CD84 Human, Sf9

What is CD84 and what cellular functions does it regulate?

CD84 (SLAMF5) is a type I transmembrane glycoprotein belonging to the SLAM family of cell-surface receptors. It contains two extracellular Ig domains (a membrane-proximal Ig constant domain and a membrane-distal Ig variable domain) and its cytoplasmic tail contains two copies of an Immunoreceptor Tyrosine-based Switch Motif (ITSM) . CD84 functions as a modulator of immune responses, particularly in leukocyte activation. It mediates homophilic adhesion through its N-terminal Ig domain, forming dimers similar to other SLAM family members . In macrophages, CD84 modulates TLR-induced responses by increasing MAPK phosphorylation and NF-κB activation, subsequently regulating cytokine production patterns by enhancing TNF-α and MCP-1 secretion while reducing IL-10 and IL-6 production .

Which cell types express CD84 in humans?

CD84 shows a broad expression pattern across human hematopoietic cells. It is expressed on:

• T and B lymphocytes (with highest expression on memory cells)

• T follicular helper (TFH) cells (with particularly elevated expression)

• Monocytes and macrophages

• Granulocytes

• Dendritic cells (DCs)

• Mast cells

• Hematopoietic stem cells (HSCs)

Expression levels vary by cell type and activation state, with innate-like lymphocytes and specific T cell subsets showing higher expression levels . Expression is upregulated during certain maturation stages of lymphocyte development, indicating stage-specific functional roles .

What expression systems are most effective for producing recombinant human CD84?

While the search results don't specifically address Sf9 insect cell expression of CD84, effective expression systems for SLAM family receptors typically include:

• Mammalian cell expression systems: These provide appropriate post-translational modifications, particularly the extensive glycosylation that CD84 undergoes. Common cell lines include HEK293 and CHO cells.

• Insect cell expression systems: Sf9 or High Five cells using baculovirus vectors can be advantageous for higher yield protein production while maintaining most post-translational modifications. For CD84, which is highly glycosylated, researchers should consider that insect cells produce simpler glycosylation patterns than mammalian cells.

The methodology should be selected based on downstream applications. For functional studies requiring proper folding and glycosylation, mammalian systems may be preferable. For structural studies requiring higher protein yields, the Sf9 system may offer advantages.

What are the critical considerations when designing plasmid constructs for CD84 expression?

When designing CD84 expression constructs, researchers should consider:

• Domain selection: The extracellular domain contains two Ig-like domains responsible for homophilic binding. For interaction studies, expressing just the extracellular region may be sufficient.

• Signal peptide optimization: Ensure the native or optimized signal peptide is included for proper trafficking.

• Affinity tags placement: C-terminal tags are generally preferred as N-terminal tags may interfere with the N-terminal Ig domain's homophilic interactions.

• Glycosylation sites: CD84 is highly glycosylated , so mutations of glycosylation sites should be carefully considered as they may affect folding and function.

• Codon optimization: For expression in Sf9 cells, codon optimization for insect cell preference can improve yield.

How does CD84 regulate macrophage activation and cytokine production?

CD84 functions as a modulator of TLR-induced responses in macrophages. Experimental evidence shows:

• Enhanced signaling: Transfection of CD84 in RAW-264.7 macrophages increases MAPK phosphorylation and NF-κB activation upon LPS stimulation .

• Differential cytokine regulation: CD84 expression increases LPS-induced TNF-α and MCP-1 secretion while significantly reducing IL-10 and IL-6 production .

• Tyrosine-dependent mechanisms: This modulatory effect is specifically mediated by Y300 within the second ITSM of CD84 .

• Knock-down effects: CD84 knock-down in bone marrow-derived macrophages (BMDMs) decreases TNF-α and IL-6 production following LPS activation .

These findings suggest CD84 plays a role in fine-tuning macrophage inflammatory responses, potentially influencing the balance between pro- and anti-inflammatory cytokine production.

What is the relationship between CD84 and PD-L1 expression in immune cells?

CD84 has been shown to regulate PD-L1 expression in multiple immune cell types:

• Direct regulation: Activation of CD84 on multiple myeloma cells induces PD-L1 expression, while blocking CD84 lowers PD-L1 mRNA and protein levels .

• Effect on myeloid cells: CD84 activation on CD14+ cells from healthy donors increases surface PD-L1 expression .

• Impact on MDSCs: CD84 activation elevates PD-L1 mRNA levels in both monocytic (M-MDSCs) and granulocytic (G-MDSCs) myeloid-derived suppressor cells from multiple myeloma patients. Conversely, blocking CD84 reduces PD-L1 expression and diminishes MDSC immunosuppressive function .

• Signaling mechanism: CD84 stimulation increases phosphorylation of AKT and S6, which are known regulators of PD-L1 expression .

These findings suggest CD84 contributes to creating an immunosuppressive microenvironment by upregulating PD-L1 expression on various immune cell populations.

What are effective approaches for studying CD84 homophilic interactions?

Studying CD84 homophilic interactions (where CD84 binds to itself) requires specialized techniques:

• Protein crystallography: This has successfully revealed the crystal structure of human CD84, showing a 2-fold homophilic dimer similar to other SLAM family members .

• Surface plasmon resonance (SPR): This can determine binding kinetics and affinity of CD84-CD84 interactions using purified proteins.

