|Mutation Type||critical splice donor site (1 bp from exon)|
|Coordinate||147,366,918 bp (GRCm38)|
|Base Change||C ⇒ T (forward strand)|
|Gene Name||FMS-like tyrosine kinase 3|
|Synonym(s)||Flt-3, CD135, Flk-2, wmfl, Flk2|
|Chromosomal Location||147,330,741-147,400,489 bp (-)|
|MGI Phenotype||Mice functionally null for this gene display abnormal lymphopoiesis. Homozygous ENU mutant mice are sensitive to infection by mouse cytomegalovirus.|
|Amino Acid Change|
|Institutional Source||Beutler Lab|
|Gene Model||not available|
|Predicted Effect||probably null|
|Phenotypic Category||decrease in NK cell response, decrease in NK cells, growth/size, hematopoietic system, immune system, MCMV susceptibility|
|Alleles Listed at MGI|
|Mode of Inheritance||Autosomal Recessive|
|Local Stock||Embryos, Sperm, gDNA|
|Last Updated||05/13/2016 3:09 PM by Anne Murray|
The warmflash (wmfl) phenotype was identified among ENU-mutagenized G3 mice in a screen for susceptibility to mouse cytomegalovirus (MCMV) (MCMV Susceptibility and Resistance Screen) (1). 100% of wmfl homozygotes died when infected with 2 x 105 pfu of MCMV, a normally sublethal inoculum (Figure 1A). Five days after infection with 105 pfu of MCMV wmfl homozygotes showed increased viral titers in the spleen and liver comparable to those observed in BALB/c mice (Figure 1B and 1C). Wmfl homozygotes also failed to control lymphocytic choriomeningitis virus (LCMV) (clone 13) infection (Figure 1D).
36 hours post-MCMV infection, exaggerated production of tumor necrosis factor (TNF)-α (Figure 2A) and IL-6 were noticeable in the serum of wmfl homozygotes, possibly driven by an increased viral load. In contrast, the level of interferon (IFN)-γ was significantly reduced (Figure 2B), and levels of interleukin (IL)-12p70 and type I IFN were moderately but not significantly reduced in the serum of wmfl mice relative to those of WT control mice (Fig 2C and 2D). The wmfl mutation did not impair virus recognition or alter TLR-mediated signaling in macrophages in vitro.
Host defense against MCMV depends on the function of NK cells that respond within the first two days of infection by expanding, producing IFNγ, and mediating cytotoxicity (2;3). Relative to controls, splenic NK1.1+CD3ε- cells were reduced in both percentage and number in wmfl homozygotes (Figure 3A), while bone marrow NK cells were slightly but not significantly reduced in percentage. The disproportionate scarcity of wmfl NK cells in the spleen suggested a defect in NK cell development that may contribute to MCMV susceptibility. The earliest inducible NK cell activation marker, CD69, acts as a costimulatory molecule for activation and proliferation. NK cells from wmfl mice underexpressed CD69 at 6 hours post MCMV infection, but upregulated expression to WT levels by 24 hours after infection (Figure 3B). In addition, a reduced percentage of wmfl NK cells produced IFNγ 24 hours following MCMV infection, but by 36 hours post-infection, comparable percentages of WT and wmfl NK cells were IFNγ+ (Figure 3C). Thus, the increased MCMV susceptibility of homozygous wmfl mice might potentially result in part from a functional defect in wmfl NK cells in which both activation marker expression and IFNγ production in response to MCMV infection are delayed.
Several tests demonstrated normal intrinsic function of wmfl NK cells. When stimulated in vitro by plate-bound antibodies against activating NK cell receptors such as NK1.1, NKp46 or Ly49D, or exposed to IL-12 or IL-12 plus IL-18, wmfl NK cells produced IFNγ and degranulated as well as WT NK cells. In vivo, wmfl NK cells were competent to kill Tap1-/- splenocytes as efficiently as WT NK cells (Figure 3D). Moreover, when NK cells from wmfl homozygotes or WT mice were transferred into Rag1-/-Il2rg-/- recipient mice (lacking B and T lymphocytes and NK cells), wmfl NK cells induced serum levels of IFNγ equivalent to those induced by WT NK cells 36 hours after MCMV infection (Figure 3E). When provided with either wmfl or WT NK cells, recipient mice survived for over 10 days after MCMV infection, demonstrating that wmfl NK cells are as competent as WT NK cells in protecting mice from MCMV-induced death. Thus, despite their diminished number in mice, wmfl NK cells are intrinsically capable of acquiring full functionality in response to activating stimuli in vitro and in vivo.
