SLAMF6 Antibody, FITC conjugated

What is SLAMF6 and what cellular functions does it regulate?

SLAMF6 (Signaling Lymphocyte Activation Molecule Family member 6) is a self-ligand receptor of the SLAM family expressed on a wide variety of hematopoietic cells. It functions as a T cell co-receptor that modulates the activation and differentiation of immune cells, playing crucial roles in both innate and adaptive immune responses. SLAMF6 activities are primarily controlled by the presence or absence of small cytoplasmic adapter proteins, specifically SH2D1A/SAP and SH2D1B/EAT-2 . The receptor participates in multiple immune functions, including:

• Regulation of T cell activation and proliferation through colocalization with the CD3 complex

• Promotion of T cell differentiation into helper T-cell Th17 phenotype, resulting in increased IL-17 secretion

• Recruitment of RORC to the IL-17 promoter

• Participation in natural killer T (NKT) cell lineage development and differentiation in conjunction with SLAMF1

• Modulation of germinal center formation and B-cell tolerance

SLAMF6 can transmit either positive or negative signals depending on its cellular context, compartmentalization, and the presence of adapter proteins, making it a nuanced regulator of immune cell function.

How does SLAMF6 compartmentalization affect T cell functions?

The spatial distribution and compartmentalization of SLAMF6 on the cell surface significantly impact its functional outcomes. Research has demonstrated that T cell activity is enhanced when SLAMF6 colocalizes with the CD3 complex . This enhancement operates through several mechanisms:

• Co-immunoprecipitation analyses reveal that SLAMF6 interacts with proteins essential for signaling downstream of the T cell receptor, suggesting shared signaling pathways between the two receptors .

• When SLAMF6 is engaged in close proximity to CD3 (plate-bound SLAMF6 condition), T cell proliferation increases compared to CD3 stimulation alone .

• Conversely, when SLAMF6 is spatially separated from CD3 during receptor engagement (soluble SLAMF6 condition), T cell proliferation is inhibited .

What are the key molecular interactions of SLAMF6 in immune signaling?

SLAMF6 engages in several critical molecular interactions that determine its signaling outcomes:

• Homophilic interaction in trans between SLAMF6 molecules on different cells serves as the primary activation mechanism .

• SLAMF6 associates with the CD3 complex, sharing downstream signaling proteins, which explains the enhanced T cell activation when these receptors cluster together .

• The receptor's cytoplasmic domain interacts with adapter proteins SH2D1A/SAP and SH2D1B/EAT-2, which dictate the nature of subsequent signaling .

• In NK cells, positive signaling involves phosphorylation of VAV1, and activation depends primarily on SH2D1B rather than SH2D1A .

• In the absence of SH2D1A/SAP, SLAMF6 can transmit negative signals to CD4+ T cells and NKT cells, likely through increased association with phosphatase PTPN6/SHP-1 via its immunoreceptor tyrosine-based switch motifs (ITSMs) .

These interactions collectively determine whether SLAMF6 will enhance or inhibit immune responses in different cellular contexts.

What types of SLAMF6 antibodies are available for research and how do they differ?

Various SLAMF6 antibodies are available for research applications, each with specific properties and applications:

• Monoclonal vs. Polyclonal: Monoclonal antibodies offer higher specificity for particular epitopes, while polyclonal antibodies (like the rabbit polyclonal ab213690) recognize multiple epitopes and may provide greater sensitivity .

• Species Reactivity: Most commercially available antibodies target mouse or human SLAMF6, with limited cross-reactivity between species .

• Conjugation Status: Antibodies may be unconjugated or conjugated to various fluorophores (FITC, PE, APC) or enzymes for different detection methods .

• Functional Properties: Some antibodies are designed for detection only (suitable for flow cytometry, immunohistochemistry), while others have functional effects (agonistic or antagonistic) .

• Bispecific Antibodies: Specially designed bispecific antibodies targeting both CD3 and SLAMF6 can promote receptor clustering and enhance T cell activation .

For immunohistochemistry applications, research has employed antibodies like the rabbit polyclonal antibody (ab213690) that targets a synthetic peptide within mouse Slamf6 aa 50-150 conjugated to Keyhole Limpet Haemocyanin .

How can FITC-conjugated SLAMF6 antibodies be optimally used in flow cytometry?

