CD2 Antibody

What is CD2 and what cellular populations express it?

CD2 is a 50-55 kDa transmembrane glycoprotein that functions as a cell surface receptor. In humans, CD2 is expressed on all mature T cells, approximately 95% of thymocytes, and most natural killer (NK) cells . Notably, expression patterns differ between species - in mice, B lineage cells from the pre-B cell stage to mature B cells also express CD2, unlike in humans . CD2 functions as an adhesion and activation molecule, interacting with CD58 (LFA-3) in humans and CD48 in mice .

What types of CD2 antibodies are available for research purposes?

Researchers have access to several types of CD2 antibodies:

CD2 antibodies target various epitopes, including the ligand-binding region (T11 epitope) and other functional domains, allowing for diverse experimental applications .

What are the primary research applications for CD2 antibodies?

CD2 antibodies serve multiple research purposes:

• Flow cytometric analysis of lymphocyte populations

• Western blot detection of CD2 protein (showing bands at approximately 48 kDa under reducing conditions)

• Immunohistochemical staining of lymphoid tissues

• Blocking CD2-mediated adhesion and signaling in functional assays

• Studying T cell development and activation mechanisms

• Therapeutic applications in transplantation and autoimmune disease models

How should CD2 antibodies be optimized for flow cytometry analysis?

For optimal flow cytometric analysis with CD2 antibodies:

• Titrate antibodies carefully - the recommended starting concentration is 0.25 μg per test or 5 μl per 10^6 cells in 100 μl suspension .

• For human PBMCs and tumor samples, implement an Fc-receptor blocking step using Human TruStain FcX (5 μl per sample) in PBS with 0.5% BSA for 10 minutes at room temperature .

• Prepare antibody mixes in ice-cold PBS/0.5% BSA and incubate cells on ice in the dark for 20 minutes .

• Include appropriate positive controls (T cell-enriched populations) and negative controls (B cell lines like Raji for human samples) .

• When analyzing data, compare CD2 expression across different lymphocyte subsets, as expression levels vary with activation state and cell type .

What controls are essential when using CD2 antibodies in Western blot analysis?

Essential controls for Western blot analysis with CD2 antibodies include:

• Positive control samples: Jurkat and MOLT-4 human acute T cell leukemia cell lines reliably express CD2 .

• Negative control samples: HeLa human cervical epithelial carcinoma and Raji human Burkitt's lymphoma cell lines do not express CD2 .

• Loading control: GAPDH or similar housekeeping proteins should be detected to verify equal loading across lanes .

• Molecular weight verification: Under reducing conditions, human CD2 appears at approximately 48 kDa in standard Western blot or 77-87 kDa in Simple Western™ systems .

Always run experiments under appropriate reducing conditions and use PVDF membranes for optimal results with Immunoblot Buffer Group 1 .

What are the critical parameters for immunohistochemical detection of CD2?

For successful immunohistochemical detection of CD2:

• Sample preparation: Both paraffin-embedded and frozen tissue sections can be used, with appropriate fixation and antigen retrieval methods .

• Antibody concentration: Begin with 3 μg/mL for paraffin-embedded tissues, adjusting based on signal-to-background ratio .

• Incubation conditions: Optimal results typically require 1 hour at room temperature followed by appropriate detection systems .

• Positive control tissues: Human tonsil provides excellent positive control tissue due to abundant T cells .

• Expected staining pattern: CD2 staining should appear as membrane-associated signal on lymphocytes, particularly in T cell-rich zones of lymphoid tissues .

How do different anti-CD2 monoclonal antibodies affect T cell function?

Anti-CD2 monoclonal antibodies can exert complex, sometimes opposing effects on T cell function:

• Some antibodies like UMCD2 demonstrate dual functionality:

  • When combined with anti-T11 antibodies, they can be mitogenic for T cells

  • When added to other T cell stimuli (IL-2 or anti-CD3), they inhibit T cell responses

• Mechanistic differences exist between stimulatory and inhibitory effects:

  • Stimulatory combinations typically induce increased cytoplasmic calcium

  • Inhibitory effects may operate through calcium-independent mechanisms

• Epitope specificity determines functional outcomes:

  • Antibodies binding the T11 (ligand-binding) region of CD2 often block E-rosetting with sheep erythrocytes

  • The T11 epitope appears to contain multiple subepitopes with distinct functional properties

What has been learned about CD2 regulation through CRISPR-Cas9 screening approaches?

