CD58 Antibody

What is CD58 and why is it significant in immunology research?

CD58 (LFA-3) is a heavily glycosylated surface protein (40-70 kDa) belonging to the immunoglobulin superfamily. It exists in multiple forms: a transmembrane variant, a GPI-linked form, and soluble CD58 (sCD58). CD58 is widely expressed on both hematopoietic and non-hematopoietic cells, including leukocytes, erythrocytes, endothelial cells, and fibroblasts .

Its significance lies in its role as the ligand for CD2 on T cells and NK cells, making it critical for:

• Immunological synapse formation

• T cell adhesion and activation

• Antigen-independent and dependent interactions between T cells and antigen-presenting cells

• Cytokine production and cytotoxicity

CD58-CD2 interaction provides essential co-stimulatory signals for optimal T cell responses, making it a key molecule in both normal immune function and pathological states .

What are the optimal protocols for CD58 antibody applications in flow cytometry?

For effective flow cytometry with CD58 antibodies:

• Sample preparation: Use fresh cells when possible. For peripheral blood, collect with anticoagulant, isolate mononuclear cells by density gradient centrifugation, and wash with PBS containing 2% FBS.

• Antibody titration: Though many commercial antibodies recommend 5 μL (0.5 μg) per test for 10^5-10^8 cells in 100 μL final volume , optimal concentration should be determined empirically for each application.

• Staining procedure:

  • Resuspend cells in staining buffer (PBS with 2% FBS)

  • Add pre-titrated CD58 antibody (common clones: TS2/9, AICD58, 1C3)

  • Incubate for 30 minutes at 4°C in the dark

  • Wash twice with staining buffer

  • Resuspend in appropriate buffer for analysis

• Controls: Always include:

  • Unstained cells for autofluorescence

  • Isotype control (matching the CD58 antibody's isotype)

  • FMO (fluorescence minus one) controls for multicolor panels

• Gating strategy: CD58 shows broad expression patterns, so proper gating on target populations is essential. For lymphocytes, initial gating on FSC/SSC is recommended, followed by exclusion of dead cells .

How can researchers validate CD58 antibody specificity for experimental applications?

Multiple validation approaches should be employed:

• Positive and negative controls:

  • Use cell lines with known CD58 expression (high: Raji, Nalm6; low/negative: specific CD58-knockout lines)

  • Include CD58-negative tissues in immunohistochemistry applications

• Genetic validation:

  • Compare staining between wild-type and CD58 knockout cells generated by CRISPR/Cas9

  • Examine cells with CD58 overexpression systems

• Peptide blocking experiments:

  • Pre-incubate antibody with recombinant CD58 protein

  • Observe elimination of specific staining

• Antibody cross-validation:

  • Compare results using multiple antibody clones targeting different CD58 epitopes

  • Validate with orthogonal methods (e.g., flow cytometry results with Western blot analysis)

• Functional validation:

  • Test if antibody blocks CD58-CD2 interactions in functional assays

  • Some clones (like AICD58) are known blocking antibodies in LFA-3 mediated adhesion

These approaches collectively provide robust validation of antibody specificity before proceeding with complex experiments .

What are the best fixation and antigen retrieval methods for CD58 immunohistochemistry?

Optimal protocols for CD58 immunohistochemistry vary by tissue type and application:

• Fixation options:

  • Formalin-fixed, paraffin-embedded (FFPE) tissues: 10% neutral buffered formalin for 24-48 hours

  • Fresh frozen sections: Brief fixation with 4% paraformaldehyde (10 minutes)

• Antigen retrieval methods:

  • Heat-induced epitope retrieval (HIER) with citrate buffer (pH 6.0) is typically effective

  • EDTA buffer (pH 9.0) may yield better results for some antibody clones

  • Protease-induced retrieval is generally not recommended for CD58

• Protocol specifics for FFPE tissues:

  • Deparaffinize and rehydrate sections

  • Perform antigen retrieval

  • Block endogenous peroxidase (3% H₂O₂) and non-specific binding (5% normal serum)

  • Incubate with primary CD58 antibody (1:50-1:200 dilution range)

  • Detect using appropriate secondary antibody system

  • Counterstain, dehydrate, and mount

• Visualization systems:

  • For brightfield: HRP-DAB systems work well

  • For fluorescence: Alexa Fluor conjugates provide strong signal with low background

Results should show membrane localization of CD58 in positive cell populations, with lymphocytes in lymphatic nodules showing particularly distinct membrane staining .

How can researchers design experiments to investigate CD58's role in tumor immune evasion?

