CD300A Antibody

What is CD300A and why is it significant in immune checkpoint research?

CD300A is a 60 kDa glycoprotein member of the immunoglobulin superfamily, also known as LMIR1 (in rodents), CMRF-35H, IRp60, CLM-8, and MAIR-I. The molecular structure consists of a 163 amino acid extracellular domain (ECD) with one Ig-like V-type domain, a 21 amino acid transmembrane segment, and a 98 amino acid cytoplasmic domain containing three immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and one non-canonical ITIM . CD300A functions as an inhibitory receptor on various immune cells including natural killer (NK) cells, eosinophils, mast cells, neutrophils, plasmacytoid dendritic cells, and various T cell subsets . Its significance stems from its role as an immune checkpoint that negatively regulates immune cell activation through phosphatase recruitment, making it a promising target for immunotherapeutic interventions in cancer and infectious diseases.

How does the molecular mechanism of CD300A inhibitory signaling compare to other immune checkpoints?

When CD300A binds to its ligands, primarily phosphatidylserine (PS) and phosphatidylethanolamine (PE), it induces phosphorylation of tyrosine residues in its cytoplasmic ITIMs. This phosphorylation leads to the recruitment of phosphatases SHIP, SHP-1, and SHP-2, which then dephosphorylate key molecules in activation signaling pathways . Unlike PD-1 and CTLA-4 that primarily regulate T cell responses, CD300A has broader inhibitory effects across multiple immune cell types. Furthermore, CD300A is distinctive in recognizing phospholipid ligands rather than protein ligands. The inhibitory cascade initiated by CD300A engagement effectively dampens immune cell functions, including cytotoxicity, cytokine production, and degranulation, providing an additional layer of immune regulation that can be exploited therapeutically .

What experimental approaches best demonstrate CD300A's interaction with phosphatidylserine?

To demonstrate CD300A's interaction with phosphatidylserine (PS), researchers employ several methodological approaches:

• PS Exposure Systems: Implementation of stimulation systems where PS is exposed on the outer surface of malignant cells allows observation of CD300A engagement .

• Binding Assays: Direct binding assays using recombinant CD300A protein and PS-containing liposomes can quantitatively measure interaction affinity.

• Functional Inhibition Studies: The functional impact of PS-CD300A interaction can be demonstrated by comparing NK cell cytotoxicity against PS-exposing targets with and without CD300A blockade .

• Competitive Inhibition: Using PS-binding proteins like Annexin V to compete with CD300A for PS binding can confirm specificity of the interaction.

• Phosphatase Recruitment Measurement: Detection of phosphatase recruitment (SHIP, SHP-1, SHP-2) to CD300A following PS exposure provides mechanistic validation of signaling initiation.

Research has shown that PS exposure significantly hampers NK cell function, and blocking PS-CD300A interaction enhances immune cell activity against targets like hematologic malignancies .

What is the significance of CD300A polymorphisms in research design and interpretation?

CD300A exhibits a significant single nucleotide polymorphism (rs2272111) that results in either arginine (CD300A R111) or glutamine (CD300A Q111) at position 111 in the Ig-like domain . This polymorphism has important implications for research:

• Antibody Selection: Different antibody clones may have varying affinities for the R111 versus Q111 variants, necessitating careful selection of detection antibodies .

• Population Differences: The frequency of these variants differs across populations, potentially explaining inconsistent results across studies with different demographic compositions.

• Functional Differences: The polymorphism may affect binding affinity to ligands or interaction with downstream signaling molecules, altering inhibitory potency.

• Therapeutic Response Prediction: Variant-specific differences may predict differential responses to CD300A-targeted therapies.

When designing CD300A research, investigators should consider genotyping samples or selecting antibodies that recognize both variants to avoid misinterpretation of results. Monoclonal antibodies specific to either CD300A R111 or CD300A Q111 or both have been developed to address this challenge .

What are the optimal methods for detecting CD300A expression across different immune cell populations?

