CD58 Antibody

What is CD58 and what is its significance in immune cell interactions?

CD58, also known as lymphocyte function-associated antigen 3 (LFA-3), is a heavily glycosylated surface glycoprotein (40-70 kDa) widely expressed on hematopoietic and non-hematopoietic cells. It serves as a costimulatory receptor that plays a crucial role in the immunological synapse formation. CD58's natural ligand is CD2, which is primarily expressed on T cells and NK cells . The CD2-CD58 interaction is fundamental for multiple immune processes, including:

• T cell and NK cell activation and proliferation

• Cell adhesion and recognition between immune cells and their targets

• Triggering intracellular signaling cascades in both T/NK cells and target cells

• Supporting cytolytic activity against neoplastic cells

The interaction between CD58 and CD2 optimizes and supplements the proliferative response mediated through TCR/CD3 signaling, making it an essential component of effective immune responses .

What are the primary experimental applications for CD58 antibodies?

CD58 antibodies are utilized in multiple research applications with distinct methodological considerations:

CD58 antibodies can be particularly valuable in studying immunological synapse formation, T cell activation mechanisms, and tumor cell recognition by cytotoxic lymphocytes .

How do CD58 antibodies help distinguish between membrane-bound and soluble CD58?

Researchers can employ different methodological approaches to distinguish between membrane-bound CD58 and its soluble form (sCD58):

For membrane-bound CD58:

• Flow cytometry with non-permeabilized cells detects surface expression

• IHC shows characteristic membrane localization, as seen in lymphatic nodules of human tonsil tissues

• Western blot of membrane fractions reveals the 55-70 kDa glycoprotein

For soluble CD58 (sCD58):

• ELISA of cell culture supernatants, serum, or urine samples

• Immunoprecipitation followed by Western blot of soluble fractions

• Functional assays comparing effects of purified sCD58 versus membrane-bound CD58 in T cell activation assays

It's important to note that sCD58 functions as an immunosuppressive factor by competing with membrane-bound CD58 for CD2 binding. At high concentrations, sCD58 can bind to CD2-positive cells and inhibit rosette formation of human T cells with erythrocytes, potentially disrupting cell-cell adhesion and recognition in vivo .

What factors influence CD58 antibody detection in Western blot experiments?

When working with CD58 antibodies in Western blot applications, researchers should consider several technical factors that influence detection quality:

• Glycosylation heterogeneity: CD58 is a heavily glycosylated protein with observed molecular weights ranging from 55-70 kDa despite a calculated molecular weight of only 28 kDa . This glycosylation pattern can vary between cell types.

• Sample preparation: Optimal lysis buffers should contain suitable detergents (e.g., RIPA or NP-40) to efficiently extract both transmembrane and GPI-anchored CD58 isoforms. Heat denaturation conditions may need optimization.

• Reducing vs. non-reducing conditions: Some epitopes on CD58 may be sensitive to reducing agents, potentially affecting antibody recognition.

• Positive controls: Including known CD58-expressing cell lines such as Raji, Jurkat, TF-1, or HeLa cells as positive controls is essential for validating antibody performance .

• Antibody dilution optimization: A recommended starting range of 1:500-1:2000 should be tested, with optimal concentration determined empirically for each experimental system .

The heterogeneity in CD58 detection may also reflect biological variables such as differential expression or post-translational modifications influenced by the cellular microenvironment or activation state.

What are the optimal conditions for immunohistochemical detection of CD58?

For successful immunohistochemical detection of CD58 in tissue sections, the following protocol optimizations are recommended:

• Antigen retrieval: Primary recommendation is TE buffer at pH 9.0, with citrate buffer at pH 6.0 as an alternative if needed . This step is critical for unmasking CD58 epitopes that may be modified during fixation.

• Antibody dilution: Begin with a dilution range of 1:200-1:800 and titrate to determine optimal concentration for specific tissue types .

• Incubation conditions: For polyclonal antibodies (e.g., 10878-1-AP), overnight incubation at 4°C often produces optimal results with minimal background staining .

• Detection system: HRP-DAB systems effectively visualize CD58 expression, particularly for localizing it to the plasma membrane of lymphocytes in lymphatic nodules .

• Counterstaining: Hematoxylin provides effective nuclear counterstaining that helps contextualize CD58 expression within tissue architecture .

Researchers should anticipate strong membrane staining in tissues with high CD58 expression, including human intrahepatic cholangiocarcinoma tissue, ovarian cancer tissue, and skin cancer tissue .

