SLAMF6 Antibody, HRP conjugated

What is SLAMF6 and what biological functions does it serve?

SLAMF6, also known as SLAM family member 6, NTB-A, or CD352, is a member of the signaling lymphocyte activation molecule family that functions as a co-receptor expressed on T cells. It plays a critical role in immune regulation, particularly in T cell activation and function. SLAMF6 has been reported to be involved in maintaining B-cell tolerance in germinal centers and in preventing autoimmunity . Importantly, research suggests that SLAMF6 functions as an inhibitory immune receptor, as its absence enables more powerful CD8+ T cells to effectively eradicate tumors . The protein can have dual functions, depending on its interactions with adaptor proteins like SAP and phosphatases SHP-1 and SHP-2, which bind to tyrosines on the cytoplasmic tail of the receptor .

How does HRP conjugation affect the applications of SLAMF6 antibodies?

The horseradish peroxidase (HRP) conjugation of SLAMF6 antibodies primarily enhances their utility in enzymatic detection methods. This conjugation enables direct detection in assays without requiring secondary antibodies, thereby simplifying experimental workflows and potentially reducing background signal. The HRP-conjugated SLAMF6 antibody is particularly suitable for ELISA applications , allowing for efficient detection of SLAMF6 in various sample types. The conjugation does not interfere with the antibody's specificity for the target protein, as the HRP molecule is typically attached to the Fc region of the antibody while leaving the antigen-binding sites unaffected.

What are the optimal storage and handling conditions for SLAMF6 antibody, HRP conjugated?

For optimal performance and longevity of the SLAMF6 antibody, HRP conjugated, proper storage conditions are crucial. According to product specifications, the antibody should be shipped at 4°C. Upon receipt, short-term storage should be at -20°C, while long-term storage is recommended at -80°C . It's important to avoid repeated freeze-thaw cycles as these can compromise antibody activity. The antibody is typically supplied in a buffer containing 0.03% Proclin 300, 50% Glycerol, and 0.01M PBS at pH 7.4 , which helps maintain stability during storage and use.

How does SLAMF6 compartmentalization affect T cell functional responses?

The spatial organization of SLAMF6 on the T cell surface significantly influences its functional impact on T cell activation. Research has demonstrated that T cell activity is enhanced when SLAMF6 colocalizes with the CD3 complex . This enhancement occurs through several mechanisms:

• When SLAMF6 clusters with CD3 in the immunological synapse, it results in increased T cell proliferation, upregulation of activation markers (CD25 and PD-1), and enhanced cytokine secretion (IL-2 and IFN-γ) .

• Conversely, when SLAMF6 is spatially separated from CD3 during activation (such as when stimulated with immobilized anti-CD3 but soluble anti-SLAMF6 antibodies), T cell activation is inhibited .

• Co-immunoprecipitation analysis has revealed that SLAMF6 interacts with proteins essential for signaling downstream of the T cell receptor, suggesting the two receptors share downstream signaling pathways .

This compartmentalization effect provides a mechanistic explanation for the dual functions of SLAMF6 in T cell regulation, with its location relative to CD3 determining whether it delivers activating or inhibitory signals.

What mechanisms underlie SLAMF6's role in tumor immunity?

SLAMF6 has emerged as an important checkpoint receptor in tumor immunity. Several key mechanisms have been identified:

• SLAMF6-deficient CD8+ T cells demonstrate enhanced anti-tumor activity in vivo, suggesting that SLAMF6 normally functions as an inhibitory checkpoint that restrains T cell-mediated tumor clearance .

• In contrast to wild-type cells, SLAMF6-deficient T cells show stronger activation and more complete acquisition of effector phenotypes when stimulated through cognate TCRs .

• In melanoma models, SLAMF6-deficient T cells demonstrated superior tumor killing capabilities and induced more durable tumor regression compared to wild-type cells .

• Mechanistically, SLAMF6 deficiency leads to a compensatory increase in LAG-3 expression, suggesting interconnected regulatory pathways . Importantly, combining SLAMF6 deficiency with LAG-3 blockade resulted in synergistic enhancement of anti-tumor responses, with faster tumor reduction and elimination .

These findings position SLAMF6 as a potential therapeutic target in cancer immunotherapy, particularly in combination strategies targeting multiple checkpoint receptors.

How do SLAMF6-deficient T cells differ from wild-type in phenotype and function?

SLAMF6-deficient T cells exhibit distinct phenotypic and functional characteristics compared to their wild-type counterparts:

• Following activation, SLAMF6-deficient T cells display a more complete shift toward effector and effector memory phenotypes, with significantly fewer cells remaining in the naive CD62L^high/CD44^low state .

• SLAMF6-deficient cells demonstrate stronger activation profiles, including enhanced cytokine production and proliferative capacity .

