IL9 Antibody

Basic Research Questions

• What is IL-9 and why are IL-9 antibodies important in immunological research?

  IL-9 was first identified in the late 1980s as a T-cell and mast cell growth factor, initially termed P40 based on its molecular weight. It's a 14 kDa peptide encoded by a 144 amino acid protein (including leader sequence) . The human IL9 gene is located on chromosome 5, while in mice it's found on chromosome 13 .

  IL-9 antibodies are crucial research tools because IL-9 plays multiple roles in both physiological and pathological immune responses:

  • Acts as a growth factor for T cells

  • Enhances IgE production in B cells

  • Induces mucus production by epithelial cells

  • Promotes mast cell accumulation in tissues

  • Alters barrier function in intestines

  • Contributes to asthma and allergic responses

  Using specific antibodies allows researchers to neutralize IL-9 activity or detect its presence, providing insight into its role in various immunological conditions.

• Which cell types produce IL-9 and how does this affect experimental design?

  IL-9 is produced by multiple immune cell types, which must be considered when designing experiments:

  • Th9 cells (primary producers, differentiated from naive CD4+ T cells with TGF-β and IL-4)

  • Th2 cells

  • Th17 cells

  • Regulatory T cells (Tregs)

  • Type 2 innate lymphoid cells (ILC2s)

  • Mast cells

  • Eosinophils

  • Natural killer T (NKT) cells

  When designing experiments to study IL-9, researchers should consider:

  1. Including appropriate stimuli to activate relevant cell populations

  2. Using cell-specific markers to identify which population is producing IL-9

  3. Employing intracellular cytokine staining techniques to detect IL-9 production

  4. Considering the tissue microenvironment, as IL-9 production may vary depending on location

• What are the key applications for IL-9 antibodies in immunological research?

  IL-9 antibodies are versatile tools with several research applications:

  • Neutralization studies: Blocking IL-9 activity in vivo to assess its role in disease models

  • Flow cytometry: Detecting intracellular IL-9 in various immune cell populations

  • Immunohistochemistry/Immunofluorescence: Visualizing IL-9 expression in tissue sections

  • Western blotting: Detecting IL-9 protein in cell or tissue lysates

  • ELISA: Quantifying IL-9 levels in serum or cell culture supernatants

  • Immunoprecipitation: Isolating IL-9 and associated proteins

  For optimal results, researchers should validate antibodies for specific applications and consider experimental controls such as isotype-matched antibodies .

Advanced Research Applications

• How should researchers implement IL-9 neutralizing antibodies in allergic inflammation models?

  When using IL-9 neutralizing antibodies in allergic inflammation models, careful experimental design is crucial:

  Dosage determination:

  • Perform dose-finding studies (e.g., 0.3 mg, 1.2 mg, 4.8 mg)

  • Measure circulating antibody levels by ELISA to confirm in vivo presence

  • Establish optimal timing of administration relative to allergen challenge

  Administration protocol:

  • Initiate treatment before allergen challenge (day -1) for preventive studies

  • For therapeutic studies, begin after established inflammation

  • Consider intraperitoneal injection for systemic effect

  Assessment parameters:

  • Measure symptom scores and behavioral changes

  • Quantify allergen-specific IgE levels

  • Assess tissue eosinophilia and mast cell numbers

  • Analyze cytokine expression profiles (IL-4, IL-5, IL-13)

  • Evaluate T cell subpopulations including Th2 and Treg cells

  Anti-IL-9 antibody treatment has been shown to decrease allergic symptoms, OVA-specific IgE levels, eosinophil counts, and inhibit Th2 responses in allergic rhinitis models .

• What methodological considerations are important when detecting IL-9 in different tissue types?

  Detection of IL-9 in different tissues requires adapting methods to tissue-specific challenges:

  For peripheral blood:

  • Use intracellular cytokine staining following PMA/ionomycin stimulation

  • Pre-titrate antibodies on Th2-polarized cells as positive controls

  • Use protein transport inhibitors (e.g., monensin) during stimulation

  • Utilize the Intracellular Fixation & Permeabilization Buffer Set for optimal staining

  For lung tissue in asthma models:

  • Consider dual immunofluorescence to co-localize IL-9 with cell-type markers

  • Use antigen retrieval for formalin-fixed tissues

  • Include isotype controls to assess non-specific binding

  • Compare IL-9 expression patterns between asthmatic and healthy control tissues

  For tumors:

  • Optimize tissue digestion protocols to preserve cell surface markers

  • Consider single-cell approaches for heterogeneous tumor microenvironments

  • Use flow cytometry to quantify IL-9+ cell populations

  • Perform comparative analysis between tumor and adjacent normal tissue

  Western blotting considerations:

  • Expect variability in observed molecular weight (15-30 kDa) due to glycosylation

  • Use positive controls such as recombinant IL-9 or transfected cell lysates

• How do different IL-9 antibody clones compare in their binding characteristics and applications?