For comprehensive CD300A detection across immune cell populations, researchers should employ multiple complementary methods:

For flow cytometry, optimal analysis includes proper compensation, isotype controls, and comparison of expression levels across activated vs. resting states. In clinical samples, CD300A expression analysis should incorporate correlation with clinical parameters, as heightened CD300A expression has been associated with shorter survival and NK cell exhaustion phenotypes .

What methodological approaches provide the most effective CD300A functional blockade?

Effective CD300A functional blockade can be achieved through several approaches, each with specific methodological considerations:

• Monoclonal Antibodies: The most direct approach uses blocking antibodies like TX49, which has been validated in both in vitro and in vivo settings . For in vitro studies, a concentration of 10μg/ml is typically effective after 4-hour pre-incubation prior to functional assays. For in vivo applications, a dosage of 10μg/kg body weight administered intravenously at days 0, 2, 4, and 6 post-intervention has demonstrated efficacy .

• Ligand Blocking: Preventing PS-CD300A interaction by masking PS using Annexin V or other PS-binding molecules represents an alternative strategy.

• Genetic Approaches: siRNA knockdown or CRISPR/Cas9 knockout of CD300A provides more complete abrogation of CD300A function, though care must be taken to confirm specificity given the homology with other CD300 family members.

• Soluble CD300A: Recombinant soluble CD300A extracellular domain can act as a decoy to capture ligands before they interact with cell-surface CD300A.

Proper controls are essential, including isotype antibodies for blocking studies and scrambled siRNA for genetic approaches. Functional readouts should include multiple parameters (cytotoxicity, cytokine production, degranulation) to comprehensively assess the impact of CD300A blockade .

What are the critical quality control parameters for CD300A antibody preparation and storage?

Maintaining CD300A antibody quality requires strict adherence to preparation and storage protocols:

Critical quality control parameters include:

• Freeze-Thaw Management: Use a manual defrost freezer and avoid repeated freeze-thaw cycles, as this significantly degrades antibody quality .

• Reconstitution Procedure: Follow manufacturer-specific guidelines for buffer composition and reconstitution volumes to ensure optimal antibody activity.

• Sterility Maintenance: Work in sterile conditions to prevent microbial contamination that could degrade the antibody or introduce artifacts in experiments.

• Functional Validation: Regularly test antibody functionality using positive control samples with known CD300A expression or through functional blocking assays.

• Lot-to-Lot Consistency: When receiving new antibody lots, perform parallel testing with previous lots to ensure consistent performance before deploying in critical experiments.

These measures ensure experimental reproducibility and reliable results in CD300A research applications .

How should researchers design CD300A-focused NK cell functional assays?

Designing robust CD300A-focused NK cell functional assays requires careful consideration of multiple parameters:

• Target Cell Selection: Choose target cells that differentially expose PS/PE (CD300A ligands) on their surface. Hematologic malignancy cell lines that naturally expose high levels of PS provide excellent models .

• Blocking Strategies: Implement CD300A blocking using monoclonal antibodies (10μg/ml) with appropriate isotype controls. Pre-incubate NK cells with blocking antibodies for 4 hours before initiating functional assays .

• Readout Parameters: Include multiple functional readouts:

  • Cytotoxicity (51Cr release, calcein retention, or flow-based assays)

  • Degranulation (CD107a surface expression)

  • Cytokine production (IFN-γ, TNF-α)

  • Expression of lysis-associated proteins

• Controls:

  • Positive controls: NK cells stimulated with IL-2/IL-15

  • Negative controls: NK cells with inhibitory receptor engagement

  • Isotype antibody controls: To account for non-specific antibody effects

  • Target-only controls: To measure spontaneous death/release

• Time Course: Evaluate both short-term (4 hours) and long-term (24-72 hours) effects to capture immediate cytotoxicity and sustained functional changes.

Research has shown that blocking PS-CD300A signals significantly enhances NK cell expression of lysis-related proteins and effector cytokines, augmenting their ability to lyse hematologic malignancies .

