CD84 Antibody

What is CD84 and what cell types express this antigen?

CD84 is a member of the signaling lymphocyte activation molecule (SLAM) family, functioning as a self-binding immunoreceptor. It is a highly glycosylated cell surface glycoprotein of approximately 64-82 kDa that predominantly appears on:

• B lymphocytes and B cell lines (including pre-B cell lines, but not plasma cell lines)

• Monocytes and macrophages (strongly expressed on tissue macrophages)

• Platelets (high expression)

• T cells (lower levels, preferentially on CD45RO+ T cells)

• Granulocytes (lower levels)

CD84 expression patterns vary across disease states, with significant differences observed between healthy donors and patients with conditions like multiple myeloma or chronic lymphocytic leukemia .

What applications are most suitable for CD84 antibodies in immunological research?

Based on validated applications across multiple sources, CD84 antibodies are suitable for:

For flow cytometry applications, researchers should note that CD84 expression is particularly strong on B cells and monocytes, making these populations good positive controls. When analyzing T cells, focusing on CD45RO+ populations will yield more consistent detection .

How should samples be prepared for optimal CD84 detection in flow cytometry?

For optimal detection of CD84 in flow cytometric applications:

• Use freshly isolated cells when possible; cryopreserved samples may show reduced expression

• For whole blood applications: Use 5μl of antibody per 100μl of whole blood

• For isolated cell suspensions: Use 5μl per million cells in 100μl staining volume

• Include appropriate blocking step (Fc block) to prevent non-specific binding

• When analyzing T cells, consider co-staining with CD45RO to identify the T cell subset with highest CD84 expression

• Use appropriate compensation controls when using PE, FITC or other fluorochrome-conjugated anti-CD84 antibodies

Multiple sources confirm that CD84 detection benefits from minimal processing time between sample collection and staining to preserve surface epitopes .

How does CD84 expression differ between healthy individuals and patients with hematological malignancies?

Research has demonstrated significant differences in CD84 expression across hematological conditions:

Interestingly, while multiple myeloma cells themselves express low levels of CD84, they induce strong expression of CD84 on cells in their microenvironment through secretion of macrophage migration inhibitory factor (MIF). This creates an important distinction between intrinsic CD84 expression and microenvironment-induced expression .

What is the relationship between CD84 activation and immune checkpoint regulation?

CD84 plays a significant role in regulating immune checkpoint molecules, particularly the PD-1/PD-L1 axis:

• CD84 activation on CLL and MM cells upregulates PD-L1 expression

• CD84 activation on stromal cells and myeloid cells in the tumor microenvironment increases PD-L1 expression

• CD84 signaling in T cells enhances PD-1 expression

• Blocking CD84 with antagonistic antibodies reduces PD-L1 expression on malignant cells and their microenvironment

• CD84 blockade reduces expression of T cell exhaustion markers (PD-1, LAG-3, CTLA-4)

The molecular pathway involves CD84-mediated activation of AKT and S6 phosphorylation, which are known regulators of PD-L1 expression. This suggests CD84 as a potential upstream target for immune checkpoint modulation .

How can researchers effectively block CD84 homophilic interactions in experimental settings?

To block CD84 homophilic interactions in research settings:

• Antibody-based approaches:

  • Use B4 blocking monoclonal antibody, which has been validated to antagonize CD84 signaling

  • Clone CD84.1.21 has been shown to partially block CD84-Ig binding to lymphocytes

  • Effective concentrations typically range from 5-20 μg/ml depending on the experimental system

• Genetic approaches:

  • siRNA knockdown of CD84 is effective in cell lines (demonstrated in 5TGM1 myeloma model)

  • CRISPR/Cas9 system has been used to establish CD84 knockout in THP1 cells

  • CD84^-/- mouse models are available for in vivo studies

• Experimental readouts to confirm effective blockade:

  • Decreased PD-L1 expression on target cells

  • Reduced phosphorylation of downstream signaling molecules (pAKT, pS6)

  • Enhanced T cell proliferation and function in co-culture systems

Studies have validated these approaches in both human primary samples and mouse models .

What methodological approaches should be used to investigate CD84's role in modulating the immunosuppressive tumor microenvironment?