Based on recent findings identifying CD58 as a critical factor in tumor immune evasion, researchers should consider these experimental approaches:

• Genetic manipulation systems:

  • Generate isogenic tumor cell lines with CRISPR/Cas9-mediated CD58 knockout

  • Create CD58 re-expression models in naturally CD58-deficient tumor lines

  • Develop inducible CD58 systems to study dynamic effects

• In vitro immune evasion assays:

  • Co-culture experiments comparing wild-type vs. CD58-deficient tumor cells with:

    • CAR T cells (measuring expansion, degranulation, cytokine release, cytotoxicity)

    • NK cells (assessing natural killing activity)

    • Regular T cells (evaluating activation markers, proliferation)

  • Growth competition assays with mixed populations to measure selective advantage

• Signaling pathway analysis:

  • Investigate JAK2/STAT1 pathway activation in CD58-deficient vs. wild-type cells

  • Examine PDL1 and IDO expression levels and their correlation with CD58 status

  • Study the LYN/CD22/SHP1 axis as downstream effectors

• Immunological synapse visualization:

  • Use confocal microscopy to quantify immunological synapse formation between T cells and CD58+ vs. CD58- tumor cells

  • Measure recruitment of key signaling molecules to the synapse

• In vivo models:

  • Xenograft models comparing CD58+ vs. CD58- tumors treated with immunotherapy

  • Analysis of tumor-infiltrating lymphocytes and their functional status

This comprehensive approach has revealed that CD58 loss induces suboptimal immunological synapse formation with immune cells, impairing their expansion, cytokine production, and cytotoxic function, ultimately leading to therapy resistance .

What methodological considerations are important when studying the relationship between CD58 alterations and response to immunotherapy?

When investigating CD58's impact on immunotherapy response, several methodological considerations are crucial:

In recent DLBCL studies, CD58 mutation rate was 9.1%, and copy number loss rate was 44.7%, with these alterations significantly correlating with reduced response to R-CHOP therapy and inferior survival. Similar methodological approaches can be applied to study other immunotherapies including checkpoint inhibitors and CAR-T cells .

How should researchers approach experiments investigating CD58's role in CAR T-cell therapy resistance?

Based on emerging evidence of CD58's importance in CAR T-cell responses, researchers should consider this experimental framework:

• Pre-clinical models:

  • In vitro systems:

    • Co-culture of CD19 CAR T-cells with CD58-proficient vs. CD58-deficient B-cell lines

    • Measure CAR T-cell expansion, cytokine production, and cytotoxicity

    • Assess formation of immunological synapses between CAR T cells and target cells

  • In vivo models:

    • Xenograft mouse models comparing CD58+ vs. CD58- tumors

    • Measure tumor growth, CAR T-cell persistence, and tumor-infiltrating lymphocyte phenotypes

    • Test combination strategies (e.g., CD58-CD2 pathway activators with checkpoint inhibitors)

• Mechanistic investigations:

  • Study the impact of CD58 loss on:

    • Immunological synapse formation and stability

    • CAR T-cell signaling cascades

    • CAR T-cell metabolic reprogramming

    • Exhaustion marker expression (PD-1, TIM-3, LAG-3)

• Resistance bypass strategies:

  • Test direct CD2 agonists to bypass CD58 requirement

  • Evaluate combination with PDL1 blockade or IDO inhibitors

  • Assess engineered CAR designs less dependent on costimulatory signals

• Translational components:

  • Measure CD58 status in patient samples before CAR T-cell therapy

  • Track CD58 alterations in cases of CAR T-cell resistance or relapse

  • Correlate CD58 status with clinical outcomes

Research has demonstrated that CD58 loss causes inefficient immunological synapse formation with CAR T cells, impairing their activation, expansion, and cytotoxic function. Combining CD58-CD2 costimulatory signaling activation with PD-L1 blockade or IDO inhibition has shown promise in sensitizing CD58-deficient tumors to CAR T-cell therapy .

What techniques should be employed to study soluble CD58 (sCD58) in disease contexts?

Soluble CD58 (sCD58) requires specialized techniques for accurate detection and functional assessment:

• Quantification methods:

  • ELISA: Develop sandwich ELISA using capture and detection antibodies specific to non-overlapping CD58 epitopes

  • Immunoprecipitation followed by Western blot: For samples with low sCD58 concentration

  • Multiplex assays: Include sCD58 in cytokine/soluble receptor panels to assess correlations with other immune mediators

• Sample considerations:

  • Collection: Standardize collection protocols to minimize artificial release from cells

  • Processing: Process samples rapidly at 4°C to prevent ex vivo generation

  • Storage: Aliquot and store at -80°C with minimal freeze-thaw cycles

  • Controls: Include matched controls (age, gender) for accurate comparison

• Functional assessment:

  • T cell assays: Measure impact of purified or patient-derived sCD58 on T cell activation