Dendritic cells (DCs) produce cytokines that promote cytotoxicity and proliferation by NK cells. In both bone marrow and spleen, the numbers of wmfl CD11c+ cells were significantly lower than those of WT mice, but percentages of wmfl CD11c+ cells were similar to those of WT mice (Figure 4A). Among conventional DCs (cDCs, CD8α–CD11b+CD11c+ plus CD8α+CD11b-CD11c+ cells) and plasmacytoid DCs (pDCs; CD11c+B220+PDCA-1+), the number of wmfl cDCs was reduced in bone marrow and spleen, and both the number and percentage of pDCs were significantly reduced in bone marrow and spleen, compared to those of WT mice (Figure 4B and 4C). However, mice depleted of pDCs do not recapitulate the MCMV susceptibility of wmfl mice (4), demonstrating that the reduction in pDCs cannot by itself account for the MCMV susceptibility of wmfl mice.
Conventional and CD8α+ DCs are infected by MCMV (5;6) and respond by producing type I IFN, IL-12 (7;8), and surface IL-15Rα (9-12) that drive the NK response against MCMV. Wmfl DCs produced reduced amounts of IL-12p40, type I IFN, and surface IL-15Rα relative to WT DCs infected with MCMV (Figure 5A-C). Similarly, wmfl DCs produced reduced IL-12p40, type I IFN and surface IL-15 in response to stimulation with either TLR7 or TLR9 ligands (Figure 5A-C). Notably, poly I:C, a TLR3 ligand, induced similar concentrations of type I IFN in cultures of wmfl and WT DCs. TLR4 stimulation with LPS elicited no cytokine responses from either wmfl or WT DCs. No differences between DCs from wmfl homozygotes and WT mice were observed in the upregulation of costimulatory molecules CD40 or CD86. However, wmfl DCs showed a moderate defect, similar in magnitude to that of DCs from Unc93b13d/3d mice (13), in an in vivo assay for T cell proliferation dependent on cross-presentation by DCs (Figure 5D).
Co-culture experiments indicated that wmfl DC-mediated NK cell activation was impaired in response to TLR ligands and MCMV infection. Upregulation of CD69 expression and IFNγ production were both determined strictly by the DC genotype rather than the NK cell genotype: whereas DCs from WT mice supported normal IFNγ production (Figure 6) and CD69 expression by either wmfl or WT NK cells, DCs from wmfl mice were unable to do so. These effects were observed in response to MCMV infection, TLR7 or TLR9 stimulation. Poly I:C induced normal levels of IFNγ in NK cells cultured with wmfl DCs, consistent with the finding that poly I:C elicited WT levels of type I IFN and IL-15Rα expression in wmfl DCs. Importantly, the same number of mixed DCs, with identical expression of immunophenotypic markers (CD11c+; CD11b+; PDCA1+; CD8+), were obtained from control and wmfl donors, ruling out a difference in DC numbers as the cause of impaired NK cell activation in these co-culture experiments.
The spleens and lymph nodes, but not thymi, of wmfl homozygotes were consistently smaller and showed reduced cellularity compared to those of control mice. Wmfl homozygotes were also slightly smaller than control mice of equivalent age and sex.
|Nature of Mutation|
The warmflash mutation was mapped to Chromosome 5, and corresponds to a G to A transition at position 33572 of the Flt3 genomic sequence (Genbank genomic region NC_000071 for linear genomic sequence of Flt3).
<--exon 9 intron 9--> exon 10--> 33558 GAGGATGGGTACAG GTGAGACACACAG… CATATCTAAATTTTGC 399 -E--D--G--Y--S --I--S--K--F--C- deleted
The mutation (red) destroys the donor site of intron 9 (blue), and results in the usage of an alternative donor site six nucleotides away in exon 9 (gray shading). This causes the in-frame deletion of 2 amino acids (YS) from exon 9. Flt3 contains 24 exons.
