CD300C Human, Sf9

What is CD300c and how does it function within the CD300 family?

CD300c belongs to the CD300 family of receptors that regulate various immune cell processes. Unlike its inhibitory counterpart CD300a, CD300c functions as an activating receptor. It contains a short cytoplasmic tail and a negatively charged amino acid residue in its transmembrane region that allows association with adaptor molecules FcεRIγ and DAP12 . Although CD300c was the first molecule identified in this receptor family, its function in primary cells has only recently been elucidated .

CD300c shares significant sequence homology with B7 family members, suggesting evolutionary relationships with these important immune regulatory molecules . The receptor has a potential role in inflammatory responses, particularly in monocytes, where cross-linking with specific antibodies induces calcium mobilization, upregulation of costimulatory molecules, and production of inflammatory cytokines .

What is the expression pattern of CD300c on human immune cells?

CD300c has a distinct expression pattern on human immune cells. Using the specific monoclonal antibody clone TX45, researchers have determined that within freshly isolated blood leukocytes, monocytes are the primary cells expressing CD300c on their surface . This contrasts with CD300a, which is more broadly expressed on various immune cells including NK cells, mast cells, B and T lymphocytes, macrophages, and granulocytes .

CD300c is also expressed on professional antigen-presenting cells (APCs), including:

• Monocytes (highest expression)

• Macrophages (differential expression based on polarization)

• Dendritic cells (variable expression)

• B cells

Interestingly, CD300c is uniquely expressed on CD56 bright Natural Killer cells following specific cytokine stimulation, indicating a specialized role in this NK cell subset .

How can researchers differentiate between CD300a and CD300c?

• Specific antibodies: The monoclonal antibody clone TX45 specifically recognizes CD300c but not CD300a, making it valuable for differential detection .

• Molecular structure differences: CD300a has a long cytoplasmic tail with three classical ITIMs and one non-classical ITIM, while CD300c has a short cytoplasmic tail .

• Functional assays: CD300a elicits inhibitory signals capable of suppressing multiple immune cell functions, whereas CD300c triggers activating signals .

• Real-time PCR: Using specific primers (e.g., PPH07153A for human CD300c), researchers can quantify CD300c mRNA relative to housekeeping genes like β-actin .

Flow cytometry protocols for differentiating these receptors typically involve:

• Using staining buffer containing 1% human AB serum to block Fc receptors

• Incubating cells with fluorochrome-conjugated antibodies for 30 minutes on ice

• Extensive washing to remove unbound antibodies

• Analysis using flow cytometers such as FACSCalibur or LSRII

What methodologies are effective for expressing CD300c in Sf9 insect cell systems?

Sf9 insect cells provide an excellent system for expressing human CD300c for functional and structural studies. The methodology involves:

• Vector construction: The CD300c extracellular domain is typically cloned into baculovirus expression vectors such as pFastBac or pAcGP67, often with a C-terminal His-tag or Fc-fusion for purification purposes.

• Transfection and viral amplification:

  • Transfect Sf9 cells with the recombinant bacmid DNA

  • Collect P1 viral stock after 72 hours

  • Amplify to generate high-titer P2 and P3 stocks

  • Optimize viral titer to prevent cell lysis during expression

• Expression conditions:

  • Infect Sf9 cells at a density of 1.5-2.0 × 10^6 cells/ml

  • Culture at 27°C with gentle shaking (120-140 rpm)

  • Harvest after 48-72 hours post-infection

  • Monitor expression using western blot or flow cytometry

• Protein purification:

  • Clarify supernatant by centrifugation (10,000×g, 30 min)

  • Perform affinity chromatography using Ni-NTA (for His-tagged) or Protein A/G (for Fc-fusion)

  • Further purify by size exclusion chromatography

Sf9-expressed CD300c has been successfully used in binding assays to investigate ligand interactions, as mentioned in the research where starved Sf9 cells were utilized in CD300 binding studies .

How do cytokines regulate CD300c expression on immune cells?

CD300c expression is dynamically regulated by various cytokines, with cell type-specific responses:

• On CD56 bright NK cells:

  • IL-2 and IL-15 treatment significantly induce CD300c expression exclusively on CD56 bright NK cells

  • This upregulation requires STAT5 signaling

  • IL-4 inhibits CD300c expression

  • IL-2 secreted from activated CD4+ T cells specifically induces CD300c expression on CD56 bright NK cells

• On monocytes:

  • TLR ligands dynamically regulate CD300c expression

  • LPS (TLR4 ligand) and flagellin (TLR5 ligand) modulate CD300c at both protein and mRNA levels

  • Time-course experiments show that CD300c expression changes within hours of stimulation

Experimental methodology for studying cytokine effects includes:

• Culture of PBMCs (1-2 × 10^6/ml) with specific cytokines or TLR ligands

• Time-course analysis (typically 6, 12, 24, and 48 hours)

• Flow cytometric analysis of surface expression

• Real-time quantitative PCR for mRNA quantification

• Western blot analysis of signaling mediators like STAT5 phosphorylation

What approaches are used to identify and characterize CD300c ligands?

