CD8 Antibody

What is the biological function of CD8 in T cell immunity?

CD8 functions as an integral membrane glycoprotein that plays essential roles in immune responses. In T cells, it primarily serves as a coreceptor for MHC class I molecule:peptide complexes. CD8 interacts simultaneously with the T-cell receptor (TCR) and MHC class I proteins presented by antigen-presenting cells (APCs), recruiting the Src kinase LCK to the vicinity of the TCR-CD3 complex. This initiates different intracellular signaling pathways that lead to lymphokine production, motility, adhesion, and activation of cytotoxic T-lymphocytes (CTLs) . This mechanism enables CTLs to recognize and eliminate infected cells and tumor cells.

In natural killer (NK) cells, CD8A homodimers at the cell surface provide a survival mechanism allowing conjugation and lysis of multiple target cells. CD8A homodimer molecules also promote the survival and differentiation of activated lymphocytes into memory CD8+ T cells .

Which anti-CD8 antibody clones are commonly used in research?

Several antibody clones have been characterized for research applications:

Anti-human CD8α antibodies:

• OKT8 (unconjugated or allophycocyanin-conjugated)

• SK1 (unconjugated, FITC-conjugated or PE-conjugated)

• MCD8 (unconjugated)

• 32/M4 (unconjugated)

• C8/144B (unconjugated)

• DK25 (allophycocyanin-conjugated)

Anti-human CD8β antibody:

• 2ST8.5H7 (unconjugated or PE-conjugated)

Anti-mouse CD8 antibodies:

• CT-CD8a (anti-CD8α)

• 53.6.7 (anti-CD8α)

• KT112 (anti-CD8β)

• CT-CD8b (anti-CD8β)

The selection of the appropriate antibody depends on the specific research application and experimental design.

How are CD8 antibodies used in tissue immunohistochemistry studies?

CD8 antibodies are extensively used in immunohistochemistry (IHC) to identify and quantify CD8+ T cells in tissue sections. The methodology typically involves:

• Tissue preparation: Fixation in formalin and embedding in paraffin

• Sectioning: Cutting thin (4-5μm) tissue sections

• Antigen retrieval: Often using Tris/EDTA pH 9.0 buffer

• Primary antibody incubation: Using anti-CD8 antibodies at optimized dilutions (e.g., 1/50 to 1/100)

• Detection system: Employing secondary antibodies and chromogenic or fluorescent detection

• Counterstaining and mounting

For example, paraffin-embedded human tonsil tissue can be effectively stained for CD8+ T cells using antibody clone C8/144B at 1/100 dilution . This approach allows researchers to analyze the distribution and density of CD8+ T cells in various tissues, including tumor microenvironments, which has significant implications for understanding immune responses in cancer and infectious diseases.

How do different anti-CD8 antibodies affect T cell activation and function?

Research has revealed significant heterogeneity in the functional consequences of CD8 binding by different antibody clones. In a comprehensive study examining seven monoclonal anti-human CD8 antibodies on six human CD8+ T cell clones with five different specificities, six of the seven antibodies tested did not activate CD8+ T cells. In contrast, one antibody (OKT8) induced effector function in all CD8+ T cells examined .

Similarly, the anti-mouse CD8 antibodies CT-CD8a and CT-CD8b activated CD8+ T cells despite having opposing effects on pMHCI tetramer staining . This observed heterogeneity provides an explanation for the apparent incongruities observed in previous studies and should be considered when interpreting results generated with these reagents.

The ability of antibody-mediated CD8 engagement to deliver an activation signal underscores the importance of CD8 in CD8+ T cell signaling. Early studies showed that preincubation with anti-CD8 antibodies can block conjugate formation between effector and target cells and inhibit CD8+ T-cell activation in response to cognate pMHCI presented on the target cell surface, providing key evidence for CD8's importance in T-cell activation .

How can anti-CD8 antibodies enhance peptide-MHC tetramer staining?

Some anti-CD8 antibodies, particularly OKT8, have been found to enhance TCR/pMHCI on-rates and, consequently, improve pMHCI tetramer staining and visualization of antigen-specific CD8+ T cells . This property makes these antibodies valuable tools for detecting low-frequency or low-avidity antigen-specific T cells.

The methodology for enhancing tetramer staining involves:

• Preincubating T cells with anti-CD8 antibody (e.g., OKT8) for approximately 25 minutes on ice

• Staining with cognate PE-conjugated tetramer (25 μg/ml) at 37°C for 15 minutes

• Performing subsequent staining with viability dyes and additional markers

What is the relationship between CD8+ T cell avidity and differentiation during infection?

Recent research has revealed that direct sensing of interferon-γ (IFN-γ) by CD8+ T cells coordinates avidity and differentiation during infection. IFN-γ promotes the expansion of low-avidity T cells, allowing them to overcome the selective advantage of high-avidity T cells, while reinforcing high-avidity T cell entry into the memory pool .

This dual effect reduces the average avidity of the primary response and increases that of the memory response. IFN-γ in this context is mainly provided by virtual memory T cells, an antigen-inexperienced subset with memory features .

