Antithymocyte Sera Research Articles

Effect of early versus delayed calcineurin inhibitor treatment on delayed graft function

IN RENAL TRANSPLANTATION, there is some controversy regarding the impact of delayed graft function (DGF) on long-term outcome. DGF, which usually ranges between 10% and 50%, makes the management of patients more complex, hinders the diagnosis of rejection, prolongs hospital stay, increases the financial cost of transplantation, and reduces graft survival rates. Acute rejection occurs more frequently producing a negative impact on long-term graft survival. The risk factors for DGF are advanced donor age and prolonged cold ischemia time. The introduction of calcineurin inhibitors (CNI), either cyclosporine (CsA) or tacrolimus (Tac), as the mainstay of immunosuppression has significantly decreased acute rejection rates and improved 1-year renal transplant survival. However, a side effect of CNI, acute nephrotoxicity due to adverse hemodynamic effects, may increase the rate of DGF. Polyclonal antibodies (PCA)–antithymocyte (ATG) or antilymphocyte (ALG) sera used for induction therapy reduce the risk of acute rejection episodes, reverse steroidresistant rejection, and improve graft survival. The administration of PCA in sequential immunosuppression for patients afflicted with DGF reduces the incidence of first rejection episodes, although both graft and patient survivals are not statistically different. In this study, we compared a conventional immunosuppressive protocol of CNI and mycophenolate mofetil (MMF) plus steroids with CNI, MMF, steroids, and PCA in renal transplants with delayed graft function.

Molecular and cellular mechanisms of donor cell-induced tolerance.

The induction of immunologic tolerance to solid organ allografts is a subject of intense investigation because of the morbidity and mortality associated with standard immunosuppressive therapy. One method that is currently in clinical and preclinical testing involves the transient ablation of recipient T cells using polyclonal antithymocyte sera or monoclonal anti-CD4/CD8 antibody treatment, followed by the posttransplant administration of donor bone marrow cells or of donor peripheral lymphoid populations. Recent studies in our laboratory have shown that the molecular and cellular basis of the prolongation of graft survival by donor cell administration depends on the cellular compartment from which the donor cells were derived. We provide here a brief review of these data followed by new data suggesting that the mode of peripheral and central selection is also dependent on the source from which the donor cells were derived.

Induction therapy with rabbit antithymocyte sera reduces rejection episodes in immunologically low-risk living donor renal transplant recipients*1

Induction therapy with rabbit antithymocyte sera reduces rejection episodes in immunologically low-risk living donor renal transplant recipients*1

Induction therapy with rabbit antithymocyte sera reduces rejection episodes in immunologically low-risk living donor renal transplant recipients

Induction therapy with rabbit antithymocyte sera reduces rejection episodes in immunologically low-risk living donor renal transplant recipients

Induction therapy with rabbit antithymocyte sera reduces rejection episodes in immunologically low-risk living donor renal transplant recipients

Induction therapy with rabbit antithymocyte sera reduces rejection episodes in immunologically low-risk living donor renal transplant recipients

Metastasizing rat nephroblastoma. A rodent Wilms' tumour model.

In newborn F344 rats, immunosuppressed with antithymocyte sera and xenografted with various human tumours (lymphomas and melanomas), regular development of nephroblastomas was observed. Transplantation experiments and chromosome analysis proved the rat origin of the tumours. The histological appearance of these rat nephroblastomas closely resemble that of the human Wilms' tumour, in which three basic components: blastema, stroma and immature epithelium can be distinguished. With serial subcutaneous-, lung-subcutaneous transplantations in immunocompetent newborn animals a spontaneously metastatic line to the lung was selected. In adult hosts, lung metastasis occurred only following orthotopic (subrenal capsule) implantation. The histogenesis and the morphological features of this rat nephroblastoma and its metastases are described.

