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Although cyclosporine has improved results of organ transplantation, treatment regimens using multiple agents are being evaluated both experimentally and clinically in attempts to diminish its often profound nephrotoxicity; some therapies act synergistically by differential inhibition of distinct steps of the rejection cascade. The effects on graft function of a full dose or a subclinical dose of CsA, ART-18, a monoclonal antibody (mAb) directed against the IL-2 receptor expressed on activated host cells, and a combination of low-dose CsA and ART-18, have been tested in rat recipients of both heart and kidney allografts. Renal graft function was assessed by several classic techniques; heart function by isolated perfusion methods. Full-dose CsA and combination treatment were most effective in both organ graft systems, with at least one-third of grafts surviving indefinitely. At seven days after transplantation, glomerular filtration rates and renal plasma flow of all grafted recipients were decreased as compared with normal; at 14 days, function in the best treatment groups had improved toward that of isografts. Similarly, cardiac output and stroke work index of best treatment groups were comparable to that of isografts. These functional studies complement previously reported immunological and immunohistological findings stressing that synergy occurs between subclinical doses of CsA and anti-IL-2-R mAb in two rat organ graft systems.

Cyclosporine has been used as primary immunosuppression in renal allograft recipients in our unit for the past decade. The overall clinical experience and long-term effects of the agent are reviewed. There were 461 consecutive recipients of kidney grafts; 379 received grafts from cadaver donors (CAD) and 82 from living related donors (LRD). Four separate clinical protocols were used sequentially using progressively decreasing doses of CyA; azathioprine was added in group 4 recipients of LRD grafts, and in patients receiving secondary CAD grafts (group 5). The patient mortality rate was less than 5%, with sepsis being the prime contributor. The majority of kidney grafts were lost within the first 2 months after operation; those that never functioned were found almost invariably to have been irreversibly rejected. During the subsequent years of follow-up, attrition of CAD grafts was significantly greater than LRD grafts. In contrast, the attrition rate of primary and secondary CAD grafts was the same after the first 3 months, emphasizing the importance of early immunologic graft destruction. Primary nonfunction occurred in 49% of CAD kidneys and 17% of LRD grafts; however 71% of initially nonfunctioning LRD grafts never functioned at all compared to 34% of CAD grafts, the majority of such organs undergoing fulminate rejection. Individual graft loss after 1 year was almost inevitably due to chronic rejection; there were no differences in long-term allograft function among the treatment groups. Although CyA has improved overall results of kidney transplantation, chronic rejection remains a major unresolved problem.

Unlabelled: LBNF1 cardiac allografts (Tx) are rejected within 36 hr in LEW rats sensitized with BN skin Tx 7 days earlier, compared to 8-day rejection in unmodified hosts. Treatment with cyclosporine or ART-18, an anti-interleukin 2 receptor (IL-2[R]) mAb monoclonal antibody, abrogates accelerated rejection and prolongs mean Tx survival to 42 days and 16 days, respectively. ART-18 given in concert with subtherapeutic dose of CsA extends survival to 25 days. These studies were designed to dissect the early mechanisms leading to ART-18 and/or CsA-mediated abrogation of accelerated Tx injury. No effect of concomitant mAb administration upon CsA through levels was noted in Tx recipients conditioned with both modalities. The beneficial effect of ART-18+CsA treatment was also unrelated to CsA-induced diminished host responses to mAb, as shown by ELISA. In the biodistribution studies 125I-labeled ART-18 accumulated preferentially into host lymphoid tissues and Tx itself and away from the blood. In animals that were concomitantly given "cold" CsA, the clearance of labeled ART-18 from the blood increased further, as did mAb sequestration into the Tx. The sensitized hosts developed high titers of complement-dependent cytotoxic (CDC) antibodies, which peaked at the time of actual Tx loss. ART-18 or CsA alone inhibited CDC, whereas combined therapy decreased further the humoral effects of sensitization. The cell-mediated lymphocytotoxicity assay revealed a similar pattern. CsA, ART-18, and combination therapy each modulated the deposition of IgG, IgM, and C3 in Tx, as shown by immunohistology. However, only CsA or ART-18+CsA therapy abolished or markedly decreased elaboration of IL-2, interferon gamma, and tissue necrosis factor alpha., In Conclusion: (1) the adjunctive low dose of CsA potentiates the inhibitory effects of ART-18 upon humoral and cellular responses, leading to accelerated rejection of cardiac Tx in presensitized rats; (2) the synergistic interaction between both modalities that results in the inhibition of lymphokine production is critical and correlates with long-term Tx survival; (3) the biodistribution patterns of mAb are important--an increased blood level of mAb does not necessarily translate into its higher therapeutic efficacy.

