INTRODUCTION
FMS-like tyrosine kinase 3 (Flt3) is expressed on hematopoietic
progenitors and terminally differentiated dendritic cells (DCs). The
receptor binds to Flt3 ligand (Flt3L) which is produced by hematopoietic
and non-hematopoietic tissues, with CD4+ T cells known
to secrete high amounts (1). Upon Flt3L stimulation, Flt3 is activated
(phosphorylated) and signals via the mitogen-activated protein kinase
(MAPK)(2), phosphatidylinositol 3 kinase (PI3K)(3) and signal
transducers and activators of transcription 3 (STAT3) pathways(4).
Flt3-dependent activation of these pathways is critical for DC
generation (3–5). When administered to mice (6, 7) or humans (8, 9),
Flt3L expands DCs and their progenitors. Conversely, mice lacking Flt3
(Flt3 -/-) (10) or Flt3L
(Flt3L -/-)(11) have significant defects in
hematopoiesis with reduced DCs observed (7, 11, 12). While the role of
Flt3 in DC differentiation is well established why terminally
differentiated DCs retain their high expression of Flt3 remains a
curiosity. Given Flt3L is constitutively present, albeit at low levels
(13), how Flt3/Flt3L signaling impacts DC function, in particular
antigen presentation, is of interest. Flt3 is proposed to regulate cDC
division (7), bone marrow-derived DC survival (14) and expression of
stimulatory genes in tumor-infiltrating conventional DC1 (cDC1) (15).
Given the increasing interest in the manipulation of Flt3/Flt3L in the
context of vaccination and tumor immunotherapy (16), it is important to
understand how Flt3 signaling impacts terminally differentiated DCs.
One approach to investigate how Flt3 signaling impacts DC function is to
examine DCs with constitutive Flt3 signaling. The Flt3-internal tandem
duplication (Flt3-ITD) mutation leads to constitutive ligand-independent
receptor activation (17–21) and occurs in approximately 20% of
patients with acute myeloid leukemia (AML) (22–24).
Flt3ITD/ITD mice develop splenomegaly, leukocytosis
and monocytosis attributed to increased survival and cell cycling of
bone marrow progenitor cells (21). Flt3ITD/ITD and
Flt3+/ITD mice have cell-intrinsic gene-dosage
dependent increases in cDCs, plasmacytoid DCs (pDC) and DC progenitors
(25). Analysis of Flt3ITD/+ mice suggests these DCs
exhibit a gene expression profile indicative of immune tolerance (25).
However, this has yet been confirmed by functional studies.
Flt3ITD/+ mice also possess expanded numbers of
noncanonical (NC) cDC. These cells, also known as
“Cx3CR1+ CD8+” or
“CD8 look-alike” DCs (25–27), have characteristics of cDC and pDCs
and have been identified in humans as “Axl+Siglec6+ (AS DC/DC5)” (28) and “pre-DC” (29).
Murine NC DCs are a population within transitional DCs (tDC) that share
a continuum of phenotypes associated with pDCs and cDC2 (30). In humans,
NC cDCs stimulate allogenic T cells (28, 29); however, little is known
about their antigen presentation capacity.
Here, we have performed an in-depth analysis of DCs isolated from
Flt3ITD/ITD mice and addressed how constitutive Flt3
signaling impacts DC antigen presentation capacity and function.
Constitutive Flt3-ITD signaling results in altered DC function. DCs
isolated from Flt3ITD/ITD mice have altered
populations with changes in the display of cell surface molecules,
cytokine secretion, antigen uptake, proteolysis and endosomal pH
culminating in significant alterations in antigen presentation.