Murine Flt3 is a 1000 amino acid protein of the class III receptor tyrosine kinase (RTK) subfamily that includes the CSF-1 receptor (also known as FMS), KIT, and platelet-derived growth factor receptor α (PDGFRα) and β. Flt3 possesses an N-terminal signal sequence followed by an extracellular domain of 542 amino acids containing five Ig-like domains (Figure 7). The extracellular domain contains nine putative N-linked glycosylation sites and 22 cysteine residues. A twenty amino acid transmembrane domain is followed by a intracellular juxtamembrane (JM) domain and a tyrosine kinase domain (14;15). Human and mouse Flt3 are 86% identical at the amino acid level (16).
Although the 3D structure of the extracellular domain of Flt3 has not been determined, that of the related KIT extracellular domain provides insights into the mechanism of ligand-induced activation of KIT and other RTKs containing five or seven extracellular Ig-like domains. The KIT ectodomain adopts an elongated serpentine shape consisting of five Ig-like domains (D1-D5) that exhibit a typical immunoglobulin superfamily fold (Figure 8) (17). Each Ig-like domain is composed of eight β strands assembled into a β sandwich consisting of two antiparallel β sheets. D1, D2, and D3 mediate binding to a dimer of two SCF molecules, resulting in dimerization of two KIT ectodomains. SCF binding causes very few structural alterations in D1, D2, and D3, but D4 and D5 dramatically alter their configurations to permit lateral homotypic D4-D4 and D5-D5 interactions between two KIT molecules leading to receptor phosphorylation and activation. The mechanism of KIT activation may be shared by the related Flt3 receptor.
The kinase domains of class III RTKs are divided into two regions separated by an inserted sequence that varies in length between members and is thought to contribute to substrate specificity. For Flt3, the insertion consists of 77 mostly hydrophilic amino acids (14). Otherwise, the kinase domain contains the conserved subdomain sequences typical of tyrosine kinases. Internal tandem duplication (ITD) of sequences within the JM is common among human FLT3 mutations associated with acute myeloid leukemia (AML, see Background), and this, in addition to mutations inserting, substituting and deleting amino acids from the JM result in homodimerization and constitutive phosphorylation of Flt3 (18;19). In addition, mutations of aspartic acid 835 in the activation loop of Flt3 are also found in AML patients, and cause constitutive tyrosine phosphorylation and confer transforming ability to mutant Flt3 (20).
Flt3 kinase activity is thought to be regulated by inhibitory intramolecular interactions between the kinase domain and the JM domain. The crystal structure of the intracellular portion of Flt3 reveals that the entire JM domain interacts with distinct regions of the kinase domain, stabilizing the inactive kinase conformation (Figure 9) (21). In addition to the inhibitory JM-kinase domain interaction, the activation loop of Flt3 lies in an inhibitory conformation blocking the ATP-binding site and the active site. Ligand binding results in phosphorylation of the activation loop, opening the ATP-binding and kinase active sites.
The wmfl mutation causes the deletion of tyrosine 402 and serine 403, located in the fourth Ig-like domain of Flt3.
Flt3 is primarily expressed by hematopoietic stem cells (14). Flt3 mRNA is detected in the brain, thymus (medullary area of fetal and newborn thymus), spleen (red pulp), lymph node (paracortical regions), bone marrow, and placenta (labyrinthine trophoblasts) (14;22;23). In the thymus, Flt3 expression is restricted to the immature CD4-CD8- Thy1lo/IL-2R- cells (14); in the bone marrow, expression is restricted to early progenitors, including CD34+ cells with high levels of c-KIT expression (24). Flt3 appears to exist as both 140 kd and 160 kd species; the 160 kd protein is an N-glycosylated form of the smaller species and is localized on the cell membrane (25;26).
