Flt3 Ligand Human, HEK

What is the molecular structure of human Flt3 Ligand and how does it compare to other cytokines?

Human Flt3 Ligand (also known as FL, Flt3L, FLT3LG) is an alpha-helical cytokine that promotes the differentiation of multiple hematopoietic cell lineages. The mature protein consists of a 158 amino acid extracellular domain (ECD) with a cytokine-like domain and a juxtamembrane tether region, a 21 aa transmembrane segment, and a 30 aa cytoplasmic tail . It functions as a noncovalently-linked dimer expressed by T cells and bone marrow and thymic fibroblasts . Structurally, Flt3 Ligand is related to M-CSF (Macrophage Colony-Stimulating Factor) and SCF (Stem Cell Factor), sharing similar tertiary structural elements despite limited sequence homology .

Within the ECD, human Flt3 Ligand shares 71% and 65% amino acid sequence identity with mouse and rat Flt3 Ligand respectively, which explains the observed cross-species activity in experimental systems . The human and mouse proteins demonstrate functional cross-reactivity, an important consideration when designing in vitro and in vivo experiments . When expressed in HEK293 cells, the protein typically appears as a 27-34 kDa monomer under reducing conditions and 23-30 kDa under non-reducing conditions due to extensive glycosylation .

What post-translational modifications are present in HEK293-derived Flt3 Ligand?

HEK293-derived human Flt3 Ligand undergoes extensive post-translational modifications, most notably glycosylation. The protein carries approximately 12 kDa of N- and O-linked carbohydrates, contributing significantly to its molecular mass . These glycosylation patterns enhance protein solubility, protect against proteolytic degradation, and influence receptor recognition.

When analyzed by SDS-PAGE, HEK293-expressed Flt3 Ligand appears as bands at 27-34 kDa under reducing conditions and 23-30 kDa under non-reducing conditions . This glycosylation profile is characteristic of proteins expressed in mammalian systems and is important for maintaining proper folding and biological activity. The molecular weight difference between reduced and non-reduced forms reflects the contribution of disulfide bonds to the protein's tertiary structure.

For researchers studying structure-function relationships, it's important to note that enzymatic deglycosylation (using PNGase F for N-linked and O-glycosidase for O-linked glycans) followed by mobility shift analysis can provide insights into the contribution of glycans to protein characteristics. Additionally, mass spectrometry approaches can provide detailed mapping of specific glycosylation sites.

What is the biological activity range for recombinant human Flt3 Ligand?

The biological activity of recombinant human Flt3 Ligand from HEK293 cells is typically measured using proliferation assays with the BaF3 mouse pro-B cell line transfected with mouse Flt3 receptor. Based on multiple product specifications, the ED50 (effective dose for 50% maximal response) ranges from 0.15-4.8 ng/mL, with most preparations showing activity in the 0.3-3.0 ng/mL range . Some manufacturers report specific activity in international units, with values of minimally 6.00 × 10^5 IU/mg .

This variation in reported activity ranges stems from differences in production methods, purification strategies, and assay conditions. For standardization across experiments, researchers should establish dose-response curves using reference standards whenever possible. It's important to note that Flt3 Ligand by itself often does not stimulate robust proliferation of early hematopoietic cells but synergizes with other cytokines like IL-3, GM-CSF, and SCF to induce growth and differentiation .

When working with primary human hematopoietic progenitor cells, higher concentrations (10-100 ng/mL) are often used to induce dendritic cell differentiation or expansion of CD34+ cells. The precise concentration should be optimized for each specific application and cell type.

How should Flt3 Ligand be reconstituted and stored for optimal activity?

For optimal activity and stability of lyophilized recombinant human Flt3 Ligand, follow these methodological guidelines:

Reconstitution procedure:

• Briefly centrifuge the lyophilized vial before opening to collect all material at the bottom.

• For carrier-free preparations, reconstitute to 100-250 μg/mL in sterile PBS . For research requiring absence of carriers, use sterile water containing 0.1% endotoxin-free recombinant human serum albumin (HSA) as a stabilizing protein .

• Gently swirl or rotate the vial to ensure complete dissolution; avoid vigorous vortexing which can denature the protein.