A 10-day course with ART-18, a mouse monoclonal antibody (mAb) directed against the rat interleukin 2 receptor (IL-2R), prolongs the survival of (LEW x BN)F1 cardiac allografts in LEW recipients to approximately 3 weeks (acute rejection = 8 days, P less than 0.001). We examined host responses against ART-18 idiotype (Id) and mouse immunoglobulin in recipients immunomodulated with ART-18 mAb. Treatment with ART-18 elicited high titers of anti-Id antibodies 14 days after transplantation. However, naive rats given ART-18 before transplantation showed strong anti-Id responses as early as day 4 after engraftment, coinciding with abrogation of the treatment effect (graft survival, approximately 10 days). Preimmunization with irrelevant mouse IgG, which elicited high titers of anti-IgG, did not influence the efficacy of ART-18 upon graft survival (17 days). The use of cyclosporin A (CsA) in conjunction with ART-18 prior to transplantation suppressed the anti-Id response and led to dramatic graft prolongation (greater than 58 days), with two of five grafts surviving indefinitely. This striking effect of CsA plus ART-18 pretreatment did not depend upon CsA per se, as grafts were rejected within 12 days in animals pretreated with CsA alone; in both groups CsA trough levels were comparable. Moreover, administration of CsA before transplantation in concert with control IgG (instead of ART-18) prompted rejection within 2-4 weeks. Thus, discrete interaction(s) between anti-IL-2R mAb and CsA prior to engraftment induces partial host unresponsiveness/tolerance to anti-IL-2R mAb treatment following transplantation and suppresses the neutralizing anti-Id responses, which results in long-term/permanent graft acceptance. This study provides a strategy to overcome the anti-Id response mounted by graft recipients that otherwise limits the efficacy of anti-IL-2R mAb treatment.

Untreated anephric LEW rats die ca. 9 days following transplantation of LBNF1 kidney allografts. Although treatment with ART-18, a mouse antirat IL-2R mAb (300 micrograms/kg/day x 10 days), prolonged graft survival to ca. 3 weeks, the severely impaired renal function was comparable to untreated controls (creatinine levels 3-5 mg/dl). In contrast, simultaneous infusion of ART-18 and a very low dose of CsA (0.75 mg/kg x 10 days), marginally effective on its own, resulted in survival of greater than 45 days; the grafts exhibited relatively good function comparable to that in rats treated with full-dose (15 mg/kg/day) CsA. This beneficial biological effect did not depend upon elevated CsA trough levels in animals conditioned with both modalities. The CD4:CD8 ratio at the graft site was lowest (0.3-0.4) in recipients treated with ART-18 + CsA. Synergy between the two agents has been demonstrated by adoptive transfer studies in which nonspecific suppression has been conferred selectively by cells infiltrating kidney grafts in rats given ART-18 and CsA in concert but not separately (LBNF1 and WF test cardiac allograft survival ca. 12 days). In contrast, suppression in the recipient spleens was donor-specific; both CD4 and CD8 cells prolonged test graft survival. Immunohistological evaluation of renal allografts revealed that therapy with ART-18 or low-dose CsA alone failed to deplete IL-2R+ cells and prevent production of IL-2, IFN-g, and TNF. In contrast, the frequency of infiltrating IL-2R+ cells and elaboration of endogenous cytokines in non-uremic hosts receiving combination therapy was greatly depressed, stressing again synergistic interaction between ART-18 and CsA. Additionally, markedly reduced class II antigen induction, XL-fibrin deposition, and glomerulitis may also contribute to prolonged survival and satisfactory function of kidney allografts in this animal group.

