Recombinant Human T-lymphocyte activation antigen CD86 (CD86), partial (Active)

What is the molecular structure of CD86 and how does it affect experimental design?

CD86 is a glycosylated protein of approximately 70 kDa, composed of 329 amino acids (about 37 kDa) with a single transmembrane domain and a cytoplasmic domain . The protein exists in multiple isoforms with varying molecular weights:

Human CD86 Isoforms (P42081):

• Isoforms 1, 2 (P42081-1, 3): 37.0~37.6 kDa

• Isoform 3 (P42081-2): 12.8 kDa

• Isoform 4 (P42081-4): 31.2 kDa

• Isoforms 5, 6 (P42081-5, 6): 24.7~28.4 kDa

When designing experiments with recombinant CD86, researchers should account for the partial active form (typically amino acids 24-247) which has a theoretical molecular weight of 26.69 kDa but appears at 40-57 kDa on SDS-PAGE due to glycosylation . For optimal experimental outcomes, reconstitute lyophilized protein in deionized sterile water (0.1-1.0 mg/mL) and add 5-50% glycerol for long-term storage at -20°C .

How does CD86 function in T cell activation pathways?

CD86 serves as a ligand for two proteins on T cells: CD28 and CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) . This dual-binding capability creates a balanced immune response through distinct signaling pathways:

• CD28 Pathway (Activating):

  • Provides essential costimulatory signals for T cell activation, proliferation, and interleukin-2 production

  • Critical for naive T cell activation within the first 24 hours

  • Transmits positive signals through NF-κB pathway activation

• CTLA-4 Pathway (Inhibitory):

  • Negatively regulates T cell activation

  • Competes with CD28 for binding to CD86 with higher affinity

  • Crucial for immune homeostasis and self-tolerance

The temporal expression of CD86 is significant - it's expressed earlier in immune responses than CD80 (B7-1), making it particularly important in initial T cell activation decisions between immunity and anergy .

What are the differential expression patterns of CD86 across B cell subtypes?

Research demonstrates distinct CD86 expression patterns across B cell populations :

Table: Mean CD86 Expression (MFI) Across B Cell Subtypes

How is CD86 expression regulated during immune cell activation and what methods measure this effectively?

CD86 expression is rapidly upregulated on B cells following activation through various mechanisms :

• Activation triggers:

  • Cross-linking with Ig receptors

  • Cytokine stimulation

  • Innate immune stimulation

• Cell-specific regulation:

  • Monocytes: Low basal expression, upregulated by IFN-γ stimulation

  • Dendritic cells: Constitutively expressed with modulation by maturation signals

  • B cells: Baseline expression varies by subtype with rapid upregulation capacity

For accurate measurement of CD86 expression, researchers should employ multiple complementary techniques:

• Flow cytometry: Gold standard for quantification using anti-CD86 monoclonal antibodies (clones IT2.2 or 2D10.4) at 5-10 μl per 10^5 cells in PBS containing 5% human serum . Analyze using mean fluorescence intensity (MFI) values to quantify expression levels.

• Quantitative RT-PCR: For transcriptional analysis, though the correlation between mRNA and protein expression is frequently low in primary CD4+ T cells .

• Immunocytochemistry: For visualization of cellular localization.

• Western blot: For protein expression in cell lysates.

What is the reciprocal relationship between CD86 and immune checkpoint regulators in B cell function?

Research has revealed a divergent expression pattern between CD86 (activation marker) and BTLA (immune checkpoint regulator) across B cell subtypes :

Reciprocal Expression Pattern:

• Naïve and transitional B cells: Lowest CD86, highest BTLA expression

• Plasmablasts and memory B cells: Higher CD86, lower BTLA expression

• Switched memory cells: Most "activation permissive" with highest CD86 and lowest BTLA among memory subsets

This inverse relationship creates a regulatory balance where:

• Inhibitory BTLA signals maintain naïve B cells in a controlled state until antigen activation

• CD86 upregulation occurs more rapidly than BTLA downregulation during B cell development

• The combined expression pattern determines the activation threshold and functional capabilities of each B cell subtype

These patterns suggest CD86 may enable rapid B cell responses while BTLA provides longer-term regulation, with important implications for understanding immune dysregulation in disease states .

How should researchers design co-stimulation blockade experiments to study CD86 function?