What techniques can accurately differentiate between CD300A and other CD300 family members?

Differentiating between highly homologous CD300 family members requires specialized techniques:

• Specific Monoclonal Antibodies: Select antibodies validated for specificity against CD300A rather than other family members. For example, clone 232612 has been validated for human CD300A specificity .

• Western Blot Analysis: CD300A can be identified at approximately 60 kDa under non-reducing conditions using specific antibodies . Comparing molecular weights can help distinguish between family members.

• PCR-Based Discrimination:

  • Design primers targeting unique regions of CD300A sequence

  • Perform melting curve analysis to confirm amplification specificity

  • Consider digital PCR for absolute quantification

• Expression Pattern Analysis: CD300A has a distinct expression pattern across immune cell subsets that differs from other family members. Flow cytometric analysis of multiple immune populations can help confirm specificity.

• Functional Validation: CD300A contains inhibitory ITIMs while some family members contain activating motifs. Functional assays demonstrating inhibition rather than activation provide additional confirmation of CD300A-specific effects.

• Genetic Verification: siRNA or CRISPR targeting unique CD300A sequences with validation of knockdown/knockout specificity provides definitive confirmation.

These approaches, particularly when used in combination, ensure accurate attribution of effects to CD300A rather than related family members.

How does CD300A modulation affect NK cell metabolic programming and exhaustion phenotypes?

CD300A engagement has significant impacts on NK cell metabolic programming and exhaustion phenotypes, with important implications for anti-tumor immunity:

• Metabolic Reprogramming: CD300A signaling likely interferes with the metabolic shift from oxidative phosphorylation to glycolysis that typically accompanies NK cell activation, though this mechanism requires further investigation.

• Exhaustion Phenotype Induction: Clinical data show that heightened CD300A expression correlates with an "exhausted" phenotype of intratumoral NK cells in patients with hematologic malignancies and solid tumors . This exhaustion phenotype is characterized by:

  • Reduced expression of activation markers

  • Decreased production of effector cytokines

  • Impaired cytotoxic potential

  • Altered metabolic function

• Reversibility Through Blockade: Importantly, blocking PS-CD300A signals with antibodies significantly amplifies the expression of lysis function-related proteins and effector cytokines in NK cells, suggesting the exhaustion phenotype can be reversed through therapeutic intervention .

• Survival Impact: In xenograft models, CD300A overexpression shortened the survival of hematologic malignancy-xenografted mice by impairing transplanted NK cells, providing in vivo evidence of CD300A's negative impact on anti-tumor immunity .

These findings suggest that CD300A represents a novel metabolic immune checkpoint that drives NK cell dysfunction in the tumor microenvironment, making it a promising target for reinvigorating NK cell-based cancer therapies.

What role does CD300A play in engineering next-generation allogeneic cell therapies?

CD300A has emerged as a critical molecule for enhancing allogeneic cell therapies, particularly in addressing NK cell-mediated rejection:

• Problem of NK Alloreactivity: When donor HLA class I is eliminated to prevent T-cell alloreactivity, this strategy also unleashes NK cell alloreactivity due to loss of inhibitory signaling .

• CD300A as Universal Protection: An engineered CD300A agonist ligand (CD300A TASR) has been developed as a new class of NK inhibitory ligand that can universally protect HLA-deficient allogeneic T cells from NK cell-mediated rejection .

• Superior Performance: In focused screens, CD300A TASR outperformed leading alternative strategies, including CD47 and HLA-E, and was uniquely capable of universally protecting allogeneic T cells against a large human cohort (45/45 donors) spanning diverse demographics and NK cell phenotypes .

• Enhanced CAR-T Efficacy: A model allogeneic T-cell therapy coexpressing an anti-CD19 chimeric antigen receptor and CD300A TASR, produced using multiplexed nonviral integration, exhibited enhanced B-cell killing potency under allogeneic immune pressure .

