CD244 Human, Sf9

What is CD244 and what are its main structural characteristics?

CD244 (also known as 2B4/SLAMF4) is an immunoregulatory receptor belonging to the Signaling Lymphocyte Activation Molecule (SLAM) family. Structurally, human CD244 contains an extracellular segment with two immunoglobulin (Ig)-like domains and a cytoplasmic domain with four immunoreceptor tyrosine-based switch motifs (ITSMs) . Notably, humans express two isoforms of CD244 via differential splicing of hnRNA, both with identical intracellular domains containing four ITSMs. These isoforms differ extracellularly by the presence or absence of five amino acids between the immunoglobulin V and C2 domains, with the shorter isoform demonstrating increased affinity for CD48 and enhanced calcium flux .

Why is CD244 considered a potential therapeutic target in immuno-oncology?

CD244 shows significant therapeutic potential due to its role in immune exhaustion within the tumor microenvironment. Research demonstrates that CD244 is expressed on multiple immune cell types critical for anti-tumor immunity, including exhausted CD8+ T cells, dendritic cells (DCs), and myeloid-derived suppressor cells (MDSCs) . Importantly, CD244 knockout mice show significantly impaired tumor growth of head and neck squamous cell carcinoma (HNSCC), and interventional treatment with anti-CD244 monoclonal antibodies impairs established tumor growth while increasing tumor-infiltrating CD8+ T cells . These findings position CD244 as a promising target for cancer immunotherapy, especially as current checkpoint inhibitors like PD-1 and CTLA-4 blockers remain suboptimal for many patients .

How does the Sf9 expression system benefit the production of functional human CD244 protein?

The Sf9 baculovirus expression system provides several advantages for producing human CD244 for research applications:

What are the optimal conditions for expressing and purifying human CD244 in Sf9 cells?

Successful expression of human CD244 in Sf9 cells requires careful optimization of multiple parameters:

The purification protocol should include clarification of culture supernatant by centrifugation, primary capture via affinity chromatography, and at least one polishing step (typically size exclusion) to ensure high purity and homogeneity .

What methods are recommended for functional validation of Sf9-expressed human CD244?

Functional validation of recombinant CD244 should employ multiple complementary approaches:

• Biochemical validation: SDS-PAGE and Western blotting using anti-CD244 antibodies (such as C1.7) to confirm protein identity and purity .

• Binding assays: Surface plasmon resonance or bio-layer interferometry to measure binding kinetics to CD48, with expected KD values in the nanomolar range. The shorter isoform should demonstrate higher affinity .

• Cell-based functional assays: Assess the ability of recombinant CD244 to:

  • Inhibit production of proinflammatory cytokines in human dendritic cells (when crosslinked)

  • Affect IFNγ production in CD8+ T cells (with effects dependent on CD244 concentration)

  • Modulate NK cell cytotoxicity against target cells

• Structural validation: Circular dichroism to confirm proper folding and stability of the protein.

How can researchers accurately assess the activating versus inhibitory functions of CD244?

The dual functionality of CD244 requires sophisticated experimental design:

• Concentration-dependent effects: Titrate CD244 concentrations, as research shows that low-intermediate CD244 expression correlates with activating signaling, while high CD244 expression correlates with inhibitory signaling in both NK cells and CD8+ T cells .

• Adapter molecule assessment: Quantify levels of key signaling molecules including SAP (SH2D1A), EAT-2 (SH2D1B), and phosphatases (SHP-1, SHP-2) in the experimental system, as their relative abundance determines signaling outcomes .

• Cell-specific readouts: For NK cells, measure cytotoxicity and IFNγ production; for CD8+ T cells, assess proliferation, cytokine production, and degranulation markers (CD107a, perforin); for DCs, evaluate proinflammatory cytokine production .

• Signaling pathway analysis: Use phospho-flow cytometry or Western blotting to track phosphorylation events in the CD244 signaling cascade.

How can researchers effectively investigate CD244's role in the tumor microenvironment?

Investigating CD244 in the tumor context requires a multi-faceted approach:

• Comprehensive immune profiling: Compare CD244 expression levels across multiple immune cell types (CD8+ T cells, NK cells, DCs, MDSCs) in tumors versus peripheral blood or healthy tissues. Research shows significantly increased CD244 expression on tumor-infiltrating immune cells compared to peripheral counterparts .

• Correlation with exhaustion markers: Analyze co-expression of CD244 with other exhaustion markers such as PD-1, LAG-3, and TIM-3 on CD8+ T cells. Studies demonstrate that CD244 expression correlates with PD-1 expression on tumor-infiltrating CD8+ T cells .

