Flt3-ITD expands canonical and noncanonical splenic DC populations.
DC populations were analyzed in Flt3+/+ and Flt3ITD/ITD littermates. Three cDC1 subpopulations were analyzed: CD103- CD86-(hereafter referred to as noncanonical “NC cDC1”) (26), CD103+ CD86+ (“DP cDC1”) and CD103- CD86+ (“SP cDC1”), in addition to cDC2. All DCs were significantly increased in Flt3ITD/ITD mice, in agreement with previous studies (25) (Figure 2A ). Specifically, Flt3ITD/ITDcDC1, cDC2 and pDC were expanded 8.3, 4.1 and 4.2-fold respectively. For Flt3ITD/ITD cDC1 populations, a 12.8-fold increase in NC cells, 14.8-fold increase in DP cDC1 and 2.7-fold increase in SP cDC1 was observed. A gene-dosage dependent decrease in surface Flt3 was detected for Flt3ITD/ITD cDC1, cDC2 and pDC (25, 35) (Figure 2B ). Total Flt3 protein and Flt3 tyrosine phosphorylation status was examined following in vitro Flt3L (100 ng/mL) stimulation and Flt3 immunoprecipitation. Increased Flt3 tyrosine phosphorylation was observed for surface (higher MW) Flt3 in Flt3+/+ and Flt3+/ITD cDCs at 10 and 30 minutes. Flt3+/ITD and Flt3ITD/ITD cDCs exhibited ligand-independent tyrosine phosphorylation of intracellular (lower MW) Flt3 in the absence of Flt3L. In addition, only intracellular Flt3 exhibited tyrosine phosphorylation in Flt3ITD/ITD cDCs (Figure 2C, Supplementary Figure 2 ).