How can CD58 antibodies be validated for functional blocking studies?

Validation of CD58 antibodies for functional blocking studies requires multiple experimental approaches:

• T cell proliferation assays: Effective blocking antibodies will neutralize the proliferative effect of recombinant CD58 on CD3+ T cells in a dose-dependent manner. The neutralization dose (ND50) typically ranges from 3-12 μg/mL in the presence of CD3 stimulation .

• Rosette formation inhibition: Functional CD58 blocking antibodies should inhibit rosette formation between T lymphocytes and erythrocytes, which depends on CD2-CD58 interactions .

• Cytotoxicity assays: Blocking antibodies can be assessed for their ability to modulate NK cell or cytotoxic T lymphocyte (CTL) killing of target cells. In functional assays, CD58 blockade typically reduces the cytolytic activity of effector cells .

• Dose-response curves: Establishing a clear dose-response relationship is critical for determining optimal antibody concentrations for functional studies. For example, the AICD58 clone has been validated as an effective blocking antibody for LFA-3 mediated adhesion .

• Controls: Include isotype controls and, where possible, comparative studies with other established CD58 blocking antibodies to confirm specificity.

A properly validated blocking antibody should produce consistent, concentration-dependent effects across multiple experimental systems and cellular contexts.

How do CD58 antibodies contribute to investigating the immunological synapse?

CD58 antibodies provide valuable tools for studying the complex architecture and dynamics of the immunological synapse (IS) through several methodological approaches:

• Immunofluorescence microscopy: CD58 antibodies can be used to visualize the spatial organization of CD58 within the IS during T/NK cell interactions with target cells. This allows researchers to track CD58 recruitment to the contact zone and its colocalization with other key molecules.

• Super-resolution microscopy: Labeled CD58 antibodies enable nanoscale imaging of CD58 distribution within IS microdomains, revealing clustering patterns that correlate with functional outcomes.

• Live cell imaging: Fab fragments derived from CD58 antibodies can monitor real-time redistribution of CD58 during IS formation without interfering with functional interactions.

• Functional perturbation: Blocking CD58 antibodies selectively disrupt CD2-CD58 interactions within the IS, allowing researchers to dissect the specific contribution of this interaction to IS stability and signaling. The CD2-CD58 interaction is a critical component of the IS that induces activation and proliferation of T/NK cells while triggering intracellular signaling in both lymphocytes and target cells .

• Proximity ligation assays: CD58 antibodies can be used in combination with antibodies against potential signaling partners to detect protein-protein interactions within the IS at molecular resolution.

These approaches collectively help elucidate how CD58-CD2 interactions contribute to IS formation, stability, and downstream signaling events that influence immune cell activation and effector functions.

What methodologies utilize CD58 antibodies to study immune evasion in tumor microenvironments?

CD58 antibodies facilitate several experimental approaches to investigate immune evasion mechanisms involving CD58 in tumor contexts:

• Expression analysis in tumor tissues: IHC with CD58 antibodies can reveal altered expression patterns in cancer cells compared to normal tissues. For example, CD58 antibodies can detect expression in human intrahepatic cholangiocarcinoma, ovarian cancer, and skin cancer tissues .

• Functional blocking studies: CD58 blocking antibodies help determine whether CD58-CD2 interactions are required for effective anti-tumor immunity in different cancer models. The anisomycin-mediated enhancement of NK cytotoxicity against hepatocellular carcinoma cells can be potently impaired by blocking CD58, suggesting its critical role in NK-mediated immunotherapy .

• Soluble CD58 detection: CD58 antibodies can quantify levels of immunosuppressive sCD58 in tumor microenvironments, which may contribute to immune evasion by interfering with CD2-CD58 interactions. Altered accumulation of sCD58 may lead to immunosuppression of T/NK cells in the tumor microenvironment .

• Correlation studies: Combining CD58 antibody staining with markers of T cell infiltration or activation status can reveal associations between CD58 expression and immune cell function in tumor tissues.

• Treatment response monitoring: CD58 antibodies can track changes in expression following immune checkpoint blockade or other immunotherapies, potentially identifying CD58 as a resistance mechanism or response biomarker.

These methodologies collectively help establish how CD58-CD2 interactions influence anti-tumor immunity and whether targeting this pathway could enhance immunotherapeutic outcomes.

