CLINICAL IMMUNOSUPPRESSION - KIDNEY EARLY

T. Kuzuya¹, M. Kato², K. Iwasaki³, M. Haneda³, Y. Miwa³, T. Kobayashi³, K. Uchida⁴, K. Yamada^1
1Pharmacy, Nagoya University Hospital, Nagoya/JAPAN, ²Kidney Center, Nagoya Daini Red Cross Hospital, Nagoya/JAPAN, ³Applied Immunology, Nagoya University School of Medicine, Nagoya/JAPAN, ⁴, Nagoya Daini Red Cross Hospital, Nagoya/JAPAN

Body: Introduction: Pharmacodynamic (PD) monitoring strategies constitute novel approaches for optimization of immunosuppressive therapy. Until now there are many approaches for PD monitoring of calcineurin inhibitors (CNIs) using T-cell responses such as cytokine expression or T cell proliferation. However, little is known about B-cell responses with these agents. The purpose of this study was to evaluate the effect of cyclosporine A (CsA) on B-cell responses using peripheral blood mononuclear cells (PBMC) and assess its clinical applicability. Methods: PBMC obtained from three healthy subjects (three times at the different day) were stained with carboxy fluorescein diacetate succimidyl ester (CFSE), and incubated with CsA in 96 well with anti-IgM antibody or phytohemagglutinin (PHA) stimulation. After incubation for 3 days in 5% CO₂ at 37^oC, the cells were stained with allophycocyanin mouse anti-human CD19 monoclonal antibody or phycoerythrin-conjugated CD3. Three-color flow cytometry was performed on a FACS Calibur dual-laser cytometer (Becton Dickinson, Mountain View, CA). Concentration of CsA and the rate of the inhibition of cell proliferation were plotted and analyzed by the sigmoid Emax model using GraphPad Prism 4 (GraphPad Software, San Diego, CA). The degree of inhibition observed at the different CsA concentrations was estimated by a 50% inhibitory concentration (IC50) and the maximum percentage inhibition which is obtained by the subtracting the bottom value from 100%. Statistical analysis was performed using one way ANOVA. p values <0.05 were considered statistically significant. Results: B-cell proliferations were inhibited in relation to an increase in CsA concentration not only stimulated by PHA but also anti-IgM antibody and the relationships could be evaluated by the inhibitory sigmoid Emax model. Each value of IC50 with anti-IgM antibody showed specificity among the subjects (mean ± SEM; 19.1 ± 2.1, 7.6 ± 1.0, 13.1 ± 1.4 ng/mL, p<0.05), although the maximum percentage inhibition showed no significant different (8.5 ± 1.9, 7.2 ± 4.8, 5.2 ± 0.6 %, p = 0.7). The value of IC50 with PHA stimulated T-cell showed significant different among the subjects (35.0 ± 2.1, 58.3 ± 1.6, 94.6 ± 1.7 ng/mL, p<0.001). And the value of IC50 of B-cell was lower than that of T-cell.Conclusion: The effect of CNIs on B-cell is not well understood, but this study indicated that CsA sufficiently inhibited anti-IgM antibody stimulated B-cellproliferation. And one of the PD parameters (IC50) showed a interindividual variability among the subjects. Antibodies against HLA and MHC class I related chain A antigens is well known to be closelyrelate to both acute vascular rejection and chronic rejection. It is a possibility that this PD assay could be a useful tool on optimal and safe immunosuppressive therapy.

Disclosure: All authors have declared no conflicts of interest.