CD2 FITC Antibody

What is CD2 and why is it an important immunological research target?

CD2 is a 50 kDa cell surface glycoprotein receptor expressed by the majority of thymocytes, all mature T cells, and a subset of NK cells, but not B lymphocytes. It functions as a pan T-cell marker and serves as a ligand for CD58 in humans, playing crucial roles in T cell adhesion and activation . CD2 is significant for immunological research because:

• It stabilizes adhesion between T cells and antigen-presenting or target cells

• Its expression varies across T cell subsets, with memory T-cells showing higher expression than naïve T-cells and regulatory T cells

• The CD2-CD58 interaction has been demonstrated to play a role in anti-tumor immune responses

• Anti-CD2 treatments have shown clinical utility in conditioning the immune system and treating transplant rejection

These characteristics make CD2 an essential marker for identifying and studying T cell populations and their functional states in various immunological research contexts.

How should researchers determine the appropriate concentration of CD2 FITC antibody for flow cytometry?

Determining the optimal concentration of CD2 FITC antibody requires titration to balance specific signal versus background. Most manufacturers provide pre-titrated recommendations as starting points:

• For RPA-2.10 clone: 5 μL (0.25 μg) per test (where a test is defined as staining a cell sample in a final volume of 100 μL)

• For other clones: 5 μL per 10^6 cells in 100 μL suspension or 5 μL per 100 μL of whole blood

A methodological titration approach includes:

• Preparing a single-cell suspension of target cells (e.g., PBMCs)

• Creating 2-fold serial dilutions of the antibody

• Using a fixed number of cells (e.g., 1×10^6 cells) per tube

• Staining cells with different antibody concentrations

• Analyzing by flow cytometry, evaluating signal-to-noise ratio

Cell numbers can range from 10^5 to 10^8 cells/test, but researchers should adjust antibody amount proportionally if significantly altering cell numbers . Always include unstained, isotype, and Fluorescence Minus One (FMO) controls for proper interpretation.

What are the key differences between various clones of CD2 antibodies?

Different clones of CD2 antibodies vary in their binding properties, applications, and performance characteristics:

RPA-2.10 clone:

• Species reactivity: Human, non-human primates, pigs

• Applications: Optimized for flow cytometric analysis

• Functional properties: Blocks mixed lymphocyte reaction

• Excitation/Emission: 488 nm/520 nm (when FITC-conjugated)

B-E2 clone:

• Species reactivity: Human

• Applications: Flow cytometry, immunohistochemistry

• Immunogen: Human thymocytes

• Isotype: Mouse IgG2b Kappa

When selecting between clones, researchers should consider:

• Epitope specificity: Different clones may bind to distinct epitopes on CD2

• Binding affinity: Variation in affinity affects sensitivity and signal intensity

• Cross-blocking: Some clones may compete for the same or overlapping epitopes

• Functional effects: Some antibodies may be neutralizing or activating

• Application suitability: A clone optimized for flow cytometry may not work well for immunoprecipitation or Western blotting

What cell types express CD2 and at what relative levels?

CD2 expression varies across immune cell populations with distinct patterns useful for identification and characterization:

T cell lineage:

• Thymocytes: CD2 is expressed on a majority of thymocytes during development

• Mature T cells: All peripheral blood T cells express CD2

• Memory vs. naïve T cells: Memory T cells exhibit higher CD2 expression levels than naïve T cells

• Regulatory T cells (Tregs): Tregs typically show relatively lower CD2 expression compared to effector T cells

Other immune cells:

• NK cells: A subset of NK cells express CD2

• B cells: CD2 is not expressed on B lymphocytes

• Monocytes/macrophages: Generally negative for CD2

Expression level changes:

• T cell activation induces increased CD2 expression

• Activation enhances lateral mobility of CD2 on the cell surface

• During immune reconstitution (e.g., after anti-CD2 treatment), naïve T cells recover more rapidly than memory subsets

This differential expression pattern makes CD2 valuable for distinguishing between T cell subpopulations and monitoring changes in T cell activation states.

How does CD2 expression change during T cell activation and what are the functional implications?

CD2 expression is dynamically regulated during T cell activation with important functional consequences:

Expression changes during activation:

• Upregulation: T cell activation induces increased CD2 expression at the cell surface

• Mobility changes: Activation enhances lateral mobility of CD2 molecules on the membrane

• Clustering: Upon engagement with CD58, CD2 molecules cluster at sites of cell-cell contact

• These changes facilitate more efficient interactions with CD58 on antigen-presenting cells, strengthening the immunological synapse

Functional implications:

• Enhanced adhesion: Increased CD2 expression improves T cell-APC interactions

• Signal amplification: CD2 clustering contributes to intracellular signaling cascades

• Positive feedback: The CD2-CD58 interaction strengthens as activation proceeds, creating a reinforcing mechanism

• Subset-specific effects: Memory T cells with higher CD2 expression show enhanced sensitivity to CD2-mediated signals

Experimental data has shown that upon anti-CD2 treatment:

• CD4+ and CD8+ memory subsets are substantially depleted

• Naïve T-cells and Tregs are relatively spared

• This pattern correlates with CD2 expression levels across these populations

Understanding these dynamic changes enables researchers to:

• Track T cell activation states

• Distinguish between naïve and memory populations

• Design targeted immunomodulatory strategies

How can CD2 FITC antibodies be used in combination with other markers for comprehensive immune cell phenotyping?