Investigating CD84's role in the immunosuppressive tumor microenvironment requires multi-faceted approaches:

• Cell type-specific analysis:

  • Isolate distinct cellular populations (malignant cells, MDSCs, T cells, stromal cells)

  • Use multiparameter flow cytometry to simultaneously assess CD84 and immune checkpoint molecules

  • Employ cell sorting to obtain pure populations for functional studies

• Co-culture systems with selective blockade:

  • Establish tri-culture systems (tumor cells + myeloid cells + T cells)

  • Perform selective CD84 blockade on specific cell populations

  • Measure outcomes including T cell proliferation, cytokine production, and cytotoxicity

• Transcriptional and signaling analysis:

  • Assess changes in myeloid differentiation genes in response to CD84 modulation

  • Monitor pathways regulating MDSC accumulation and function

  • Quantify PD-L1/PD-1 expression changes at both mRNA and protein levels

• In vivo validation approaches:

  • Use CD84 knockout bone marrow chimeras to distinguish microenvironment vs. tumor cell effects

  • Treat tumor-bearing animals with CD84 blocking antibodies

  • Assess changes in immune cell infiltration, activation status, and tumor burden

Research has shown this integrated approach can reveal how CD84 bridges between malignant cells and their microenvironment to create immunosuppressive conditions .

How does CD84 blockade affect myeloid-derived suppressor cell (MDSC) development and function?

CD84 plays a critical role in MDSC biology, with blockade affecting multiple aspects of these immunosuppressive cells:

• Effects on MDSC differentiation:

  • CD84 activation leads to upregulation of genes regulating differentiation to both M-MDSCs and G-MDSCs

  • CD84 blockade reduces the accumulation of MDSCs in tumor microenvironments

  • Transcriptional changes affecting myeloid differentiation pathways occur following CD84 inhibition

• Impact on suppressive function:

  • CD84 blockade significantly reduces PD-L1 expression on both M-MDSCs and G-MDSCs at both mRNA and protein levels

  • Antagonizing CD84 on sorted MDSCs reduces their suppressive activity against T cells

  • CD84 inhibition leads to increased CD8+ T cell division and IFN-γ secretion in MDSC co-culture systems

• Methodological considerations for studying these effects:

  • MDSC populations should be carefully defined (M-MDSCs: Ly6G-, Ly6C+, CD11b+, CD11c- and G-MDSCs: Ly6G+, Ly6Clo, CD11b+, CD11c-)

  • Both transcriptional (mRNA) and surface protein expression changes should be monitored

  • Functional suppression assays are essential to confirm biological relevance

These findings indicate CD84 as a regulator of both MDSC development and their immunosuppressive capacity .

What are the technical considerations when evaluating CD84's role in different hematological malignancies?

When investigating CD84 across different hematological malignancies, several technical considerations are critical:

• Disease-specific expression patterns:

  • MM cells express low levels of CD84 but induce high expression in their microenvironment

  • CLL cells express higher intrinsic levels of CD84

  • Expression patterns differ between MM, MGUS, and healthy controls

• Sample preparation considerations:

  • Bone marrow aspirates require processing within 24 hours to maintain surface antigen integrity

  • Use of enzymatic digestion for solid tissue samples may affect CD84 epitope detection

  • Patient-derived samples show greater variability than cell lines

• Experimental design challenges:

  • Distinguish between direct effects on malignant cells versus indirect effects through microenvironment

  • Account for heterogeneous expression across patient samples

  • Consider the role of CD84-induced MIF secretion as a confounding factor

• Functional assay selection:

  • Chromium release assays can assess T cell-mediated killing of malignant cells

  • 7AAD staining provides an alternative measure of cell death

  • BrdU incorporation quantifies effects on proliferation

These considerations help researchers design robust experiments that account for the complex biology of CD84 across different hematological conditions .

What are the signaling mechanisms downstream of CD84 that regulate immune function?

CD84 initiates complex signaling cascades that regulate immune cell function through multiple pathways:

• Proximal signaling events:

  • CD84 activation leads to phosphorylation of its immunoreceptor tyrosine-based switch motifs (ITSMs)

  • This creates docking sites for adapter proteins including SH2D1A/SAP and SH2D1B/EAT-2

  • In myeloid cells, CD84 signaling implicates FES and PTPN6/SHP-1 (independent of SH2D1A and SH2D1B)

• Downstream pathway activation:

  • CD84 stimulation significantly increases phosphorylation of AKT and S6

  • These pathways directly regulate PD-L1 expression

  • In macrophages, CD84 enhances LPS-induced MAPK phosphorylation and NF-κB activation

• Cell type-specific signaling differences:

  • In platelets, homophilic interactions enhance interferon gamma secretion via a SH2D1A-dependent pathway

  • In mast cells, CD84 negatively regulates high-affinity immunoglobulin epsilon receptor signaling

  • In dendritic cells, CD84 positively regulates macroautophagy via stabilization of IRF8 by inhibiting TRIM21-mediated proteasomal degradation

• Methodological approaches to study CD84 signaling:

  • Phospho-flow cytometry to detect activation of signaling intermediates

  • Biochemical analysis of phosphorylation states of downstream targets

  • Co-immunoprecipitation to identify interacting partners

  • CRISPR/Cas9 knockout followed by rescue with CD84 mutants lacking specific signaling motifs

Understanding these signaling mechanisms provides insight into potential therapeutic targeting strategies for CD84 in various disease contexts .