Single-Center Analysis of Impact of Immunosuppressive Therapy Upon Renal Allograft Survival in the Black Population

The effect of race upon renal allograft survival is controversial. Between 1981 and 1987, at Vanderbilt University Medical Center, 448 patients (75 black, 373 white) received azathioprine, 3 mg/kg daily; prednisone, 30 mg daily; and intravenous antithymocyte sera, 0.2 mL/kg/day for 14 days, after transplantation. Prednisone doses were decreased gradually to 10 mg daily within 6 months of transplantation. Azathioprine was maintained at doses of 2 to 3 mg/kg/daily; lower doses were administered if significant myelosuppression occurred. One-year graft survival was 72% and 85% among black and white recipients, respectively (P less than .01). Two hundred thirty-six patients have been treated with azathioprine (3 mg/kg initially tapered during the first week to 1.5 to 2 mg/kg); prednisone, 30 mg daily; and cyclosporine, 10 mg/kg per day. Cyclosporine therapy was begun after recipient serum creatinine levels had decreased below 3 mg/dL. Before therapy was initiated and until levels of cyclosporine were maintained between 150 and 200 ng/mL (whole blood), antithymocyte serum was administered. This immunosuppressive protocol resulted in 1-year graft survival of 90% and 87% in black and white recipients, respectively. Not only was graft loss markedly reduced, but the interracial difference noted before the use of cyclosporine was no longer evident. The type of immunosuppressive therapy used clearly affected 1-year allograft survival among black recipients. The combination of azathioprine, cyclosporine, and prednisone resulted in improved graft survival overall, but had the most significant effect among blacks.

POLYCLONAL RABBIT GAMMA GLOBULINS AGAINST A HUMAN CYTOTOXIC CD4+ T CELL CLONE

Antihuman lymphocyte rabbit (or horse) gamma globulins used in recipients of organ transplantation are prepared against thymocytes or immortalized cell lines, the only two sources so far allowing enough antigen preparation. These cells lack, however, the surface determinants characteristic of alloreactive blasts involved in the rejection process. We have derived long-term cultures of a panel of alloreactive (untransformed) clones from a rejected kidney. Among them, clone 1E7 has been chosen as a cytotoxic CD4+ (CD2+ CD3+ TCR alpha beta+) clone proliferating against HLA-DR8 targets. This clone (clonality assessed on T cell receptor genomic rearrangements) has been grown using weekly stimulations with the kidney donor-derived EBV cell line and recombinant IL-2. Clone cultures have been adapted to mass production after optimization of culture conditions satisfying pharmaceutical requirements. This procedure warranted a reproducible source of antigen since the functional and phenotypic characteristics of the immunizing 1E7 cells remained identical through the life span of the culture. In addition, the study of the total growth capacity of 1E7 cells showed consistent expansion until the 40th cell cycle, ensuring a progeny that will satisfy the large-scale requirement for a clinical trial. Rabbits were injected with 100 x 10(6) 1E7 cells (21, 14, and 7 days before bleeding). Sera were depleted of agglutinin by red blood cell absorption and globulin antiblast (GAB) prepared by SO4Na precipitation and ion exchange chromatography; 50% complement-mediated target cell lysis and 50% inhibition of E rosette formation and alloproliferation were obtained at GAB dilutions of about 1:250-1:500. Prescreened on cynomolgus monkeys, GAB could significantly prolong skin grafts when given prophylactically. Finally GAB have been used in human recipients of kidney grafts for prophylaxis of early rejection. Results of this pilot study are given in a separate report in this issue. In conclusion we have, for the first time, set up a large-scale preparation of polyclonal globulin against a normal human alloreactive clone, and this new reagent should present several advantages over classic antilymphocyte or antithymocyte sera because it contains specificities against activation antigens and has less crossreactivity variation among batches.

Immunotherapy for virus infection.