Several studies have demonstrated the contradictory role of IFN-γ on CD8+ T cell proliferation, differentiation, and effector functions . This complexity highlights the importance of understanding cytokine networks in shaping CD8+ T cell responses and has implications for vaccine design and immunotherapeutic approaches.

What experimental protocols are used to study anti-CD8 antibody effects on T cell function?

Based on published methodologies, several experimental approaches are commonly employed:

T cell activation assays:

• Mix 5×10^4 T cells with anti-CD8 antibodies at appropriate concentrations

• Include conditions with and without secondary crosslinking (using 5 μl anti-mouse IgG antibody)

• Incubate overnight at 37°C in a 5% CO₂ atmosphere

• Include appropriate positive controls:

  • Target cells pulsed with 10^-7 M cognate peptide

  • 10 μg/ml anti-human CD3 antibody (UCHT1)

  • 50 ng/ml PMA and 1 μg/ml ionomycin

Antibody fragment analysis:
To determine which antibody components mediate observed effects, researchers can generate and test:

• Fab fragments (monovalent antigen-binding fragment)

• F(ab')₂ fragments (bivalent antigen-binding fragments without Fc region)

• Fc' fragments (crystallizable fragment)

These approaches allow detailed investigation of the mechanisms by which anti-CD8 antibodies influence T cell function and can help resolve apparent contradictions in the literature.

How should researchers interpret heterogeneous results when using anti-CD8 antibodies?

When encountering variable or contradictory results with different anti-CD8 antibodies, researchers should consider:

• Antibody clone specificity: Different antibody clones bind to different epitopes on the CD8 molecule, potentially leading to distinct functional outcomes. For example, while most anti-CD8 antibodies tested did not activate T cells, OKT8 induced effector function in all CD8+ T cells examined .

• CD8 dependency of T cells: Considerable heterogeneity exists between different CD8+ T cells in terms of their ability to activate in the presence of anti-CD8 antibodies. This led to the classification of T cells as either CD8-dependent or CD8-independent .

• Experimental conditions: Factors such as antibody concentration, presence of crosslinking agents, temperature, and timing can significantly influence results.

• T cell population heterogeneity: The composition and activation state of the T cell population being studied can affect responses to anti-CD8 antibodies.

To address these issues, researchers should:

• Test multiple antibody clones under standardized conditions

• Include appropriate positive and negative controls

• Consider the specific research question when selecting antibodies

• Clearly report the specific antibody clone, format, and experimental conditions used

How does CD8+ T cell phenotype relate to antigen recognition and TCR engagement?

The relationship between antigen recognition, TCR engagement, and resulting CD8+ T cell phenotype has been studied using advanced technologies like Antigen-TCR Pairing and Multiomic Analysis of T-cells (APMAT). This integrated experimental-computational framework allows for high-throughput capture and analysis of CD8+ T cells with paired antigen, TCR sequence, and single-cell transcriptome data .

A recent study utilizing APMAT to analyze CD8+ T cells from HLA A*02:01 COVID-19 participants revealed that distinct physicochemical features of antigen-TCR pairs strongly associate with both T cell phenotype and T cell persistence. This suggests that CD8+ T cell phenotype following antigen stimulation is at least partially deterministic, rather than solely the result of stochastic biological processes .

These findings have important implications for understanding how antigen properties and TCR sequences influence the functional diversity and efficacy of CD8+ T cell responses, with potential applications in vaccine design and immunotherapy development.

How do CD8+ T cells contribute to SARS-CoV-2 immunity?

Research on a B cell-depleted lymphoma patient with chronic SARS-CoV-2 infection has provided insights into the role of CD8+ T cells in viral control and mutation. In this patient with defective antibody responses, researchers observed a potential association between SARS-CoV-2 mutations and CD8+ T cell alterations, suggesting possible contributions of CD8+ T cells in the evasion of SARS-CoV-2 from host immunity .

Analysis of this patient's immune response showed:

• T cells were predominantly CD8-positive (60%)

• Many CD8+ T cells (31%) co-expressed activation markers CD38 and HLA-DR

• CD4+ T cells showed less activation

• Increased peripheral CD8+ T cell numbers were observed immediately before certain viral mutations arose (T325K and T4164I)

• These patterns suggested CD8+ T cell-driven immunoevasion

While it is accepted that neutralizing antibodies protect from infection and provide correlates of protection against severe disease, virus-exposed individuals sometimes develop specific T cells in the absence of a detectable antibody response. The role of CD8+ T cells is further supported by:

• Rhesus macaque models where CD8+ T cell depletion abrogated protective immunity against SARS-CoV-2 rechallenge when antibody levels waned

• Mouse models where T cell responses elicited by vaccination were protective against severe disease

• Human studies showing CD8+ T cell responses to immunodominant nucleoprotein epitopes associated with less severe disease

These findings suggest that potential impacts of CD8+ T cells on SARS-CoV-2 mutations, particularly in those with humoral immunodeficiency, warrant further investigation to inform vaccine design strategies.

What methodologies are used to analyze tumor-infiltrating CD8+ T lymphocytes?

