FLT3LG Antibody

Basic Research Questions

• What is FLT3LG and what is its primary biological function?

  FLT3LG is a protein encoded by the FLT3LG (or FLT3L) gene that stimulates the proliferation of early hematopoietic cells by activating the FLT3 receptor tyrosine kinase . It functions as a key regulator of dendritic cell (DC) development, particularly plasmacytoid DCs and CD8-positive classical DCs and their CD103-positive tissue counterparts . FLT3LG serves as a crucial bridge between innate and adaptive immunity by influencing the maturation of dendritic and B cells . The protein exists in both membrane-bound and soluble forms, with glycosylation sites that affect its biological activity . It synergizes with multiple colony stimulating factors and interleukins to regulate hematopoiesis and immune cell activation .

• What are the cellular sources of FLT3LG expression in normal tissues?

  Single-cell sequencing data indicates that FLT3LG is predominantly expressed in CD4+ and CD8+ T lymphocytes under normal physiological conditions, as demonstrated in peripheral blood mononuclear cell (PBMC) datasets . Its receptor, FLT3, is mainly expressed on dendritic cells, creating a system where T cell-derived FLT3LG can influence dendritic cell development and function . Under pathological conditions such as acute myeloid leukemia (AML), the expression pattern of FLT3LG significantly changes, with notably reduced expression in T cells compared to healthy controls . This differential expression in health versus disease makes FLT3LG an important marker for studying immune dysregulation.

• What applications are FLT3LG antibodies commonly used for?

  FLT3LG antibodies are utilized across multiple techniques in research settings. Primary applications include:

  • Western blotting (WB) for protein expression analysis

  • Immunohistochemistry on paraffin-embedded tissues (IHC-P) for localization studies

  • Immunocytochemistry/Immunofluorescence (ICC/IF) for cellular localization

  • Enzyme-linked immunosorbent assays (ELISA) for quantification of FLT3LG in biological samples

  • Flow cytometry for analyzing expression on specific cell populations

  These techniques collectively enable researchers to characterize FLT3LG expression, localization, and function in various experimental contexts, from basic biology to disease models .

Advanced Research Questions

• How do mutations in FLT3LG affect its binding to FLT3 receptor and subsequent signaling?

  Mutations in FLT3LG can significantly alter its binding affinity and bioactivity. For example:

  The L27P mutation in FLT3LG (Flt3Lg-L27P) results in:

  • At least a 10-fold higher ED50 compared to wild-type FLT3LG when produced in CHO cells

  • Reduced bioactivity while maintaining target specificity

  • Reduced recycling of the FLT3 receptor, which affects downstream signaling

  These mutational effects have been leveraged for therapeutic applications. For instance, chimeric antigen receptors (CARs) incorporating mutated FLT3LG-L27P (Flt3m-CAR) maintain their specificity for FLT3 targets but with reduced ligand bioactivity, potentially creating safer immunotherapy constructs that minimize off-target effects .

  Researchers investigating FLT3LG mutations should employ binding affinity assays, functional T cell activation assays, and receptor recycling studies to fully characterize how specific mutations affect the FLT3LG-FLT3 signaling axis .

• What are the methodological considerations for developing oncolytic viruses expressing FLT3LG?

  Developing oncolytic viruses expressing FLT3LG requires several methodological considerations:

  1. Vector design: Bacterial artificial chromosome (BAC) systems have been successfully used to generate recombinant oncolytic viruses expressing FLT3LG . The process involves:

     • Construction of a shuttle vector containing the FLT3LG open reading frame

     • Incorporation of appropriate promoters (e.g., CMV promoter)

     • Addition of restriction sites (e.g., BamH1, Not1) and kozak sequences for optimal expression

     • Confirmation of insertion by sequencing

  2. Selection of viral backbone: Herpes simplex virus-1 (HSV-1) derivatives like G47Δ have been used successfully as oncolytic backbones for FLT3LG expression