• Allow the solution to stand for 5-10 minutes at room temperature before aliquoting.

Storage recommendations:

• Store lyophilized protein at -20°C to -80°C until the expiry date, or at room temperature for up to 2 weeks .

• After reconstitution, prepare single-use aliquots in low-binding microcentrifuge tubes to avoid freeze-thaw cycles.

• For short-term use (within 1 week), store reconstituted protein at 4°C .

• For long-term storage (up to 6 months), keep reconstituted aliquots at -20°C to -80°C .

Researchers should validate protein activity after reconstitution using a functional assay such as BaF3-FLT3 cell proliferation. The presence of carrier proteins (like albumin) can significantly extend the functional half-life in dilute solutions.

What synergistic effects occur when combining Flt3 Ligand with other cytokines?

Flt3 Ligand exhibits multiple synergistic interactions with other cytokines, significantly enhancing its biological effects in hematopoietic cell development:

• With IL-3, GM-CSF, and SCF: Flt3 Ligand synergizes to promote the mobilization and myeloid differentiation of hematopoietic stem cells . This involves cooperative activation of multiple signaling pathways. For experimental approaches, sequential or simultaneous cytokine treatment protocols can be employed, with optimal concentrations determined through dose-matrix experiments.

• With IL-2, IL-6, IL-7, and IL-15: Flt3 Ligand cooperates to induce NK cell development from CD34+ progenitors . This synergy is particularly pronounced with IL-15, where Flt3 Ligand primes progenitors by upregulating IL-15 receptor components.

• With IL-3, IL-7, and IL-11: Flt3 Ligand enhances terminal B cell maturation . This involves sequential developmental stages where Flt3 Ligand acts early to expand lymphoid progenitors, while IL-7 directs B-lineage commitment.

• With GM-CSF and/or IL-4: Synergistically drives dendritic cell differentiation from monocytes or CD34+ cells. This combination is widely used in DC generation protocols.

It's important to note that Flt3 Ligand by itself does not stimulate robust proliferation of early hematopoietic cells but requires these synergistic interactions with other cytokines to induce growth and differentiation . When designing experiments utilizing these synergistic effects, researchers should carefully optimize cytokine concentrations and treatment timing, as sequence-dependent effects have been documented in multiple systems.

How does Flt3 Ligand influence dendritic cell development?

Flt3 Ligand plays a central role in dendritic cell (DC) development and is crucial for steady-state production of both conventional DCs (cDCs) and plasmacytoid DCs (pDCs) from hematopoietic progenitors . FLT3LG induces the expansion of monocytes and immature dendritic cells as well as early B cell lineage differentiation . The absence of FLT3L results in significantly reduced DC numbers across multiple tissues, demonstrating its non-redundant role in DC homeostasis .

At the molecular level, Flt3 Ligand binds to the FLT3 receptor (CD135), inducing receptor dimerization and activation of intrinsic tyrosine kinase activity. This triggers phosphorylation of multiple intracellular domains and activates several downstream signaling cascades, including JAK2/STAT3/STAT5, PI3K/AKT, and MEK/ERK pathways . These signaling events upregulate key transcription factors essential for DC lineage specification and development.

In experimental applications, Flt3 Ligand (100 ng/mL) supplemented cultures of bone marrow or CD34+ cells generate heterogeneous DC populations that more closely resemble steady-state DCs compared to GM-CSF-derived DCs. In vivo administration of Flt3 Ligand dramatically expands DC populations in multiple tissues, providing a valuable experimental tool to study DC biology.

What role does Flt3 Ligand play in FLT3-mutated AML and how can this inform therapeutic approaches?

In FLT3-mutated acute myeloid leukemia (AML), particularly those with internal tandem duplications (FLT3-ITD), Flt3 Ligand (FL) plays a complex role that has significant implications for therapy. FL levels often increase substantially during chemotherapy and after FLT3 inhibitor treatment, potentially contributing to treatment resistance . This occurs through FL-mediated activation of residual wild-type FLT3 receptors, which can partially compensate for inhibited mutant signaling.

