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Background: Recent studies suggest that the appearance of anti-HLA antibodies in the early posttransplant period is associated with an increased incidence of acute and chronic rejection months later. However, very little is known about the prevalence of anti-HLA antibodies at the time that the rejection episodes are diagnosed. The purpose of this study was to analyze retrospectively 420 sera from 263 renal allograft recipients who were readmitted to the hospital for any reason between 1989 and 1998 in order to determine if a correlation existed between the presence of donor-specific anti-HLA antibodies and graft rejection., Methods: Sera were assayed for IgG HLA class I and II antibodies by ELISA. The ELISA results were analyzed using contingency tables with Fisher's exact test and compared with mismatched antigens in the donor., Results: Antibodies to donor HLA class I molecules in the posttransplant sera were extremely rare, occurring in only 6 of the 420 sera (1.4%) analyzed. Antibodies to donor class II antigens were slightly more common, occurring in 25 of the 420 sera (6%). In 21 of these 25 cases (84%), the presence of donor-specific HLA class II antibodies was associated with episodes of either acute (n=14) or chronic rejection (n=7). Five patients had antibodies to both class I and class II donor antigens, and all five of them lost their grafts to rejection., Conclusion: Although the presence of donor-specific HLA antibodies presented a significant risk for acute or chronic rejection, 77% of all acute and chronic rejections occurred in patients without detectable HLA antibodies.

The improvement of graft survival over the past decade has mainly been due to the development of more highly specific immunosuppressive agents, such as cyclosporine (CsA) and FK506. CsA and FK506 inhibit T cell activation by interfering with the calcium-mediated pathway, one of two pathways needed for T cell activation. The other pathway, mediated by protein kinase C (PKC), is not currently a target of any clinically used immunosuppressive agent. The purpose of this study was to assess the immunosuppressive properties of Ro 31-8220, a member of a new family of potent and selective PKC inhibitors. Peripheral blood mononuclear cells were isolated from the blood of normal human donors and utilized in a series of standard immunological assays. Three discrete activation events were inhibited by Ro 31-8220: mitogen-induced interleukin (IL)-2 production (IC50 80 nM), IL-2-dependent T lymphoblast proliferation (IC50 350 nM), and IL-2Ralpha (CD25) expression (control cells were 83% CD25+, mean fluorescence intensity = 163 +/- 4, 400-nM-treated cells were 56% CD25+, mean fluorescence intensity = 130 +/- 7). Noninhibitory doses of CsA (8 nM) or FK506 (0.2 nM) suppressed mitogen-induced IL-2 production by 60-80% when combined with a noninhibitory dose (25 nM) of Ro 31-8220, indicating the potent synergy between these agents. The ability of Ro 31-8220 to inhibit both early and late activation events and to synergize with CsA/FK506 suggests that this family of compounds has great potential as immunosuppressive agents and as probes with which to elucidate the role of PKC in T cell activation.

AM1 (M17) is the major metabolite of cyclosporine found in the blood of human transplant recipients, and trough levels of this derivative exceed those of the parent compound approximately two-fold. Studies performed in vitro indicate that AM1 retains only 10-20% of the biological activity of the parent compound, but very little is known about its in vivo immunosuppressive effects. We therefore developed a rapid and sensitive method, based on the rejection of allogeneic L1210 (H-2d) leukemia cells by C57BL/6 (H-2b) mice, to assess the immunosuppressive activity of AM1 in vivo. Rejection of the leukemia allograft was determined by analyzing the spleens from mice injected intravenously with 10(5) L1210 cells for the presence of H-2Kd-positive cells by flow cytometry using an FITC-conjugated monoclonal anti-H-2Kd antibody. Nonimmunosuppressed mice rejected the allogeneic cells and survived indefinitely. Spleens from these mice were virtually free of H-2Kd-positive cells (0.51 +/- 0.21%) by day 7. In contrast, C57BL/6 mice treated with 10 mg/kg/day s.c. of CsA all died from the L1210 challenge (mean survival time of 9 +/- 1 days). Spleens from mice treated in this manner contained 11.02 +/- 3.31% H-2Kd-positive cells on day 7. There was a direct correlation between the dose of CsA administered (7.5-50 mg/kg/day) and the percentage of H-2Kd-positive cells in the spleen. We then compared the immunosuppressive activity of AM1 and CsA in this model. AM1 was purified from the urine of CsA-treated renal allograft recipients by a combination of preparative adsorption-desorption chromatography and preparative elution high-performance liquid chromatography. AM1 at a dose of 10 mg/kg/day exhibited no demonstrable immunosuppressive effect, and trough levels of AM1 on day 7 were only 36 +/- 4 ng/ml. Increasing the dose of AM1 to 50 mg/kg/day resulted in only 1.05 +/- 0.16% H-2Kd-positive cells in the spleens (P = NS) and a mean trough level of 221 +/- 27 ng/ml. In contrast, mice treated with 50 mg/kg/day of CsA exhibited 17.7 +/- 2.9% H-2Kd-positive cells in their spleens and a mean trough CsA level of 3036 +/- 277 ng/ml. The half-life of a single subcutaneous dose of 10 mg/kg of AM1 (4.6 hr) was significantly shorter than that of CsA (9.7 hr) in mice. Compared with CsA, the lack of immunosuppressive effect of AM1 in vivo therefore appears to be due to a combination of decreased immunosuppressive activity and increased rate of clearance in mice.