  3. Functional considerations: FLT3LG-expressing oncolytic viruses work through multiple mechanisms:

     • Direct viral oncolysis of tumor cells

     • Enhanced recruitment of dendritic cells and T cells to the tumor microenvironment

     • Improved antigen presentation and T cell activation

     • Potential synergy with other immunomodulatory approaches (e.g., CTLA-4 blockade, cytokine delivery)

  4. Combination strategies: FLT3LG-expressing viruses have shown enhanced efficacy when combined with other immunomodulatory approaches, such as:

     • Co-delivery with viruses expressing chemokines like MIP-1α

     • Combination with RANTES or IL-18, which enhance DC recruitment or Th1 immunity, respectively

  These approaches leverage FLT3LG's ability to increase numbers of both intratumoral dendritic cells and cytotoxic T lymphocytes, potentially improving tumor control in experimental models .

• How can researchers effectively quantify FLT3LG in complex biological samples?

  Quantifying FLT3LG in complex biological samples requires careful methodological approaches:

  1. ELISA-based methods: Commercial kits (e.g., Mouse/Rat Flt-3 Ligand/FLT3L Quantikine ELISA Kit) have been validated for quantifying FLT3LG in serum, urine, and tissue homogenates . Sample processing typically involves:

     • For tissues: Homogenization using a rubber tip and 70 μm cell strainer

     • For urine/serum: Direct application to the assay following standard dilution protocols

  2. Flow cytometry: For cellular FLT3LG detection:

     • Single-cell suspensions should be prepared from tissues

     • Appropriate antibodies (e.g., CD135 (FLT3) monoclonal antibody-APC) with proper isotype controls

     • Gating strategy should include individual single-color controls for compensation adjustment

     • Data acquisition using flow cytometry platforms and analysis with software like Flowjo

  3. Gene expression analysis: qPCR approaches for FLT3LG mRNA quantification:

     • RNA extraction using TRIzol reagent

     • cDNA synthesis using appropriate RT kits

     • qPCR using SYBR Green or similar techniques

     • Normalization to housekeeping genes (e.g., β-actin)

     • Calculation of relative expression using the 2^(-ΔΔCT) method

  4. Validation and controls: For all methods, researchers should include:

     • Positive controls (recombinant FLT3LG at known concentrations)

     • Negative controls (samples known not to express FLT3LG)

     • Multiple technical and biological replicates

     • Statistical analysis to determine significance (p-values < 0.05 considered statistically significant)

• What is the significance of FLT3LG's interaction with other cytokines in experimental design?

  FLT3LG exhibits significant synergistic interactions with multiple cytokines, which has important implications for experimental design:

  1. Synergistic cytokine combinations:

     • FLT3LG synergizes with IL-3, G-CSF, GM-CSF, and EPO to enhance cell proliferation effects

     • Collaboration with IL-7 and IL-11 promotes hematopoietic progenitor differentiation and long-term cloning

     • Combined with KIT ligand and IL-3, FLT3LG promotes myeloid-derived suppressor cell (MDSC) proliferation

  2. Experimental considerations:

     • When studying FLT3LG, researchers should consider the broader cytokine milieu, particularly in complex systems like the tumor microenvironment

     • Bioinformatic analyses have shown significant positive correlations between FLT3LG and cytokines such as TNF, CXCR3, and CCL4

     • Following BCG treatment, multiple cytokines beyond FLT3LG are elevated, including IL-1β, IL-8, IL-15, IL-18, CCL2, and CCL3

  3. Methodological implications:

     • Experimental designs should include appropriate cytokine controls

     • Blocking specific cytokines or their receptors may help delineate FLT3LG-specific effects

     • Combination treatments in therapeutic contexts should be carefully designed to leverage potential synergies

     • Multiplex cytokine assays rather than single-cytokine quantification may provide more comprehensive insights into complex biological responses

  These interactions underscore the importance of considering the broader cytokine network when designing experiments to study FLT3LG function and when developing FLT3LG-targeted therapeutic approaches.