CD2 FITC antibodies can be strategically combined with other markers to create comprehensive immunophenotyping panels for detailed characterization of immune cell subsets.

T cell subset identification panel:

Methodological considerations:

• Panel design:

  • Consider spectral overlap between fluorochromes

  • Place CD2 FITC in a channel that minimizes compensation issues with PE or other green fluorochromes

  • Include markers that allow clear discrimination between populations

• Sample preparation protocol:

  • Follow consistent staining procedures

  • For intracellular markers like FOXP3, use appropriate fixation/permeabilization kits

  • Ensure sufficient washing steps to reduce background

• Control strategy:

  • Include FMO controls for markers with continuous expression patterns

  • Use known positive and negative populations as internal controls

  • Include appropriate isotype controls when evaluating new markers

By combining CD2 FITC with these marker panels, researchers can achieve detailed characterization of immune cell subsets in various experimental and clinical settings.

What technical considerations are essential when incorporating CD2 FITC antibodies into multicolor flow cytometry panels?

When incorporating CD2 FITC antibodies into multicolor flow cytometry panels, several technical factors must be considered:

Spectral characteristics and compensation:

• FITC excitation maximum: 495-498 nm (optimally excited by 488 nm blue laser)

• FITC emission maximum: 519-524 nm

• Primary spillover concerns: PE, BB515, Alexa Fluor 488

• Compensation strategy: Include single-stained controls for each fluorochrome

Brightness considerations:

• FITC has moderate brightness compared to other fluorochromes

• CD2 is typically expressed at sufficient levels for detection with FITC

• For dim antigens, consider brighter fluorochromes like PE or APC

Panel design recommendations:

• Marker-fluorochrome pairing strategy:

  • Pair high-expression markers with dim fluorochromes

  • Pair low-expression markers with bright fluorochromes

  • CD2 is generally well-expressed on T cells, making FITC appropriate

• Clone selection considerations:

  • Ensure the CD2 clone (e.g., RPA-2.10, B-E2) doesn't have known issues with your application

  • Be aware that some clones may compete for overlapping epitopes

  • Note that anti-CD2 BV786 (RPA-2-10) and RH-CD2 show partial competitive binding to the CD2 epitope

• Titration importance:

  • Always titrate antibodies individually before combining in a panel

  • Optimal concentrations may differ when antibodies are combined due to fluorochrome interactions

• Buffer formulation:

  • Use buffers with protein (0.5-2% BSA/FBS) to reduce non-specific binding

  • Consider adding Fc receptor blocking reagents

  • Maintain consistent buffer composition across experiments

How does CD2 blockade affect T cell function in experimental models?

CD2 blockade through anti-CD2 monoclonal antibodies produces distinct immunomodulatory effects in experimental models:

Cellular depletion patterns:

• Preferential depletion of CD4+ and CD8+ memory T cell subsets

• Naïve T cells and regulatory T cells (Tregs) are relatively spared

• These effects correlate with differential CD2 expression levels across T cell subsets

Mechanism of action:

• Direct blockade of CD2-CD58 interactions, disrupting T cell adhesion and costimulation

• Complement-dependent cytolysis (CDC) contributes to cell depletion in some models

• Blockade of CD2-mediated signaling affects T cell activation pathways

Functional effects in experimental systems:

• Concentration-dependent inhibition of mixed lymphocyte reactions (MLRs)

• Reduction in T cell proliferative responses

• Secondary lymphoid organ examination reveals differences between peripheral blood and tissue effects:

  • Lymph node examination shows less significant lymphocyte depletion compared to peripheral blood

  • This suggests compartmental differences in antibody distribution or effect

Reconstitution kinetics:

• Early immune reconstitution is observed for naïve T cells

• Memory T cell counts remain depressed for extended periods (>1 week)

• This differential recovery pattern provides a window for selective immunomodulation

Experimental models demonstrating clinical relevance:

• Reversal of kidney allograft acute rejections

• Prevention of steroid and antithymocyte globulin (ATG) resistant rejections

• Reduced acute rejection rates compared to standard-of-care when used as induction therapy

What are the implications of CD2-CD58 interactions in anti-tumor immune responses?

The CD2-CD58 interaction pathway plays an important role in anti-tumor immunity, with significant implications for understanding tumor immune evasion and developing novel immunotherapies:

Role in normal T cell-tumor cell interactions:

• CD2 on T cells interacts with CD58 on target cells, including potential tumor cells

• This interaction strengthens adhesion between cytotoxic T cells and their targets

• The interaction also provides costimulatory signals that enhance T cell activation

Tumor immune evasion mechanisms:

• Reduced CD58 signaling is associated with immune escape of tumor cells in various hematological and lymphoid malignancies

• Restoration of the CD58 signal promotes an anti-tumor response

• CD58 alterations are now recognized as immune checkpoint-like mechanisms

Therapeutic implications:

• CD2-CD58 pathway as a target for immunotherapy development:

  • CD2 agonism approaches to enhance T cell activation

  • CD58 restoration strategies to directly reverse immune evasion

  • Bispecific engagers to redirect T cells via CD2

  • Combination therapies to enhance other checkpoint inhibitors

• Biomarker potential:

  • CD58 expression/mutation status as predictor of response to immunotherapy

  • CD2 expression patterns on tumor-infiltrating lymphocytes as prognostic indicator

Research shows that following cytomegalovirus (CMV) infection, the CD2-CD58 interaction may have additional roles in antiviral immune responses, suggesting broader implications for understanding anti-pathogen immunity .