Most first-generation FLT3 inhibitors (like quizartinib) demonstrate higher potency against FLT3-ITD than wild-type FLT3. Consequently, increased FL levels can competitively reduce inhibitor binding to wild-type FLT3 and activate downstream signaling pathways, particularly ERK1/2 and AKT . In contrast, newer FLT3 inhibitors with equivalent potency against both wild-type and mutant FLT3, such as gilteritinib, demonstrate efficacy even in the presence of elevated FL .

In studies examining gilteritinib's effect on FLT3 signaling, it was found to inhibit both FLT3 wild-type and FLT3-ITD to a similar degree in HEK293 and Ba/F3 cells, and similarly suppressed FLT3 downstream signaling molecules (including ERK1/2 and STAT5) in both the presence and absence of FL in MOLM-13 cells . Co-crystal structure analysis showed that gilteritinib bound to the ATP-binding pocket of FLT3 . These results suggest that gilteritinib has therapeutic potential in FLT3-mutated AML patients with FL overexpression .

For research into therapeutic approaches, experimental models using FL-expressing leukemia cells in xenograft systems can mimic the elevated FL conditions observed in patients, providing more predictive preclinical evaluation of novel therapeutics .

How do Flt3 Ligand inhibitors function and what are their research applications?

Flt3 Ligand inhibitors represent an emerging class of research tools with distinct mechanisms and applications. Unlike FLT3 receptor tyrosine kinase inhibitors (TKIs) that target the intracellular domain, these molecules inhibit the extracellular interaction between Flt3 Ligand and the FLT3 receptor.

High-throughput screening has identified small molecular weight compounds that competitively inhibit the binding of fluorescently labeled Flt3 Ligand to FLT3-overexpressing cells . In one notable study, a total of 679 small molecular weight ligands (1.4%) were confirmed to strongly inhibit (>75%) the binding of fluorescent-labeled FLT3 ligand (FL cytokine) to FLT3 overexpressed in HEK-293 cells, at two different concentrations (5 and 20 μM) . Concentration-response curves obtained for 111 lead-like molecules confirmed the unexpected tolerance of the FLT3 extracellular domain for low molecular weight druggable inhibitors exhibiting submicromolar potencies, chemical diversity, and promising pharmacokinetic properties .

These inhibitors have several important research applications:

• Probing FLT3 signaling biology: Unlike genetic knockouts or receptor TKIs, these inhibitors allow temporal control over FLT3 activation, enabling precise studies of pathway dynamics.

• Neurological research: Some Flt3 Ligand inhibitors have shown efficacy in models of neuropathic pain, suggesting FLT3 signaling plays a role in neuroinflammation . Further investigation of one hit confirmed inhibitory properties in dorsal root ganglia neurons and in a mouse model of neuropathic pain .

• Hematopoiesis studies: Selective inhibition of Flt3 Ligand provides tools to dissect its specific contributions within complex cytokine networks.

For researchers interested in using these tools, validation experiments should include confirming target engagement (e.g., competitive binding assays) and establishing functional consequences in relevant cell systems before application to complex biological questions.

What factors can affect the bioactivity of Flt3 Ligand in experimental settings?

Multiple factors can significantly impact the bioactivity of recombinant human Flt3 Ligand in experimental settings, requiring careful methodological consideration:

Physical and chemical factors:

• Temperature fluctuations: Repeated freeze-thaw cycles can dramatically reduce activity. Maintain single-use aliquots at -80°C for optimal preservation.

• Protein concentration: Dilute solutions (<10 μg/mL) without carrier proteins show accelerated activity loss through surface adsorption to storage vessels. Maintain stock solutions at ≥50 μg/mL or add carrier proteins (0.1-0.5% BSA or HSA).

• pH fluctuations: Activity is optimal between pH 6.8-7.4, with significant reductions outside this range.

• Oxidation: Methionine and cysteine residues are susceptible to oxidation, potentially affecting receptor binding.

Biological matrices:

• Proteases in biological samples: Serum, tissue homogenates, and some cell culture media contain proteases that can degrade Flt3 Ligand.

• Soluble FLT3 receptor: Biological samples may contain variable levels of soluble FLT3 receptor that can sequester Flt3 Ligand.

• Endogenous Flt3 Ligand: Background levels in serum or conditioned media may affect dose-response relationships.