  • CD2 binding studies: Assess competitive binding between membrane CD58 and sCD58

  • Rosette inhibition assays: Test sCD58's ability to inhibit CD2-CD58 mediated cell-cell adhesion

• Clinical correlation analyses:

  • Disease activity: Correlate sCD58 levels with inflammatory markers and disease scores

  • Treatment response: Monitor changes in sCD58 during therapy

  • Prognostic value: Assess sCD58 as a biomarker for disease outcomes

Studies have shown that sCD58 is present in human serum, urine, and synovial fluid of rheumatoid arthritis patients. At high concentrations, sCD58 can bind to CD2-positive cells and inhibit rosette formation of human T cells with erythrocytes, potentially disturbing cell-cell adhesion and recognition in vivo .

What approaches can resolve discrepancies in CD58 antibody detection between different experimental platforms?

When faced with discrepancies between detection methods, researchers should implement this systematic troubleshooting approach:

• Antibody-specific factors:

  • Epitope mapping: Different antibody clones recognize distinct epitopes that may be differentially accessible across methods

  • Antibody format: Native antibodies may perform differently than conjugated versions

  • Fixation sensitivity: Some epitopes are destroyed by certain fixatives or denatured in Western blotting

• Sample preparation differences:

  • Flow cytometry: Preserves native conformation but may suffer from internalization during processing

  • Western blotting: Denatures proteins, potentially destroying conformational epitopes

  • IHC/IF: Fixation and antigen retrieval methods critically affect epitope accessibility

  • ELISA: May detect both membrane-bound and soluble forms

• Resolution strategies:

  • Cross-validation protocol:

    1. Split samples and process using multiple methods

    2. Use at least three antibody clones recognizing different epitopes

    3. Compare results qualitatively and quantitatively

  • Control ladder:

    1. Include samples with known CD58 expression levels (high, medium, low)

    2. Create standard curves using recombinant CD58 when possible

    3. Normalize results to these standards across platforms

• Interpretation framework:

  • Consider CD58 isoforms (GPI-linked vs. transmembrane) that may be differentially detected

  • Evaluate alternative splicing that might affect epitope presence

  • Assess post-translational modifications that could mask epitopes

  • Document method-specific sensitivity thresholds

When discrepancies persist, biological validation through functional assays (e.g., CD2-binding capacity) provides the definitive assessment of biologically relevant CD58 expression .

How can single-cell analysis technologies be optimized for studying CD58 expression heterogeneity within tumors?

Single-cell technologies offer unprecedented insights into CD58 expression heterogeneity, requiring specific optimization approaches:

• Single-cell RNA sequencing protocols:

  • Sample preparation: Optimize tissue dissociation to minimize epitope damage

  • Cell sorting strategies: Consider index sorting to correlate protein expression with transcriptome

  • Computational analysis: Develop algorithms to detect CD58 splice variants and correlate with cellular phenotypes

  • Integration approaches: Combine with TCR sequencing to link CD58 expression to T-cell clonal expansion

• Single-cell protein analysis:

  • Mass cytometry (CyTOF): Develop CD58 antibody conjugates compatible with metal isotope labeling

  • CODEX multiplexed imaging: Optimize CD58 antibody panel for spatial expression analysis

  • Flow cytometry: Design high-dimensional panels to simultaneously assess CD58 with immune checkpoint molecules

• Spatial transcriptomics adaptations:

  • Probe design: Create specific probes for CD58 mRNA variants

  • Spatial correlation: Analyze CD58 expression in relation to tumor-immune interfaces

  • Multiplex approaches: Combine with immunofluorescence to correlate transcript with protein

• Protocol considerations:

  • Fresh vs. frozen tissue: Compare CD58 detection efficiency

  • Fixation impact: Evaluate different fixatives on CD58 epitope preservation

  • Antibody validation: Confirm specificity in the single-cell context with knockout controls

Recent studies have employed single-cell RNA-seq to demonstrate that CD58 expression in tumor cells negatively correlates with CD8+ T-cell exhaustion/dysfunction status, providing insights not possible with bulk analysis methods .

What experimental design best evaluates the therapeutic potential of targeting the CD58-CD2 axis in cancer immunotherapy?