Identifying CD300c ligands has been challenging, but several methodological approaches have proven effective:

• Recombinant protein binding assays:

  • Generate CD300c-Ig fusion proteins

  • Label with fluorochromes (e.g., Alexa Fluor 488)

  • Perform binding assays on potential target cells

  • Analyze using flow cytometry

• Lipid binding assays:

  • Prepare liposomes with specific phospholipids (PE, PS, PC, etc.)

  • Incubate with CD300c-Ig fusion proteins

  • Detect binding through ELISA or flow cytometry-based methods

• Competitive binding experiments:

  • Pre-incubate cells with phospholipid-binding proteins (e.g., MFG-E8, duramycin)

  • Perform CD300c binding assays

  • Analyze inhibition patterns to identify lipid specifics

• Cell-based screening:

  • Test CD300c binding to cells in different states (live, apoptotic, activated)

  • Use annexin V and propidium iodide to correlate binding with cell death markers

  • Investigate the role of divalent cations by using EDTA or EGTA

Research suggests that similar to CD300a, which binds to phosphatidylethanolamine (PE) and phosphatidylserine (PS), CD300c may interact with phospholipids exposed during apoptosis or cellular activation .

How can researchers functionally characterize CD300c in different experimental systems?

Functional characterization of CD300c requires multiple experimental approaches:

• Cross-linking experiments:

  • Use specific antibodies like clone TX45 to cross-link CD300c on cell surfaces

  • Measure calcium mobilization using calcium-sensitive dyes

  • Analyze upregulation of activation markers (e.g., CD86)

  • Quantify cytokine production using ELISA or cytometric bead arrays

• Gene silencing approaches:

  • Transfect monocytes or other CD300c-expressing cells with specific siRNA

  • Typically use 300nM siRNA concentration

  • Verify knockdown efficiency by flow cytometry and qPCR

  • Analyze functional consequences in relevant assays

• Combined stimulation assays:

  • Stimulate cells with TLR ligands (e.g., LPS) alone or in combination with CD300c engagement

  • Measure inflammatory cytokine production

  • This approach revealed that LPS-mediated cytokine production by monocytes is enhanced when CD300c is simultaneously engaged

• NK cell functional assays:

  • Isolate CD56 bright NK cells

  • Engage CD300c with specific antibodies

  • Measure degranulation (CD107a expression)

  • Analyze chemokine and cytokine secretion profiles

  • Results show that CD300c cross-linking enhances the ability of CD56 bright NK cells to degranulate and produce cytokines

What are the methodological considerations when using CD300c-Fc fusion proteins for immunotherapy research?

CD300c-Fc fusion proteins have significant potential for immunomodulation, particularly for T cell-mediated diseases. Key methodological considerations include:

• Design and expression:

  • Optimal design includes the extracellular domain of CD300c fused to the Fc portion of human or mouse IgG

  • Expression systems include mammalian cells (for glycosylation) or Sf9 cells (for higher yield)

  • Purification typically involves Protein A/G affinity chromatography followed by size exclusion

• Functional validation:

  • T cell proliferation assays using CFSE dilution

  • Mouse splenic cells labeled with CFSE and cultured with anti-CD3 antibody and graded doses of CD300c-Ig protein

  • Analysis of proliferation in both CD4+ and CD8+ T cells

  • Assessment of activation markers (CD69) expression

• In vivo models:

  • Graft-versus-host disease (GVHD) models have shown that CD300c-Fc administration attenuates disease progression

  • Dosing regimens typically involve 100-200 μg per injection

  • Treatment schedules determine efficacy and longevity of effect

  • Multiple parameters should be monitored: survival, clinical scores, histopathology, and T cell function

• Mechanism investigation:

  • Identification of the putative counter-receptor on T cells (expression increases upon activation)

  • Analysis of signaling pathways affected by CD300c-Fc treatment

  • Determination of effects on cytokine patterns and T cell differentiation

Research has shown that CD300c-Fc fusion proteins significantly inhibit the proliferation, activation, and cytokine production by both CD4 and CD8 T cells in vitro, suggesting therapeutic potential for T cell-mediated diseases .

