CD1A Monoclonal Antibody

What is CD1A and why is it an important research target?

CD1A is a 49 kDa sialoglycoprotein and member of the CD1 family of non-classical MHC class I-like proteins. It is non-covalently associated with β2-microglobulin, which appears necessary for efficient folding and surface expression . CD1A functions as an antigen-presenting protein that binds self and non-self lipid and glycolipid antigens and presents them to T-cell receptors on natural killer T-cells . Its importance as a research target stems from its restricted expression pattern on specific cell types (cortical thymocytes, Langerhans cells, dendritic cells) and certain malignancies, making it valuable for both diagnostic and therapeutic applications .

Which cell types typically express CD1A?

CD1A is predominantly expressed on:

• Cortical thymocytes (strongly expressed)

• Langerhans cells in the epidermis

• Dendritic cells including interdigitating cells

• Certain T-cell leukemias and lymphomas

• Occasionally in some subtypes of acute myeloid leukemia (FAB subtypes M4 and M5)

CD1A is notably absent on peripheral blood T and B lymphocytes, monocytes, granulocytes, platelets, and erythrocytes . Freshly isolated bone marrow and blood dendritic cells are CD1A negative, as are most antigen-presenting cells resident in lymphoid organs .

What are the primary applications for CD1A monoclonal antibodies in research?

CD1A monoclonal antibodies are widely used in:

• Flow cytometry for identification and enumeration of CD1A-positive cells

• Immunohistochemistry (both frozen and paraffin-embedded tissues)

• Western blotting for protein detection

• Diagnostic imaging of CD1A-positive tumors

• Evaluating dendritic cell maturation and function

• Studying T-cell development in the thymus

• Identification of Langerhans cells in tissue samples

How should CD1A antibodies be selected for specific applications?

Selection criteria should be based on:

For optimal selection, researchers should consider:

• The epitope recognized (CD1A has four different epitopes designated as groups A, B, C, and D)

• Whether internalization of the antibody-CD1A complex is desirable (occurs at 37°C)

• The isotype's compatibility with secondary detection systems

What tissue preparation methods are optimal for CD1A immunohistochemistry?

For optimal CD1A detection in tissues:

Frozen Sections:

• Fix briefly in acetone or 4% paraformaldehyde

• Avoid over-fixation which can mask epitopes

• Block endogenous peroxidase activity if using HRP detection systems

• Include appropriate blocking step to minimize non-specific binding

Paraffin-Embedded Sections:

• Heat-induced epitope retrieval is essential, preferably using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

• For clone O10, use at 1:100-1:200 dilution after heat-induced epitope retrieval

• For clone L21-A, recommended dilution is 1:100-1:200 for FFPE sections

• DAB (3,3'-diaminobenzidine) is commonly used as chromogen, with hematoxylin counterstaining

Positive controls should include human skin (Langerhans cells) or thymus (cortical thymocytes) .

How can CD1A monoclonal antibodies be applied in cancer research and immunotherapy?

CD1A monoclonal antibodies have significant potential in cancer research and immunotherapy:

Diagnostic Applications:

• Identification and classification of T-cell leukemias, particularly the cortical subtype of T-ALL

• Diagnosis of Langerhans Cell Histiocytosis (LCH), where CD1A expression is a defining characteristic

• Monitoring disease progression and treatment response

Therapeutic Applications:

• Development of fully human anti-CD1A antibodies like CR2113 that demonstrate:

  • Potent antibody-dependent cell cytotoxicity (ADCC)

  • Moderate complement-dependent cytotoxicity (CDC)

  • Specific anti-tumor activity against CD1A-expressing malignancies

• Potential for antibody-drug conjugates targeting CD1A-positive tumors

• Chimeric antigen receptor (CAR) T-cell therapy targeting CD1A

Research indicates that human anti-CD1A mAbs like CR2113 have advantages over murine antibodies (like NA1/34), including higher binding affinity and reduced immunogenicity. Surface plasmon resonance analysis showed that CR2113 has superior binding kinetics compared to murine antibodies .

What challenges exist in studying CD1A+ dendritic cells in lymph nodes and tumor microenvironments?

