SIRPG Antibody

What is the function of SIRPG in human T-cells and why is it important for immunological research?

SIRPG (Signal Regulatory Protein gamma, also known as CD172g) is a transmembrane glycoprotein exclusively expressed on human T-cells. Unlike other SIRP family members, SIRPG is the only member expressed on T-cells and is notably absent in rodents, making it uniquely relevant to human immunology .

SIRPG positively regulates T-cell activation through its interaction with CD47. Research has demonstrated that this interaction enhances antigen-specific T-cell proliferation and affects cell-cell adhesion . The importance of SIRPG lies in its role in modulating immune responses, with evidence showing that genetic variants in SIRPG correlate with autoimmune diseases like type 1 diabetes and multiple sclerosis .

In experimental settings, SIRPγ has been observed to undergo spatial reorganization at the immune synapse, although this process appears to be independent of its interaction with CD47 . Functionally, SIRPγ expression varies with T-cell differentiation, making it a valuable marker for studying T-cell development and activation states .

What are the recommended applications for SIRPG antibodies in experimental protocols?

Based on validated research protocols, SIRPG antibodies are effectively utilized in several experimental applications:

• Flow Cytometry (FACS): Most commonly used to quantify SIRPG expression on T-cells, particularly when analyzing expression patterns across different T-cell subpopulations. Multiple validated clones are available (e.g., OX-119, LSB2.20) for surface and intracellular staining .

• Western Blotting (WB): Effective for detecting SIRPG protein expression and analyzing different SIRPG isoforms. This application is valuable when examining protein expression levels in various cell types or experimental conditions .

• ELISA: Useful for quantitative detection of SIRPG in solution or for studying SIRPG-CD47 interactions. Both direct and competitive ELISA formats have been employed .

• Immunoprecipitation (IP): Applied to isolate SIRPG from complex protein mixtures for downstream applications .

• Immunocytochemistry (ICC): Used to visualize the cellular localization of SIRPG, particularly valuable when studying its distribution at the immune synapse .

• Confocal Microscopy: Implemented for co-localization studies between SIRPG and CD47, offering insights into spatial reorganization during T-cell activation .

How should researchers select the appropriate anti-SIRPG antibody clone for specific experimental applications?

When selecting anti-SIRPG antibody clones, researchers should consider:

• Epitope Recognition: Different clones recognize distinct epitopes within SIRPG. For example, clone 7H3A2 targets amino acids 29-360, while others may target different regions . This is particularly important when studying specific domains or isoforms of SIRPG.

• Validated Applications: Evidence from published studies indicates that certain clones perform better in specific applications:

  • Clone OX-119 has been extensively validated for flow cytometry

  • Clone LSB2.20 works well for surface staining, though recent research has revealed it may not be fully characterized for all applications

  • For Western blotting, polyclonal antibodies targeting amino acids 101-150 have shown good results

• Cross-Reactivity: Consider species reactivity—some antibodies are human-specific while others cross-react with monkey or bovine SIRPG .

• Conjugation Requirements: Based on experimental design, select appropriate conjugated antibodies (PE, FITC, BV421) for multicolor flow cytometry or unconjugated antibodies for applications requiring secondary detection .

• Blocking Capability: Some antibodies like KWAR23 (originally characterized as anti-SIRPα) have been shown to block SIRPG-CD47 interactions and can be valuable for functional studies .

How do genetic variants of SIRPG impact experimental design and data interpretation?

The SNP rs2281808, particularly the TT variant, significantly affects SIRPG expression and function in T-cells, requiring specific methodological considerations:

• Genotyping Protocol: Researchers should incorporate rs2281808 genotyping when studying SIRPG, as this intronic SNP between exons 5 and 6 directly influences SIRPG expression levels. The TT variant correlates with reduced SIRPγ expression on T-cells (TT < CT < CC genotypes) .

• Flow Cytometry Analysis Adjustments: SIRPγ expression shows a bimodal distribution on CD8 T-cells, particularly pronounced in CT carriers. Researchers should adjust gating strategies to account for SIRPγ^high and SIRPγ^low populations, with approximately 37.4% ± 11 of CD8 T-cells being SIRPγ^low in CT carriers versus 21.8% ± 12 in CC carriers .

• Functional Assays Interpretation: Data indicates that SIRPγ^low CD8 T-cells exhibit heightened effector functions with lower activation thresholds. When comparing functional responses between samples, researchers should consider the distribution of SIRPγ^high versus SIRPγ^low T-cells, as this could explain variability in results .

