CD63 Antibody

What is CD63 and why is it significant in immunological research?

CD63 is a member of the tetraspanin (TM4SF) family, a 238 amino acid protein with a molecular mass of approximately 25.6 kDa before post-translational modifications . It plays crucial roles in cell-matrix adhesion, cell migration, hemostasis, and innate immune responses . CD63's significance stems from its widespread expression across multiple immune cell types and its dynamic regulation during cell activation. In its native form, CD63 contains four transmembrane domains and undergoes extensive glycosylation, resulting in apparent molecular weights between 40-60 kDa in most experimental conditions . Research interest in CD63 has intensified due to its dual immunomodulatory roles and potential as a therapeutic target in allergic and inflammatory conditions.

How does CD63 expression vary across different cell types and activation states?

CD63 exhibits distinctive expression patterns across various immune cells. It was first identified in platelet granules, appearing on the surface only upon platelet activation . In neutrophils, CD63 resides in azurophilic granules under resting conditions but translocates to the cell surface following activation, making it a reliable marker for granule release . CD63 is also found on monocytes/macrophages and is weakly expressed on resting granulocytes, T lymphocytes, and B lymphocytes .

In mast cells and basophils, CD63 serves as a degranulation marker, with surface expression significantly increasing upon IgE-mediated activation . This differential expression pattern makes CD63 particularly valuable for monitoring cellular activation states across multiple immune cell lineages, especially in flow cytometry and immunohistochemical applications.

What are the primary applications of CD63 antibodies in immunological research?

CD63 antibodies serve multiple critical functions in research settings:

Beyond detection, CD63 antibodies serve as experimental tools to modulate immune cell functions. Anti-CD63 monoclonal antibodies can inhibit T-cell proliferation and cytokine production in peripheral blood mononuclear cells (PBMCs) . They can also suppress IgE-dependent allergic reactions by inhibiting mast cell degranulation and adhesion to extracellular matrix proteins like fibronectin and vitronectin .

How do CD63 antibodies modulate T-cell activation pathways?

The relationship between CD63 and T-cell activation represents a complex area of investigation. Anti-CD63 monoclonal antibodies like COS3A have been shown to inhibit CD3-mediated T-cell proliferation and CD25 expression, but interestingly, this effect depends on the presence of monocytes . When purified T-cells are used instead of PBMCs, the suppressive effect is not observed, suggesting an indirect mechanism of action .

Research has revealed that anti-CD63 antibodies downregulate IL-2 and IFN-γ production by T-cells while simultaneously upregulating IL-10 secretion by monocytes . This monocyte-derived IL-10 appears to be the primary mediator of T-cell suppression, as neutralizing IL-10 with anti-IL-10 antibodies restores T-cell responsiveness . This complex interplay between CD63 signaling, monocyte activation, and T-cell function represents an important area for researchers investigating immune regulation and potential therapeutic targets for autoimmune disorders.

What mechanisms underlie anti-CD63 antibody effects on mast cell degranulation?

Mechanistic studies have revealed that anti-CD63 does not affect early FcεRI signaling events such as global tyrosine phosphorylation or calcium mobilization . Instead, it specifically impairs the Gab2-PI3K signaling pathway, which is essential for both degranulation and adhesion processes . This selective inhibition of downstream signaling explains why anti-CD63 antibodies preferentially inhibit degranulation in cells adhering to extracellular matrix proteins like vitronectin and fibronectin but not in cells attached to fibrinogen or maintained in suspension .

How can CD63 antibodies be utilized to investigate integrin-tetraspanin interactions?

CD63 forms complexes with multiple integrin molecules, including the α3, α4, and α6 chains of β1 integrins, as well as with VLA-3, VLA-6, CD11/CD18, and various kinases including phosphatidylinositol 4-kinase and tyrosine kinases . These interactions position CD63 as a critical regulator of integrin-mediated adhesion and signaling.

