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 ).