During a search for molecular components that regulate the development and proliferation of hematopoietic stem cells, Flt3 was identified from an enriched stem cell population by PCR amplification of common tyrosine kinase sequences (14). Flt3 was also cloned independently from mouse testis (15). Like other RTKs, ligand binding to Flt3 results in receptor dimerization, autophosphorylation, and recruitment and phosphorylation of substrates to initiate a downstream signaling. The ligand for Flt3, Flt3 ligand (Flt3L), is a type I transmembrane protein with similar overall structure to stem cell factor (the ligand for c-KIT) and colony stimulating factor-1 (CSF-1) (27;28). Flt3L may also be released as a soluble homodimeric protein that can induce tyrosine phosphorylation of Flt3 and proliferation of Flt3-transfected BAF/BO3 cells (27-29). It is a weak proliferative stimulus on its own, but Flt3L synergizes with other hematopoietic growth factors (e.g. CSF, IL-11, IL-12, stem cell factor) to promote growth and/or differentiation of bone marrow progenitors (30). Flt3L is expressed in cells of the hematopoietic bone marrow microenvironment, including bone marrow fibroblasts (31), and in myeloid, B- and T-cell lines (32). Flt3L is also detected in heart, spleen, lung, liver, skeletal muscle, kidney, testis, placenta and pancreas, although its function in these tissues is not understood (28). Interestingly, Flt3L mRNA is not detected in either mouse or human brain despite Flt3 expression there.
Both Flt3 and Flt3L have been knocked out by targeted deletion in mice, providing some understanding of their function in vivo. Flt3-/- mice are born healthy and with normal mature hematopoietic cell populations, including myeloid and lymphoid cells of the spleen and thymus, and monocytes, granulocytes and erythrocytes of the bone marrow (33). However, the percentage of bone marrow B220+ cells is reduced in Flt3-/- mice (20.6±1.0 in Flt3-/- vs. 30.8±1.8 in Flt3+/-), but only in the pro B and pre B cell populations and not in immature or mature B cell populations. In vitro, the potential of Flt3-/- cells to generate B cells is reduced, while myeloid cells are unaffected. However, when transplanted into irradiated hosts in long-term competitive reconstitution experiments in vivo, Flt3-/- bone marrow cells have a reduced capacity to repopulate multiple hematopoietic cell populations, including myeloid cells of bone marrow and spleen, thymic and splenic T cells, and bone marrow and splenic B cells.
In contrast to the Flt3-/- phenotype, Flt3l-/- mice display a distinct array of defects. Flt3l-/- mice have a reduced leukocyte cellularity in the peripheral blood (white blood cells and lymphocytes reduced, platelets normal, neutrophils increased), as well as reduced cellularity of bone marrow (immature B lymphocytes and myeloid cells reduced), spleen (B and T cells reduced) and lymph node (B and T cells reduced) (34). Myeloid precursors are reduced in number, and B lymphocyte precursors are reduced in both number and frequency in the bone marrow of Flt3l-/- mice. Natural killer (NK) cells are also reduced in number in Flt3l-/- mice, and these cells have a reduced ability to lyse Yac-1 cells upon stimulation with poly I:C (34;35). In addition, dendritic cells (DC, CD11c+ CD8α+ and CD11c+ CD8α-) (34) and plasmacytoid dendritic cells (pDC, CD11c+ B220+) (36) are reduced in Flt3l-/- spleen (~8-10-fold), thymus (~3-7 fold) and lymph nodes (~3-9 fold), although the DC present are able to stimulate proliferation of allogeneic naïve T cells in a mixed lymphocyte reaction (34). These findings are consistent with the observations that daily Flt3 administration results in an increase in DC numbers in mice (37;38), and that oral administration of SU11657, a Flt3 kinase inhibitor, reduces pDC and DC levels in spleen approximately 5-6 fold (36). Perhaps surprisingly, Flt3l-/- mice were not found to have an increased frequency of infection, although susceptibility to specific infectious agents was not tested (34).
Although not all of the same phenotypes were examined, the reasons for phenotypic differences between Flt3-/- and Flt3l-/- mice are not known. The major differences are the decreased leukocyte cellularity and myeloid precursors in Flt3l-/- but not Flt3-/- mice. Strain-dependence of the phenotypes is a possibility (Flt3l-/- mice were on C57BL/6 background; Flt3-/- mice were on 129/Sv/Ev background), but another factor could be possible redundancy of Flt3 or Flt3L with other receptor tyrosine kinases or ligands that may compensate for the absence of Flt3/Flt3L in knockout mice. Consistent with this hypothesis, c-kit, Flt3 double mutant mice have more severe defects than either single mutant alone, with reductions in bone marrow and thymic cellularity, and reduced numbers of myeloid precursors, similar to the Flt3l-/- phenotype (33). However, Flt3L is not known to bind other receptors.