Researchers face several challenges when studying CD1A+ dendritic cells:

Technical Challenges:

• Maintaining dendritic cell viability during tissue processing

• Low frequency of CD1A+ cells in many tissues requiring enrichment techniques

• Variable CD1A expression depending on dendritic cell maturation state

• Manual evaluation of dendritic cells involves problems of interobserver variation

Biological Complexities:

• CD1A expression is dynamic and dependent on the functional state of dendritic cells

• CD1A expression is high when cells are capturing antigen and down-regulated during antigen presentation

• The significance of CD1A+ dendritic cell infiltration differs according to tumor histology and primary site

• Strong confounding between CD1A+ dendritic cell infiltration and lymph node metastasis has been observed in some cancers

How do different CD1A antibody clones compare in terms of epitope recognition and binding kinetics?

Different CD1A antibody clones recognize distinct epitopes with varying binding characteristics:

Surface plasmon resonance analysis revealed that CR2113 has superior binding kinetics compared to the murine NA1/34 antibody. When bound to CD1A, these antibodies can be internalized at 37°C, which is an important consideration for antibody-drug conjugate development .

What controls are necessary when using CD1A antibodies in flow cytometry and immunohistochemistry?

Proper controls are essential for reliable CD1A antibody experiments:

Flow Cytometry Controls:

• Positive Control: MOLT-4 human acute lymphoblastic leukemia cell line (known to express CD1A)

• Negative Controls:

  • Isotype control matched to the CD1A antibody (e.g., mouse IgG1 for HI149 clone)

  • Known CD1A-negative cell lines or peripheral blood lymphocytes

• Fluorescence Minus One (FMO) controls when using multiple fluorochromes

• Titration of antibody concentration to determine optimal signal-to-noise ratio

Immunohistochemistry Controls:

• Positive Tissue Controls:

  • Human skin (Langerhans cells)

  • Thymus (cortical thymocytes)

• Negative Tissue Controls:

  • Lymph node (except for dendritic cells)

  • Peripheral blood cells

• Technical Controls:

  • Isotype control antibody

  • No primary antibody control

  • Antigen blocking/peptide competition controls for specificity verification

How can researchers troubleshoot weak or absent CD1A staining in immunohistochemistry?

When encountering weak or absent CD1A staining, consider these troubleshooting approaches:

For paraffin sections, heat-induced epitope retrieval is critical for CD1A detection. For clone L21-A, recommended dilution is 1:100-1:200 for FFPE sections with appropriate antigen retrieval . If assessment of CD1A-positive cells remains difficult, consider counterstaining with nuclear dyes to better visualize cell morphology .

What factors affect CD1A expression levels in dendritic cells, and how should these be considered in experimental design?

CD1A expression in dendritic cells is influenced by multiple factors that researchers should consider:

Developmental Factors:

• CD1A is absent on freshly isolated bone marrow and blood DC precursors

• Expression increases during dendritic cell differentiation from monocytes

• Expression levels vary between different dendritic cell subpopulations

Functional State:

• CD1A expression is high when cells are capturing antigen

• Expression is down-regulated during antigen presentation

• Maturation state affects expression levels

Experimental Manipulations:

• Culture conditions affect CD1A expression in in vitro-generated dendritic cells

• Cytokine treatments (IL-4, GM-CSF) influence expression levels

• Activation signals can modulate CD1A levels

Tissue-Specific Factors:

• Microenvironmental cues affect CD1A expression

• Expression patterns differ between different anatomical locations

• Disease states can alter expression levels

When designing experiments involving CD1A, researchers should:

• Standardize dendritic cell isolation and culture protocols

• Consider the maturation/activation state of dendritic cells

• Include appropriate time points to account for dynamic expression

• Use additional markers in conjunction with CD1A to better define cell populations

• Be aware that freezing and thawing cycles may affect CD1A detection

How should researchers interpret CD1A expression in the context of cancer prognosis and immune response?