• Implementation Strategy: Genetic engineering approaches incorporating CD300A TASR expression can be integrated into current manufacturing processes for allogeneic therapies.

This innovative application of CD300A biology represents a universal solution to NK alloreactivity, potentially broadening the population that could be effectively treated by next-generation allogeneic cell therapies .

How can CD300A targeting strategies be integrated with other immune checkpoint inhibitors?

Integration of CD300A targeting with other immune checkpoint inhibitors offers promising combinatorial approaches:

• Complementary Checkpoint Coverage: CD300A primarily regulates NK cells and innate immune functions, while checkpoints like PD-1/PD-L1 and CTLA-4 primarily regulate T cell responses. Combining these approaches could provide broader immune reactivation.

• Synergistic Potential: Blocking CD300A enhances NK cell functions including cytokine production and cytotoxicity, which could synergize with T cell-focused checkpoint inhibitors by:

  • Increasing tumor antigen release through enhanced NK-mediated tumor lysis

  • Promoting dendritic cell activation through NK-derived cytokines

  • Enhancing NK-T cell cross-talk through improved NK function

• Implementation Approaches:

  • Sequential administration: CD300A blockade followed by standard checkpoint inhibitors

  • Concurrent administration: Simultaneous administration of multiple blocking antibodies

  • Bispecific antibody development: Single molecules targeting both CD300A and other checkpoint molecules

• Considerations for Combined Targeting:

  • Potential for increased immune-related adverse events

  • Optimal timing and dosing for each component

  • Patient selection based on expression patterns of multiple checkpoints

Preclinical research suggests that CD300A blockade significantly enhances NK cell lytic function , providing strong rationale for combination with T cell-targeting checkpoint inhibitors to achieve comprehensive immune reactivation against malignancies.

What is the significance of CD300A in infectious disease immunology research?

CD300A plays a crucial role in infectious disease immunology, particularly in regulating phagocyte functions and T cell responses:

• Parasite Infection Models: In Leishmania infection models, CD300A receptor blocking enhances the early clearance of parasites from visceral organs . This effect appears mediated through multiple mechanisms:

  • Enhanced phagocyte effecter functions

  • Improved cytokine production

  • Better antigen presentation

• Impact on T Cell Differentiation: CD300A blockade benefits antigen-experienced CD4+CD44+ and CD8+CD44+ T cells, enabling them to acquire more pro-inflammatory cytokine-producing phenotypes .

• Memory Formation Enhancement: CD300A receptor blocking enhances the conversion of CD4+ T effector cells to memory phenotypes (CCR7highCD62Lhigh) by 1.6-fold and 1.9-fold at 14 and 21 days post-infection, respectively .

• Experimental Design Approach:

  • For in vivo studies: anti-CD300A antibodies (10μg/kg body weight) administered intravenously at days 0, 2, 4, and 6 post-infections

  • For in vitro studies: cells pre-treated with anti-CD300A antibodies (10μg/ml) for 4 hours before infection

• Methodological Controls:

  • Isotype antibody controls

  • Direct testing of antibody effects on parasite viability

  • Control for PS exposure on parasites using duramycin

These findings indicate that CD300A is an important regulator of host phagocytic cell functions and T cell differentiation against pathogens, highlighting its potential as a therapeutic target in infectious diseases .

What are the correlations between CD300A expression and clinical outcomes in different malignancies?

Research has revealed significant correlations between CD300A expression and clinical outcomes across various malignancies:

• Survival Impact: Heightened CD300A expression correlates with shorter survival in patients with hematologic malignancies (HMs) and solid tumors, as revealed through analysis of The Cancer Genome Atlas (TCGA) database .

• NK Cell Exhaustion: High CD300A expression is associated with an "exhausted" phenotype of intratumoral NK cells, characterized by reduced effector function and cytokine production .

• Tumor Infiltration Patterns: Increased infiltration of CD300A-positive lymphocytes is observed within tumors compared to peri-tumoral tissues, suggesting specific recruitment or induction in the tumor microenvironment .

