Lynette J. Dumble

T SUPPRESSOR CELLS INDUCED BY TRANSFUSIONS AND CYCLOSPORINE : STUDIES IN THE MURINE CARDIAC ALLOGRAFT MODEL

Pregraft transfusion combined with immunosuppression at the time of grafting improves the survival of clinical and experimental allografts. The mechanisms responsible for this effect were investigated in the murine model of cardiac transplantation, combining transfusions 7 to 30 days prior to transplantation with cyclosporine 100 mg/kg, 7 to 20 days pregraft or on days 0, 4, and 6 after grafting. Pregraft DST, third-party blood, and CsA all improved graft survival in the BALB/c-to-CBA donor-recipient combination. In animals treated with DST at 14 days pregrafting, 4/9 grafts survived for greater than 100 days. In those given C57BL/6 blood, or CsA on days 0, 4, 6 postgraft, 1/9 grafts survived for greater than 100 days. When 10(7) spleen cells from DST-treated CBA mice with long-surviving BALB/c heart grafts were transferred to naive CBA mice that then received a BALB/c heart 24 hr later, the transferred cells prolonged graft survival, with all grafts functioning at greater than 40 days, and 4/7 at greater than 100 days. Selective removal of T cells from the spleen cell population prior to transfer showed that L3T4+ T cells, but not Ly-2+ T cells, were required to maintain BALB/c allografts. Combining a short course of CsA with DST was more effective than either treatment alone. The most effective combined treatment was DST at day -14 with 100 mg/kg CsA given on days 0, 4, and 6 postgrafting (8/10 grafts survived greater than 100 days). This treatment also induced splenic suppressor T cells of the L3T4+ Ly-2- phenotype. These results clearly show that L3T4+ splenic T suppressor cells are induced by donor-specific blood transfusion with or without CsA treatment, and that these cells play a role in maintaining long-term tolerance to allografts in the mouse heart transplant model.

The immunosuppressive properties of new oral prostaglandin E1 analogs

Prostaglandins play an important role in cell-mediated immune responses. Their clinical use has been limited by poor oral bioavailability, short half-lives, and significant toxicity profiles. We studied the immunosuppressive properties of new, synthetic, prostaglandin E1 (PGE1) methyl ester analogs (misoprostol, enisoprost) with oral bioavailability using an allogeneic in vitro immunoassay. Our results show that the PGE1 analogs suppress alloproliferative responses and supplement the immunosuppressive activity of cyclosporine and methylprednisolone. Moreover, we demonstrate that addition of recombinant interleukin-2 to the PGE1 analogs restores alloimmune responsiveness and the expression of surface class II antigen and IL-2 receptors on responder lymphocytes. These studies, together with preliminary in vivo data in rodents and man, suggest that the new synthetic oral PGE1 analogs may provide therapeutic efficacy in clinical transplantation and a variety of immunologically mediated diseases.

Adverse influence of recipient lymphoid resistance to in vitro immunosuppression on the outcome of kidney transplants.

The study investigated whether preoperative in vitro sensitivity of lymphocytes from potential renal transplant recipients could identify patients at increased risk of acute rejection following transplantation and immunosuppression with cyclosporine, azathioprine, and prednisolone. Mixed lymphocyte culture responses were measured preoperatively in the presence of methylprednisolone, CsA, and antithymocyte globulin, and without immunosuppressive agents in 50 transfused recipients of primary cadaver renal transplants. Patients were classified as sensitive if all three immunosuppressive agents produced more than 50% inhibition of their MLC responses, and as resistant if one or more agents failed to produce 50% inhibition. All patients received postoperatively a standardized triple immunosuppressive regimen. Acute rejection was confirmed histologically and treated with Pred with or without ATG or monoclonal antibody OKT3. A total of 29 patients (58%) were sensitive and 21 (42%) were resistant; 4 patients were resistant to 3 agents, 5 were resistant to MP and ATG, 6 were resistant to MP and CsA, and 6 were resistant to MP alone. Sensitive and resistant groups did not differ in age, sex, transfusion history, HLA A, B and DR mismatches or duration of follow-up. The resistant group had a higher rate of graft loss from acute rejection (chi 2 = 6.0, d.f. = 1, P less than 0.02), more episodes of acute rejection (chi 2 = 8.7, d.f. = 3, P less than 0.05), and a higher proportion of patients in whom reflux nephropathy was the cause of renal failure (chi 2 = 18.3, d.f. = 1, P less than 0.001). The resistant group also had a higher proportion of highly sensitized patients and higher serum creatinine concentrations than the sensitive group, although the differences did not reach statistical significance. The study indicates that patients at high risk of acute rejection of renal allografts can be identified by a pretransplant in vitro assay, a finding that could influence recipient selection and immunosuppression.