Understanding of virus-induced immune response disease and the related immunopathology has its roots in the studies reported by Rowe (1954). He showed that suppression of immune responses changed the ordinarily lethal, acute infection with lymphocytic choriomeningitis virus (LCMV) to a persistent infection in susceptible mice. The subsequent work of many investigators who used neonatal thymectomy, genetically athymic mice, irradiation, antilymphoid drugs, or antithymocyte sera, etc. (reviewed in Buchmeeer et al. 1980) extended the concept of immune response-mediated injury during viral infection. Thereafter, the role of lymphocytes in mediating virus-induced immunologic injury was delineated. First, experiments performed independently by Lundstedt (1969) and Oldstone et al. (1969) showed that lymphocytes obtained 6–9 days after an acute LCMV infection or primary inoculation killed LCMV-infected targets in vitro. Next, such lymphocytes were found to be of thymic origin and to bear Thy 1.2 markers of their surfaces (Cole et al. 1973; Marker and Volkert 1973). Gilden et al. (1972a, b) showed that mice which survived an ordinarily lethal dose of LCMV owing to immunosuppression developed acute lymphocytic choriomeningitis (LCM) disease and died when reconstituted with syngeneic, immune T-lymphocytes. Zinkernagel and Doherty (1974 a) defined the need for two signals between the cytolytic T-lymphocyte and its infected target, namely, virus specificity and H-2 restriction, so that killing of LCMV-infected cells could proceed.

Effect of infectious bursal disease on natural killer cell activity and mitogenic response of chicken lymphoid cells: role of adherent cells in cellular immune suppression.

A pathogenic isolate of infectious bursal disease virus (IBDV) caused persistent and extensive lesions in the bursa but mild and transient lesions in the thymuses of chickens of lines 63 and P. The effect of IBDV on two cellular immune functions, namely, natural killer cell cytotoxicity and mitogenic response, was studied. The natural killer cell activity was not consistently influenced, but the virus, during the first 2 weeks of infection, caused transient depression of the blastogenic response of spleen cells to phytohemagglutinin. Studies on mitogenic hyporesponsiveness revealed that the functional impairment was mediated by a suppressor cell that shared several characteristics with macrophages; i.e., the suppressor cell was adherent to plastic, was phagocytic, and resisted treatment with antithymocyte and antibursa cell sera. Removal of suppressor cells from the spleens of virus-infected chickens resulted in restoration of the mitogenic response of cells. Further, in mixing experiments, the suppressor cell isolated from the spleens of virus-infected chickens also inhibited the mitogenic response of normal spleen cells. We concluded that reduced mitogenic response of lymphocytes in IBDV-infected chickens was not due to a lack of functional T-cells, as suggested previously by others, but was due to macrophage-like suppressor cells. The suppressor cells, although present in certain normal chickens, became activated during early stages of IBDV infection.

SUCCESSFUL RENAL TRANSPLANTATION IN PATIENTS WITH T-CELL REACTIVITY TO DONOR

A positive crossmatch due to T-cell reactivity against donor cells is considered a strong contraindication to renal transplantation because of the risk of graft loss from rejection. However, the significance of T-cell reactivity before but not at the time of transplantation is unknown. To determine whether transplantation can be successful under these circumstances, graft survival was observed in 15 highly sensitised patients whose T cells were reactive to donor sera before but not at the time of transplantation. All patients have been followed up for at least 1 year post transplant. Immunosuppression was by azathioprine, prednisone, and rabbit antithymocyte sera. 9 (60%) have functioning grafts and a mean serum creatinine of 1·6 mg/dl. Early non-function occurred in 12 patients. One graft was lost to early acute humoral rejection and two others to chronic rejection. 14 of the 15 had a fall in reactivity to a panel of normal lymphocytes before transplant. 4 of the 15 had donor-specific B-cell antibodies at the time of transplantation and 3 of these lost their grafts because of rejection.