Researchers can leverage CD63 antibodies to:

• Immunoprecipitate CD63-integrin complexes to study their composition and stoichiometry

• Investigate how CD63 clustering affects integrin activation states

• Determine how CD63-integrin interactions influence downstream signaling pathways

• Assess how these interactions vary across different cell types and activation conditions

The capacity of anti-CD63 antibodies to inhibit cell adhesion to specific extracellular matrix proteins like fibronectin and vitronectin, but not fibrinogen, provides a valuable experimental approach to dissect the selective involvement of CD63 in different integrin-mediated adhesion processes .

What are the optimal detection conditions for CD63 by Western blot?

Western blot detection of CD63 requires careful consideration of sample preparation and experimental conditions:

When analyzing CD63 by Western blot, researchers should be aware that the extensive glycosylation of this protein results in a diffuse band between 40-60 kDa under typical conditions . Treatment with N-glycanase yields a sharper band at approximately 25 kDa, reflecting the protein's core molecular weight . Additionally, some antibody clones recognize CD63 only in its non-reduced form, emphasizing the importance of maintaining disulfide bonds for epitope integrity .

How should researchers optimize immunohistochemical staining for CD63?

Successful immunohistochemical detection of CD63 requires attention to several critical parameters:

• Epitope Retrieval: Heat-induced epitope retrieval using basic pH buffers (e.g., Antigen Retrieval Reagent-Basic) is essential for optimal staining in paraffin-embedded tissues . This process uncovers epitopes masked during fixation and embedding procedures.

• Antibody Concentration: A concentration range of 0.3-25 μg/mL is typically effective, but this should be optimized for each tissue type and antibody clone . Start with the manufacturer's recommended concentration and adjust based on signal-to-noise ratio.

• Incubation Conditions: Standard protocols recommend primary antibody incubation for 1 hour at room temperature, but this may be extended to overnight at 4°C for weaker signals .

• Detection Systems: High-sensitivity detection systems like HRP-polymer antibodies (e.g., VisUCyte™ HRP Polymer) provide superior results compared to traditional secondary antibody approaches .

• Interpretation: CD63 staining typically appears on cell membranes and in cytoplasmic granules, with subcellular localization varying by cell type and activation state . In lung cancer tissue, for example, specific staining localizes to cell membranes in certain cell populations .

What controls are essential when using CD63 antibodies in flow cytometry?

Flow cytometric applications of CD63 antibodies require rigorous controls to ensure data validity:

• Isotype Controls: Match the isotype, species, and conjugation of the CD63 antibody precisely to control for non-specific binding. For example, when using Rabbit Anti-Human CD63 Monoclonal Antibody, an appropriate rabbit IgG isotype control should be employed at the same concentration .

• Fixation/Permeabilization Controls: Since CD63 has both surface and intracellular expression, comparing staining in permeabilized versus non-permeabilized samples helps distinguish these populations. FlowX FoxP3/Transcription Factor Fixation & Perm Buffer Kit has been validated for this purpose .

• Positive Cell Type Controls: Include cell types known to express CD63 strongly (e.g., activated platelets, monocytes) to confirm antibody functionality .

• Negative Cell Type Controls: Include cell populations with minimal CD63 expression or compare resting versus activated cells to demonstrate specificity .

• Blocking Controls: Pre-incubation with unlabeled anti-CD63 before adding labeled anti-CD63 can confirm epitope specificity through signal reduction .

How should researchers interpret contradictory immunomodulatory effects of CD63 antibodies?

The literature reports seemingly contradictory roles for CD63 in immune regulation, functioning as both an inhibitor and co-stimulator of T-cell responses . These apparent contradictions can be resolved through careful experimental design and data interpretation.

The inhibitory effect of anti-CD63 antibodies on T-cell proliferation is observed primarily in mixed cell populations (PBMCs) rather than in purified T-cells, suggesting an indirect mechanism . This is mediated through increased IL-10 production by monocytes, which subsequently suppresses T-cell responses . In contrast, direct effects on T-cells might manifest as co-stimulatory under different experimental conditions.

When interpreting CD63 antibody effects, researchers should:

• Consider the cellular composition of the experimental system

• Measure multiple parameters beyond proliferation (cytokine production, activation markers)

• Evaluate the timing of CD63 engagement relative to TCR stimulation

• Assess how adhesion status might influence outcomes

• Determine whether observed effects are direct or mediated through secondary messengers

These considerations help reconcile apparently contradictory data and provide a more nuanced understanding of CD63's complex role in immune regulation.