• Transcript Analysis Strategy: The rs2281808 T variant affects alternative splicing of SIRPG, resulting in altered isoform distributions. RNA-seq analysis using Event Analysis methods can resolve specific SIRPG transcript isoforms and quantify their relative abundance between genotypes .

• Disease Association Studies: When investigating SIRPG in autoimmune conditions, researchers should account for the significantly greater frequency of rs2281808 T genetic variant in T1D (22% vs. 4% in healthy donors) and RRMS subjects, as this could confound interpretation of disease-specific effects .

What are the optimal protocols for studying SIRPG-CD47 interactions in experimental settings?

Based on published methodologies, the following optimized protocols have been validated for investigating SIRPG-CD47 interactions:

• ELISA-Based Binding Assays:

  • For SIRPG-CD47 binding studies, immobilize recombinant human SIRPγ at 2 μg/ml in carbonate buffer (pH 9.2)

  • Pre-incubate biotinylated human CD47 (3 μg/ml) with test antibodies at room temperature for 15 minutes

  • Incubate the pre-incubated mixture on immobilized SIRPγ overnight at room temperature

  • Detect with streptavidin-peroxidase using conventional methods

• Flow Cytometry Interaction Studies:

  • Isolate PBMCs by Ficoll from blood samples

  • Pre-incubate cells with human Fc receptor-binding inhibitor (1/50 dilution)

  • Stain with anti-SIRPγ antibodies for 30 minutes at 4°C

  • Co-stain with lineage markers (CD3+ for T lymphocytes, CD14+ for monocytes)

  • Analyze on a flow cytometer after appropriate gating strategies

• Confocal Microscopy for Co-localization:

  • Stain Jurkat or primary T-cells with anti-SIRPγ (e.g., LSB2.20) and anti-CD47 mAbs for 30 minutes at 4°C in the dark

  • Fix cells with 4% PFA for 10 minutes at room temperature

  • Permeabilize with PBS + 0.1% Triton for 5 minutes at room temperature

  • Counterstain with DAPI for 5 minutes

  • Mount with ProLong and allow 48 hours for polymerization

  • Analyze using confocal microscopy with appropriate software for colocalization analysis (e.g., ImageJ with coloc2 plugin)

• Blocking Studies:

  • The anti-SIRPα clone KWAR23 has been identified as a Pan anti-SIRP mAb that efficiently blocks both SIRPα and SIRPγ interactions with CD47

  • This antibody can be used at 10 μg/ml in functional assays to block SIRPG-CD47 interactions

How can researchers accurately distinguish between different SIRPG isoforms in experimental settings?

SIRPG produces multiple transcript isoforms via alternative splicing, all encoding potentially functional proteins. To distinguish between these isoforms:

• Transcript Isoform Detection:

  • Implement isoform-specific qPCR assays with fluorescently labeled probes targeting unique exon junctions for each isoform

  • For comprehensive analysis, use RNA-seq with Event Analysis to count reads mapping uniquely to specific SIRPG transcript isoforms

  • Research has validated probes specifically for SIRPG isoforms 1 and 2, isoform 3, and isoform 4

• Protein Isoform Identification:

  • Use immunoblotting with increasing amounts of protein lysate (10-30 μg) from T-cells

  • Separate proteins on 4-12% Bis-Tris gels

  • Published research has identified distinct molecular weights for different isoforms: isoform 1 (42 kDa), isoform 2 (38 kDa), and isoform 4 (30 kDa)

  • Note that protein corresponding to isoform 3 (18 kDa) was not detectable in Jurkat T-cells despite transcript detection

• Functional Characterization:

  • CRISPR/Cas9 targeting of alternatively spliced exons can help determine isoform-specific functions

  • Research shows that targeting one alternatively spliced exon in SIRPG can eliminate all SIRPγ expression in Jurkat T cells

  • Assess the contribution of specific isoforms to T-cell functions by comparing cells with CRISPR-modified SIRPG to wild-type cells

• Genotype Correlation:

  • The rs6043409 genotype significantly affects isoform distribution, with the minor A allele associated with reduced expression of isoforms 1 and 2 compared to the major G allele

  • Consider genotyping for accurate interpretation of isoform expression patterns

What are common challenges when detecting SIRPG in human samples and how can they be addressed?