How can researchers investigate the effects of CD58 expression modulation using CD58 antibodies?

Several experimental strategies utilizing CD58 antibodies can elucidate the functional consequences of CD58 expression modulation:

• Expression monitoring following treatments: CD58 antibodies enable quantification of expression changes after exposure to various stimuli. For instance, anisomycin treatment dramatically elevates CD58 expression on hepatocellular carcinoma cells, while UV-B irradiation decreases CD58 expression on EBV-transformed B cells .

• Correlation with functional outcomes: By combining CD58 expression analysis with functional assays, researchers can establish relationships between expression levels and biological responses. For example, blockade of CD58 impairs anisomycin-mediated enhancement of NK cytotoxicity, demonstrating CD58's importance in NK-mediated immunotherapy .

• Genetic manipulation validation: After genetic modification of CD58 (knockdown, knockout, or overexpression), antibodies confirm the success of manipulation at the protein level before proceeding to functional studies.

• Comparative analysis across treatment conditions:

• Relationship to soluble CD58: CD58 antibodies can differentiate between membrane-bound and soluble forms, allowing investigation of how modulation of one form affects the other and subsequent immune responses.

These methodologies enable researchers to comprehensively analyze how CD58 expression changes influence immune cell activation, adhesion, and effector functions in various physiological and pathological contexts.

Why might researchers observe variable molecular weights for CD58 in Western blot analyses?

The variable molecular weights observed for CD58 in Western blot analyses (typically ranging from 40-70 kDa) result from several biological and technical factors:

• Post-translational modifications: CD58 is heavily glycosylated, contributing significantly to its apparent molecular weight. The calculated molecular weight of the CD58 protein backbone is approximately 28 kDa, but glycosylation increases this to 55-70 kDa in most experimental systems .

• Isoform heterogeneity: CD58 exists in both transmembrane and GPI-anchored forms, which may display slightly different electrophoretic mobility patterns .

• Cell type-specific variations: Different cell types may express CD58 with varying glycosylation patterns. For example, CD58 from lymphoid tissues may show different banding patterns compared to CD58 from epithelial cells.

• Sample preparation effects: Harsh denaturation conditions or the presence of reducing agents can affect protein conformation and potentially alter migration patterns.

• Proteolytic processing: Partial proteolysis during sample preparation may generate lower molecular weight fragments detected by some antibodies.

When interpreting Western blot results, researchers should validate CD58 detection using positive control samples from established CD58-expressing cell lines such as Raji, Jurkat, TF-1, HeLa cells, or human lymph tissue .

What common issues affect CD58 antibody performance in immunohistochemistry?

Several common challenges can impact CD58 antibody performance in immunohistochemical applications:

• Inadequate antigen retrieval: CD58 epitopes may be masked during fixation, particularly with formalin. While TE buffer at pH 9.0 is recommended as the primary retrieval method, some tissues may require alternative approaches (such as citrate buffer at pH 6.0) .

• Fixation artifacts: Overfixation can irreversibly mask epitopes, while inadequate fixation may fail to preserve tissue architecture. The fixation duration and conditions should be standardized across experimental samples.

• Background staining: Non-specific binding may occur in tissues with high endogenous peroxidase activity or when using suboptimal blocking conditions. Proper blocking steps and inclusion of appropriate negative controls are essential.

• Antibody concentration: Inappropriate antibody dilution can lead to either weak specific staining or excessive background. Titration within the recommended range (1:200-1:800 for IHC) is necessary for each new tissue type .

• Detection system sensitivity: Some applications may require amplification steps (such as tyramide signal amplification) to detect low-level CD58 expression.

• Tissue-specific variations: CD58 expression patterns differ across tissues. While strong membrane staining is expected in lymphocytes of lymphatic nodules , other tissues may require optimization of staining protocols.

To troubleshoot these issues, researchers should systematically test different antigen retrieval methods, antibody concentrations, and detection systems while always including appropriate positive controls (such as tonsil tissue, which reliably expresses CD58) .

How should researchers interpret functional assay results when using CD58 blocking antibodies?

When interpreting functional assay results with CD58 blocking antibodies, researchers should consider these methodological principles:

• Dose-response relationships: Establish clear dose-response curves, as effective blocking antibodies typically show concentration-dependent effects. The neutralization dose (ND50) for inhibiting CD58-induced T cell proliferation generally ranges from 3-12 μg/mL .