How can researchers distinguish between direct and indirect effects of anti-CD63 antibodies?

Determining whether anti-CD63 antibody effects are direct or indirect requires systematic experimental approaches:

• Cell Purification Studies: Compare antibody effects on mixed populations versus purified cell types. For example, anti-CD63 inhibits T-cell proliferation in PBMCs but not in purified T-cells, indicating an indirect mechanism .

• Cytokine Neutralization: Block potential mediators to determine their role. Neutralizing IL-10 reverses the inhibitory effect of anti-CD63 on T-cell proliferation, confirming its role as a mediator .

• Signaling Pathway Analysis: Determine which pathways are affected by anti-CD63. Anti-CD63 does not affect early FcεRI signaling events like calcium mobilization but impairs the Gab2-PI3K pathway, suggesting selective rather than global inhibition .

• Time-Course Experiments: Monitor the temporal relationship between antibody binding and functional outcomes. Delayed effects often indicate indirect mechanisms involving intermediate mediators.

• Co-culture Systems: Use transwell or conditioned media approaches to determine if soluble factors are involved in mediating anti-CD63 effects between different cell populations.

What explains the differential effects of anti-CD63 antibodies in adhesion-dependent versus suspension conditions?

The striking difference in anti-CD63 antibody effects between adherent cells and cells in suspension provides important insights into CD63 biology. In mast cells, anti-CD63 inhibits FcεRI-mediated degranulation by approximately 46% when cells are adherent but shows no significant effect when cells are in suspension .

This adhesion dependency can be explained by several factors:

This adhesion dependency has important implications for experimental design and data interpretation, as results may vary dramatically depending on whether cells are studied in adherent or suspension conditions.

What therapeutic potential do CD63 antibodies hold for allergic and inflammatory conditions?

Anti-CD63 antibodies have demonstrated promising effects in suppressing IgE-dependent allergic reactions in experimental models, suggesting potential therapeutic applications . Their ability to inhibit mast cell degranulation and adhesion to specific extracellular matrix components positions them as candidates for treating allergic disorders where mast cell hyperactivity plays a central role .

Several lines of evidence support their therapeutic potential:

• Anti-CD63 inhibits FcεRI-mediated degranulation in mast cells adherent to extracellular matrix proteins relevant to inflammatory microenvironments .

• The inhibitory effect on degranulation correlates with impaired adhesion, suggesting a dual mechanism that could enhance therapeutic efficacy .

• Anti-CD63 specifically impairs the Gab2-PI3K pathway without affecting global tyrosine phosphorylation or calcium mobilization, indicating a selective mechanism with potentially fewer side effects .

• In vivo models have confirmed that anti-CD63 antibodies can inhibit FcεRI-mediated allergic reactions, validating their potential for translation to clinical applications .

Future research should focus on optimizing antibody specificity, evaluating long-term effects, and developing delivery methods that target relevant tissue microenvironments where mast cell-matrix interactions occur.

How might advanced technologies enhance CD63 antibody applications in research?

Emerging technologies present exciting opportunities to expand CD63 antibody applications:

• Single-Cell Analysis: Combining CD63 antibodies with single-cell RNA sequencing or CyTOF can reveal heterogeneity in CD63 expression and function across cell populations and activation states .

• Super-Resolution Microscopy: Techniques like STORM or PALM can visualize CD63-containing microdomains and their dynamic interactions with partners like integrins at unprecedented resolution.

• CRISPR-Cas9 Epitope Engineering: Precise modification of CD63 epitopes can generate cells expressing variants that interact differentially with antibodies, enabling structure-function studies.

• Bispecific Antibodies: Developing antibodies that simultaneously target CD63 and relevant partners could provide new tools to study and manipulate tetraspanin-protein complexes.

• Intravital Imaging: CD63 antibodies adapted for in vivo imaging could track dynamic changes in CD63 expression during immune responses in living organisms.

These technological advances will not only enhance our understanding of CD63 biology but may also reveal new potential applications for CD63-targeting therapeutics in inflammatory and immune-mediated conditions.