• These cells show differential expression of exhaustion markers. While PD-1, CD244 (SLAMF4), and TIM-3 levels are similar to wild-type cells after prolonged activation, SLAMF6-deficient T cells exhibit higher expression of LAG-3, suggesting a compensatory regulatory mechanism .

• Functionally, SLAMF6-deficient T cells demonstrate superior anti-tumor efficacy in vivo, with the ability to induce more durable tumor regression in melanoma models .

These differences highlight how SLAMF6 normally constrains T cell activation and effector function, with its absence unleashing more potent anti-tumor T cell responses.

What protein interactions occur downstream of SLAMF6 signaling?

SLAMF6 engages a complex network of protein interactions that mediate its signaling effects:

• Co-immunoprecipitation and mass spectrometry analyses have identified key proteins that interact with SLAMF6, including those essential for TCR signaling .

• SLAMF6 associates with SRC tyrosine kinases (LCK and FYN) that are critical for T cell receptor signaling, suggesting a mechanism by which SLAMF6 can modulate TCR signal transduction .

• The interactome analysis revealed that SLAMF6, even when spatially removed from CD3, remains associated with many signaling proteins essential for TCR signaling, including LCK, FYN, and ZAP70 .

• This "steal" of signaling molecules away from the TCR complex may contribute to the inhibitory effect observed when SLAMF6 is engaged separately from CD3 .

• Downstream kinase prediction analysis indicates that different activation conditions (SLAMF6 clustered with versus separated from CD3) engage distinct downstream signaling pathways, despite both conditions signaling via the TCR .

Understanding these protein interactions provides insight into how SLAMF6 can either enhance or inhibit T cell activation depending on its spatial organization relative to the TCR-CD3 complex.

What are the implications of combined SLAMF6 and LAG-3 targeting in immunotherapy?

Research has revealed promising synergistic effects when targeting both SLAMF6 and LAG-3:

• SLAMF6-deficient T cells exhibit compensatory upregulation of LAG-3, suggesting interconnected regulatory pathways .

• Blocking LAG-3 on SLAMF6-deficient lymphocytes significantly increases their cytokine secretion, whereas it has minimal effect on wild-type cells .

• In vivo studies demonstrated that combining SLAMF6 deficiency with LAG-3 blockade resulted in faster and more complete tumor regression in melanoma models .

• This combination led to a three-fold increase in IFN-γ production compared to SLAMF6 deficiency alone, indicating enhanced effector function .

These findings suggest that dual targeting of SLAMF6 and LAG-3 represents a promising strategy for enhancing anti-tumor immunity, potentially overcoming resistance mechanisms that might develop with single-checkpoint blockade approaches.

What validation approaches should be used for SLAMF6 antibody in experimental systems?

When using SLAMF6 antibody, HRP conjugated, in research applications, proper validation is essential:

• Specificity testing: Confirm antibody specificity using positive and negative control samples. For human SLAMF6, the recombinant protein used as immunogen (amino acids 248-331) can serve as a positive control, while samples known to lack SLAMF6 expression can be negative controls.

• Titration experiments: Determine optimal antibody concentration through titration experiments to achieve maximum signal with minimal background. This is particularly important for ELISA applications where the HRP conjugation is utilized .

• Blocking experiments: Perform pre-adsorption with the immunizing peptide to confirm specificity of detection.

• Cross-reactivity assessment: While the antibody is reported to be human-specific , testing for potential cross-reactivity with homologous proteins or with SLAMF6 from other species may be necessary depending on the experimental system.

• Functional validation: For studies involving SLAMF6 modulation, confirm that antibody binding affects expected downstream signaling pathways, such as those involving SAP and SHP phosphatases .

How can researchers effectively design experiments to study SLAMF6 compartmentalization?

Based on recent research methodologies , the following approaches can be employed to study SLAMF6 compartmentalization effects:

• Comparative stimulation conditions: Set up parallel stimulation conditions where:
  a. SLAMF6 and CD3 cluster together (using plate-bound anti-CD3 and anti-SLAMF6 antibodies)
  b. SLAMF6 is spatially separated from CD3 (using plate-bound anti-CD3 but soluble anti-SLAMF6 antibodies)

• Functional readouts: Measure multiple parameters to assess functional outcomes, including:

  • T cell proliferation using cell tracking dyes

  • Surface activation marker expression (CD25, PD-1)

  • Cytokine production (IL-2, IFN-γ)

  • Cell counts over time

• Protein interaction analysis: Perform co-immunoprecipitation followed by mass spectrometry to identify proteins that interact with SLAMF6 under different compartmentalization conditions .

• Microscopy approaches: Use confocal or super-resolution microscopy to directly visualize SLAMF6 localization relative to CD3 and other TCR components during T cell activation.

• Bispecific antibody testing: Engineer bispecific antibodies targeting both SLAMF6 and CD3 to force co-localization and assess functional consequences .