Increasing numbers of sensitized patients are either precluded from receiving an allograft or experience accelerated rejection which may be refractory to conventional therapy. Using a rat model, we have shown that accelerated (24 hr) rejection of LBN cardiac Tx in LEW rats sensitized with BN skin grafts 7 days earlier, could be prevented by treatment with cyclosporine (15 mg/kg/day x7 days, Tx survival about 42 days) or ART-18, an anti-IL-2R mAb (300 micrograms/kg/day x10 days i.v., Tx survival about 16 days). In this study, we evaluated intragraft mechanisms responsible for these effects by immunoperoxidase localization of relevant humoral mediators (IgG, IgM, C3, cross-linked fibrin), graft infiltrating cells (GIC), and associated cytokines (IL-2, IFN-g, tumor necrosis factor [TNF], or cytokine receptors (IL-2R). Tx rejected in fulminating fashion by 24 hr in sensitized hosts showed extensive and progressive endothelial deposition of IgG, C3, and fibrin from 2 hr, followed by an influx of neutrophils at 3 hr, and peak numbers of GIC by 18 hr (88.8 +/- 20.3 leukocytes/field). At 18 hr, GIC consisted of neutrophils (26%), T cells (20%, greater than 90% of which were OX-8+), and monocytes/macrophages (53%), whereas B cells were absent. By 18 hr, up to 20% of GIC were IFN-g+, 10% were IL-2R+, and 10% were IL-2+, consistent with labeling of 20% of cells with OX-22. Widespread endothelial and mononuclear cell labeling for TNF and the procoagulant molecular tissue factor (TF) were also noted. In contrast to untreated grafts, CsA treatment essentially abolished intragraft Ig, C3, and fibrin deposition. Moreover, despite dense cell infiltration at 24 hr (total GIC 55.3 +/- 13.4/field), analysis of CsA-treated Tx showed markedly decreased neutrophils (0.5%), with increased T cells (35%) and similar proportions of macrophages (66%). In addition to the reduction in neutrophils, Ig and C3, fewer IL-2R+ (6%) and OX-22+ (3%) cells, considerably less TNF and TF, and almost no IL-2+ or IFN-g+ GIC (less than 1%) were detected. Surprisingly, ART-18 treatment also greatly decreased but did not abolish endothelial deposition of C3, IgG, or IgM, whereas widespread endothelial and mononuclear labeling for fibrin, TNF, and TF remained. In addition, GIC (about 54.8 +/- 16.1/field) contained only moderately reduced numbers of neutrophils (31%) and the proportions of T cells (27%) and macrophages (49%) were generally comparable to those of rejecting Tx in untreated rats.(ABSTRACT TRUNCATED AT 400 WORDS)

One hundred thirty two patients who received cadaver kidney transplants in three Boston transplant centers were analyzed to demonstrate the immunosuppressive effect of cyclosporine and steroid therapy versus conventional immunosuppression. Of these, 35 patients received the cyclosporine and steroid regimen and 97 received conventional immunosuppression. The number of patients with preformed reactive antibody was greater in the cyclosporine group; however, a greater number of patients were diabetic in the conventional group. The incidence of acute tubular necrosis was significantly higher in the cyclosporine group. There was no significant difference in transplant or patient survival between these groups.

Cyclosporine, a cyclic endecapeptide of fungal origin, has recently been released for use in clinical transplantation. Trials in kidney, heart, liver and bone marrow recipients were encouraging: 1-year graft survival rates were 70% to 80% for kidney and heart recipients, and 60% to 65% for liver allograft recipients. Cyclosporine is also effective in treating bone marrow recipients with acute graft-versus-host disease. The drug selectively inhibits T-helper cell production of growth factors essential for B cell and cytotoxic T-cell differentiation and proliferation, while allowing expansion of suppressor T-cell populations. Drug absorption varies greatly, necessitating monitoring of drug level and individualization of therapy. Nephrotoxicity is the most frequent side effect of cyclosporine. An increased incidence of B-cell lymphomas seen when cyclosporine was used in conjunction with cytotoxic agents or anti-lymphocyte globulin has very rarely been observed when concomitant immunosuppression has been limited to low-dose corticosteroids. Lower initial doses of cyclosporine, followed by more rapid tapering may reduce the incidence of nephrotoxicity without compromising improved graft outcome.

Serial changes in phenotype, cell cycle, and functional behavior of lymphocyte subpopulations occurring both during acute rejection in unmodified hosts and in long-surviving heterotopic cardiac allografts in rats treated with cyclosporine (CsA) were studied. Using flow cytometry, RNA and DNA content of cells was examined during various phases of cell activation. In animals acutely rejecting their grafts, numbers of cells infiltrating the grafts and in host spleen in G1 phase (higher RNA content) increased, starting from day 3, and peaked by 5-6 days posttransplantation, and numbers of cells in S/G2/M phase (higher DNA content) remained stable. Similar, although slightly delayed changes were noted in CsA-treated recipients. The ratio of T helper (Th) to T suppressor/cytotoxic (Ts/c) phenotype cells infiltrating acutely rejecting grafts by day 3, was 1.6; it inverted abruptly to 0.7 by days 5-6, suggesting a preponderance of Ts/c during the later stages of allograft rejection. Ratio inversion occurred slightly later in host spleen and later in peripheral blood. Similarly, treatment with CsA produced a transient depression of Th, with recovery of the Th: Ts/c ratio during weeks 3-4 following transplantation. Adoptive transfer studies were then performed to investigate the functional significance of the T cell subsets. Survival of test grafts was prolonged significantly (ca. 14 days, P less than 0.001) when cells infiltrating grafts and spleen were transferred during inversion of Th:Ts/c; before that period, test graft survival was shortened in a second-set manner. These experiments suggest that suppressor cells may be responsible for resolution of acute rejection, as well as for host unresponsiveness seen after CsA treatment, and they represent an important homeostatic host mechanism following immunological stimulation.