• Therapeutic Implications: Blocking PS-CD300A signals significantly enhances NK cell function against malignant cells, suggesting potential therapeutic benefits of CD300A blockade .

• In Vivo Validation: CD300A overexpression shortened the survival of HM-xenografted mice by impairing transplanted NK cells, providing direct evidence for CD300A's negative impact on anti-tumor immunity .

These findings collectively position CD300A as both a prognostic marker and a potential therapeutic target in malignancies, particularly those where NK cell function is critical for disease control.

What are the most common causes of inconsistent CD300A antibody staining results?

Inconsistent CD300A antibody staining can stem from several technical and biological factors:

• Polymorphic Variants: CD300A has a single nucleotide polymorphism (rs2272111) resulting in either arginine (R111) or glutamine (Q111) variants . Using antibodies that preferentially recognize one variant will produce inconsistent results in genetically diverse sample sets.

• Epitope Accessibility Issues: The CD300A epitope may be masked by ligand binding (PS/PE) or conformational changes, particularly in samples with high levels of apoptosis or cellular stress.

• Variable Expression Levels: CD300A expression fluctuates based on cellular activation state and environment. Inconsistent handling or activation of samples can lead to variable expression.

• Technical Variables:

  • Inadequate fixation or permeabilization protocols

  • Suboptimal antibody concentration (typical working concentration: 2 μg/mL)

  • Buffer composition variations

  • Fc receptor-mediated non-specific binding

• Alternative Splicing: CD300A has alternatively spliced isoforms that may lack the Ig-like domain or contain only the cytoplasmic domain , potentially affecting epitope presence.

To mitigate these issues, researchers should validate antibodies across multiple sample types, include appropriate controls, optimize staining protocols for specific applications, and consider using antibodies specifically developed to recognize both polymorphic variants .

How can researchers troubleshoot non-specific binding in CD300A Western blot applications?

Non-specific binding in CD300A Western blot applications can be addressed through several methodological refinements:

• Optimized Blocking: Use 5% non-fat dry milk or BSA in TBS-T for 1-2 hours at room temperature. For particularly problematic samples, consider alternative blocking agents such as casein or commercially available blocking buffers.

• Buffer Selection: Use Immunoblot Buffer Group 1 for CD300A detection, as specified in published protocols . This buffer composition has been validated for specific CD300A detection.

• Membrane Considerations:

  • PVDF membranes are recommended for CD300A detection

  • Reduce auto-fluorescence by brief methanol treatment if using fluorescent detection

  • Consider low-fluorescence membranes for fluorescent detection systems

• Antibody Parameters:

  • Optimize primary antibody concentration (2 μg/mL recommended for MAB2640)

  • Extend incubation time while reducing antibody concentration

  • Use HRP-conjugated secondary antibodies specific to the primary antibody species (e.g., Anti-Rat IgG for MAB2640)

• Technical Adjustments:

  • Include non-reducing conditions as specified for CD300A detection

  • Increase washing duration and number of washes (minimum 3x15 minutes with TBS-T)

  • Prepare fresh buffers to avoid contamination issues

• Controls:

  • Include positive control (human granulocyte lysates)

  • Run negative control samples (cell lines not expressing CD300A)

  • Include secondary-only control lane to identify non-specific secondary binding

These approaches should improve the specificity of CD300A detection at its expected molecular weight of approximately 60 kDa .

What strategies can improve CD300A antibody-based immunoprecipitation efficiency?

