Selective Expression of the MAPK Phosphatase Dusp9/MKP-4 in Mouse Plasmacytoid Dendritic Cells and Regulation of IFN-ß Production.

Plasmacytoid dendritic cells (pDCs) efficiently produce large amounts of type I IFN in response to TLR7 and TLR9 ligands, whereas conventional DCs (cDCs) predominantly secrete high levels of the cytokines IL-10 and IL-12. The molecular basis underlying this distinct phenotype is not well understood. In this study, we identified the MAPK phosphatase Dusp9/MKP-4 by transcriptome analysis as selectively expressed in pDCs, but not cDCs. We confirmed the constitutive expression of Dusp9 at the protein level in pDCs generated in vitro by culture with Flt3 ligand and ex vivo in sorted splenic pDCs. Dusp9 expression was low in B220(-) bone marrow precursors and was upregulated during pDC differentiation, concomitant with established pDC markers. Higher expression of Dusp9 in pDCs correlated with impaired phosphorylation of the MAPK ERK1/2 upon TLR9 stimulation. Notably, Dusp9 was not expressed at detectable levels in human pDCs, although these displayed similarly impaired activation of ERK1/2 MAPK compared with cDCs. Enforced retroviral expression of Dusp9 in mouse GM-CSF-induced cDCs increased the expression of TLR9-induced IL-12p40 and IFN-ß, but not of IL-10. Conditional deletion of Dusp9 in pDCs was effectively achieved in Dusp9(flox/flox); CD11c-Cre mice at the mRNA and protein levels. However, the lack of Dusp9 in pDC did not restore ERK1/2 activation after TLR9 stimulation and only weakly affected IFN-ß and IL-12p40 production. Taken together, our results suggest that expression of Dusp9 is sufficient to impair ERK1/2 activation and enhance IFN-ß expression. However, despite selective expression in pDCs, Dusp9 is not essential for high-level IFN-ß production by these cells.

Unique requirements for reactivation of virus-specific memory B lymphocytes.

Memory B cells (MBCs) are rapidly activated upon Ag re-exposure in vivo, but the precise requirements for this process are still elusive. To address these requirements, T cell-independent reactivation of MBCs against virus-like particles was analyzed. As few as 25 MBCs are sufficient for a measurable Ab response after adoptive transfer. We found that MBCs were reactivated upon antigenic challenge to normal levels after depletion of macrophages, CD11c(+) dendritic cells, and matured follicular dendritic cells. Furthermore, MBC responses were possible in TNF/lymphotoxin α double-deficient mice after partial normalization of lymphoid architecture by means of long-term reconstitution with wild-type bone marrow. Activation did not occur when chimeric mice, which still lack all lymph nodes and Peyer's patches, were splenectomized prior to MBC transfer. Together with our finding that MBC responses are weak when Ag was administered within minutes after adoptive MBC transfer, these results strongly suggest that MBCs have to occupy specific niches within secondary lymphoid tissue to become fully Ag-responsive. We provide clear evidence that MBCs are not preferentially resident within the splenic marginal zones and show that impaired homing to lymphoid follicles resulted in significantly diminished activation, suggesting that reactivation of MBCs occurred inside lymphoid follicles. Furthermore, comparison of virus-specific MBC T cell-independent reactivation versus primary T cell-independent type II B cell activation revealed unique requirements of MBC activation.