Fifty seven recipients of renal allografts initially treated with CsA and low-dose prednisone were switched to azathioprine and low-dose steroids. Ten had prolonged (greater than 28 days) allograft nonfunction after transplantation (group 1), 8 had ongoing, poorly controlled rejection (group 2), and 39 had stable functioning grafts (group 3). With a mean follow-up period of 5 +/- 3 months after conversion, 50 grafts remained functional including 6 of 10 in group 1, 6 of 8 in group 2, and 38 of 39 in group 3. Thirty-seven (65%) had improved function, 12 (21%) had stable function, and 8 (14%) experienced declining renal function. Three of these latter 8 patients required reinstitution of CsA therapy. There were 20 episodes of acute rejection in 18 patients; one graft lost function because of acute rejection unresponsive to therapy. Reasons for the 6 other graft losses were persistent primary nonfunction in 3 patients from group 1, untreated rejection in 2 patients who had multiple prior rejection episodes while on CsA, and chronic rejection in one patient. Although renal function has improved or stabilized in the majority (86%) of individuals changed to azathioprine therapy, there was substantial risk of acute rejection (32%) complicating this procedure. Patients most likely to benefit from conversion to azathioprine therapy are those with prolonged graft nonfunction after transplantation and those with serum creatinines greater than 2.0 mg/dl.

Mice sensitized with alloantigens and treated with cyclosporin A (CsA) were incapable of generating antigen-specific cytolytic lymphocytes (CL). Lymphocytes from these CsA-treated animals could not be reactivated upon exposure to the same alloantigens in mixed lymphocyte culture (MLC), whereas their response to a third-party antigen remained intact, suggesting a long-lasting and specific effect of CsA. After being irradiated, these lymphocytes from CsA-treated animals were added to normal MLC and were shown to prevent normal lymphocytes from becoming cytolytic in a dose-dependent and antigen-nonspecific fashion. These suppressor cells were not detected in mice receiving CsA only, indicating that CsA did not induce but rather permitted the expression of suppressor cells possibly generated by allosensitization. The suppressor cells appeared to be T lymphocytes, because treatment with anti-Thy-1.2 antibody and C abrogated their suppressive activity. The present results suggest that activation and/or sparing of suppressor cells by CsA may account for the long-lasting unresponsiveness seen in CsA-treated animals.

We have reviewed the results of patient survival and transplant function of the last 100 recipients of renal allografts treated with cyclosporine (CyA) plus low-dose steroids since November 1981; the follow-up varies between 3 months and 2 years. A group of 56 individuals transplanted between January 1980 and October 1982 and immunosuppressed with azathioprine (Aza) and steroids were used as comparison. There were five deaths among 100 patients treated with CyA and two among 56 treated with Aza. There were, however, marked differences in allograft function. Using actuarial curves, 2-year allograft survival from 24 living, related, one haplotype matched donors was 83%, as compared to an unsatisfactory 60% graft function among 24 nonrandomized, comparable, Aza-treated recipients. The 2-year actuarial survival of 76 allografts from cadaver donors was 76%; that of 36 grafts in patients treated with Aza, 48%. Interestingly, function of first cadaver allografts was 84% at 2 years, far better (p less than 0.002) than cadaver graft function (58%) in patients who had been previously transplanted; these latter results are comparable to Aza-treated cadaver recipients. Side effects and complications of this difficult drug, as well as its benefits, have been stressed in this article.

We have analyzed the adjunctive effect of subtherapeutic doses of cyclosporine (CsA, 1.5 mg/kg/day x 7 or 14 days) on cardiac allograft survival in actively and passively enhanced rats. This CsA dose, one tenth of the effective dose, when administered after, but not before, transplantation into enhanced hosts produced permanent graft acceptance; cardiac allografts survive c. 25 days in recipients enhanced only and 1 week in untreated animals. Adoptive transfer of spleen T cells of OX8+ or W3/25+ phenotype from long-term (greater than 200 days) graft recipients prolonged donor-specific test graft survival in naive rats (c. 16 days and c. 14 days, respectively, P less than 0.001) and delayed rejection in reconstituted B rats from 7 days to 21-23 days (P less than 0.001). Indeed, both T subsets were separately equally potent and with no overlap responsible for the suppressor activity. The phenotypic profile of the immune cells in the maintenance phase of enhanced or enhanced + CsA-treated recipients was comparable to naive or isografted controls as demonstrated by flow cytometry and immunohistologic studies. Furthermore, the activation status of the graft infiltrate in long-term survivors was similar regardless of the initial immunosuppressive protocol. CsA contributed selectively to the enhancing regimen in the induction phase of unresponsiveness, diminishing the cellularity of graft infiltrate and preventing intragraft T cell activation. These studies stress synergy between subtherapeutic doses of CsA and immunologic active/passive enhancement, 2 immunosuppressive modalities that spare T cells with suppressor capabilities but differ in the inhibition of T helper cell activation.