Genome-wide CRISPR-Cas9 loss-of-function screening has revealed key regulators of CD2 expression:

• Primary regulators identified in Jurkat T cells include:

  • CD2 itself (self-regulation)

  • BAP1 (BRCA1-associated protein-1), a nuclear deubiquitinating enzyme essential for thymocyte development

  • SUZ12, a component of the polycomb repressive complex 2 (PRC2)

• Validation experiments confirmed:

  • BAP1 knockout significantly reduces CD2 expression at both protein and mRNA levels

  • SUZ12 knockout also reduces CD2 expression but to a lesser extent than BAP1 loss

• T cell activation modulates CD2 expression patterns:

  • Wild-type Jurkat T cells show a 1.8-fold increase in CD2 expression upon stimulation

  • BAP1 knockout T cells show impaired CD2 upregulation upon stimulation (only reaching 21% CD2+ cells vs. nearly 100% in wild-type)

These findings suggest epigenetic regulatory mechanisms play critical roles in controlling CD2 expression, with potential implications for understanding T cell activation and development .

What are the pharmacokinetic and pharmacodynamic profiles of therapeutic anti-CD2 antibodies?

Studies of therapeutic anti-CD2 antibodies reveal important pharmacokinetic and pharmacodynamic properties:

• The humanized IgG1κ monoclonal antibody siplizumab and its rat parent BTI-322:

  • Both cause rapid, transient depletion of CD2+, CD3+, CD4+, CD8+ lymphocytes and NK cells

  • Immune reconstitution occurs more rapidly with BTI-322 compared to siplizumab

  • Lymph node T cell depletion reaches approximately 45% at doses >0.6 mg/kg

  • Lymph node architecture restoration requires two weeks to two months

• Key differences between antibodies:

  • Siplizumab demonstrates longer half-life than BTI-322

  • BTI-322 administration results in more adverse events than siplizumab

  • Siplizumab is species-specific, reacting only with human and chimpanzee cells

These profiles provide crucial insights for designing therapeutic protocols using anti-CD2 antibodies in transplantation and autoimmune disease settings .

Why might flow cytometry with CD2 antibodies yield inconsistent results?

Inconsistent flow cytometry results with CD2 antibodies may stem from several factors:

• Technical considerations:

  • Inadequate antibody titration (optimal concentration is typically ≤0.25 μg per test)

  • Insufficient blocking of Fc receptors, particularly important with mixed cell populations

  • Improper compensation when using multiple fluorochromes

  • Sample preparation issues (cell death, aggregation)

• Biological variables:

  • CD2 expression varies with T cell activation state (1.8-fold increase upon stimulation)

  • CD2 expression differs between lymphocyte subsets

  • Species differences (mouse B cells express CD2, unlike human B cells)

  • Internalization of CD2 after antibody binding

• Protocol optimization:

  • Cell numbers should be empirically determined (range from 10^5 to 10^8 cells/test)

  • Post-acquisition gating strategies must account for different expression levels across cell types

  • Fresh vs. frozen samples may show different staining patterns

What factors influence the specificity of CD2 antibodies in immunohistochemistry?

Several factors affect CD2 antibody specificity in immunohistochemistry:

• Antibody characteristics:

  • Purity level (should exceed 90% as determined by SDS-PAGE)

  • Aggregation (should be less than 10% as determined by HPLC)

  • Clone-specific binding properties

  • Monoclonal vs. polyclonal antibodies

• Tissue preparation:

  • Fixation method and duration

  • Antigen retrieval techniques

  • Block effectiveness

  • Section thickness

• Protocol optimization:

  • Primary antibody concentration (typically starting at 3 μg/mL)

  • Incubation time and temperature

  • Detection system sensitivity

  • Washing stringency

For optimal specificity, use antigen affinity-purified antibodies with post-manufacturing filtration (0.2 μm) and validate results with known positive and negative control tissues .

How should researchers interpret variations in CD2 expression across different T cell subsets?

When analyzing variations in CD2 expression across T cell subsets:

• Baseline expression patterns:

  • All mature T cells should express CD2, though at varying levels

  • Approximately 95% of thymocytes express CD2

  • Most NK cells express CD2, potentially at different levels than T cells

• Activation-dependent changes:

  • T cell stimulation typically increases CD2 expression 1.2-1.8 fold

  • Different stimulation protocols (PMA, ionomycin, or combinations) result in varying upregulation levels

  • CD2 expression changes may correlate with other activation markers

• Functional correlations:

  • CD2 expression appears co-regulated with stemness in some contexts

  • CD2 functional responses may differ between naive and memory T cells

  • Expression patterns may change in pathological conditions

• Regulatory mechanisms:

  • Epigenetic regulators like BAP1 and SUZ12 significantly impact CD2 expression levels

  • Transcriptional regulation occurs at the mRNA level, as confirmed by qPCR analysis