• Cell adhesion assays: Cells expressing CD84 can be tested for homophilic adhesion using aggregation assays or flow-based adhesion systems.

• FRET/BRET techniques: These can detect CD84-CD84 interactions in live cells by tagging CD84 molecules with complementary fluorescent or bioluminescent proteins.

• Site-directed mutagenesis: Creating point mutations in the N-terminal Ig domain can identify specific residues involved in homophilic binding.

For these studies, properly folded and glycosylated CD84 protein is critical, making the expression system choice (mammalian vs. insect) an important consideration.

How can researchers effectively study the CD84 signaling cascade?

CD84 signaling is complex and involves multiple downstream pathways. Research approaches include:

• Phosphoprotein analysis: Detecting phosphorylation of ITSMs in the CD84 cytoplasmic domain and recruitment of SH2-containing proteins (SAP, EAT-2) using phospho-specific antibodies and co-immunoprecipitation .

• SH2 domain interaction assays: Pull-down experiments with SH2 domains to identify binding partners and their binding requirements.

• Knockout/knockdown studies: CRISPR/Cas9 or siRNA approaches to eliminate CD84 expression and observe signaling consequences, as demonstrated in THP1 cells .

• Pathway analysis: Monitoring activation of downstream signaling molecules, particularly MAPK, NF-κB, AKT, and S6, following CD84 stimulation .

• Receptor clustering assays: Using anti-CD84 antibodies to induce receptor clustering and trigger signaling events.

• Protein stability assessment: Analyzing how CD84 signaling affects the stability of key transcription factors, as seen with IRF8 in dendritic cells .

How is CD84 implicated in cancer immunology?

CD84 appears to play significant roles in the tumor microenvironment across multiple cancer types:

• Multiple myeloma (MM): CD84 expression is elevated on MM cells compared to precursor conditions like MGUS. More significantly, CD84 is strongly upregulated on cells within the MM microenvironment, particularly CD14+ myeloid cells .

• Immunosuppressive mechanisms: CD84 regulates PD-L1 expression on both tumor cells and myeloid cells in the tumor microenvironment, contributing to T cell exhaustion and immune evasion .

• Melanoma: SLAMF9, another SLAM family member, has been identified in tumor-associated macrophages (TAMs) from melanoma, suggesting SLAM family receptors may broadly influence tumor-associated immunity .

• Therapeutic targeting: Blocking CD84 reduces the immunosuppressive capacity of myeloid cells and enhances T cell responses, suggesting potential immunotherapeutic applications .

Research examining CD84 as both a biomarker and therapeutic target in cancer is still developing, with particular promise in addressing immune evasion mechanisms.

What methodological approaches are most effective for targeting CD84 in disease models?

Several approaches have shown efficacy in targeting CD84 in experimental disease models:

• Blocking antibodies: The B4 blocking monoclonal antibody effectively reduces CD84-mediated effects, including PD-L1 upregulation, demonstrating utility in functional studies .

• siRNA knockdown: Effective for transient CD84 silencing in primary cells, as demonstrated in monocyte-derived dendritic cells .

• CRISPR/Cas9 gene editing: Creating CD84-deficient cell lines, such as THP1 cells with CD84 knockout, provides stable models for studying CD84 functions .

• Transgenic expression: Overexpression of CD84 in cell lines like RAW 264.7 allows gain-of-function studies to assess downstream effects .

• Recombinant protein competition: Soluble CD84 extracellular domain can potentially disrupt CD84-CD84 interactions without activating signaling.

The choice of approach depends on the specific research question, with consideration for whether inhibition of CD84-CD84 interactions or blockade of downstream signaling is the primary goal.

What are common challenges in expressing and purifying human CD84 protein?

Researchers working with CD84 may encounter several challenges:

• Glycosylation heterogeneity: CD84 is highly glycosylated, which can result in protein heterogeneity and may complicate structural studies or affect binding assays.

• Protein stability: The extracellular domain may have stability issues outside the membrane context, potentially requiring optimization of buffer conditions or addition of stabilizing mutations.

• Functional conformation: Ensuring the recombinant protein maintains native conformation, particularly for the homophilic binding interface.

• Expression yield: Mammalian expression systems often provide lower yields than insect cell systems, requiring optimization of culture conditions and harvest timing.

• Tag interference: Affinity tags may interfere with CD84 functions, necessitating careful tag design and placement, with potential tag removal for functional studies.

How can researchers validate CD84 antibodies for different applications?

Proper antibody validation is critical for CD84 research and should include:

• Positive and negative controls: Testing on cells with known high expression (e.g., activated T cells, macrophages) versus CD84-negative or CD84-knockout cells.

• Cross-reactivity assessment: Testing against related SLAM family members, particularly important for polyclonal antibodies.

• Application-specific validation:

  • For flow cytometry: Titration to determine optimal concentration and comparison with isotype controls

  • For Western blotting: Confirmation of expected molecular weight (~73 kDa), accounting for glycosylation

  • For immunoprecipitation: Verification of ability to capture native CD84

  • For functional studies: Confirmation of neutralizing or activating capacity

• Epitope mapping: Understanding which domain the antibody recognizes, as antibodies against different epitopes may have distinct functional effects.