The mechanism of action of cyclosporin A (CsA) in inhibiting the induction of alloreactive cytolytic T lymphocytes (CTL) in mixed-lymphocyte culture (MLC) was investigated. CsA at concentrations of 10(-3) to 10(-1) micrograms/ml completely prevented the generation of CTL. However, the addition of culture supernatants from mitogen-activated lymphocytes to MLC not only significantly reversed the suppressive effect of CsA but also fully restored the reactivity of lymphocytes already treated with CsA. By measuring the presence of a soluble helper mediator (SHF) in MLC supernatants, we found that CsA-treated lymphocytes produced no SHF, possibly interleukin 2 (IL-2). The effect of CsA on receptors for IL-2 was subsequently studied and it was found that the binding capacity of 125I-labeled IL-2 to lymphocytes was not altered by the presence of CsA. These findings suggest that the prevention of helper cells from producing SHF, rather than the inhibition of the response of effector cells to SHF, is a possible explanation for the immunosuppression mediated by CsA.

LEW rat recipients of (LEW X BN)F1 strain heterotopic cardiac transplants treated with cyclosporine A (CsA) (15 mg/kg/day intramuscularly, 7 days) retain grafts indefinitely despite drug withdrawal. Donor-specific suppressor T cells that are active in passive transfer experiments have been harvested from long-term CsA-treated hosts. Although CsA is known to inhibit in vitro cytokine release, the in vivo effects of the CsA on the lymphokine cascade are not known. We investigated the action of the drug upon spontaneous and mitogen-induced interleukin 1 (IL 1), interleukin 2 (IL 2), and interleukin 3 (IL 3) release by spleen cells obtained from the following groups of rats: 1) normal, i.e., untreated and ungrafted; 2) grafted, acutely rejecting; 3) grafted, actively treated; and 4) under CsA-induced state of "tolerance." The results demonstrate that in vivo CsA therapy inhibits monocyte (IL 1 release) as well as lymphocyte function (IL 2 and IL 3 release) only during the inductive phase (the 7 days of treatment). During the "tolerant" phase, mitogen (Con A and LPS)-induced release of interleukins was quantitatively similar to that noted in normal animals. In contrast, a remarkable increase in the spontaneous production of IL 3 was observed in the "tolerant" group. Because cytokine release is not inhibited in the "tolerant" state, our data strongly support the concept that maintenance of the state of unresponsiveness is governed by the emergence of suppressor cells. The correlation of increased spontaneous production of IL 3 during this period leads us to postulate that this interleukin may be implicated in the activation or clonal expansion of suppressor cells, and hence may play a role in graft tolerance.

(LEW X BN)F1 cardiac allografts are rejected acutely (7 days) in unmodified LEW rats, yet survive indefinitely following cyclosporine (CsA) treatment (15 mg/kg im for 7 days) or in T-cell-deprived (B) recipients. Using these models, the function of T helper cells (Th) in the maintenance phase of CsA-mediated long-term graft survival was examined. With monoclonal antibody immunoaffinity fractionation techniques, Th (W3/25+OX8-) were separated from spleens of CsA-treated hosts 3-4 weeks after grafting (CsA-Th), from specifically sensitized (s-Th), or from normal ungrafted (n-Th) rats. Adoptive transfer of 60 X 10(6) CsA-Th into B recipients produced rejection of donor-specific, but not third-party grafts in 21 +/- 7 days, comparable to s-Th (17 +/- 4 days), but faster than n-Th (4-5 weeks, P less than 0.025). CsA-Th recombined with T cytotoxic/suppressor phenotype (CsA-Tc/s, OX8+W3/25-) in numbers contained in 100 X 10(6) CsA-T cells were ineffectual, even when supplemented with exogenous interleukin 2-rich conditioned medium (IL-2CM); in contrast 100 X 10(6) s-T cells + IL-2CM inevitably caused acute rejection in B hosts (11 +/- 3 days). Increasing numbers of Th incrementally to 100 X 10(6) augmented the effectiveness of s-Th (rejection in 13 +/- 2 days), but did not improve potency of CsA-Th (20 +/- 2 days). Suppressor activity produced by small numbers of contaminating CsA-Tc/s (c. 0.4%, 4-5 X 10(5) cells in 100 X 10(6) CsA-Th) accounted for extended graft survival in B recipients, as this small number of CsA-Tc/s transferred into untreated syngeneic rats increased test graft survival to c. 16 days (P less than 0.001). IL-2 production by spleen cells, depressed during CsA treatment, returned to normal levels 2-3 weeks following drug withdrawal, whereas transfer of CsA-Th into B recipients induced a shift of IL-2 levels from dramatically depressed to normal, findings suggesting normal IL-2 production by CsA-Th. This report demonstrates that an unresponsive state in CsA-treated animals is achieved despite the presence of fully potent donor-specific Th. Active suppressor activity plays a critical role in the maintenance phase of graft survival in rats treated transiently with CsA.