FITC-conjugated SLAMF6 antibodies are valuable tools for flow cytometric analysis of SLAMF6 expression and localization. For optimal use in flow cytometry:

• Titration: Determine the optimal antibody concentration by titrating the antibody to achieve maximum separation between positive and negative populations while minimizing non-specific binding.

• Sample Preparation:

  • Isolate PBMCs or relevant cell populations using standard density gradient protocols

  • Adjust cell concentration to 1-5 × 10^6 cells/mL in appropriate buffer (PBS with 0.5-2% BSA or FBS)

  • Minimize exposure to light after adding FITC-conjugated antibodies to prevent photobleaching

• Staining Protocol:

  • Include appropriate isotype controls to determine background fluorescence

  • For multi-color flow cytometry, use compensation controls to adjust for spectral overlap

  • When used in panels, consider that FITC signal may be affected by cellular autofluorescence

• Analysis Considerations:

  • SLAMF6 expression can vary between cell subsets; therefore, use appropriate gating strategies

  • For studies examining SLAMF6 compartmentalization, combine flow cytometry with imaging techniques for comprehensive analysis

  • When evaluating SLAMF6 occupancy after antibody treatments, be aware that bound therapeutic antibodies may block binding of detection antibodies

Research has demonstrated that FITC-conjugated anti-SLAMF6 antibodies can be effectively combined with other markers to analyze expression patterns across different immune cell populations, particularly in studies of chronic lymphocytic leukemia and other hematological malignancies .

What controls should be included when using SLAMF6 antibodies in experimental settings?

Proper controls are essential when using SLAMF6 antibodies to ensure data reliability and interpretability:

• Antibody-Specific Controls:

  • Isotype Controls: Include appropriate isotype-matched control antibodies conjugated to the same fluorophore (FITC) to assess non-specific binding

  • Blocking Controls: Pre-incubation with unconjugated anti-SLAMF6 should prevent subsequent binding of FITC-conjugated SLAMF6 antibodies

  • Titration Series: Establish optimal antibody concentration by testing a range of dilutions

• Sample-Specific Controls:

  • Positive Controls: Include cell types known to express high levels of SLAMF6 (e.g., activated T cells)

  • Negative Controls: Include cell types with minimal SLAMF6 expression or SLAMF6-knockout cells

  • Secondary-Only Controls: For indirect detection methods, include samples treated only with secondary reagents

• Experimental Design Controls:

  • When studying the effects of SLAMF6 modulation, include both plate-bound and soluble antibody conditions to differentiate between clustering and spatial separation effects

  • For functional studies, compare different antibody clones that may have distinct functional properties

  • For therapeutic studies, include both single-agent and combination treatments to assess synergistic effects

• Time Course Controls:

  • When monitoring receptor occupancy, assess multiple time points after antibody administration to account for the antibody half-life (approximately 6-8 days for mouse IgG2a)

  • In studies examining receptor internalization or modulation, include both short-term and long-term time points

Research has shown that SLAMF6 receptors may remain occupied by therapeutic antibodies for extended periods, affecting subsequent detection and requiring careful timing of assessments .

How should experiments be designed to study SLAMF6 compartmentalization effects?

Designing experiments to study SLAMF6 compartmentalization requires careful consideration of several factors:

• In Vitro Receptor Engagement Approaches:

  • Plate-Bound vs. Soluble Antibody Conditions: To study the effect of SLAMF6 clustering with CD3, compare plate-bound anti-SLAMF6 (coated alongside anti-CD3) versus soluble anti-SLAMF6 (added to cultures with plate-bound anti-CD3) .

  • Bead-Based Systems: Design antibody-conjugated beads with anti-CD3, anti-SLAMF6, or anti-CD3 + anti-SLAMF6 to control receptor proximity .

  • Fc Cross-linking: Use secondary antibodies to cluster primary antibodies bound to cell surface receptors, creating controlled receptor aggregates .

• Bispecific Antibody Approaches:

  • Generate bispecific antibodies targeting both CD3 and SLAMF6 to promote receptor proximity and clustering .

  • Validate bispecific antibody binding using ELISA assays with appropriate cell lysates (e.g., SLAMF6-expressing Raji cells vs. SLAMF6 KO Jurkat cells) .

• Imaging Techniques:

  • Transfect cells with fluorescently tagged constructs (e.g., LifeAct mCherry for actin and GFP-SLAMF6) to visualize receptor localization at the immunological synapse .