Enhancing CD300A antibody-based immunoprecipitation requires attention to several critical parameters:

• Lysis Buffer Optimization:

  • Use mild non-ionic detergents (0.5-1% NP-40 or Triton X-100) to preserve protein-protein interactions

  • Include phosphatase inhibitors to preserve ITIM phosphorylation status

  • Add protease inhibitor cocktail to prevent degradation

  • Consider including 10% glycerol for protein stability

• Antibody Selection and Coupling:

  • Choose antibodies validated for immunoprecipitation applications

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

  • Consider covalent coupling of antibodies to beads to prevent co-elution of antibody chains

  • For difficult samples, try different antibody clones targeting different epitopes

• Incubation Parameters:

  • Extend incubation time (overnight at 4°C) to improve capture efficiency

  • Use gentle rotation rather than aggressive shaking

  • Optimize antibody-to-lysate ratio through titration experiments

• Washing Procedures:

  • Implement stringent washing with increasing salt concentrations

  • Include detergent in wash buffers to reduce non-specific interactions

  • Perform at least 4-5 wash cycles with large volumes

• Elution Strategies:

  • For phosphorylation studies, use phosphopeptide elution to maintain phosphorylation status

  • Consider native elution methods for downstream functional assays

  • For mass spectrometry applications, optimize elution buffers for compatibility

• Verification Approaches:

  • Confirm successful immunoprecipitation by immunoblotting for CD300A

  • Verify co-immunoprecipitation of known interactors (e.g., SHP-1, SHP-2, SHIP)

These strategies should improve both the specificity and yield of CD300A immunoprecipitation, facilitating downstream analyses of interaction partners and post-translational modifications.

How should CD300A antibody performance be validated across different sample types?

Comprehensive validation of CD300A antibody performance across different sample types requires a systematic approach:

• Multi-sample Type Validation Matrix:

• Genetic Controls:

  • Use CD300A knockout/knockdown models as negative controls

  • Include CD300A-overexpressing systems as positive controls

  • Compare polymorphic variant samples (R111 vs. Q111)

• Cross-reactivity Assessment:

  • Test antibody against other CD300 family members to confirm specificity

  • Evaluate species cross-reactivity if working with both human and animal models

• Quantitative Performance Metrics:

  • Signal-to-noise ratio across sample types

  • Linearity of detection across expression ranges

  • Lot-to-lot consistency evaluation

  • Reproducibility across technical replicates

• Application-specific Validation:

  • For functional blocking studies: confirm inhibition of PS-CD300A interaction

  • For polymorphism detection: verify discrimination between R111 and Q111

  • For clinical applications: establish concordance with clinical parameters

Thorough validation across diverse sample types ensures reliable and reproducible results in CD300A research, particularly important given its variable expression across cell types and disease states .

What considerations are critical when developing or selecting antibodies against specific CD300A polymorphic variants?

Development or selection of antibodies against specific CD300A polymorphic variants requires careful consideration of several critical factors:

• Epitope Selection:

  • Target regions containing the polymorphic residue (position 111)

  • Design peptide immunogens that specifically incorporate either R111 or Q111

  • Consider structural modeling to identify accessible epitopes

• Validation Strategy:

  • Test against cell lines or recombinant proteins expressing each variant

  • Verify specificity using flow cytometry, ELISA, and Western blot

  • Confirm variant discrimination using samples of known genotype

• Cross-reactivity Testing:

  • Evaluate binding to other CD300 family members

  • Test against closely related proteins from different species

  • Assess potential cross-reactivity with common contaminants

• Application-specific Optimization:

  • For flow cytometry: optimize fluorochrome conjugation and titration

  • For Western blot: test under reducing and non-reducing conditions

  • For functional studies: verify blocking capacity

• Production Considerations:

  • Select appropriate antibody format (monoclonal vs polyclonal)

  • Choose optimal host species to minimize background in target applications

  • Consider humanization for potential therapeutic applications

• Clinical Translation Potential:

  • Evaluate correlation with disease outcomes for each variant

  • Assess predictive value for response to CD300A-targeted therapies

  • Consider development of companion diagnostics for variant detection

Successful development of monoclonal antibodies specific to either CD300A R111 or CD300A Q111 or both has been achieved , enabling researchers to analyze CD300A genotype by flow cytometry and supporting the development of tailored therapeutic approaches that account for this significant polymorphism.