We have evaluated the effects of CsA, methylprednisolone (MP), 6-mercaptopurine (6-MP), and FK506 on T cell-dependent and T cell-independent immunoglobulin production. FK506 and 6-MP were potent inhibitors of IgG and IgM production by PWM-stimulated peripheral blood mononuclear cells, which depend on the presence of T cells. CsA was less effective in this system and MP actually enhanced IgG and IgM production. In order to assess the direct effects of these various immunosuppressive agents on B cells, we utilized human B cell lines representing different stages of B cell differentiation. The B cell lines CESS and SKW6.4 exhibit increased production of IgG and IgM, respectively, in response to interleukin-6. These cells represent activated, but not fully differentiated, B cells. CsA inhibited IL-6-induced IgG production by CESS cells by 64% at 100 ng/ml and 6-MP inhibited this response by 82% at 250 ng/ml. Neither CsA nor 6-MP effectively inhibited IL-6-induced IgM production by SKW6.4 cells. MP at 250 ng/ml inhibited IL-6-induced IgG production by 89%, but enhanced IL-6-induced IgM production more than two-fold. FK506 did not inhibit IL-6-induced IgG or IgM production, suggesting that it has no direct effect on the ability of B cells to respond to this differentiation factor. These experiments clearly demonstrate that CsA, MP and 6-MP have direct inhibitory effects on the response of human B cells to IL-6. In contrast, FK506 has no direct effect on these B cells lines, but is more potent than the other agents at inhibiting T cell-dependent immunoglobulin production.

The in vitro biological activity of cyclosporine (CsA) and four of its metabolites (M1, M8, M17, and M21) was determined. M1, M17, and M21 are primary metabolites, while M8 is a secondary metabolite derived from either M1 or M17. The order of inhibitory activity in production assays was phytohemagglutinin (PHA), concanavalin A (ConA), mixed lymphocyte culture (MLC), and interleukin-2 (IL-2) CsA greater than M17 greater than M1 greater than M21 much greater than M8. In the PHA assay, CsA was significantly more inhibitory than M17, but in Con A and MLC assays, the inhibitory activity of M17 approached that of CsA. More importantly, M17 and M1 inhibited the production of IL-2 in the MLC to the same extent as CsA. M21 was significantly less inhibitory than either M17 or M1, and M8 appeared to be largely devoid of biological activity. These experiments demonstrate that single hydroxylations of amino acids 1 (M17) and 9 (M1) do not significantly affect the ability of the molecule to block IL-2 production, but hydroxylation of both amino acids renders the molecule virtually inactive. In addition, the presence of the N-methyl group on amino acid 4 appears to be very important, since removal of this group (M21) greatly diminishes the immunosuppressive activity.

Cyclosporine levels by radioimmunoassay (RIA) and high-performance liquid chromatography (HPLC) were monitored in serial blood samples (n = 177) from 11 renal allograft recipients. HPLC analysis revealed three primary metabolites of CsA (M17, M1, and M21) in peak and trough blood samples; M17 was the preponderant metabolite. In 4 patients on whom serial metabolite assays were performed, M17 was found in the blood at 86-2004 ng/ml; M1 and M21 were found at up to 100 ng/ml. The immunosuppressive properties of purified metabolites M1, M17, M21, and M8 (which was not detected in the blood) were compared with CsA. M17--and, to a lesser extent, M1 and M21--were found to inhibit the in vitro response of human mononuclear cells in the mixed leukocyte culture and in mitogen (phytohemagglutinin [PHA], concanavalin A [Con A], and pokeweed mitogen [PWM]) assays at 1000 ng/ml. M8 exhibited no in vitro inhibitory activity. M17 was further tested at 10-1000 ng/ml in PHA and mixed lymphocyte culture (MLC) assays. M17 had considerably less inhibitory activity (12-43%) than CsA (18-70%) in the PHA assay. However, in MLC experiments M17 blocked the proliferative response by 39-72% at 100-800 ng/ml, which approached the degree of inhibition exhibited by CsA (63-87%). In 34 of 37 (92%) patient blood samples, the level of metabolite M17 was found to exceed the parent drug level and could not be measured accurately by RIA. The observed in vitro immunosuppressive activity of metabolites (particularly M17) and their presence in the blood of renal allograft recipients suggest a possible role for these metabolites in the immunopharmacology of CsA.