  • Establish co-culture systems (e.g., Jurkat T cells with antigen-loaded Raji B cells) to study physiological receptor organization during cell-cell interactions .

• Functional Readouts:

  • Measure T cell proliferation using standard techniques like CFSE dilution or 3H-thymidine incorporation

  • Assess activation marker expression (CD25, PD-1) by flow cytometry

  • Quantify cytokine production (IL-2, IFN-γ) by ELISA or intracellular staining

  • Perform biochemical analysis through co-immunoprecipitation and mass spectrometry to identify SLAMF6-interacting proteins under different compartmentalization conditions

This multi-faceted approach allows comprehensive assessment of how SLAMF6 localization impacts its functional outcomes in T cell biology.

What are the optimal protocols for immunoprecipitation studies with SLAMF6 antibodies?

For effective immunoprecipitation studies with SLAMF6 antibodies, researchers should follow these optimized protocols:

• Cell Preparation and Stimulation:

  • Culture T cells expressing tagged SLAMF6 (e.g., V5-tagged SLAMF6 in Jurkat cells)

  • Stimulate cells under different conditions (e.g., plate-bound vs. soluble antibodies) to assess compartmentalization effects

  • Use 5-10 × 10^6 cells per immunoprecipitation condition to ensure adequate protein yield

• Cell Lysis:

  • Lyse cells in ice-cold buffer containing 1% NP-40 or similar non-denaturing detergent

  • Include protease and phosphatase inhibitors to preserve protein interactions

  • Perform lysis for 30 minutes on ice with periodic gentle mixing

  • Clear lysates by centrifugation (14,000 × g, 10 minutes, 4°C)

• Immunoprecipitation:

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

  • Add anti-tag antibodies (e.g., anti-V5) or anti-SLAMF6 antibodies to the cleared lysates

  • Incubate overnight at 4°C with gentle rotation

  • Add protein A/G beads and incubate for an additional 2-4 hours

  • Wash beads 4-5 times with lysis buffer containing reduced detergent concentration

• Analysis:

  • For mass spectrometry analysis, elute proteins and process according to standard protocols

  • For Western blot analysis, elute proteins by boiling in SDS sample buffer

  • Include appropriate controls: input lysate, isotype control immunoprecipitation, and beads-only control

Research has demonstrated that this approach can effectively identify SLAMF6-interacting proteins, revealing that key regulatory signaling proteins remain associated with SLAMF6 in the soluble condition, potentially contributing to the inhibitory effect observed when SLAMF6 is separated from CD3 .

How do different SLAMF6 antibody immobilization methods affect experimental outcomes?

Different immobilization methods for SLAMF6 antibodies significantly impact experimental outcomes by altering receptor clustering and subsequent signaling:

• Plate-Bound Immobilization:

  • Method: Anti-SLAMF6 antibodies are pre-coated onto culture plates, often alongside anti-CD3

  • Outcome: Promotes SLAMF6 clustering with CD3, enhancing T cell proliferation, activation marker expression (CD25, PD-1), and cytokine production (IL-2, IFN-γ)

  • Mechanism: Facilitates recruitment of shared signaling components, amplifying downstream signal transduction

• Soluble Antibody Addition:

  • Method: Anti-SLAMF6 antibodies are added in solution to cultures with plate-bound anti-CD3

  • Outcome: Results in inhibition of T cell proliferation and reduced activation compared to plate-bound conditions

  • Mechanism: Causes spatial separation of SLAMF6 from CD3, potentially sequestering key signaling molecules away from the CD3 complex

• Antibody-Conjugated Beads:

  • Method: Antibodies are conjugated to microbeads in different combinations (anti-CD3, anti-SLAMF6, or anti-CD3 + anti-SLAMF6)

  • Outcome: Co-conjugation of anti-CD3 and anti-SLAMF6 on the same beads enhances IL-2 release compared to anti-CD3 alone, while mixing separate anti-CD3 and anti-SLAMF6 beads fails to augment T cell activity

  • Advantage: Allows precise control over receptor clustering geometry

• Bispecific Antibody Approach:

  • Method: Bispecific antibodies simultaneously targeting CD3 and SLAMF6 are generated through co-expression of monovalent constructs

  • Outcome: Enhances T cell activation by promoting clustering of SLAMF6 with CD3 at the time of receptor engagement

  • Application: Potential therapeutic approach for amplifying T cell responses

The experimental outcomes demonstrate that physical proximity of SLAMF6 to CD3 is a critical determinant of its co-stimulatory versus inhibitory functions, highlighting the importance of receptor compartmentalization in immune cell signaling .

