DISCUSSION
Flt3 is critical for DC development, but it is unclear how Flt3 expression and signaling impacts terminally differentiated cDCs. In this study we investigated Flt3 expression and determined how constitutive Flt3 signaling downstream as a result of the ITD mutation impacts DC number, phenotype and function.
In agreement with previous studies (31, 39), we identified Flt3 expression by WT splenic cDC and pDC populations. To our knowledge, this is the first time both surface and intracellular Flt3 protein in primary cDC1, cDC2 and pDC have been measured. Interestingly, surface Flt3 protein in pDCs migrates more slowly by SDS-PAGE than Flt3 expressed by cDC1 and cDC2. There are no described isoforms for murine Flt3. Therefore it is unlikely that pDC express an alternative Flt3. Flt3 is N-linked glycosylated (34, 35), and the larger form observed in pDCs likely represents Flt3 that is more heavily decorated with carbohydrate moieties. While beyond the scope of this study, Flt3 glycosylation in DCs has yet to be investigated and as such further investigations are required to fully characterise Flt3 in pDC.
We observed regulation of Flt3 expression during cDC activation, with increased surface Flt3 following activation in vitro. In contrast, surface Flt3 is reduced when splenic cDCs are activated by different inflammatory stimuli in vivo . It is likely the in vivo reduction is due to increased serum Flt3L in response to TLR stimulation (40) that engages and downregulates Flt3. Flt3/Flt3L signaling promotes survival of terminally differentiated DCs, with increased apoptosis observed in bone marrow derived DC treated with Flt3 tyrosine-kinase inhibitors (14). Therefore, upregulation of surface Flt3 during inflammation, as evidenced under in vitro conditions, likely increases cDC responsiveness to Flt3L, and promotes their survival and immunogenic potential in vivo .
We identified Flt3ITD/ITD DCs undergoing Flt3L-independent Flt3 signaling. This correlated with increased numbers of splenic cDC and pDC populations in Flt3ITD/ITDmice. In agreement with previous studies (25), the largest expansion was NC cDC1. This differs with Flt3L administration to Flt3+/+ mice, which expands canonical cDC1 populations (25, 26). While previous studies have assessed the transcriptional profiles of DC populations in Flt3-ITD mice (25), the functional consequences have not been previously examined. Our data demonstrate that constitutive Flt3 signaling downstream of Flt3-ITD, leads to aberrant splenic DC phenotype and function. Elevated MHC II surface expression was observed on Flt3ITD/ITD DC (except NC cDC1). Similarly, Flt3ITD/ITD NC cDC1 and cDC2 displayed increased surface MHC I. Flt3ITD/ITD cDC2 exhibited increased antigen uptake, more rapid antigen proteolysis and more rapid antigen access to acidic, proteolytic intracellular compartments. These changes culminated in improved MHC II presentation of cell-associated antigen, but not soluble antigen, by these cells. Flt3-ITD promotes basal autophagy in cancer cell lines (non-DC) and Flt3-ITD+ patient samples, while inhibition or knockdown of Flt3-ITD reduces autophagic flux (41). This may account for elevated CD4+ T cell priming since LC3-associated phagocytosis (LAP) and autophagy contribute to MHC II presentation (42). Future experiments investigating autophagy in Flt3ITD/ITD DCs are of interest, particularly given its role in cDC antigen presentation (43, 44).
Our investigation of how constitutive Flt3 signaling impacts DC antigen presentation outcomes identified that MHC I cross-presentation of both cell-associated and soluble OVA was largely unchanged for Flt3ITD/ITD DC subsets, with the exception of NC cDC1. Here we show Flt3ITD/ITD NC cDC1 cannot cross-present cell-associated OVA but are capable of cross-presenting soluble OVA. To date, the immunological function of NC cDC1s is elusive. These cells were initially considered DC progenitors (29). More recent analysis, however, renders this unlikely (45). Human NC cDC1s can stimulate allogenic T cells (28–30) and they accumulate with pDC in influenza-infected lung (45). Our analysis, in agreement with others (26, 45), shows Flt3ITD/ITD NC cDC1s have impaired IL-12 production. This may stem from reduced surface DEC205 which would render them less responsive to CpG (46). The rare frequency of Flt3+/+ NC cDC1s means our analysis does not permit us to conclude whether these are intrinsic functions of NC cDC1 or whether the cells are displaying altered function due to constitutive Flt3-ITD signalling.
In conclusion, we have demonstrated that splenic primary DCs regulate Flt3 and are responsive to Flt3L. Constitutive Flt3 signaling, due to the Flt3-ITD mutation, leads to significant changes to splenic DC poulations with the development of cDCs that have an altered phenotype, including increased MHC expression, pro-inflammatory cytokine secretion and improved MHC II antigen presentation of cell-associated antigen. These data highlight the impact of Flt3 signalling on DC biology which is of particular interest given the increasing use of Flt3L as an adjuvant in settings of vaccination and immunotherapy. In addition, this research assists in characterizing the phenotype and function of DCs in patients that express the Flt3-ITD mutation and to assess their contribution to leukemogenesis.