What is the potential role of CD300c in inflammatory and autoimmune diseases?

CD300c's role as an activating receptor on monocytes and its ability to enhance inflammatory cytokine production suggest significant implications for inflammatory and autoimmune conditions:

• Inflammatory disorders:

  • CD300c engagement on monocytes enhances LPS-induced inflammatory cytokine production

  • This synergistic effect may contribute to excessive inflammation in sepsis or chronic inflammatory conditions

  • Cross-linking of CD300c induces calcium mobilization and upregulation of costimulatory molecules like CD86, potentially amplifying inflammatory cascades

• Autoimmune diseases:

  • CD300c-Fc fusion proteins inhibit T cell immunity and attenuate GVHD in mice

  • This suggests potential therapeutic applications in T cell-mediated autoimmune conditions

  • The inhibitory effect occurs for both CD4+ and CD8+ T cells, indicating broad immunomodulatory potential

• Balance with inhibitory CD300a:

  • CD300a recognizes phosphatidylethanolamine and phosphatidylserine on apoptotic cells

  • This interaction regulates the removal of dead cells, a process crucial for preventing autoimmunity

  • The balance between activating CD300c and inhibitory CD300a may be disturbed in autoimmune conditions

Research approaches to investigate CD300c in disease models include:

• Flow cytometric analysis of CD300c expression in patient samples

• Correlation of expression levels with disease activity scores

• Functional assays comparing healthy versus disease-associated monocytes

• Therapeutic targeting in animal models using CD300c-Fc fusion proteins or antibodies

How do the experimental methodologies for CD300c differ between human and mouse systems?

Understanding the differences between human and mouse CD300c systems is crucial for translational research:

Key methodological considerations when transitioning between systems:

• Antibody selection must account for species-specific epitopes

• Fusion protein design may require optimization for each species

• Signaling pathway analysis may reveal species-specific differences

• In vivo models should consider the evolutionary differences in receptor distribution and function

Research has shown that human CD300c-Fc fusion proteins can effectively modulate mouse T cell responses, suggesting sufficient cross-species conservation of functional interactions .

What are the current technical challenges in CD300c research and how can they be addressed?

Several technical challenges exist in CD300c research, each requiring specific methodological approaches:

• Distinguishing CD300c from CD300a:

  • Challenge: High sequence homology (>80%) between CD300a and CD300c

  • Solution: Use clone TX45 antibody specific for CD300c

  • Alternative: Develop molecular approaches using unique sequence regions

  • Future direction: Generation of additional specific monoclonal antibodies

• Ligand identification:

  • Challenge: Limited knowledge of natural ligands for CD300c

  • Solution: Systematic screening of phospholipids and cell surface molecules

  • Approach: Use recombinant CD300c-Fc fusion proteins in binding assays

  • Control: Compare with known CD300a ligand interactions (PE and PS)

• Functional redundancy:

  • Challenge: Potential functional overlap with other activating receptors

  • Solution: Combinatorial knockdown/knockout approaches

  • Analysis: Comprehensive phenotypic and functional readouts

  • Interpretation: Consider compensatory mechanisms when interpreting results

• Expression system optimization:

  • Challenge: Protein folding and post-translational modifications

  • Solution for structural studies: Sf9 insect cell expression provides high yield

  • Solution for functional studies: Mammalian expression preserves glycosylation

  • Validation: Confirm biological activity of recombinant proteins in functional assays

• Translational relevance:

  • Challenge: Extrapolating from in vitro findings to in vivo significance

  • Solution: Develop relevant animal models

  • Approach: Correlate findings with human disease samples

  • Future direction: Single-cell analysis to capture heterogeneity in expression and function

What signaling pathways are activated downstream of CD300c?

CD300c functions as an activating receptor with distinct signaling mechanisms:

• Adaptor molecule associations:

  • CD300c associates with the adaptor molecules FcεRIγ and DAP12

  • This association occurs through a negatively charged amino acid residue in the transmembrane region

  • The adaptor molecules contain immunoreceptor tyrosine-based activation motifs (ITAMs)

• Early signaling events:

  • Cross-linking CD300c with specific antibodies induces calcium mobilization

  • This indicates activation of phospholipase C-γ (PLC-γ) and IP3-mediated calcium release

  • The calcium flux can be measured using fluorescent calcium indicators in flow cytometry or microscopy

• Downstream pathways:

  • NF-κB activation leads to production of inflammatory cytokines

  • MAPK pathways regulate cellular activation and differentiation

  • The exact contribution of each pathway requires inhibitor studies or phospho-flow cytometry

• Functional outcomes:

  • Upregulation of costimulatory molecules (e.g., CD86)

  • Production of inflammatory cytokines

  • Enhanced response to TLR ligands like LPS

Experimental approaches to study CD300c signaling include:

• Phospho-specific flow cytometry to track kinase activation

• Immunoprecipitation to identify protein-protein interactions

• Inhibitor studies to delineate pathway contributions

• Genetic approaches (mutation of key residues) to determine critical signaling motifs

How can researchers study the interaction between CD300c and its ligands?

