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.