Experimental design considerations:

• Cell responsiveness: Receptor expression levels vary substantially between cell types and culture conditions. FLT3 receptor can be downregulated after continued exposure to Flt3 Ligand, affecting response kinetics in long-term cultures.

• Timing of addition: The biological effect of Flt3 Ligand may depend on the differentiation stage of target cells and the presence of other cytokines.

• Readout selection: Different biological effects (proliferation, differentiation, survival) may have different dose-response relationships and kinetics.

Implementing quality control steps, such as regular activity testing with reference cell lines, can help ensure consistent experimental outcomes when working with this cytokine.

What cell types respond to Flt3 Ligand and what methodologies can detect this response?

Flt3 Ligand primarily affects cells expressing the tyrosine kinase receptor FLT3 (CD135), which includes early hematopoietic stem and progenitor cells, dendritic cell precursors, and certain leukemic blasts. To detect cellular responses to Flt3 Ligand, researchers can employ multiple methodologies:

Cell types responding to Flt3 Ligand:

• Hematopoietic stem and progenitor cells: Flt3 Ligand synergizes with other cytokines to promote their mobilization and differentiation .

• Dendritic cell precursors: FLT3LG induces the expansion of monocytes and immature dendritic cells .

• B cell progenitors: Flt3 Ligand supports early B cell lineage differentiation .

• NK cell precursors: Flt3 Ligand cooperates with IL-2, IL-6, IL-7, and IL-15 to induce NK cell development .

• Leukemic blasts expressing FLT3: Particularly relevant in AML with FLT3 mutations .

Detection methodologies:

• Proliferation assays: The standard bioassay uses BaF3 cells transfected with mouse Flt3 receptor, with ED50 values typically ranging from 0.150-4.80 ng/mL .

• Phosphorylation analysis: Western blotting for phosphorylated FLT3 receptor and downstream signaling molecules (ERK1/2, STAT5, AKT) following short-term stimulation with Flt3 Ligand .

• Flow cytometry: Phenotypic analysis of expanded populations using lineage-specific markers.

• Colony formation assays: Methylcellulose-based colony assays with CD34+ cells to assess progenitor expansion and differentiation capacity.

• Gene expression analysis: qRT-PCR or RNA-seq to measure transcriptional changes in FLT3-regulated genes following stimulation.

Each method provides unique insights into the biological effects of Flt3 Ligand, making a multi-parameter approach optimal for comprehensive characterization.

How can Flt3 Ligand be used in hematopoietic stem cell expansion protocols?

Flt3 Ligand plays a crucial role in hematopoietic stem cell (HSC) expansion protocols, particularly for research and potential clinical applications. A methodological approach includes:

Protocol design considerations:

• Starting population: Purified CD34+ cells from cord blood typically show greater expansion potential than those from adult bone marrow or peripheral blood.

• Cytokine combinations: Flt3 Ligand (50-100 ng/mL) should be combined with SCF (50-100 ng/mL) as a core cocktail, supplemented with TPO (20-50 ng/mL) to maintain long-term repopulating potential.

• Medium composition: Serum-free formulations supplemented with L-glutamine and antibiotics provide defined conditions for reproducible expansion.

Implementation methodology:

• Initial seeding at 1-5 × 10^4 CD34+ cells/mL in appropriate medium with cytokines.

• Culture maintenance with half-medium changes every 2-3 days, replenishing cytokines at each feeding.

• Assess expansion by monitoring total cell numbers, CD34+ cell enumeration by flow cytometry, and functional assays (colony formation).

• For optimal expansion, maintain cultures at relatively low density (< 1 × 10^6 cells/mL) and consider using gas-permeable culture vessels for improved oxygenation.

Assessment metrics:

• Quantity: Fold-expansion of total nucleated cells and CD34+ cells (typically 20-100 fold for CD34+ cells over 7-14 days).

• Quality: Maintenance of primitive immunophenotype and multilineage differentiation potential.

• Functionality: Retention of colony-forming capacity, particularly maintenance of mixed and high-proliferative potential colonies.

This approach maximizes the expansion of hematopoietic progenitors while preserving stem cell characteristics for subsequent experimental or therapeutic applications.