As mentioned above (Protein Prediction), mutations in FLT3 are frequently found in patients with acute myeloid leukemia (AML). FLT3 is expressed at high levels in 70-100% of AML cases, and activating mutations in FLT3 occur in about one-third of such cases [reviewed in (39)]. There are two main classes of FLT3 activating mutations. Internal tandem duplications (ITD) consist of in-frame duplications of variable lengths of sequence encoding the juxtamembrane domain, and tyrosine kinase domain (TKD) mutations are usually single base substitutions or small insertions/deletions in the activation loop of the kinase domain. Thus, while Flt3 signaling is required for DC development, increased signaling can also (likely in combination with other genetic lesions) lead to the rapid proliferation of abnormal cells in the bone marrow to cause AML.
The signaling pathways activated by Flt3 signaling are many and studies using cell lines (e.g. IL-3-dependent Ba/F3 cells) expressing a chimeric receptor with the extracellular domain of CSF-1 receptor and the transmembrane and cytoplasmic domain of Flt3 demonstrate that PI-3 kinase (PI3K) Shc, Grb2, Vav, Fyn and Src are components of the Flt3 signaling pathway (40). These results have been recapitulated using Ba/F3 cells expressing either human or murine Flt3 and stimulated with Flt3L, and have been extended to include the Ras/MAPK and Stat5 pathways in Flt3 signaling (41-43). Studies using mutant FLT3 (containing activating mutations found in AML) implicate the Src family kinase Lyn, MAPK and STAT5 in activated FLT3 signaling (44;45). Recently, analysis of hematopoietic-derived cell-specific knockouts of Stat3 demonstrated that Stat3 is required for Flt3-mediated DC development (46). In these mice, the hematopoietic stem cell population is normal, and Flt3L treatment is able to induce a 2- to 3-fold accumulation of common lymphoid (CLP) and myeloid progenitors (CMP). However, Flt3L fails to induce common DC precursors in the Stat3-deficient mice, indicating that Stat3 is required in the Flt3L pathway during the transition from CLP/CMP to common DC precursors.
Flt3 is a hematopoietic receptor tyrosine kinase important for the development of many immune cells, including T cells, B cells, NK cells, and DCs (33;34). Binding of its ligand Flt3L results in receptor activation and stimulates the growth of progenitor cells in the bone marrow and blood. Bone marrow cells from Flt3wmfl/wmfl mice were unresponsive to the proliferation and survival signals delivered by Flt3L in vitro (Figure 10). Based on these data, the Flt3wmfl allele is presumed to encode a non-functional protein. The location of the wmfl lesion suggests that it may abrogate ligand binding.
Flt3 signaling is well known for its role in DC ontogeny (34;36-38;47;48). Flt3 signaling is thought to contribute predominantly to DC differentiation, while other factors such as GM-CSF drive DC function during inflammation and infection (49;50). Consistent with previous studies, Flt3wmfl/wmfl mice had reduced numbers of DCs. However, the function of Flt3 is not restricted to the differentiation of precursors to the DC identity per se. DCs that survived to maturity in an Flt3-deficient background, while expressing a variety of DC markers, lacked the full functionality of DCs that developed in the presence of Flt3. The most dramatic difference in performance of Flt3wmfl/wmfl DCs was seen in the failure of these cells to respond to MCMV infection or ligands for TLR7 or TLR9 by producing IL-12, type I IFN, and IL-15Rα.
The principal defect underlying MCMV susceptibility caused by the Flt3wmfl mutation thus appears to be an impaired ability of DCs to assist in the activation of NK cells, which exist in diminished numbers in Flt3wmfl/wmfl mice. The reduction in NK cell numbers is not likely, by itself, to account for the death of Flt3wmfl/wmfl homozygotes following sublethal inoculation with MCMV. NK cells from Flt3wmfl/wmfl mice exhibited full intrinsic functionality, and delays in Flt3wmfl/wmfl NK cell activation and function were observed only under conditions in which NK cells required activation by DCs, e.g., during TLR stimulation or MCMV infection in vivo, and in NK-DC co-cultures. Recent data that Flt3L-induced expansion of NK cells in the absence of infection requires a CD11chi DC population expressing IL-15 (51) further support the idea that Flt3 signaling, by an unknown mechanism, renders DCs competent to stimulate NK cell activation and expansion.