• Functional assessment: Measure spontaneous production of immunosuppressive mediators by CD244-expressing cells isolated from tumors versus peripheral tissues. CD244-high cells show increased expression of immunosuppressive molecules .

• Genetic approaches: Compare tumor growth kinetics in wild-type versus CD244-/- mice, as demonstrated in HNSCC models showing significantly impaired tumor growth in knockout animals .

• Interventional studies: Test anti-CD244 monoclonal antibodies in established tumor models to assess effects on tumor growth, immune cell infiltration, and activation status .

What experimental strategies can resolve contradictory findings in CD244 signaling research?

Contradictory findings regarding CD244 function can be addressed through systematic investigation:

• CD244 density analysis: Quantitatively measure CD244 expression levels, as research consistently shows that CD244 receptor concentration affects whether activating or inhibitory signaling occurs. In CD8+ T cells with high CD244 expression, blocking CD244 increases IFNγ production (reflecting inhibitory signaling), while in cells with low-intermediate CD244 expression, blocking decreases IFNγ production (reflecting activating signaling) .

• Adapter molecule profiling: Comprehensively assess the expression and availability of key signaling molecules in your experimental system:

• Competitive binding assessment: Evaluate the competitive interactions between signaling molecules, as studies show that only one molecule associates with the ITSM at a time, and the presence of SAP prevents binding of inhibitory phosphatases .

• ITSM-specific analysis: Investigate the role of individual ITSMs, as mechanistic models show that different ITSMs preferentially bind different signaling molecules (e.g., in human NK cells, the first, second, and fourth ITSMs of CD244 activate NK-mediated cytotoxicity by binding SAP, while the third ITSM binds phosphatases and inhibits NK cytotoxicity) .

How can Sf9-expressed CD244 be utilized for therapeutic antibody development?

Sf9-expressed CD244 provides an excellent platform for therapeutic antibody development:

• Immunogen preparation: Use purified recombinant CD244 from Sf9 cells as an immunogen for antibody generation, ensuring proper folding and epitope presentation.

• Epitope mapping: Perform comprehensive epitope mapping to identify antibodies that bind to functionally important regions, particularly those that might block CD244-CD48 interactions or induce conformational changes affecting signaling.

• Functional screening: Test antibody candidates in:

  • Binding inhibition assays to assess ability to block CD244-CD48 interaction

  • Cell-based assays measuring reversal of exhaustion in CD8+ T cells

  • Cytokine production assays with dendritic cells

  • NK cell cytotoxicity assays

• In vivo validation: Evaluate promising antibodies in tumor models, measuring:

  • Tumor growth inhibition

  • Changes in immune cell infiltration and phenotype

  • Combination effects with established checkpoint inhibitors

• Humanization strategies: For antibodies showing therapeutic potential, develop humanization strategies to reduce immunogenicity while maintaining binding and functional properties.

What are common challenges in CD244 expression and how can they be addressed?

Researchers often encounter several challenges when working with CD244:

How should researchers interpret conflicting CD244 functional data across different experimental systems?

When reconciling conflicting data, consider these methodological factors:

• Experimental system differences: Compare results between in vitro cell lines, primary cells, and in vivo models. CD244 function has been shown to differ dramatically between these systems.

• Expression level effects: Normalize results to CD244 expression levels, as high versus low expression correlates with inhibitory versus activating functions. Flow cytometric quantification using standardized beads can provide absolute expression values .

• Adapter molecule availability: Measure and report levels of key signaling molecules (SAP, EAT-2, phosphatases) in your experimental system, as their relative abundance determines signaling outcomes .

• Stimulation conditions: Standardize stimulation protocols. For CD8+ T cells, anti-CD3/CD28 stimulation affects CD244 function differently than antigen-specific stimulation .

• Temporal dynamics: Consider time-course experiments, as CD244 signaling may show different effects at different time points post-stimulation.

What critical controls should be included when studying CD244 in cancer immunotherapy research?

Robust CD244 research requires comprehensive controls:

• Genetic controls: Include CD244-/- cells or mice as negative controls. Studies have shown that CD244-/- mice have significantly impaired tumor growth in HNSCC models .

• Antibody controls: Use isotype controls matched to anti-CD244 antibodies; include Fab fragments to distinguish between blocking and crosslinking effects.

• Expression level controls: Compare cells with different CD244 expression levels (high vs. low) as functional outcomes differ based on expression density .

• Combination controls: When testing CD244 targeting with other checkpoint inhibitors, include single-agent controls to assess potential synergistic or antagonistic effects.

• Species-specific considerations: Be cautious when extrapolating between mouse and human systems, as there are structural differences between species (mice express two isoforms with different ITSM numbers, whereas humans express two isoforms with identical intracellular domains) .