Immunohistochemical analysis of tumor-infiltrating lymphocytes (TILs) using CD8 antibodies is a widely employed approach in cancer research. The methodology typically involves:

• Tissue preparation: Formalin-fixed, paraffin-embedded tumor tissue sections

• Immunohistochemical staining: Using validated anti-CD8 antibodies (e.g., clone C8/144B)

• Quantification: Counting CD8+ cells, often in multiple high-power fields

• Comparative analysis: Often performed alongside other T cell markers like CD4

Example of quantitative analysis parameters:

• Number of CD8+ cells per high-power field or per mm²

• Distribution pattern (peritumoral vs. intratumoral)

• Proximity to tumor cells or other stromal elements

In one study, statistical analysis of CD8+ TILs showed that the number of CD8+ lymphocytes tended to be higher in one patient group compared to another, though the difference approached but did not reach statistical significance (p = 0.052) . Such analyses provide valuable insights into the tumor immune microenvironment and may have prognostic or predictive value in cancer treatment.

What factors should be considered when selecting CD8 antibodies for specific applications?

When selecting CD8 antibodies for research applications, consider these critical factors:

Application-specific considerations:

• Technique compatibility: Ensure the antibody is validated for your specific application (flow cytometry, IHC, Western blot, functional assays)

• Species reactivity: Verify compatibility with the target species (human, mouse, etc.)

• Subunit specificity: Determine whether an antibody targeting CD8α or CD8β is more appropriate (CD8 exists as either αα homodimer or αβ heterodimer)

Functional considerations:

• Effect on T cell activation: Some antibodies (e.g., OKT8) can trigger T cell effector function while others cannot

• Impact on tetramer binding: Antibodies can enhance (e.g., OKT8) or inhibit pMHCI tetramer binding

• Epitope accessibility: Consider whether the epitope remains accessible in fixed/processed samples

Technical considerations:

• Conjugation: For flow cytometry, select appropriate fluorophore conjugation

• Format: Whole IgG vs. fragments (Fab, F(ab')₂) for specific applications

• Concentration optimization: Perform titration experiments to determine optimal working concentration

By carefully evaluating these factors, researchers can select the most appropriate CD8 antibody for their specific experimental needs, leading to more reliable and interpretable results.

How can researchers optimize CD8 antibody staining protocols for different tissue types?

Optimizing CD8 antibody staining protocols for different tissue types requires systematic adjustment of several parameters:

Antigen retrieval optimization:
Different tissues may require specific antigen retrieval methods:

• Heat-induced epitope retrieval using Tris/EDTA pH 9.0 buffer works well for spleen tissue

• Citrate buffer (pH 6.0) may be more suitable for other tissues

• Enzymatic retrieval may be necessary for heavily fixed samples

Antibody dilution optimization:

• Start with manufacturer's recommended dilution

• Perform titration series (e.g., 1/25, 1/50, 1/100, 1/200)

• Optimal dilution balances strong specific staining with minimal background

• For example, ab17147 has been successfully used at 1/100 for tonsil tissue and 1/50 for spleen tissue

Incubation conditions:

• Temperature (4°C, room temperature, 37°C)

• Duration (1 hour to overnight)

• Humidity control to prevent section drying

Detection system selection:

• Polymer-based systems often provide superior sensitivity for IHC

• Fluorescent detection may be preferred for multiplex staining

Positive and negative controls:

• Include known CD8+ tissues (tonsil, spleen, lymph node) as positive controls

• Use isotype controls and/or CD8-negative tissues as negative controls

Systematic optimization of these parameters will yield consistent, specific staining across different tissue types.

What are common pitfalls in CD8 antibody experiments and how can they be avoided?

Common experimental pitfalls and their solutions include:

1. False negative results:

• Problem: Inadequate antigen retrieval, particularly in formalin-fixed tissues

• Solution: Optimize antigen retrieval method (buffer, pH, duration, temperature)

• Problem: Antibody concentration too low

• Solution: Perform antibody titration; consider more sensitive detection systems

2. False positive/high background:

• Problem: Non-specific binding, particularly in tissues with high endogenous peroxidase

• Solution: Thorough blocking steps; quench endogenous peroxidase with H₂O₂

• Problem: Antibody concentration too high

• Solution: Optimize dilution; include appropriate washing steps

3. Misinterpretation of functional assays:

• Problem: Different anti-CD8 antibodies have varying effects on T cell activation

• Solution: Include multiple antibody clones; use OKT8 as positive control for activation

• Problem: Heterogeneity in CD8-dependence among T cell populations

• Solution: Characterize T cell populations for CD8-dependence before functional studies

4. Inadequate controls:

• Problem: Lack of appropriate positive and negative controls

• Solution: Include isotype controls, FMO controls (flow cytometry), and biological controls

5. Batch effects:

• Problem: Variability between experimental batches affecting reproducibility

• Solution: Process all comparative samples simultaneously; include internal reference standards

By anticipating these common issues and implementing appropriate controls and optimization steps, researchers can avoid pitfalls and generate more reliable data when working with CD8 antibodies.