How can SLAMF6 antibodies be utilized in cancer immunotherapy research?

SLAMF6 antibodies demonstrate significant potential in cancer immunotherapy research through several mechanisms:

• Direct Targeting of SLAMF6-Expressing Tumors:

  • Anti-SLAMF6 monoclonal antibodies have shown efficacy in eliminating tumor progression in murine models of Chronic Lymphocytic Leukemia (CLL) and B cell lymphoma

  • A single injection of anti-SLAMF6 antibody was capable of abrogating tumor progression in the spleen, bone marrow, and blood of SCID mice transplanted with aggressive B220+CD5+ CLL cells

• Combination Therapies:

  • Co-administration of anti-SLAMF6 antibodies with Bruton tyrosine kinase (Btk) inhibitor ibrutinib demonstrates synergistic effects, efficiently eliminating tumor cells in multiple compartments including spleen, bone marrow, liver, and peritoneal cavity

  • This synergy likely results from ibrutinib's ability to down-regulate BCR signaling, cause apoptosis of tumor cells, and mobilize CLL cells from protective niches into circulation where they are more vulnerable to antibody-mediated elimination

• Enhancement of T Cell Function:

  • Bispecific anti-CD3/SLAMF6 antibodies can promote SLAMF6 clustering with CD3, enhancing T cell activation and potentially boosting anti-tumor immune responses

  • This approach may be particularly valuable for adoptive T cell therapies where enhanced T cell function is desired

• Antibody-Dependent Cellular Cytotoxicity (ADCC):

  • Anti-SLAMF6 antibodies can mediate ADCC, though efficacy varies by tissue compartment, potentially due to differences in effector cell populations or tumor microenvironment factors

Research indicates that anti-human SLAMF6 monoclonal antibodies effectively killed human CLL cells both in vitro and in vivo, supporting further exploration of SLAMF6-targeted approaches for CLL and other B cell malignancies .

What are the challenges in developing SLAMF6-targeted therapeutic antibodies?

Developing SLAMF6-targeted therapeutic antibodies presents several significant challenges:

• Tissue-Specific Efficacy Limitations:

  • Anti-SLAMF6 antibodies show differential efficacy across tissue compartments, with reduced effectiveness in the peritoneal cavity and omentum compared to spleen, bone marrow, and blood

  • This variability appears to depend on the tumor microenvironment and may relate to differences in subpopulations of tumor cells or the inability of certain tissue-resident macrophages to induce effective Antibody-Dependent Cellular Cytotoxicity (ADCC)

• Receptor Occupation Dynamics:

  • After antibody administration, SLAMF6 receptors remain occupied for extended periods, affecting the efficacy of subsequent antibody doses

  • Following initial anti-SLAMF6 antibody injection, the receptor on blood CLL cells remained inaccessible for at least 3 days and only partially accessible after 6 days, consistent with the 6-8 day half-life of mouse IgG2a

  • This necessitates careful timing of dosing schedules in therapeutic applications

• Dual Functional Properties:

  • SLAMF6 can exert both stimulatory and inhibitory effects depending on cellular context, compartmentalization, and adapter protein expression

  • This functional duality introduces complexity in predicting therapeutic outcomes and potential side effects across different patient populations and disease states

• Format Optimization:

  • Determining the optimal antibody format (conventional, bispecific, antibody-drug conjugate) requires extensive testing

  • For bispecific antibodies targeting CD3 and SLAMF6, optimizing the binding affinity and stoichiometry is crucial for balancing effector functions with target engagement

• Combination Therapy Implementation:

  • Identifying the most effective combination therapies requires understanding complex interaction mechanisms

  • While anti-SLAMF6 with ibrutinib shows promise, optimal sequencing, dosing, and patient selection criteria need further investigation

These challenges highlight the importance of comprehensive preclinical evaluation and rationally designed clinical trials for SLAMF6-targeted therapeutic approaches.

How does SLAMF6 function differ between normal and malignant B cells?