DCs are specialized to capture and present antigenic peptides in conjunction with MHC proteins for recognition by and activation of T cells in a process known as cross-presentation. The T cell response is essential for control of LCMV, but dispensable for early control of MCMV. Impaired cross-presentation by Flt3wmfl/wmfl DCs is consistent with the reduced ability of Flt3wmfl/wmfl mice to control LCMV, and suggests that CD8+ T cell expansion or cross-presentation itself may require signals from DC-derived cytokines, including type I IFN, IL-12, and IL-15, which are produced at reduced levels by Ftl3wmfl/wmfl DCs.
The MCMV susceptibility of Flt3wmfl/wmfl mice suggested that Flt3 signaling during viral infection is necessary for DCs to fully respond to TLR stimuli. However, Flt3 depletion during infection did not affect control of viral titers, indicating that Flt3 signaling as it occurs acutely during MCMV infection is neither necessary nor sufficient to promote the DC functions that activate NK cells and permit survival (1). Thus, Flt3 signaling during DC development preconditions these cells to become competent to produce an adequate cytokine response and thus activate NK cells to full cytolytic function during viral infection. A burst of Flt3L production is induced by TLR signaling in extrahematopoietic cells; this surge in Flt3L may serve an important function in other types of infection, yet to be identified.
|Primers||Primers cannot be located by automatic search.|
Warmflash genotyping is performed by amplifying the region containing the mutation using PCR, followed by sequencing of the amplified region to detect the single nucleotide change.
warmflash (F): 5’-GCCCAAACCACAGCTCAACTTTGTCTATGC -3’
warmflash (R): 5’- AAGGAAAAGTGGCTCTCCTCACAGGTGC -3’
1) 95°C 2:00
2) 95°C 0:30
3) 56°C 0:30
4) 72°C 1:00
5) repeat steps (2-4) 29X
6) 72°C 7:00
7) 4°C ∞
Primers for sequencing
warmflash_seq(F): 5’- CCTGGTCTACAAAGGAGGTTC -3’
warmflash_seq(R): 5’- TCAACTCACAGACTGAGGGG -3’
The following sequence of 753 nucleotides (from Genbank genomic region NC_000071 for linear genomic sequence of Flt3, sense strand) is amplified:
33082 gcccaaacc acagctcaac tttgtctatg ctttctttca
33121 ttaaactgac aaatttttaa aaaaatcatg tatggtaggt ctctatgttg atcagcctgg
33181 tctacaaagg aggttctaag ttagatgggg ctagaaccat atatatatgt atatatatgt
33241 gtgtgtgtgt gtgtgtgtgt gtgtatgtat atatgtatgt atcacatata tatcataaca
33301 tgatatatgt gatatatgta tatatgatat atgtgtgtat atatgatata tataatcctt
33361 atacaaatat caatatctaa tttgtaaatt tcctttttac agaaaaaggg tttataaacg
33421 ctaccagctc gcaagaagag tatgaaattg acccgtacga aaagttctgc ttctcagtca
33481 ggtttaaagc gtacccacga atccgatgca cgtggatctt ctctcaagcc tcatttcctt
33541 gtgaacagag aggcctggag gatgggtaca ggtgagacac acagcacagc cctcctccct
33601 cgagaaggac acacacacct tccccttccc cccaacctcc cacccacccc ccatcccctg
33661 caccaccact accaccacct accccccaat tccccggccc ccatccccgg tatcgagcac
33721 ctctgtcccc gagctataga cttagccccc tcagtctgtg agttgattta ttgttgagtc
33781 agaaaactga agactgaaag caaaccgcac ctgtgaggag agccactttt cctt
Primer binding sites are underlined; sequencing primer binding sites are highlighted in gray; the mutated G is indicated in red.
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|Science Writers||Eva Marie Y. Moresco|
|Illustrators||Diantha La Vine|
|Authors||Celine Eidenschenk, Karine Crozat, Bruce Beutler|