  Different IL-9 antibody clones have distinct binding properties affecting their applications:

  Clone	Isotype	Epitope Region	Neutralizing	Recommended Applications	Notes
  MH9D1	Not specified	Unique epitope	Yes	ICFC, ELISA	Not blocked by MH9A4; pre-titrated for flow cytometry
  MH9A4	Mouse IgG2b, κ	Human IL-9	No	Flow cytometry, ELISA	Does not block IL-9 binding to receptor; useful for detection
  EPR23484-151	Rabbit IgG	C-terminus	Not specified	IP, WB, ICC/IF, Flow Cyt	Works with human, mouse, rat; validated for multiple applications
  RM9A4	Rat IgG1, κ	Not specified	Not specified	ICFC	Specifically for mouse IL-9; APC-conjugated available

  Selection considerations:

  • For neutralization studies, select antibodies with confirmed blocking activity

  • For co-staining, choose antibodies compatible with other markers in panel

  • Consider species reactivity - some antibodies are species-specific

  • Validate antibodies for specific applications before experimental use

• How can IL-9 antibodies be utilized in cancer immunotherapy research?

  IL-9 antibodies offer valuable approaches for cancer immunotherapy research:

  Experimental design considerations:

  • Use multiple tumor models to account for cancer heterogeneity (studies have used K1735M2, CT26, and F9 models)

  • Include both immunocompetent and immunodeficient mice to assess immune contribution

  • Assess IL-9 antibody efficacy as monotherapy and in combination with other immunotherapies

  • Determine optimal dosing schedule and route of administration

  • Monitor tumor growth, survival, and immune infiltration

  Investigational approaches:

  • Neutralization studies: IL-9 deficiency enabled T cell sensitization to tumors and promoted immunologic memory in mouse models

  • Targeted delivery approaches: IL-9-based immunocytokines constructed with tumor-targeting antibodies (e.g., F8 antibody targeting EDA) for localized delivery

  • Cellular mechanisms: Analyze effects on CD8+ T cells, CD4+ T cells, and other immune populations through depletion studies and adoptive transfer experiments

  Readout parameters:

  • Tumor volume measurements

  • Survival analysis

  • Quantification of tumor-infiltrating lymphocytes

  • Assessment of memory T cell responses through rechallenge experiments

  • Analysis of cytokine profiles in tumor microenvironment

• What is known about IL-9's role in T follicular helper cells and how can researchers study this interaction?

  Recent research has revealed IL-9's role in T follicular helper (Tfh) cell function:

  Key findings:
  IL-9 receptor signaling in Tfh cells promotes:

  • Development of germinal center B cells

  • Production of antigen-specific antibodies

  • Expression of Bcl6, CXCR5, IL-4, and IL-21 in Tfh cells

  Experimental approaches to study this interaction:

  1. Genetic models: Use CD4-specific IL-9 receptor-deficient mice (Il9rafl/fl with CD4-Cre)

  2. Immunization protocols:

     • Sheep red blood cell (SRBC) immunization

     • House dust mite (HDM) inhalation for allergic asthma models

  3. Analysis methods:

     • Flow cytometry to quantify Tfh cells (CD4+CXCR5+PD-1+)

     • ELISA for antigen-specific antibody titers

     • Immunohistochemistry for germinal center formation

     • RT-PCR for expression of Bcl6, IL-4, and IL-21

  4. Source identification:

     • Analyze ILC2s as potential IL-9 producers in secondary lymphoid tissues

     • Investigate interaction between ILC2s and IgD+ B cells in producing Alox5-mediated leukotrienes

• How do researchers optimize intracellular staining protocols for IL-9 detection by flow cytometry?

  Optimizing intracellular IL-9 staining requires attention to several technical factors:

  Cell stimulation:

  • Stimulate cells with PMA (50 ng/ml) and ionomycin (1 μg/ml) for 4-6 hours

  • Add protein transport inhibitor (monensin or brefeldin A) during stimulation

  • For Th9-specific detection, polarize cells with TGF-β and IL-4 before stimulation

  Fixation and permeabilization:

  • Use Intracellular Fixation & Permeabilization Buffer Set

  • Fix cells with 4% paraformaldehyde for 10-20 minutes

  • Permeabilize with 90% methanol or specialized permeabilization buffer

  Antibody selection and titration:

  • Pre-titrate antibodies to determine optimal concentration (typically 0.06 μg per 10^6 cells)

  • Include isotype controls (e.g., Rabbit monoclonal IgG for EPR23484-151)

  • Consider fluorophore brightness and panel compatibility

  Sample preparation:

  • Use 10^5 to 10^8 cells per test in 100 μL final volume

  • Maintain cold temperature during processing to preserve cellular integrity

  • Block Fc receptors before antibody staining

  Controls and validation:

  • Use transfected cells expressing IL-9 as positive controls

  • Compare staining with multiple IL-9 antibody clones

  • Include fluorescence-minus-one (FMO) controls

• What experimental approaches can evaluate the relationship between IL-9 and regulatory T cells in disease models?

