Flt3-ITD impacts cDC antigen presentation.
Next the antigen presenting capabilities of Flt3ITD/ITD cDC subsets were investigated. Due to the expansion of DCs (Figure 2 ) and regulatory T cells in Flt3+/ITD mice (25) in vitro , rather thanin vivo , antigen presentation assays were performed to enable direct comparisons of equal numbers of Flt3+/+ versus Flt3ITD/ITD cDCs in the absence of an altered extracellular environment. Spleens were harvested and cDCs isolated by sorting to purity by flow cytometry. cDCs were incubated with CellTrace-Violet (CTV)-labelled OT-I (Figure 5A) or OT-II (Figure 5B) cells in the presence of cell-associated OVA (splenocytes pulsed with OVA protein) or OVA protein alone. T cell proliferation was determined three days later. cDC1 subsets cross-present cell-associated OVA (37) whereas both cDC1 and cDC2 are capable of cross-presenting OVA protein in vitro (38). A significant defect in cross-presentation of cell-associated OVA was observed for Flt3ITD/ITD NC cDC1 in comparison to Flt3+/+ cDC1, while only minor changes were observed for MHC I presentation of OVA protein (Figure 5A ). In contrast, Flt3-ITD largely improved MHC II antigen presentation for both antigens tested, with increased OT-II proliferation observed for Flt3ITD/ITD NC, SP and cDC2 in response to cell-associated OVA, and increased presentation of OVA protein by Flt3ITD/ITD NC and SP DCs (Figure 5B ). Together, these data demonstrate that Flt3 signaling impacts MHC I and MHC II antigen presentation of both cell-associated and soluble OVA.