Studying CD300c-ligand interactions requires specialized methodologies:

• Recombinant protein production:

  • Generate CD300c extracellular domain as Fc fusion proteins

  • Express in appropriate systems (mammalian or Sf9)

  • Purify using affinity chromatography

  • Validate protein folding and functionality

• Cell-based binding assays:

  • Incubate target cells with fluorescently labeled CD300c-Ig

  • Wash extensively to remove unbound protein

  • Analyze binding by flow cytometry

  • Include controls like CD300a-Ig and control-Ig

• Biochemical binding assays:

  • Prepare liposomes with defined lipid composition

  • Perform solid-phase binding assays (ELISA-based)

  • Measure binding using detection antibodies or labeled proteins

  • Determine binding affinity and specificity

• Competitive binding experiments:

  • Pre-incubate cells or lipids with known ligand binders

  • Example: Use MFG-E8 (binds PS) or duramycin (binds PE)

  • Add CD300c-Ig and assess binding inhibition

  • This approach helped identify PS and PE as CD300a ligands

• Mutagenesis studies:

  • Generate point mutations in CD300c

  • Express mutant proteins and assess binding

  • Identify critical residues for ligand recognition

  • Compare with structure-function relationships of related receptors

This systematic approach has successfully identified phosphatidylserine and phosphatidylethanolamine as ligands for CD300a, suggesting similar methodologies may identify CD300c ligands .

What are emerging approaches for studying CD300c in complex immune environments?

Several cutting-edge approaches are emerging for studying CD300c in complex immune contexts:

• Single-cell technologies:

  • Single-cell RNA sequencing to map CD300c expression across immune populations

  • CITE-seq combining protein and transcript detection at single-cell resolution

  • Spatial transcriptomics to understand tissue-specific expression patterns

  • These approaches can reveal heterogeneity in CD300c expression and regulation

• Advanced imaging techniques:

  • Live-cell imaging of CD300c clustering during immune cell interactions

  • Super-resolution microscopy to visualize receptor organization in the membrane

  • Intravital microscopy to track CD300c-expressing cells in vivo

  • These methods provide spatial and temporal resolution of CD300c function

• CRISPR-based functional genomics:

  • CRISPR knockout of CD300c in primary human monocytes

  • CRISPR activation/inhibition to modulate CD300c expression

  • CRISPR screens to identify regulators of CD300c expression and function

  • These genetic tools allow precise manipulation of CD300c biology

• Systems biology approaches:

  • Multi-omics integration (transcriptomics, proteomics, metabolomics)

  • Network analysis of CD300c signaling pathways

  • Mathematical modeling of CD300c's role in inflammatory responses

  • These integrative methods place CD300c function in broader biological context

These emerging approaches will help address key questions about CD300c's role in immune regulation and its potential as a therapeutic target.

How might CD300c be targeted therapeutically in immune-mediated diseases?

CD300c represents a promising therapeutic target with several potential approaches:

• CD300c-Fc fusion proteins:

  • Function as decoy receptors to block endogenous CD300c-ligand interactions

  • Inhibit T cell proliferation, activation, and cytokine production

  • Attenuate graft-versus-host disease in mouse models

  • May be effective in other T cell-mediated autoimmune conditions

• Blocking antibodies:

  • Prevent CD300c from engaging its natural ligands

  • Reduce inflammatory responses in monocytes

  • Could be humanized for therapeutic development

  • May require careful epitope selection to avoid cross-reactivity with CD300a

• Agonistic antibodies:

  • Engage CD300c to promote specific immune responses

  • Could enhance anti-tumor immunity by activating NK cells

  • May be useful in vaccination strategies

  • Cross-linking CD300c enhances NK cell degranulation and cytokine production

• Small molecule modulators:

  • Target the CD300c signaling pathway

  • Offer potential advantages in tissue penetration and dosing

  • Requires detailed understanding of structure-function relationships

  • May achieve more selective modulation of specific downstream pathways

The optimal therapeutic approach will depend on the disease context, with inhibitory strategies suitable for inflammatory conditions and stimulatory approaches potentially beneficial for immune activation against infections or tumors.