  The interaction between IL-9 and regulatory T cells (Tregs) can be studied through several approaches:

  In allergic inflammation:

  • Monitor CD4+CD25+Foxp3+ Treg populations after anti-IL-9 antibody treatment

  • Assess Foxp3 and IL-10 mRNA expression in treated tissues

  • Measure Foxp3 protein levels by Western blot or flow cytometry

  Mechanistic investigations:

  • Use adoptive transfer of Tregs from IL-9-deficient or wild-type mice to evaluate functional differences

  • Perform co-culture experiments with Tregs and effector T cells in the presence/absence of IL-9

  • Analyze IL-9 receptor expression on Treg subpopulations

  In autoimmune models:

  • Administer anti-IL-9 mAb in experimental autoimmune encephalomyelitis (EAE) models

  • Assess disease incidence and severity

  • Analyze regulatory cell populations in relevant tissues

  Molecular signaling studies:

  • Investigate STAT protein activation in Tregs following IL-9 stimulation

  • Examine changes in suppressive molecules (GITR, GITRL, ICOS, ICOSL)

  • Analyze autocrine IL-9 signaling in Treg function

  Research has shown that anti-IL-9 antibody treatment enhances tolerogenic effects of regulatory T cells, suggesting IL-9 neutralization could potentially benefit allergen immunotherapy .

• How can researchers design IL-9-based immunocytokines for targeted therapy applications?

  Development of IL-9-based immunocytokines involves several methodological considerations:

  Design strategies:

  • Select appropriate antibody fragments (scFv, diabody) for tissue targeting

  • Consider optimal positioning of IL-9 within fusion protein structure

  • Design different formats to compare targeting efficiency:

    • Antibody-IL9 (N-terminal fusion)

    • IL9-antibody (C-terminal fusion)

    • Antibody-IL9-antibody (sandwiched configuration)

  Targeting moieties:

  • Use antibodies targeting disease-relevant antigens (e.g., F8 antibody targeting EDA in neovascular structures)

  • Include non-relevant antibodies (e.g., KSF antibody against hen egg lysozyme) as controls

  Production and characterization:

  • Clone assembled constructs into mammalian expression vectors

  • Express in appropriate cell lines (often HEK293 cells)

  • Purify using affinity chromatography

  • Characterize biochemical parameters:

    • Size-exclusion chromatography

    • SDS-PAGE

    • ELISA binding assays

  Functional validation:

  • Assess bioactivity using cell proliferation assays (e.g., MC/9 mast cells)

  • Compare activity with commercial recombinant IL-9

  • Evaluate in vivo biodistribution after radioiodination

  • Measure tumor:blood ratios for targeting efficiency assessment

  Research has shown that sandwich configurations (IL-9 flanked by two antibody units) may achieve superior tumor targeting with tumor:blood ratios >10:1 at 24 hours post-administration .

• What are the key considerations when investigating IL-9's role in different disease contexts?

  IL-9's diverse roles across disease contexts require tailored experimental approaches:

  In allergic diseases:

  • Compare IL-9 expression in patient samples versus healthy controls

  • Analyze IL-9 receptor expression patterns

  • Study the effect of seasonal allergens on IL-9 production

  • Investigate genetic polymorphisms in IL9 and IL9R genes

  • Consider sex-specific differences in IL-9-related phenotypes

  In cancer studies:

  • Contrast IL-9's effects in different cancer types

  • Assess how IL-9 impacts tumor microenvironment

  • Investigate the relationship between IL-9 and immunologic memory

  • Study both CD4+ and CD8+ T cell responses to IL-9 manipulation

  • Evaluate adaptive transfer of T cells from IL-9-deficient animals

  In autoimmune conditions:

  • Determine optimal timing of anti-IL-9 treatment

  • Measure impact on disease progression markers

  • Assess IL-9's role in tissue damage and remodeling

  • Analyze the balance between inflammatory and regulatory immune responses

  Methodological considerations across contexts:

  • Include both genetic approaches (IL-9 knockout) and acute interventions (neutralizing antibodies)

  • Account for potential compensatory mechanisms in germline knockout models

  • Measure multiple parameters to capture IL-9's pleiotropic effects

  • Consider timing of IL-9 intervention relative to disease phase