Lymphocytes from spleen, peripheral blood, thymus, and lymph node of naive rats, nonimmunosuppressed recipients of MHC-incompatible heart grafts, and cyclosporine-treated recipients of MHC-incompatible heart grafts were tested for their ability to augment or suppress proliferation of naive cells in an in vitro MLR co-culture assay. Rats treated with cyclosporine for only 7 days maintained their grafts indefinitely. Potent suppressor activity was found in the peripheral blood and spleen of adult naive rats. In untreated engrafted rats, increased suppressor activity was found 1 wk after transplantation and increased helper activity 2 wk after transplantation. In contrast, subnormal helper and suppressor activity was found in cyclosporine-treated rats 1 wk after transplantation. Subsequently, suppressor activity peaked at 2 to 3 wk and helper activity at 4 wk after transplantation. Beyond 5 wk, the cyclosporine-treated rat was indistinguishable from naive ungrafted rats. Two types of suppressor activity were identified that differed in buoyant density and cyclophosphamide sensitivity. Neither suppressor activity demonstrated antigen specificity. These data suggest that one role of cyclosporine in this rat model is to delay the initial helper mechanisms until generalized suppressor activity is operable. The increased antigen-nonspecific activity is only transient, presumably until the final antigen-specific mechanisms become operative.

Heterotopic, vascularized small intestine transplants were performed in inbred strains of rats to investigate the structural, functional, and immunologic consequences of intestinal transplantation with and without immunosuppression with cyclosporine (CyA). Lewis X Brown Norway F1 intestine was rejected by untreated Lewis recipients in 7 to 10 days. Structurally, rejected intestine was characterized by shortened crypts and villi lined by damaged attenuated epithelial cells. Functionally, rejection was associated with impaired epithelial active ion transport as indicated by decreased potential difference and with diminished epithelial barrier function as reflected by decreased transepithelial resistance. Administration of CyA for 7 days prevented clinical rejection and partially prevented the structural and functional defects. Lewis intestine transplanted into Lewis X Brown Norway F1 recipients caused fatal graft versus host disease (GVHD) in 9 to 17 days. Treatment with CyA for 7 days failed to prevent GVHD routinely, but prolonged administration delayed fatal GVHD until CyA was discontinued. Intestine from Lewis "B" rats made deficient of T cells by thymectomy, irradiation, and reconstitution with syngeneic T cell-depleted bone marrow failed to cause GVHD in Lewis recipients. Reconstitution of the "B" rats with T cells before transplantation restored the GVHD response. These results may be relevant in the consideration of clinical small intestinal transplantation.

Cyclosporine (CsA) immunosuppressive therapy of renal allograft recipients at Brigham and Women's Hospital yielded a 20% increase in 12- and 24-month allograft survival over azathioprine (Aza) treated controls. A trend towards increased allograft survival with better HLA-A,B, or DR matching in recipients of cadaver allografts treated with CsA was appreciated, but it fell short of statistical significance. Nephrotoxicity is common in CsA-treated patients and is manifested by a largely reversible increase in the serum creatinine. It is possible to convert patients from CsA to Aza without adverse effects on allograft survival or allograft function.

The effects of anti-IL 2R mab treatment on local GVHR and on heterotopic cardiac allograft survival in rats were investigated. Anti IL 2 mab inhibited specifically both GVHR as well as allograft rejection. IL 2R-targeted therapy was superior to that of anti-T helper/inducer cell treatment, as it did not affect T subset distribution and did not reduce the number of circulating T lymphocytes. This therapy when combined with a low subtherapeutic dose of CsA exerted a strongly synergistic effect. The results support the concept of IL 2R-targeted therapy in suppressing undesired immune reactions with limited side effects.

