Abstract

Immunosuppressive drugs used in patients (pts) after stem cell / organ transplantation (Tx) as well as in pts with autoimmune disease are known to impair the cellular immune response. This results in an increased incidence of viral infections and viral associated malignancies which has been ascribed to the effect of immunosuppressive drugs on lymphocytes. However, in vitro data indicate that immunosuppressive drugs also target Dendritic Cells (DCs), the most potent antigen-presenting cells and initiators of lymphocyte responses. So far, most studies are based on in vitro data obtained with DC culture in the presence of different concentrations of single immunosuppressive drugs. To investigate the effect of immunosuppression on DC phenotype and function in vivo, we quantitatively and qualitatively analyzed freshly isolated human BDCA-1(CD1c) positive DCs from 15 solid organ transplant (SOT) recipients under immunosuppressive treatment. The percentage of BDCA-1 positive cells among total PBMCs was not statistically different in pts vs ctrls (0,52 vs 0,65, p<0,18). BDCA-1 positive DCs were analyzed for expression of HLA class I and II, CD14, costimmulatory molecules and chemokine expression. Interestingly, CD14 was found to be significantly higher expressed on pt-DCs vs ctrl-DCs suggesting a more immature DC-phenotype. We observed a trend toward a reduced expression of HLA-DR and CD86 on pts-DCs as compared to ctrls-DCs (p=0,059). Surface profile of BDCA-1 positive DCs was also analyzed after 48h of LPS and CD40L stimulation. Here we found a marked upregulation of HLA-DR and CD86 in pts- DCs as well as ctrl-DCs. Supernatant of stimulated DCs was analyzed with cytokine capture beads for secretion of inflammatory cytokines. High secretion of IL-6, IL-1 beta and partially of TNF-alpha by stimulated DCs was observed in both groups. Other Th2 type cytokines (IL-10, IL-4, IL-5) and Th1 type cytokines like IFN-gamma and Il-2 were not significantly secreted. We additionally addressed the question if mature and functionally competent DCs could be generated ex vivo from this pts cohort. After 9 days of culture with GM-CSF, IL-4, IL-1, IL-6, TNF-alpha and PGE2 fully mature DCs could be generated. Co-culture of EBV-peptide-pulsed DCs with autologous T-cells resulted in significant expansion of EBV-specific T cells in pts and ctrls. These T cells were fully functional as shown by IFN-γ secretion detected by ELISPOT. In summary, this is the first analysis of freshly isolated BDCA-1 positive DCs from immunosuppressed pts. Our data support the notion that immunosuppressive drugs target DCs and contribute to a maturation defect of circulating blood DCs which may help to understand the mechanism of impaired cellular immune responses in immunosuppressed pts. However, ex vivo generated DCs from immunosuppressed pts do not show an impairment in phenotype and function, suggesting that they could be efficiently be used in immunotherapeutic strategies.

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