Flt3 expression by wild type splenic DCs.
Before investigating the impact of constitutive Flt3 signaling in DCs, we first investigated the expression and regulation of the Flt3 receptor in primary DCs. In agreement with previous studies (31), cell-surface Flt3 was detected on splenic cDC1, cDC2 and pDC, but not T or B lymphocytes. The highest expression was observed on cDC1 that are known to preferentially expand in response to Flt3L (32, 33) (Figure 1A ). Total Flt3 protein expression was determined for purified cell populations with two forms of Flt3 detected by immunoblotting, similar to non-DC cell lines (34, 35) (Figure 1B, Supplementary Figure 1A ). The higher molecular weight (MW, approximately 160 kDa) species is likely cell surface Flt3, given its slower migration due to being highly glycosylated. The lower MW (approximately 130 kDa) band is likely intracellular Flt3 (34, 35). Flt3 was only detected by immunoblot in cells displaying it at the cell surface; cDC1, cDC2 and pDC. The detected pattern of Flt3 varied between DC populations, with pDC expressing a higher MW species of cell surface Flt3 than cDC1 or cDC2. To determine if terminally differentiated cDC are responsive to Flt3L, primary murine splenic cDCs were isolated and stimulated with Flt3L (100 ng/mL) for 1 hour. Cell surface Flt3 expression was significantly reduced on both cDC subsets following in vitro Flt3L stimulation (Figure 1C ).
Next, Flt3 expression during cDC activation was characterized. Splenic cDCs were purified and activated in vitro with toll-like receptor 9 (TLR9) agonist CpG 1668 ODN (class B, 0.5 uM). Flt3 protein, detected by immunoblotting, showed an increase in intensity for higher MW Flt3, and a loss of lower MW Flt3, following CpG treatment (Figure 1D, Supplementary Figure 1B ). In agreement, elevated surface Flt3 was detected by flow cytometry (Figure 1E ). To investigate if this were also the case following cDC activation in vivo , mice were injected intravenously (IV) with CpG (0.2 µM) or PBS. cDCs were purified one day later and Flt3 expression determined by flow cytometry (Figure 1F ). In contrast to in vitro DC activation, a significant reduction in cell surface Flt3 was observed. Similar results were observed when DCs were isolated from mice IV injected with LPS (3 µg) or poly I:C (50 µg) (Supplementary 1C ). A short time course, with spleens harvested from mice 0 – 4 hours after CpG IV injection showed surface Flt3 loss by cDC1 as rapidly as one hour after CpG administration, while cDC2 displayed the first signs of surface Flt3 reduction four hours following CpG administration (Figure 1G ). For both in vitro and in vivo treatment with CpG, increased CD86 was used as a positive control for cDC activation (Supplementary Figure 1D ). Together, these data demonstrate that splenic DCs express and regulate Flt3 at steady-state and during activation in vitroand in vivo. The contrast in Flt3 expression between in vitro and in vivo activation is likely due to Flt3L-dependent receptor internalization following CpG treatment in vivo. This is not observed in vitro due to limited Flt3L and therefore receptor internalization.