Th2-biased immunogenicity of stimulated cpdm BMDC
The defective IL12 production (Fig. 3A) and Th2-dominant cytokine profile in cpdm mice [5] suggest that the absence of SHARPIN affects the ability of cpdm BMDC to induce T cell differentiation into effector cells. Co-culture of allogeneic naïve CD4+ T cells with WT BMDC stimulated with LPS or poly I:C elicited robust IFNγ production, whereas the concentration of IFNγ in cpdm BMDC-T cell cultures was significantly lower after LPS stimulation (Fig. 7A), indicating impaired Th1-polarizing abilities of cpdm BMDC. In addition to TLR3/4 agonists, the TLR2 ligand Pam3CYS was used since it has been shown to induce both Th1 and Th2 responses [28]–[30]. Pam3CYS-matured WT BMDC induced robust IFNγ production at a significantly higher level than cpdm BMDC (Fig. 7A). The reduced Th1 differentiation following Pam3CYS stimulation is consistent with the recent report of decreased IL12 production in cpdm macrophages following TLR2 stimulation [31]. In contrast, more Th2-specific IL4 cytokine was produced in the cpdm BMDC co-cultures than the WT control (Fig. 7B), suggesting Th2-skewed immunogenicity of cpdm BMDC. The production of Th17-specific cytokine IL17A following LPS stimulation of dendritic cells was similar between stimulated WT and cpdm BMDC cocultures (data not shown). Despite the distinct Th1- and Th2-stimulating abilities of WT and cpdm BMDC, they were equally effective in IL2 production from BMDC-T cell co-cultures except for poly I:C stimulation where WT BMDC induced more IL2 than cpdm BMDC (Fig. 7C). The Th2-biased stimulating capability of cpdm BMDC when co-cultured with allogeneic naïve CD4+ T cells is consistent with the Th2-dominant cytokine phenotype observed in cpdm mice.
10.1371/journal.pone.0031809.g007 Figure 7  Stimulated cpdm BMDC induced Th2-biased cytokine production from naïve CD4+ T cells.
WT and cpdm BMDC (5×104 cells in 0.1 mL complete medium; MHC haplotype: H-2b) were incubated with medium, 100 ng/mL LPS, 25 µg/mL poly I:C or 5 µg/mL Pam3CYS. After 24 hours, cells were washed with PBS and incubated with freshly isolated allogeneic naïve CD4+ T cells (2.5×105 cells in 0.1 mL complete medium; MHC haplotype: H-2d). After 5 days, supernatants were collected and the secretion of IFNγ, IL4, and IL2 were measured by ELISA. Negative controls are 1) stimulated BMDC without co-culture with allogeneic CD4+ T cells; 2) allogeneic CD4+ T cells without co-culture with stimulated BMDC. Samples from both negative controls had no detectable production of the aforementioned cytokines (not shown). Results are analyzed based on 2–4 mice per group. * P<0.05.

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