SLAMF1 Human

What is SLAMF1 and where is it expressed in human cells?

SLAMF1, also known as CD150, is a type I glycoprotein belonging to the SLAM subfamily of the CD2-like family of proteins. It is primarily expressed on hematopoietic cells, with distinct expression patterns in different cell types. In resting macrophages, SLAMF1 shows very low surface expression, with the major cellular pool located in the endocytic recycling compartment (ERC) . Flow cytometry analyses have demonstrated that only approximately 1% of monocytes and 4% of macrophages express SLAMF1 on their surface, while about 40% of differentiated THP-1 cells are SLAMF1 positive . Additionally, SLAMF1 is expressed on various other immune cells including cytotoxic T lymphocytes, T helper cells, NK cells, NKT cells, classical dendritic cells (cDCs), and monocytes/macrophages, though expression levels vary significantly between these cell populations .

How does SLAMF1 expression change during immune cell activation?

SLAMF1 expression is dynamically regulated during immune cell activation. Upon lipopolysaccharide (LPS) stimulation, surface expression of SLAMF1 in primary macrophages increases by more than 50% after 6 hours of stimulation, accompanied by an increase in total SLAMF1 protein expression . Various Toll-like receptor (TLR) ligands can induce SLAMF1 expression, including Pam3Cys (TLR1/2), FSL-1 (TLR2/6), R848 (TLR7 and -8), and CL075 (TLR8), with Escherichia coli being the most potent stimulator . This pattern of expression suggests that multiple TLRs control SLAMF1 expression in human cells, highlighting its potential importance in various inflammatory responses.

What distinguishes human SLAMF1 from mouse SLAMF1?

A critical difference between human and mouse SLAMF1 lies in their interaction with TRAM (Toll receptor-associated molecule). Research has demonstrated that the human SLAMF1 protein interacts with TRAM, but this interaction is not observed with mouse SLAMF1 proteins . This species-specific difference has important implications for translational research, as mouse models may not fully recapitulate the role of SLAMF1 in human immune responses, particularly in TLR4-mediated signaling pathways. Additionally, while mouse SLAMF1 has been shown to positively regulate NOX2 activity by forming a complex with beclin-1–Vps34–Vps15–UVRAG, human SLAMF1 appears to have distinct interaction mechanisms that affect its function in different cellular contexts .

How does SLAMF1 regulate TLR4-mediated signaling in human macrophages?

SLAMF1 functions as a critical regulator of TLR4-mediated signaling in human macrophages through several sophisticated mechanisms:

• TRAM adapter recruitment: SLAMF1 interacts with the Toll receptor–associated molecule (TRAM) and controls its trafficking from the endocytic recycling compartment (ERC) to E. coli phagosomes .

• Phagosomal signaling complex formation: In resting macrophages, SLAMF1 is localized to the ERC, but upon addition of E. coli, it traffics together with TRAM from ERC to E. coli phagosomes in a Rab11-dependent manner . This trafficking is essential for the formation of the TLR4 signaling complex on phagosomes.

• Enhancement of type I interferon production: SLAMF1 enhances production of type I interferon (particularly IFNβ) induced by Gram-negative bacteria through modulation of MyD88-independent TLR4 signaling .

• Domain-specific interactions: The interaction between SLAMF1 and TRAM involves specific domains: amino acids 68 to 95 of TRAM and the 15 C-terminal amino acids of SLAMF1 . These precise molecular interactions are crucial for proper signal transduction.

• LPS-enhanced binding: The interaction between endogenous SLAMF1 and TRAM is enhanced upon LPS stimulation, suggesting a dynamic regulatory mechanism responsive to bacterial stimuli .

Through these mechanisms, SLAMF1 serves as a potential target for modulating inflammatory responses against Gram-negative bacteria.

What experimental approaches can effectively detect SLAMF1-TRAM interactions?

To investigate SLAMF1-TRAM interactions, researchers can employ several methodological approaches:

• Endogenous co-immunoprecipitation (Co-IP): Using anti-SLAMF1 and anti-TRAM antibodies to precipitate protein complexes from cell lysates before and after LPS stimulation. This approach has successfully demonstrated that endogenous SLAMF1 coprecipitates with TRAM in macrophages, with enhanced interaction following LPS stimulation .

• Deletion mutagenesis: Creating deletion mutants of both SLAMF1 and TRAM to identify critical interaction domains. Studies have shown that a SLAMF1 deletion mutant (1–330) lacking just the last five C-terminal amino acids fails to interact with TRAM, pinpointing the TRAM interaction site at the very C terminus of SLAMF1 .

• Fluorescent protein tagging: Using tagged proteins (e.g., TRAM-YFP) for co-transfection experiments to visualize interactions through microscopy or for co-IP followed by western blotting .

• Colocalization analysis: Confocal microscopy combined with quantitative colocalization analysis using Manders's colocalization coefficient provides precise measurement of SLAMF1-TRAM spatial relationships during trafficking and signaling events .

• Proximity ligation assay (PLA): This technique can detect protein-protein interactions in situ with high specificity and sensitivity, which is particularly valuable for confirming interactions in their native cellular context.