Based on emerging evidence of CD58's role in immune evasion, a comprehensive experimental framework should include:

• Preclinical platform development:

  • In vitro models:

    • CD58-deficient vs. CD58-proficient isogenic tumor lines

    • Primary patient-derived tumor models with characterized CD58 status

    • 3D organoid cultures incorporating immune components

  • In vivo models:

    • Syngeneic mouse models with human CD58 knockin

    • Humanized mouse models with human immune system components

    • PDX (patient-derived xenograft) models stratified by CD58 status

• Therapeutic strategy evaluation:

  • CD58-CD2 axis targeting approaches:

    • CD2 agonistic antibodies or engineered CD58 ligands

    • Recombinant CD58 constructs for CD58-deficient tumors

    • Combination with checkpoint inhibitors (anti-PD1/PDL1)

    • IDO inhibitors to counter CD58-deficiency mechanisms

  • Readout parameters:

    • Tumor growth/regression kinetics

    • Immune cell infiltration and activation state

    • Metabolic and functional immune cell assessment

    • Long-term memory and recurrence evaluation

• Translational components:

  • Biomarker development: Standardized CD58 testing for patient stratification

  • Response prediction models: Algorithms incorporating CD58 with other immune markers

  • Resistance mechanisms: Monitoring for escape pathways during treatment

  • Patient selection strategies: Criteria for identifying optimal responders

Recent research demonstrated that direct activation of CD58-CD2 costimulatory signaling in combination with anti-PDL1 blockade or IDO inhibitor sensitized CD58-deficient DLBCL to chimeric antigen receptor T-cell therapy, providing a foundation for this approach .

What methodological approaches best characterize CD58's role in the formation and function of the immunological synapse?

Advanced imaging and functional techniques can elucidate CD58's role in immunological synapse (IS) formation:

• High-resolution imaging approaches:

  • Live cell imaging protocols:

    • Spinning disk confocal microscopy for dynamic IS formation

    • Total internal reflection fluorescence (TIRF) microscopy for molecular recruitment

    • Lattice light-sheet microscopy for 3D dynamics with minimal phototoxicity

  • Molecular tagging strategies:

    • CD58 tagged with fluorescent proteins (ensuring tag doesn't interfere with function)

    • Key signaling proteins labeled to track recruitment to CD58-rich domains

    • Calcium indicators to correlate CD58 engagement with signaling events

• Quantitative analysis frameworks:

  • IS formation metrics:

    • Measurement of CD58 clustering at the interface

    • Recruitment kinetics of associated signaling molecules

    • Size and stability of CD58-enriched domains

    • Correlation with functional outcomes (T cell activation)

  • Analytical tools:

    • Machine learning algorithms for IS pattern recognition

    • Single-molecule tracking to assess CD58 mobility

    • Spatial statistics to quantify molecular colocalization

• Functional correlation approaches:

  • Microfluidic systems: Control of cell-cell contact dynamics

  • Force measurement: Atomic force microscopy to assess CD58-CD2 binding strength

  • Calcium flux assays: Correlate with IS formation quality

  • Degranulation assessments: Link IS quality to functional outputs

• Perturbation techniques:

  • Domain-specific mutations: Identify critical CD58 regions for IS formation

  • Acute disruption: Optogenetic tools to precisely control CD58 availability

  • Spatial manipulation: Nanopatterned surfaces with controlled CD58 distribution

Studies have demonstrated that disruption of CD58 in tumor cells induces the formation of suboptimal immunological synapses with CAR T cells, leading to impaired cell expansion, degranulation, cytokine secretion, and cytotoxicity .

How can researchers effectively study CD58 genetic alterations in patient samples and their impact on immunotherapy response?

Comprehensive analysis of CD58 genetic alterations requires an integrated methodological approach:

• Sample collection and processing protocols:

  • Fresh tissue:

    • Immediate stabilization for RNA/DNA extraction

    • Single-cell isolation protocols for heterogeneity assessment

  • FFPE samples:

    • Optimized DNA extraction methods for fragmented DNA

    • Laser capture microdissection for tumor-specific analysis

  • Liquid biopsies:

    • cfDNA isolation optimized for detecting CD58 alterations

    • Circulating tumor cell enrichment strategies

• Genomic analysis techniques:

  • Targeted sequencing panels:

    • Design to cover CD58 exons, splice sites, and regulatory regions

    • Include genes in CD58-related pathways (CD2, JAK2/STAT1, LYN/CD22/SHP1)

  • Copy number analysis:

    • Array-CGH or NGS-based methods to detect CD58 deletions

    • Digital droplet PCR for precise quantification

  • Structural variant detection:

    • Long-read sequencing to identify complex rearrangements

    • Fiber-FISH for visualization of large-scale alterations

• Functional validation methods:

  • Reporter assays: Construct libraries to test impact of mutations

  • CRISPR knock-in: Introduce patient-specific mutations to cell lines

  • Protein binding studies: Assess impact on CD2 interaction

  • Patient-derived organoids: Test immunotherapy response in context

• Clinical correlation framework:

  • Prospective biomarker studies: Follow patients with CD58 alterations

  • Response prediction models: Incorporate CD58 status into algorithms

  • Longitudinal monitoring: Track emergence of CD58 alterations during treatment