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.