How does SLAMF1 contribute to cell survival through the AKT signaling pathway?

SLAMF1 promotes cell survival through the AKT signaling pathway via several interconnected mechanisms:

• Regulation of phospho-AKT levels: SLAMF1 deficiency disturbs the AKT signaling pathway, as evidenced by altered phospho-ATP levels between SLAMF1 wild-type and mutant cells .

• Differential response to AKT inhibition: SLAMF1-deficient cells show differential responses to AKT inhibitors (like MK-2206) compared to SLAMF1 wild-type cells, suggesting that SLAMF1 modulates AKT-dependent survival pathways .

• Competitive survival advantage: In mixed culture experiments, SLAMF1 wild-type cells often out-compete SLAMF1-deficient cells, particularly under stress conditions such as nutritional deficiency or acidic microenvironment .

• Pro-apoptotic protein regulation: A reverse relationship has been observed between phospho-AKT and Bim (a pro-apoptotic protein) in relation to SLAMF1 expression. SLAMF1-deficient cells show increased Bim levels under cytokine or growth factor deprivation, while SLAMF1 wild-type cells maintain low Bim levels .

• FOXO3 regulation mechanism: The activated AKT phosphorylates FOXO3, causing it to be sequestered in the cytosol, thereby inhibiting transcription of the pro-apoptotic gene Bim. SLAMF1 appears to regulate this pathway to promote cell survival under unfavorable growth conditions .

This relationship between SLAMF1 and AKT signaling provides important insights into how SLAMF1 may influence cell survival in various physiological and pathological contexts.

What evidence supports SLAMF1 as a biomarker in rheumatoid arthritis?

Multiple lines of evidence support SLAMF1 as a potential biomarker in rheumatoid arthritis (RA):

• Differential gene expression analysis: Bioinformatic analysis of datasets GSE45291 and GSE89408 identified SLAMF1 as one of four candidate biomarkers (along with CCR7, KLRK1, and TIGIT) from differentially expressed immune-related genes in RA patients .

• Animal model validation: In collagen-induced arthritis (CIA) mouse models, SLAMF1 shows consistent upregulation that correlates with disease progression .

• Tissue expression: Immunohistochemical staining demonstrates significantly higher SLAMF1 expression in diseased joints of CIA mice compared to controls .

• Immune cell expression patterns: Flow cytometry analysis shows that SLAMF1 expression on CIA mice-derived CTL cells, T helper cells, NK cells, NKT cells, classical dendritic cells, and monocytes/macrophages is significantly higher than corresponding immune cells from healthy control mice .

• Consistency with disease pathology: The elevated expression of SLAMF1 across multiple immune cell types aligns with the multi-cellular nature of RA pathogenesis, suggesting its potential role in disease development .

These findings collectively suggest that SLAMF1 may serve as a key biomarker in the development and progression of RA, potentially providing new insights for exploring disease mechanisms and therapeutic targets.

What methodologies are optimal for targeting SLAMF1 in therapeutic development?

Several methodological approaches can be employed for targeting SLAMF1 in therapeutic development:

• Monoclonal antibody development: Creating antibodies that specifically target SLAMF1 to either block or enhance its function. This approach could be particularly valuable for modulating TLR4-TRAM-TRIF inflammatory signaling in human cells .

• Domain-specific inhibitors: Designing peptides or small molecules that target the C-terminal region of SLAMF1 (specifically the last 15 amino acids) that mediates its interaction with TRAM, potentially allowing for selective inhibition of specific SLAMF1 functions without affecting others .

• Cell-specific delivery systems: Developing methods to deliver SLAMF1-targeting agents specifically to relevant cell populations (e.g., macrophages in inflammatory conditions or specific immune cells in rheumatoid arthritis) .

• Expression modulation approaches: Using RNA interference, antisense oligonucleotides, or CRISPR-based technologies to modulate SLAMF1 expression in specific cell types involved in disease pathogenesis .

• AKT pathway combination therapies: Since SLAMF1 contributes to cell survival through the AKT signaling pathway, combining SLAMF1-targeting with AKT inhibitors could provide synergistic therapeutic effects in certain contexts .

• Biomarker-guided therapy selection: Using SLAMF1 expression levels as a biomarker to select patients who might benefit from specific immunomodulatory therapies, enabling personalized medicine approaches .

The therapeutic targeting approach should be carefully selected based on whether enhancement or inhibition of SLAMF1 function is desired for a particular condition, considering its dual roles in both promoting protective immunity against bacterial infections and potentially contributing to inflammatory pathologies.

What techniques are most effective for analyzing SLAMF1 expression in different immune cell subsets?

For comprehensive analysis of SLAMF1 expression across immune cell populations, several complementary techniques have proven particularly effective:

• Multiparameter flow cytometry: This remains the gold standard for analyzing SLAMF1 (CD150) expression across multiple immune cell populations simultaneously. By combining antibodies against SLAMF1 with markers for specific immune cell subsets, researchers can quantify both the percentage of SLAMF1-positive cells and the expression level within each subset .