Pretransplant transfusion and cyclosporine-induced enhancement of rabbit skin allografts: Donor-specific versus third-party blood

Lassociation de transfusion et de cyclosporine ne semble induire une bonne facilitation que si le sang transfuse est specifique du donneur

Human renal allograft rejection is predicted by serial determinations of antibody-dependent cellular cytotoxicity.

SUMMARY The early recognition and prompt treatment of rejection may minimise damage to renal allografts. Preliminary studies showed whole blood effector cell function in a constant antibody-dependent cellular cytotoxicity (ADCC) assay system to be suppressed in recipients with stable renal function. To investigate the possible role of ADCC in the rejection process, serial estimates were performed in 29 consecutive recipients of cadaveric kidneys. The generation of ADCC, as measured in vitro, preceded the biochemical confirmation of an impending rejection episode by 3 to 5 days for 31 of 33 episodes experienced by 23 recipients. Statistical analysis (x2) demonstrated a highly significant correlation between ADCC generation as measured in vitro and subsequent episodes of graft rejection. In contrast, six recipients who did not experience rejection episodes during the first 3 months postgrafting showed no evidence of ADCC activity. Thus, ADCC may be used to identify rejection earlier than has previously been possible.

Effects of Cyclosporin A, Antilymphocyte Serum and Donor-Specific Transfusions on Murine Delayed-Type Hypersensitivity and Skin Graft Survival

The effect of immunosuppressive reagents cyclosporin A (CsA) and rabbit anti-mouse antilymphocyte serum (ALS) on the response to alloantigens was studied in inbred mouse strains. Alloantigen was given either as a cell suspension which induced a delayed-type hypersensitivity reaction (DTH), or as a full-thickness skin graft. Dose-response studies showed that DTH reactions in CBA mice sensitised to BALB/c cells were reduced to background levels when recipient mice were treated with 100 mg/kg CsA on days 0, 4 and 6 after primary alloantigenic challenge. The response to a second challenge was significantly decreased by CsA treatment during primary or secondary exposure to alloantigen and CsA was as effective as ALS in abrogating both primary and secondary DTH reactions. Survival of full-thickness grafts of BALB/c skin on CBA mice was increased from 9 to 23 days by ALS treatment on days -1 and +2, with grafts given on day 0. Long-term treatment with CsA, from day -14 to +12, also prolonged graft survival from 9 to 18 days but donor-specific transfusions, with or without concomitant ALS or CsA treatment, decreased graft survival and often sensitised the recipients. This occurred with transfusions administered from -63 to -7 days and on the day of grafting. Thus, in H-2 mismatched mice, both CsA and ALS treatments produced a state of tolerance when administered during short-term exposure to alloantigen.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of whole blood antibody dependent cellular cytotoxicity by heat aggregated human IgG.

Inhibition of the whole blood antibody dependent cellular cytotoxicity (ADCC) of a lymphoblastoid cell line by heat aggregated human IgG (HAI) is described. Optimal sensitising antibody and effector cell concentrations were established to permit the detection of 0.1 microgram/ml HAI. Conditions which avoid the ADCC inhibitory effects of normal human sera were defined. Twelve paired normal human sera were stored at -70 degrees C for prolonged (greater than 3 years) and shorter (less than 6 months) periods of time. Sensitised sheep red cells were used to determine complement depletion in serum dilutions heated at 40 degrees C, 45 degrees C, 50 degrees C. Concentrations of human IgG (1 000-0.1 microgram/ml) were prepared in foetal calf serum before heating at 63 degrees C for 30 min to aggregate the IgG. ADCC inhibitory activity, frequently characteristic of normal human serum was minimised (less than 10%) when sera were stored at -70 degrees C and heated to 50 degrees C for 30 min to inactivate serum complement. This assay provides an economical, reproducible and sensitive test for the detection of circulating IgG complexes.

Characterisation of cyclosporine-induced suppressor cells in murine delayed-type hypersensitivity responses.

The effect of Cyclosporine A (CsA) on T cell-mediated delayed-type hypersensitivity (DTH) reactions to murine alloantigens was analysed by transferring spleen cells from sensitised and suppressed mice into irradiated naive recipients. Selective removal of Thy1+, Ly1+ or Ly2+ cells prior to intravenous transfer of cells from alloantigen-sensitised mice showed that Ly1+ Thy1+ cells transferred sensitivity. Concomitant treatment of mice with alloantigen and CsA suppressed the DTH response to alloantigens, and spleen cells transferred from these suppressed mice abrogated the response of sensitised cells transferred at the same time. The suppressor cells were strain-restricted and antigen-specific with the surface phenotype Thy1+, Ly1-Ly2+.