• Context-dependent effects: The functional impact of CD58 blockade may vary depending on:

  • The activation state of effector cells

  • The presence of additional costimulatory or inhibitory signals

  • The specific cell types involved in the interaction

• Comparison with genetic approaches: When possible, compare antibody blocking results with genetic manipulation of CD58 (siRNA knockdown or CRISPR knockout) to confirm specificity of observed effects.

• Differentiation from antibody Fc effects: Include appropriate isotype controls to distinguish specific CD58 blockade from potential Fc receptor-mediated effects of the antibody.

• Time-dependent considerations: Some cellular responses to CD58 blockade may be immediate (adhesion disruption) while others develop over time (altered proliferation or cytokine production). Time-course experiments may reveal different patterns.

• Compensatory mechanisms: Prolonged CD58 blockade might induce upregulation of alternative costimulatory pathways. Extended blockade experiments should monitor potential compensatory responses.

The mixed lymphocyte reaction can be profoundly dampened by soluble CD58, and similarly, CD58 blockade may alleviate the cytotoxicity of human NK clones, highlighting the functional importance of CD58-CD2 interactions in diverse immune contexts .

How can CD58 antibodies contribute to investigating CD58's role in immunotherapeutic approaches?

CD58 antibodies enable several innovative research approaches in immunotherapy development:

• Biomarker development: CD58 antibodies can assess expression levels in tumor biopsies before and during immunotherapy, potentially identifying patients more likely to respond to certain treatments. Expression analysis in various tumor types, including intrahepatic cholangiocarcinoma, ovarian cancer, and skin cancer tissues, may reveal cancer-specific patterns .

• Combination therapy strategies: Functional studies using CD58 blocking antibodies can determine whether CD58-CD2 interactions synergize with or antagonize other immunotherapy approaches (such as checkpoint inhibitors).

• Therapeutic antibody development: Characterizing the functional effects of different CD58 antibody clones can inform the development of therapeutic antibodies that either block immunosuppressive effects or enhance costimulatory functions.

• CAR-T cell engineering: CD58 antibodies help evaluate the importance of CD58-CD2 interactions in CAR-T cell efficacy against various tumor types, potentially leading to improved CAR designs that incorporate CD2 signaling elements.

• Overcoming resistance mechanisms: CD58 antibodies can investigate whether alterations in CD58 expression contribute to resistance to existing immunotherapies, particularly in tumors that evade T cell-mediated killing.

Research indicates that the adhesion molecule CD58 is likely to be critical for NK-mediated immunotherapy, as blockade of CD58 can potently impair anisomycin-mediated enhancement of NK cytotoxicity against hepatocellular carcinoma cells . This suggests that targeting or enhancing CD58-CD2 interactions could represent a novel immunotherapeutic approach.

What techniques combine CD58 antibodies with other molecular tools to study complex immune interactions?

Advanced multi-parameter techniques that integrate CD58 antibodies with other molecular tools enable comprehensive analysis of complex immune interactions:

• Multiplex immunofluorescence imaging:

  • CD58 antibodies combined with antibodies against other immune markers allow simultaneous visualization of multiple molecules within the immunological synapse or tumor microenvironment

  • Spatial relationships between CD58 and its binding partners or downstream signaling molecules can be mapped at subcellular resolution

• Mass cytometry (CyTOF):

  • Metal-conjugated CD58 antibodies can be incorporated into large antibody panels to phenotype dozens of markers simultaneously on individual cells

  • This enables identification of specific cellular subsets where CD58 expression correlates with particular functional states or disease outcomes

• Proximity ligation assays:

  • CD58 antibodies paired with antibodies against potential interaction partners can reveal direct molecular associations in situ with high specificity

  • This technique can validate predicted protein-protein interactions involving CD58 in different cellular contexts

• CRISPR screens with CD58 antibody readouts:

  • Genome-wide CRISPR screens followed by CD58 antibody staining can identify genes that regulate CD58 expression

  • This approach can uncover novel regulatory pathways controlling CD58 in different physiological or pathological states

• Single-cell RNA-seq with protein detection:

  • CD58 antibodies conjugated to oligonucleotide barcodes can correlate CD58 protein expression with transcriptional profiles at single-cell resolution

  • This reveals relationships between CD58 expression and broader cellular states or differentiation trajectories

These integrated approaches provide multi-dimensional insights into how CD58-CD2 interactions connect to broader immune regulatory networks and how these interactions might be therapeutically manipulated in disease contexts.