• Single-cell RNA sequencing (scRNA-seq): Provides transcriptomic profiles at single-cell resolution, allowing analysis of SLAMF1 mRNA expression across all immune cell types in a sample without prior knowledge of cell subset markers.

• Imaging flow cytometry: Combines the quantitative power of flow cytometry with the visual information of microscopy, enabling visualization of SLAMF1 localization within cells while maintaining the ability to analyze multiple cell populations .

• Immunohistochemistry with multiplex staining: Allows visualization of SLAMF1 expression in tissue context, with the ability to co-stain for cell type-specific markers. This approach has been successfully used to demonstrate elevated SLAMF1 expression in diseased joints of RA models .

• Cell sorting with downstream analysis: Isolating specific immune cell subsets by fluorescence-activated cell sorting (FACS) followed by western blot or qPCR for deeper analysis of SLAMF1 expression and regulation .

Table 1: Comparison of SLAMF1 surface expression across human immune cell types

What approaches are most effective for studying SLAMF1's role in bacterial clearance?

For investigating SLAMF1's role in bacterial clearance, several methodological approaches have proven particularly valuable:

• Bacterial killing assays: Comparing the ability of SLAMF1-sufficient and SLAMF1-deficient macrophages to kill bacteria, particularly Gram-negative bacteria like E. coli, through colony-forming unit (CFU) counts at various time points .

• Trafficking analysis: Tracking SLAMF1 movement during bacterial phagocytosis using confocal microscopy and colocalization analysis with markers like Rab11. Studies have shown that SLAMF1 traffics from the endocytic recycling compartment to E. coli phagosomes in a Rab11-dependent manner .

• TRAM recruitment analysis: Examining how SLAMF1 controls the trafficking of TRAM from the endocytic recycling compartment to phagosomes containing Gram-negative bacteria, which is crucial for TLR4-mediated signaling .

• Type I interferon production measurement: Quantifying IFNβ production in response to bacterial stimulation in cells with or without SLAMF1, as SLAMF1 enhances production of type I interferon induced by Gram-negative bacteria .

• Phagosome isolation and characterization: Isolating phagosomes containing bacteria to analyze the recruitment of SLAMF1 and associated proteins to these compartments .

• CRISPR/Cas9 gene editing: Creating SLAMF1-deficient cell lines for comparative studies of bacterial clearance efficiency and signaling responses .

These approaches collectively provide a comprehensive understanding of SLAMF1's multifaceted role in bacterial recognition, phagocytosis, and clearance mechanisms, particularly through its regulation of TLR4-TRAM-TRIF signaling from phagosomes.

What are the key structural domains of SLAMF1 that mediate its functions?

SLAMF1 contains several key structural domains that are critical for its diverse functions:

• Extracellular domain: Contains an Ig-like domain that mediates homotypic interactions with SLAMF1 on other cells, as SLAMF1 belongs to a family where most members engage in homotypic interactions .

• C-terminal domain: The last 15 amino acids of SLAMF1's C-terminal region are crucial for its interaction with TRAM. Deletion studies have demonstrated that removing just the last five C-terminal amino acids (mutant 1-330) completely abolishes the interaction with TRAM, pinpointing this region as critical for TRAM binding .

• Transmembrane domain: Anchors SLAMF1 in the cell membrane and may contribute to its proper orientation for interactions with other proteins .

• Cytoplasmic domain: Contains immunoreceptor tyrosine-based switch motifs (ITSMs) that interact with SLAM associated protein (SAP) family adaptors, which are critical for downstream signaling .

• ERC localization motif: Contains structural elements that direct SLAMF1 to the endocytic recycling compartment in resting cells, which is important for its trafficking function during bacterial challenge .

Understanding these structural domains provides opportunities for targeted therapeutic approaches and deeper insights into the molecular mechanisms of SLAMF1 function in various cellular contexts.

How does SLAMF1 relate to other members of the SLAM family?

SLAMF1 is one member of a larger family of nine receptors (SLAMF1-9) with both shared and distinct characteristics:

• Shared expression patterns: Like other SLAM family receptors, SLAMF1 is expressed on hematopoietic cells, though the specific distribution across immune cell subtypes varies between family members .

• Homotypic interactions: With the exception of SLAMF4, SLAM family receptors (including SLAMF1) are homotypic in nature, meaning downstream signaling occurs when hematopoietic cells expressing the same SLAM receptor interact .

• Adaptor protein signaling: The function of SLAMF1, like other SLAM family receptors, is largely controlled via SLAM associated protein (SAP) family adaptors, which include SAP, Ewing sarcoma associated transcript (EAT)-2, and EAT-2-related transducer (ERT) .

• Unique functions: Despite family similarities, SLAMF1 has unique properties, particularly its role in TLR4-mediated signaling through TRAM interaction, which appears to be specific to SLAMF1 and not shared with other family members .

• Evolutionary conservation: While the SLAM family is conserved across species, there are important species-specific differences, such as the human-specific interaction between SLAMF1 and TRAM that is not observed with mouse SLAMF1 .

This combination of shared family characteristics and unique functional properties makes SLAMF1 an important focus for understanding immune regulation in both normal physiology and disease states.