Flt3-ITD impacts DC function
For efficient T cell priming, DCs present antigen on MHC molecules (signal 1) in the presence of costimulatory molecules (signal 2) and cytokines (signal 3). Given we observed changes in signal 2 (Figure 3 ), we next focused on signal 1 and signal 3. Antigen presentation first requires DCs to capture antigen and efficiently degrade it. Therefore, antigen uptake, endosomal pH and proteolysis were assessed. Ideally, DP, NC, and SP cDC1 would be isolated for both Flt3+/+ and Flt3ITD/ITD mice. The low number of cDC1 subpopulations in Flt3+/+ mice, however, meant this was not feasible. Therefore, Flt3ITD/ITD DP, NC, and SP cDC1 were compared with Flt3+/+ cDC1, and Flt3ITD/ITD cDC2 were compared with Flt3+/+ cDC2. First antigen uptake was examined. cDCs were incubated with OVA-Cy5 for 15 and 90 mins or dextran-A647 for 15 mins and the intracellular fluorescence determined by flow cytometry. Overall, similar uptake of either OVA protein or dextran was observed for Flt3+/+ and Flt3ITD/ITD cDC1 populations, although Flt3ITD/ITD DP cDC1 showed a trend towards reduced uptake compared with the other subsets. In contrast, OVA protein and dextran uptake by Flt3ITD/ITD cDC2 was elevated compared with Flt3+/+ cDC2 (Figure 4A, Supplementary Figure 4A ).
To investigate Flt3ITD/ITD cDC2 in more detail, their ability to undertake antigen proteolysis was examined using DQ-OVA, a self-quenched form of OVA which fluoresces as it undergoes proteolysis. Purified cDCs were pulsed with DQ-OVA, excess antigen removed and cDCs examined by flow cytometry at the indicated time points. OVA-Cy5 was used to normalize for the increased uptake of DQ-OVA by Flt3ITD/ITD cDC2 compared with Flt3+/+ cDC2. Flt3ITD/ITD cDC2 showed more rapid DQ-OVA proteolysis with the signal plateauing at 30 mins as compared with 60 or 90 mins for Flt3+/+ cDC2. This was accompanied by reduced endosomal pH for Flt3ITD/ITD cDC2 as detected by the pH sensitive probe, dextran-pHrodo, which increases fluorescence as pH decreases (Figure 4B, Supplementary Figure 4B ). Therefore, constitutive Flt3-ITD signalling enhances antigen uptake and proteolysis by cDC2.
Next, cytokine secretion (signal 3) by Flt3+/+ and Flt3ITD/ITD cDCs was assessed. Cells were sorted to purity and equal numbers were incubated with CpG and inflammatory cytokines IFNγ and GM-CSF, conditions that elicit maximal DC IL-12 production (36). Supernatants were harvested after 18 hours and concentrations of IL-6, IL-10, IL-12p70, TNF-α and MCP-1 measured using the BD Cytometric Bead Array Assay. IL-10 and MCP-1 were not detected by cDC1 or cDC2, and IL-12 was only detected for cDC1. Robust cytokine secretion was observed for Flt3+/+ cDC1 and Flt3ITD/ITD DP cDC1. In contrast, Flt3ITD/ITD NC cDC1 produced significantly reduced IL-6, TNF-α and IL-12 in comparison to other cDC1 subsets. Similarly, Flt3ITD/ITD SP cDC1 produced reduced TNF-α and IL-12 (Figure 4D ). For cDC2, Flt3ITD/ITD DCs produced increased IL-6 and similar TNF-α in comparison to Flt3+/+ DC (Figure 4E ).