Interpreting CD1A expression in cancer contexts requires nuanced analysis:

Prognostic Considerations:

• High CD1A+ dendritic cell infiltration in primary tumors has been associated with both favorable and unfavorable outcomes depending on cancer type

• In gallbladder cancer, CD1A+ dendritic cell infiltration into regional lymph nodes correlated with unfavorable clinical outcomes

• All cases with high CD1A+ dendritic cell infiltration in one study had nodal metastasis

• CD1A expression in tumor cells (as in some T-ALL) generally indicates a specific disease subtype and may influence treatment choices

Immune Response Interpretation:

• CD1A+ dendritic cells can indicate an ongoing immune response against tumors

• Their functionality rather than mere presence may be more important (mature vs. immature)

• Context matters: the same CD1A+ cell density may have different implications depending on:

  • Tumor type

  • Tumor stage

  • Location (tumor center vs. invasive margin)

  • Co-infiltrating immune cells

When analyzing CD1A expression data, researchers should:

• Quantify CD1A+ cells using standardized methods to enable cross-study comparisons

• Assess their distribution pattern (clustered vs. dispersed)

• Evaluate their morphology (mature dendritic vs. immature)

• Consider co-expression of other markers indicating maturation status

• Correlate with clinical parameters and outcomes

What are the considerations for developing CD1A-targeted therapeutic antibodies?

Development of CD1A-targeted therapeutic antibodies requires attention to several critical factors:

Target Validation:

• Confirm consistent CD1A expression in the target disease

• Evaluate CD1A density on target cells versus normal tissues

• Assess internalization capacity (critical for antibody-drug conjugates)

• Consider epitope accessibility in the disease context

Antibody Engineering:

• Human or humanized antibodies preferred over murine to reduce immunogenicity

• Isotype selection impacts effector functions:

  • IgG1 isotype typically provides strongest ADCC and CDC

  • IgG4 offers reduced effector functions if targeting normal CD1A+ cells

• Fc engineering can enhance ADCC and CDC activities

• Binding affinity optimization ensures optimal target engagement

Effector Function Selection:

• CR2113 demonstrates both ADCC and CDC capabilities

• ADCC appears more potent than CDC for anti-CD1A antibodies

• Direct apoptosis induction may be desirable for certain applications

• For diagnostic applications, minimizing effector functions may be preferred

Research with CR2113 showed modest but specific anti-tumor activity against CD1A-expressing tumors as a naked antibody, suggesting potential for enhancement through antibody-drug conjugates or other modifications .

How do CD1A antibody-based assays compare with other methods for identifying and characterizing Langerhans cells and dendritic cells?

CD1A antibody detection offers specific advantages and limitations compared to other methods:

For comprehensive characterization of dendritic cells, a multi-marker approach is recommended:

• Use CD1A as a primary marker for immature Langerhans cells and certain DC subsets

• Include maturation markers (CD83, CD86) to determine activation state

• Add lineage-specific markers for DC subset identification

• Consider functional assays to assess antigen presentation capacity

One study noted difficulty in assessing CD83-positive and CD86-positive cells because their intensity revealed by IHC was faint and dendritic shapes were unclear, highlighting the advantage of CD1A as a more robust marker for certain applications .

How is our understanding of CD1A's role in lipid antigen presentation influencing antibody development?

Recent advances in understanding CD1A's function are shaping antibody development:

CD1A functions as an antigen-presenting protein that binds self and non-self lipid and glycolipid antigens and presents them to T-cell receptors on natural killer T-cells . This specialized role in lipid antigen presentation offers unique opportunities for targeted therapies and diagnostic approaches.

Emerging Research Areas:

• Development of antibodies that modulate CD1A-restricted T cell responses

• Antibodies that can block or enhance specific lipid antigen presentation

• Targeting CD1A-lipid-T cell receptor complexes rather than CD1A alone

• Using CD1A antibodies to deliver lipid antigens to dendritic cells

These advances may lead to:

• Novel immunomodulatory therapies that specifically affect lipid antigen presentation

• Better understanding of autoimmune conditions involving lipid antigens

• More precise targeting of specific dendritic cell functions

• Therapeutic approaches that leverage the unique properties of CD1A-restricted T cells

What new technologies are enhancing the development and application of CD1A antibodies?