Cyclosporine, the first of a new generation of selective immunosuppressive agents, has already proved to be of exceptional value in human organ transplantation; however, its role in human reconstructive surgery remains to be established. The possibility that short-term treatment could be sufficient for indefinite survival of bone, muscle, nerve, and vein allografts is attractive and might provoke changes in prevailing attitudes regarding the use of allogenous tissues in reconstructive procedures. Cyclosporine may start a new line of immunosuppressive agents with more potent therapeutic effect and less potential toxicity. This new generation of drugs, together with the experience in tissue transfer amassed by plastic surgeons during the last decade, may eventually result in unprecedented possibilities in surgical reconstruction.

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.

Cyclosporine in combination with other chemical or biological immunosuppressive modalities has been useful in clinical and experimental organ transplantation. In these studies, the efficacy of adjunctive subtherapeutic doses of CsA given to immunologically enhanced heart graft recipients or to animals treated with an anti-IL-2 receptor monoclonal antibody (ART18) are described. Individually, the treatment entities are only partially effective. In rats undergoing active and passive enhancement alone, heart allograft survival was increased to 25 +/- 12 days in two-thirds, indefinitely in one-third. After ART18 treatment, grafts survive 21 +/- 1 days. Grafts are accepted permanently in animals receiving full-dose CsA (15 mg/kg X 7), but are rejected acutely (c. 7 days) when subtherapeutic doses (1.5 mg/kg X 7) are used. However, when subtherapeutic doses of CsA are given in combination with immunological enhancement or with interleukin-2-receptor-targeted therapy, graft survival increases dramatically, with permanent or markedly prolonged engraftment occurring in all instances. In the early phases of host unresponsiveness, both enhancement and IL-2R-targeted therapy, graft survival increases dramatically, with permanent or markedly prolonged engraftment occurring in all instances. In the early phases of host unresponsiveness, both enhancement and IL-2R-targeted therapy spare selectively T cells with suppressor activity in vivo; in enhanced animals, the W3/25+ subset is responsible for prolonged graft survival, the OX8+ fraction is responsible in ART18-treated animals and in CsA-treated animals. Both subpopulations show suppressor activity in the later stages of combination treatment. IL-3 production is increased significantly in these states of unresponsiveness, an observation also noted during maintenance CsA treatment; this seems to correlate with suppressor activity. Immunoperoxidase studies of the graft infiltrates emphasize the synergistic effects of combination treatments. Thus, subtherapeutic doses of CsA plus biologic host manipulations produce greatly increased graft survival by affecting selectively different host immune mechanisms.

Monoclonal antibodies to lymphokine-induced activation antigens of lymphocytes and macrophages were used to analyze the intragraft events occurring during acute rejection of rat heterotopic cardiac allografts. The cells present during untreated rejection were then compared with those present in situ after immunosuppression with the mouse anti-rat IL 2 receptor (anti-IL 2R) monoclonal antibody ART-18 or cyclosporin (CsA). Untreated rats rejected their grafts within 7 days, whereas rats receiving 10 days of i.v. ART-18 antibody therapy showed graft prolongation to more than 21 days, and rats receiving CsA for 7 days maintained their grafts indefinitely. Untreated rejection was associated with an influx of T (W3/13+) cells and macrophages (ED-1+, ED-2+). Activated mononuclear cells (IL 2R+) were identified within rejecting grafts from day 2, and their numbers peaked on days 4 to 6 when 15 to 20% of infiltrating leukocytes were IL 2R+. Double labeling studies of IL 2R+ cells present at day 6 showed surprisingly that both T cells and macrophages expressed IL 2R. In particular, although 55.8 +/- 6.9% (mean +/- SD) of IL 2R+ cells expressed the pan-T cell antigen 3/13, a similar proportion of IL 2R+ cells (49.8 +/- 8.2%) expressed the macrophage antigen ED-2. Conversely, both T cells and macrophage populations showed heterogeneity in their expression of IL 2R, because 39.2 +/- 12.2% of T cells and 31.0 +/- 13.4% of macrophages were IL 2R+. In addition, inflammatory macrophages at day 6 expressed the A1-3 antigen. Expression of this antigen by macrophages has previously been linked with development of macrophage procoagulant activity, and in this model intragraft inflammatory macrophages were closely associated with widespread deposits of fibrin. By comparison with untreated animals, rats treated with either ART-18 or CsA both lacked detectable IL 2R+ cells during the first 14 days post-transplantation (post-Tx), and showed significantly less cellular infiltration. However, although grafts of CsA-treated animals continued to remain IL 2R- and failed to stain with the macrophage activation marker A1-3, ART-18-treated rats showed increasing infiltration by both IL 2R+ mononuclear cells and A1-3+ macrophages, as well as increasing perivascular and interstitial fibrin deposition, prior to rejection by day 22. These studies document the presence of small number of activated intragraft T cells and macrophages during rat cardiac rejection, and show how CsA, and to a lesser extent anti-IL 2R therapy, inhibit this in situ activation and prolong graft survival. (ABSTRACT TRUNCATED AT 400 WORDS)