SLAMF6 demonstrates important functional differences between normal and malignant B cells:

• Expression Pattern Differences:

  • In normal B cells, SLAMF6 expression fluctuates during development and activation, with distinct patterns at different maturation stages

  • In malignant B cells, particularly in CLL, SLAMF6 expression is often altered, showing either increased or aberrant expression patterns

  • These expression differences may be exploited for selective targeting of malignant cells

• Signaling Pathway Alterations:

  • In normal B cells, SLAMF6 participates in B-cell tolerance mechanisms within germinal centers and may negatively regulate humoral immune responses in conjunction with SLAMF1 and CD84/SLAMF5

  • In malignant B cells, SLAMF6 signaling may be dysregulated, potentially contributing to tumor survival and progression

  • The balance between activating and inhibitory signals through SLAMF6 is often disrupted in B cell malignancies

• Adapter Protein Dependencies:

  • SLAMF6 function depends critically on adapter proteins SH2D1A/SAP and SH2D1B/EAT-2

  • Malignant B cells may exhibit altered expression or function of these adapter proteins, changing SLAMF6 signaling outcomes

  • In the absence of SH2D1A/SAP, SLAMF6 tends to associate with phosphatase PTPN6/SHP-1, potentially generating inhibitory signals

• Therapeutic Vulnerability:

  • Malignant B cells, particularly in CLL, demonstrate sensitivity to anti-SLAMF6 antibody treatment, resulting in tumor elimination in multiple tissue compartments

  • This therapeutic vulnerability suggests functional differences in how SLAMF6 contributes to malignant B cell survival compared to normal B cells

  • The synergistic effect observed when combining anti-SLAMF6 antibodies with ibrutinib indicates that SLAMF6 may interact with B cell receptor signaling pathways in malignant B cells

Understanding these differences is crucial for developing targeted therapeutic approaches that effectively eliminate malignant B cells while minimizing effects on normal B cell populations.

What are common pitfalls in SLAMF6 antibody-based experiments and how can they be avoided?

Researchers encounter several common pitfalls when working with SLAMF6 antibodies, but these can be avoided with appropriate strategies:

Careful attention to these potential pitfalls and implementation of appropriate controls will significantly enhance the reliability and interpretability of SLAMF6 antibody-based experiments.

How should contradictory data on SLAMF6 function be evaluated and reconciled?

Evaluating and reconciling contradictory data on SLAMF6 function requires a systematic approach:

• Context-Dependent Function Analysis:

  • SLAMF6 demonstrates dual functionality, acting as both a positive and negative regulator depending on cellular context

  • When evaluating contradictory data, carefully assess the experimental systems used, as SLAMF6 has been reported to both mediate T-cell adhesion and inhibit T-cell:B-cell interactions in different contexts

  • Consider the cell types involved, activation states, and presence of other receptors that may influence SLAMF6 function

• Adapter Protein Expression:

  • The presence or absence of adapter proteins SH2D1A/SAP and SH2D1B/EAT-2 critically determines SLAMF6 signaling outcomes

  • Analyze adapter protein expression in the experimental systems being compared

  • In the absence of SH2D1A/SAP, SLAMF6 tends to transmit negative signals to CD4+ T-cells and NKT cells, potentially explaining seemingly contradictory results

• Receptor Compartmentalization:

  • SLAMF6 function is highly dependent on its compartmentalization relative to other receptors, particularly CD3

  • Compare the methodologies used for receptor engagement (plate-bound vs. soluble antibodies)

  • Consider that contradictory findings may reflect differences in receptor clustering rather than inherent functional disparities

• Experimental Design Variations:

  • Critically evaluate differences in experimental design between studies:

    • Antibody clones and concentrations

    • Timing of measurements

    • Readout systems

    • Cell culture conditions

  • Perform side-by-side comparisons using standardized protocols when possible

• Integrate Multiple Data Types:

  • Combine functional data with molecular analyses (e.g., co-immunoprecipitation, mass spectrometry)

  • Use imaging techniques to correlate receptor localization with functional outcomes

  • Develop computational models to integrate diverse datasets and predict context-dependent behaviors

By systematically evaluating these factors, researchers can often reconcile seemingly contradictory data and develop a more nuanced understanding of SLAMF6's complex biology.

What quantitative methods are recommended for analyzing SLAMF6 expression and colocalization?

Several quantitative methods are recommended for rigorous analysis of SLAMF6 expression and colocalization:

These quantitative methods provide robust frameworks for analyzing SLAMF6 expression patterns and colocalization events, facilitating more precise understanding of SLAMF6 biology and function in different cellular contexts.