Technological innovations are advancing CD1A antibody research:

Antibody Engineering Technologies:

• Phage display libraries have successfully generated fully human anti-CD1A mAbs like CR2113

• Semi-synthetic phage display approaches allow selection based on specificity and avidity

• Surface plasmon resonance analysis provides precise binding kinetics measurements

• Confocal microscopy techniques reveal internalization dynamics of antibody-CD1A complexes

Advanced Imaging Applications:

• Multiparameter flow cytometry enables precise phenotyping of CD1A+ cells

• Multiplex immunohistochemistry allows simultaneous detection of CD1A with other markers

• Imaging mass cytometry provides spatial context for CD1A expression

• In vivo imaging using labeled CD1A antibodies offers diagnostic potential

Therapeutic Innovations:

• Development of antibody-drug conjugates targeting CD1A

• Bispecific antibodies engaging CD1A and effector cells

• CAR-T cells recognizing CD1A epitopes

• Nanoparticle delivery systems using CD1A antibodies for targeting

These technological advances are enabling more precise detection, quantification, and targeting of CD1A-expressing cells in both research and clinical applications.

What is the optimal protocol for flow cytometric analysis of CD1A expression?

Recommended Protocol for CD1A Flow Cytometry:

Materials:

• Anti-CD1A monoclonal antibody (recommended clones: HI149, NA1/34)

• Appropriate isotype control

• Cell suspension buffer (PBS with 2% FBS and 0.1% sodium azide)

• Fixation buffer (optional)

Procedure:

• Cell Preparation:

  • Collect cells (1-5 × 10^6 cells per sample)

  • Wash cells twice with cell suspension buffer

  • Resuspend at 1 × 10^7 cells/mL

• Antibody Staining:

  • Aliquot 100 μL cell suspension (1 × 10^6 cells) to flow tubes

  • Add ≤1 μg of CD1A antibody (or amount determined by titration)

  • Include separate tubes for isotype control and unstained control

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

• Washing:

  • Add 2 mL cell suspension buffer

  • Centrifuge at 350 × g for 5 minutes

  • Discard supernatant and resuspend in 0.5 mL buffer

• Analysis:

  • Analyze on flow cytometer with appropriate laser/filter settings

  • Collect at least 10,000 events

  • Gate based on forward/side scatter to exclude debris

  • Compare with isotype control to determine positive population

Tips for Optimization:

• Titrate antibody to determine optimal concentration

• For intracellular staining, include permeabilization step

• When multicolor panels are used, include FMO controls

• MOLT-4 cells serve as reliable positive control

• Peripheral blood lymphocytes can serve as negative control

What are the critical steps for successful CD1A immunohistochemistry in paraffin-embedded tissues?

Critical Protocol for CD1A Immunohistochemistry on FFPE Tissues:

Materials:

• Anti-CD1A monoclonal antibody (recommended clones: O10, L21-A)

• Antigen retrieval buffer (citrate buffer pH 6.0 or EDTA buffer pH 9.0)

• Detection system (e.g., polymer-HRP)

• DAB chromogen

• Hematoxylin counterstain

Procedure:

• Section Preparation:

  • Cut 4-5 μm sections from FFPE blocks

  • Mount on positively charged slides

  • Deparaffinize and rehydrate through xylene and graded alcohols

• Antigen Retrieval (Critical Step):

  • Heat-induced epitope retrieval using citrate buffer (pH 6.0)

  • Pressure cooker or microwave method (20 minutes)

  • Allow slides to cool in buffer for 20 minutes

• Blocking Steps:

  • Block endogenous peroxidase with 3% H₂O₂ for 10 minutes

  • Rinse in wash buffer

  • Apply protein block for 10 minutes to reduce background

• Antibody Incubation:

  • Apply primary CD1A antibody (dilution 1:100-1:200)

  • Incubate for 1 hour at room temperature or overnight at 4°C

  • Wash thoroughly (3 × 5 minutes)

• Detection and Visualization:

  • Apply appropriate secondary detection system

  • Develop with DAB for 5-10 minutes (monitor microscopically)

  • Counterstain with hematoxylin

  • Dehydrate, clear, and coverslip

Critical Considerations:

• Antigen retrieval is the most crucial step for successful CD1A detection

• Optimal antibody dilution should be determined for each new lot

• Include positive control tissue (skin or thymus) on each slide

• CD1A positive cells should show membrane and cytoplasmic staining

• Langerhans cells should display characteristic dendritic morphology