Researchers frequently encounter several technical challenges when detecting SIRPG in human samples:

• Variable Expression Levels:

  • Challenge: SIRPG expression varies with T-cell differentiation states and is genetically influenced by SNPs like rs2281808

  • Solution: Include T-cell differentiation markers (CD45RA, memory markers) in flow cytometry panels and consider genotyping samples for rs2281808 to stratify results accordingly

• Antibody Clone Specificity Issues:

  • Challenge: Some anti-SIRPγ mAbs (e.g., LSB2.20) have not been appropriately characterized for all applications

  • Solution: Validate antibody specificity using positive and negative controls (e.g., SIRPG knockout cells created by CRISPR/Cas9). Consider the Pan anti-SIRP mAb KWAR23 for blocking studies

• Distinguishing SIRPG from Other SIRP Family Members:

  • Challenge: Potential cross-reactivity with SIRPα and SIRPβ

  • Solution: Use T-cell specific gating strategies in flow cytometry since SIRPG is exclusively expressed on T cells. For protein detection methods, validate antibody specificity against recombinant SIRPα and SIRPγ proteins

• Detecting Alternatively Spliced Isoforms:

  • Challenge: Multiple SIRPG transcript isoforms exist, not all producing detectable protein

  • Solution: Use isoform-specific primers in qPCR or RNA-seq with Event Analysis. For protein detection, use gradient gels (4-12%) to resolve different molecular weight isoforms

• Intracellular versus Surface Detection:

  • Challenge: Some experimental conditions may affect SIRPG surface expression

  • Solution: Perform parallel surface and intracellular staining to capture the total SIRPG expression. Use appropriate fixation and permeabilization buffers (e.g., eBioscience Intracellular Fixation & Permeabilization Buffer Set)

How can researchers validate SIRPG antibody specificity for their experimental systems?

Proper validation of SIRPG antibody specificity is critical for accurate experimental results:

• Recombinant Protein Binding Assays:

  • Immobilize recombinant human SIRPγ (0.5-2 μg/ml) in carbonate buffer (pH 9.2)

  • Test antibody binding in range (from initial concentration at 5-10 μg/ml)

  • Include recombinant SIRPα as a control to test cross-reactivity

  • Detect binding with appropriate secondary antibodies or direct detection methods

• Cellular Validation Strategies:

  • Compare staining patterns between T-cells (SIRPG-positive) and monocytes (SIRPG-negative, SIRPα-positive)

  • Use CRISPR/Cas9-modified Jurkat cells with SIRPG knockout as negative controls

  • Implement parallel staining with multiple anti-SIRPG antibody clones targeting different epitopes

• Western Blot Validation:

  • Include both SIRPG-expressing (T-cells, Jurkat) and non-expressing cell types

  • Run gradient gels (4-12% Bis-Tris) to properly resolve SIRPG isoforms

  • Validate molecular weights against predicted values: isoform 1 (42 kDa), isoform 2 (38 kDa), and isoform 4 (30 kDa)

  • Include appropriate loading controls (γ-tubulin, GAPDH)

• Blocking and Competition Assays:

  • Pre-incubate antibodies with recombinant SIRPG before staining to demonstrate specificity

  • Perform sequential staining with unlabeled followed by labeled antibody to test for epitope competition

  • For functional blocking antibodies, validate blocking efficiency using ELISA-based SIRPG-CD47 interaction assays

• Flow Cytometry Controls:

  • Include matching isotype controls for each antibody format and fluorochrome

  • Implement FMO (Fluorescence Minus One) controls for multicolor panels

  • Use live/dead discrimination with LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit

How should researchers interpret altered SIRPG expression patterns in autoimmune disease samples?

Published research provides important guidelines for interpreting SIRPG expression data in autoimmune contexts:

What are the methodological considerations for investigating SIRPG in animal models given its absence in rodents?

SIRPG is absent in rodents, presenting unique challenges for translational research:

• Humanized Mouse Models:

  • Develop humanized immune system mice by reconstituting immunodeficient mice with human hematopoietic stem cells

  • These models allow study of human T-cells expressing SIRPG in an in vivo context

  • Flow cytometry protocols should include markers to distinguish human from murine cells

• GVHD Models as Functional Readouts:

  • Graft-versus-host disease models have been validated for studying the functional consequences of altered SIRPG expression

  • Published research demonstrates that SIRPγ^low T-cells have enhanced pathogenicity in vivo in GVHD models

  • Use standardized scoring systems to quantify disease severity and correlate with SIRPG expression levels

• In Vitro Alternatives:

  • Human cell-based assays using primary T-cells provide more relevant systems for SIRPG research

  • Co-culture systems with antigen-presenting cells can model SIRPG-CD47 interactions

  • Cell-cell adhesion assays with CRISPR/Cas9-modified Jurkat cells have demonstrated that SIRPG targeting affects conjugate formation

• Cross-Species Considerations:

  • Some SIRPG antibodies cross-react with non-human primate SIRPG

  • Non-human primate models may offer alternatives when studying SIRPG biology in vivo

  • When selecting antibodies for such studies, verify cross-reactivity with the target species

• Transgenic Expression Approaches:

  • Consider transgenic expression of human SIRPG in mouse T-cells to study its function

  • Such models should control for appropriate expression levels and patterns

  • Verify functionality of human SIRPG in the murine cellular context through binding assays with mouse CD47

How can SIRPG antibodies be utilized in studying T-cell functions in autoimmune disease patient samples?

SIRPG antibodies offer valuable tools for investigating T-cell dysfunction in autoimmunity:

• Patient Stratification Protocol:

  • Combine SIRPG phenotyping with rs2281808 genotyping to stratify patients

  • Quantify SIRPγ^high versus SIRPγ^low T-cell populations by flow cytometry

  • This approach enables correlation of SIRPG expression patterns with clinical parameters and treatment responses

• Multiparameter Flow Cytometry Panels:

  • Design panels incorporating anti-SIRPG antibodies (clones OX-119 or LSB2.20) with markers of:

    • T-cell activation (CD25, CD69)

    • Differentiation (CD45RA, CCR7)

    • Exhaustion (PD-1, CTLA-4)

    • Functional markers (IFN-γ, granzyme B)

  • This provides comprehensive phenotypic and functional profiling of SIRPG-expressing T-cells

• Functional Assays:

  • SIRPα-γ/CD47 blockade with KWAR23 impairs IFN-γ secretion by chronically activated T cells

  • Design assays measuring cytokine production, proliferation, and cytotoxicity in SIRPγ^high versus SIRPγ^low T-cells from patients

  • These assays can reveal functional consequences of altered SIRPG expression in disease contexts

• Ex Vivo T-cell Expansion:

  • When expanding patient T-cells ex vivo, monitor SIRPG expression changes during culture

  • This approach enables assessment of whether disease-associated SIRPG expression alterations are maintained in vitro

  • Consider T-cell stimulation conditions that maintain physiological SIRPG expression patterns

• Imaging Applications:

  • Apply confocal microscopy to visualize SIRPG distribution at the immune synapse in patient-derived T-cells

  • Research shows that SIRPγ spatial reorganization at the immune synapse is independent of its interaction with CD47

  • This can reveal whether altered SIRPG expression affects immunological synapse formation in autoimmune conditions

What methodological approaches should be used when studying the relationship between SIRPG genetic variants and autoimmune disease risk?

To effectively investigate SIRPG genetics in autoimmunity:

• Genotyping Strategy:

  • Implement targeted genotyping for established SIRPG risk variants (rs2281808, rs6043409)

  • Consider broader sequencing of the SIRPG locus to identify additional variants

  • Research shows rs2281808 TT variant has significantly higher frequency in T1D (22% vs 4% in healthy controls) and RRMS subjects

• Expression Quantitative Trait Loci (eQTL) Analysis:

  • Correlate genotype data with SIRPG transcript levels using qPCR or RNA-seq

  • The GTEx whole blood meta-analysis shows rs2281808 as being negatively associated with SIRPγ expression

  • This approach validates functional consequences of genetic variants

• Isoform-Specific Expression Analysis:

  • Utilize isoform-specific qPCR assays to quantify relative abundance of SIRPG transcript isoforms

  • The rs6043409 variant alters a predicted exonic splicing enhancer, resulting in significant shifts in SIRPG transcript isoform distribution

  • This methodology provides mechanistic insights into how variants affect SIRPG function

• Functional Genomics Approach:

  • Implement CRISPR/Cas9-based genome editing to recreate risk variants in T-cell lines

  • Compare functional properties of isogenic cell lines differing only in SIRPG variants

  • This strategy establishes causal relationships between variants and cellular phenotypes

• Clinical Correlation Methodology:

  • Correlate SIRPG genotypes with detailed clinical parameters in autoimmune cohorts

  • Research suggests rs2281808 is associated with early-onset diabetes patients

  • This approach helps identify genetic associations with specific disease subtypes or progression patterns