamine response was blunted. Sixty days after transplantation, mucosal PD was reduced in allografts treated with a short course of FK 506, but normal in allografts receiving continuous immunosuppression with FK 506 and in syngeneic grafts. A decrease of mucosal resistance was not a feature of rejection nor a sequel of limited FK 506 therapy. Our data indicate that allograft rejection results in a significant decrease in mucosal PD and a poor response to glutamine. Control of rejection by FK 506 preserves normal electrophysiologic responses of the allograft mucosa.     

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.