Characterization of three different human T cell membrane antigens, two being present on T lymphocyte subpopulations

Heteroantisera were raised in rabbits to thymocytes, HSB2 cells, and Sezary cells. Following absorption with Ia-positive leukemia cells, these sera appeared to be specific for different T cell antigens. Both the anti-HSB2 and the anti-Sezary sera reacted with approximately 50% and the antihymocyte serum with 100% of normal peripheral blood T lymphocytes. None of the sera reacted with B cells. The apparent molecular weights of the antigens being derected were determined by immunoprecipitation followed by SDS polyacrylamide gel eletrophoresis. A dimer of 170,000 daltons consisting of two similar 85,000-dalton polypeptide chains was immunoprecipitated by the anti-HSB2 serum whereas single polypeptides of 53,000 and 64,000 daltons were immunoprecipitated by the anti-Sezary and antithymocyte sera, respectively.

Cellular requirements for generation of thymocyte stimulatory factor and characterization of its target cell.

The cellular requirements for the generation of thymocyte stimulatory factor, TSF, were defined, and the target cell for its activity was in part characterized. TSF production, which is triggered by concanavalin A stimulation of murine spleen cells, requires both an Lyt 1+23- cell and an Ia+ non-T accessory cell. The Ia+ cell does not bear surface Ig, is not killed by treatment with antithymocyte sera and C, and is partially depleted by Sephaadex G-10 filtration. TSF-augmentation of the thymocyte mitogenic response to Con A requires Lyt 1+, Ia- thymocytes, which are present in both the cortisone-sensitive and resistant populations. The relationship between TSF and other activities produced by Con A activation of spleen cells is discussed.

Animal model for small cell carcinoma of the lung. Effect of immunosuppression and sex of mouse on tumor growth in nude athymic mice.

An animal model for the experimental study of small cell carcinoma of the lung (SCCL; human) has been developed. Tumors arising from continuous cell lines of cultured tumor cells as well as transplantable tumors of human SCCL in nude athymic mice have been characterized as to growth rate and morphology. The effect of antilymphocyte and antithymocyte sera and the sex of the host mouse were evaluated.

Induction of rat sarcomas in rats treated with antithymocyte sera after transplantation of human cancer cells.

Human cancer cells that had had high (greater than 160) tissue culture passages, when transplanted into antithymocyte-treated F344 newborn rats, caused induction of rat sarcomas in the rats within 2 or 3 subcultures, whereas human cancer cells with low (5-33) passages in vitro did not cause overt induction of rat sarcomas until after 5-10 subtransplantations. Because oncornavirus activity was not detected in either rat or human tumors, it is suggested that transforming sequences located on the human tumor cells may have been transferred to supporting rat reticulum cells in close contact with the human cancer cells.

Blastogenic potency of concanavalin-A-bound L1210 leukemic vaccine associated with its immunogenic activity.

Glutaraldehyde-treated L1210 murine leukemic cells induced immune resistance in mice when concanavalin-A (Con-A) was bound to their surface. They were not immunogenic when Con-A was separately inoculated, indicating requirement of vaccine-bound Con-A for enhancing immunogenic potency of the vaccine. Based on the finding that vaccine-bound Con-A induced in vitro blastogenesis of spleen cells 10 times as efficiently as unbound Con-A did, the association of blastogenic potency of Con-A-bound vaccine with its immunogenic activity was examined. The possibility of their association was supported by the finding that induction of immune resistance by vaccines was reduced when their blastogenic activities had been reduced by treatment of the vaccine with anti-Con-A antisera or by substituting succinyl-Con-A or other lectins for Con-A. Moreover, the fact that combined inoculations of Con-A-bound vaccine, assumed to be blastogenic to T cells, with anti-thymocyte sera, but not with cyclophosphamide or carrageenan, resulted in reduced production of resistant animals was also consistent with, but not proving, this possibility. Feasibility of this possibility was further supported by the finding that Con-A-bound vaccine did not induce immune resistance in animals that had been abrogated in blastogenic activity of spleen cells by prior administration of Con-A.

