DC10 suppression of IgG1 secretion by OVA-specific B cells is antigen-specific, but requires DC presentation of intact allergen on their cell surface.
(A) To assess the antigen specificity of DC10-dependent IgG1 plasma cell suppression, we pulsed DC10 with no antigen (no Ag), an irrelevant allergen (house dust mite; HDM), intact OVA protein or OVA peptide323-39 and assessed their abilities to inhibit IgG1 secretion as above. The DC10 and lung cells were incubated for 24 h in 96-well U-bottom plates, and then transferred into ELISPOT plates for 5 h. (B) To confirm that DC10 presentation of intact cell surface-associated allergen to B cells (versus processed allergen) is required for suppression of IgG1 production, we pulsed DC10 with OVA for 2 h and washed them, and then either used them without allowing any further time for antigen processing (- OVA process.), or we returned them to the incubator overnight to allow full allergen processing (ON OVA process.). We then assessed the abilities of both populations to suppress either OVA-specific IgG1 secretion as above (left panel) or, as a control, immunostimulatory DC (DC-LPS)-activated asthmatic CD4+ T cell proliferative responses (right panel) by use of standard 3H-thymidine uptake assays. For suppression of OVA-specific IgG1 plasma cells, the cells were co-incubated for 24 h in 96 U-bottom plate before transfer into ELISPOT plates for 5 h. Control wells for the T cell suppression assay included lung CD4+ T cells alone, DC-LPS alone, and DC-LPS and T cells, but without DC10. The DC10 that had been given time to process their allergen were unable to suppress IgG1 secretion but, as expected, were able to suppress the T cell response. This data is representative of three independent experiments (n = 4/group). Statistical analyses were performed using one-way ANOVA assays with Tukey’s post-hoc testing. *, ** and *** signify p<0.05, 0.01 and 0.001, respectively.