Immunological unresponsiveness toward skin allografts was studied in cyclosporine (CsA)-treated rats. BN skin grafts survive about 22 days and about 34 days in LEW hosts following 7 or 14 days of daily CsA treatment (15 mg/kg/day), respectively; in unmodified hosts grafts are rejected by 9 days. Indefinite (greater than 100 days) survival can, however, be produced by administering maintenance 15 mg/kg CsA every fourth day, following an initial course of the agent for 14 days. Early signs of graft rejection (hair loss, localized epidermal breakdown, and ulcerations) occurring in some animals were reversed by a CsA "pulse" (15 mg/kg/day) for 7 days, reduced gradually to the maintenance dose. CsA was equally effective when started as late as 4 days after grafting, but ineffectual when started after day 4. Once BN grafts were rejected, the agent could not prevent second-set rejection of donor-specific grafts, but significantly prolonged the survival of third-party (WF) skins. Survival of original BN grafts was unchanged by the placement of second BN grafts during both the inductive and maintenance phases; these second grafts survived as long as the original grafts. In contrast, secondary third-party (WF) grafts were promptly rejected; their destruction did not influence survival of the original grafts. Thus, indefinite survival of rat skin allografts is feasible with low maintenance doses of CsA. Graft rejection at later stages can be reversed by resuming daily therapy. Host unresponsiveness is stable and specific both during the early inductive and later maintenance phases.

Cyclosporine (CsA) is known to induce long-term survival of skin allografts, although the cellular mechanisms responsible for this effect are not well understood. To further define the effects of CsA-induced immunosuppression, we performed a morphological and immunohistological study of acute skin allograft rejection in the rat, comparing untreated and CsA-treated animals. Three significant differences were found between grafts in CsA-treated and untreated rats. First, CsA-allografts contained fewer MRC OX-8+ cytotoxic T cells than untreated allografts. Second, although the presence and numbers of infiltrating macrophages (W3/25+, W3/13- cells) was not influenced by CsA treatment, CsA treatment blocked expression of a macrophage membrane activation antigen, defined by the monoclonal antibody A1-3, which has previously been linked with development of macrophage procoagulant activity (PCA). Third, diminution in MRC OX-8+ lymphocytes and A1-3+ macrophages in CsA allografts was associated with an absence of the widespread thrombotic and necrotizing microvascular injury typical of acute rejection in untreated rats. We conclude that prolongation of skin allograft survival by CsA is related to its ability to prevent cell mediated injury to the endothelium of graft vessels, and possibly also to inhibition of macrophage PCA with consequent reduced thrombus formation.

Although cyclosporine (CsA) is a powerful immunosuppressive agent in organ transplantation, its efficacy in skin transplantation has not been examined completely. We have tested it as primary immunosuppression in a rat skin allograft model. Histoincompatible Brown-Norway skin grafts are rejected in untreated Lewis hosts within 9 +/- 1 days but survive for 22 +/- 3, 34 +/- 2, or 41 +/- 8 days after 7, 14, or 21 days of CsA treatment (15 mg/kg per day subcutaneously), respectively (p less than 0.001). Animals treated daily for 4 weeks died from drug toxicity; however, an initial 2-week course followed by a low maintenance dose (15 mg/kg every fourth day) produced indefinite (greater than 150 days) graft acceptance without side effects. The long-surviving grafts were supple, grew long hair, and showed normal histology. When the drug was stopped at any time during this maintenance period, early signs of rejection (hair loss, epidermal breakdown, and localized ulceration) occurred, which could be reversed completely by a short CsA "pulse" (15 mg/kg per day for 7 days). These experimental data support the potential application of CsA immunosuppression in human skin allotransplantation.

Forty-eight patients with stable renal function after allotransplantation have been converted from CsA/prednisone to azathioprine/prednisone to assess the short- and long-term effects upon renal function. Virtually all patients show an initial improvement in serum creatinine levels. Three patients developed chronic renal failure after 12 to 21 months, and three died of pneumonia 7, 12, and 19 months later. The mean serum creatinine level at latest follow-up (seven to 36 months) was 2.5 +/- 1.5 mg/dL for all 48 patients. Of interest, a control group of 21 patients not converted to azathioprine had serum creatinine levels of 2.5 +/- 0.8 mg/dL, over a follow-up period of five to 25 months. It is not immediately apparent that either group will have a superior overall outcome, although patients on azathioprine seem to have more of a risk for graft loss. More data are needed with various dosage schedules, and with randomized controls.