Role of the Murine Major Histocompatibility Complex in Macrophage-Mediated Cytolysis

Peritoneal cells from congeneic resistant mice infected with BCG displayed differential cytotoxicity toward tumor cells destroying more allogeneic tumor cells than syngeneic tumor cells. This observation was made regardless of the tumor cells used or the effector cell source. The responsible effector cell remained in a doubly adherent population, was sensitive to carrageenan and silica, insensitive to anti-thymocyte sera, and is probably a macrophage. Activated macrophages were capable of reacting against tumor cells as well as histoincompatible embryonic cells. These observations may indicate that macrophages are capable of discriminating cell surface components linked to the major histocompatibility complex.

Role of T Lymphocytes in Adjuvant Arthritis

Abstract The effects of treatments with cyclophosphamide (CY), hydrocortisone and anti-thymocyte sera (ATS) on the development of adjuvant arthritis (AA) were examined in WKA rats inoculated with wax D to induce AA. A single injection of 25 to 50 mg/kg of CY given 2 to 3 days before wax D inoculation caused severe arthritis with high incidence, whereas larger doses of CY were less efficient. Furthermore, the enhancing effect of CY pretreatment was abolished by passively transferred normal syngeneic thymocytes 1 day before wax D inoculation, but not by thymocytes that had been treated with ATS and guinea pig C. On the basis of these results and the previous observations on the effect of adult thymectomy and low-dose irradiation, we concluded that this enhancing effect of CY pretreatment was caused by selective depletion of suppressor T lymphocytes. Pretreatment with 12.5 mg of hydrocortisone also caused severe arthritis. This enhancing effect of hydrocortisone could also be due to the elimination of those suppressor cells. In contrast, in vivo pretreatment with ATS showed striking inhibition on the development of AA, suggesting that ATS could eliminate T lymphocytes that were responsible for eliciting this disease. Thus it appears that at least two T cell subpopulations are involved in the development of AA, one is an ATS-sensitive T2 subpopulation that is effective for induction of AA and the other is a T1 subpopulation that regulates this disease process.

Functional Heterogeneity Among the T-Derived Lymphocytes of the Mouse

Abstract The T1 subpopulation of peripheral T cells was defined in mice by its short half life, insensitivity to anti-thymocyte sera (ATS) in vivo, and slow kinetics of response to antigen. The T2 subpopulation was defined by its long life time, elimination by ATS in vivo, and rapid response to antigen. Mice containing only T1-type T cells were constructed by adult thymectomy (ATx) followed immediately by the elimination of T2 cells by ATS treatment. Immunization of these mice with SRBC led to the production of memory helper cells in the T2 subpopulation. This process depended on the presence of T1 cells and for the most part required SRBC immunization, although a few SRBC-specific T2 cells reappeared in the mice in the absence of antigen. We conclude that T1 cells can give rise to T2 cells in an antigen-driven step and that the two populations correspond to virgin and memory T cells, respectively.

Induction of specific anti-guinea pig T cell sera in rabbits

In an attempt to increase the specificity of antisera raised in rabbits against strain 2 guinea pig thymocytes and brain, the rabbits were screened for titres of natural antibodies to thymocytes and other lymphocytes. Although unimmunized rabbits commonly had moderate titres of cytotoxic antibodies to guinea pig thymocytes, occasional animals had low titres to thymocytes and moderate titres to bone marrow cells. Intravenous immunization of this latter group of rabbits with thymocytes led to the production of high titred anti-thymocyte sera which were easily made specific for thymus-derived lymphocytes (T cells) by absorption with L 2 C lymphoma, a bone marrow-derived lymphoma of strain 2 guinea pigs. Sera raised against guinea pig brain in complete Freund's adjuvant which had high titres of antibodies to both thymocytes and bone marrow cells could be made specific for T cells only with great difficulty. The cytotoxic activity of the anti-T cell serum could be absorbed by strain 2 thymocytes and brain homogenates, while high dilutions of this serum inhibited the formation of spontaneous rosettes between guinea pig lymphoid cells and normal rabbit erythrocytes.