When designing co-stimulation blockade experiments to study CD86 function, researchers should follow these methodological approaches :

• Experimental setup:

  • Use CMBLAST medium containing 1.6% L-glutamine, 3% penicillin/streptomycin, and 5% inactivated AB-type human serum in RPMI 1640

  • Culture PBMCs in polypropylene tubes at 10^7 cells/ml

  • Add anti-CD86 monoclonal antibody (clone IT2.2, 5 μg/ml) for blockade

• Essential controls:

  • Medium-only control (baseline)

  • Stimulation-only control (e.g., with TRYPO 20 μg/ml)

  • Antibody-only control (to assess antibody effects without stimulation)

  • Combined stimulation and blockade experimental condition

• Analysis parameters:

  • Measure cytokine expression (IFN-γ, IL-4, IL-17, IL-10)

  • Calculate relative expression index through ratio of anti-CD86 vs anti-CD80 blockade effects

  • Apply non-parametric statistical methods (Kruskal-Wallis with Dunn's multiple comparisons)

This approach allows for isolation of CD86-specific effects while controlling for experimental variables .

What are the critical technical considerations when using recombinant CD86 proteins in functional assays?

When utilizing recombinant CD86 proteins in functional assays, researchers must address several technical considerations to ensure reliable results :

• Expression system selection:

  • Mammalian cell systems provide proper glycosylation patterns essential for functionality

  • Sf9 Baculovirus cells are commonly used for higher yield but may have different post-translational modifications

  • The expression region typically covers amino acids 24-247, representing the extracellular domain

• Protein handling protocols:

  • Reconstitute lyophilized protein in deionized sterile water (0.1-1.0 mg/mL)

  • For storage stability, add 5-50% glycerol (final concentration)

  • Aliquot to avoid freeze-thaw cycles which compromise activity

  • Centrifuge vials briefly before opening to collect contents

• Validation checkpoints:

  • Verify protein purity (>90-95% by SDS-PAGE)

  • Confirm molecular weight (theoretical MW: 26.69 kDa; observed: 40-57 kDa due to glycosylation)

  • Test biological activity in a standardized assay before experimental use

• Carrier protein considerations:

  • For long-term storage, add carrier protein (0.1% HSA or BSA)

  • In functional assays, account for potential effects of carrier proteins

• Tag interference assessment:

  • Most recombinant CD86 proteins contain C-terminal 6xHis-tags

  • Verify that tags do not interfere with binding properties in your specific assay system

Following these technical guidelines ensures maximum activity and reproducibility in functional studies involving recombinant CD86 .

How does CD86 polymorphism influence susceptibility to immune-mediated diseases?

CD86 genetic variations significantly impact disease susceptibility, particularly the Ile179Val polymorphism (rs2681417) :

Research Evidence:
A family-based association study of two independent Danish samples (135 and 100 trios of children with atopy and their parents) demonstrated that the Ile179Val substitution in exon 5 of CD86 was significantly associated with atopic diseases . Functional analysis revealed altered costimulatory effects on cytokine production in cells expressing the variant.

Disease Associations:

• Allergic diseases:

  • Asthma susceptibility is significantly influenced by the CD86 Ile179Val variant

  • This polymorphism shows linkage to multiple atopy phenotypes including rhinitis and atopic dermatitis

  • The 3q21 locus harboring CD86 shows significant linkage to allergic disorders

• Autoimmune conditions:

  • CD86 expression variations are linked to rheumatoid arthritis activity

  • The balance of switched memory versus non-switched memory B cells (with different CD86 expression levels) differs depending on disease activity

The mechanism involves altered T cell activation thresholds, as the polymorphism affects CD86's ability to provide costimulatory signals, potentially influencing decisions between immunity and tolerance .

What is CD86's role as a biomarker in cancer and how can it be harnessed for immunotherapy?

CD86 has emerged as a significant biomarker in cancer with potential immunotherapeutic applications :

• Prognostic value:

  • In bladder cancer, CD86 serves as an immune-related prognostic biomarker

  • Higher CD86 expression correlates with immune infiltration patterns

  • CD86 expression is associated with improved response to immunotherapy

• Immune microenvironment associations:

  • CD86 expression positively correlates with CD8+ T cells and dendritic cell infiltration

  • Expression patterns correlate with other immune checkpoint molecules (CTLA-4, PDCD1LG2, IDO1, HAVCR2)

  • CD86 expression is negatively correlated with DNA methylation, suggesting epigenetic regulation

• Immunotherapeutic implications:

  • As the ligand for both CD28 and CTLA-4, CD86 represents a pivotal point for immune modulation

  • Anti-CTLA-4 therapies indirectly affect CD86 signaling pathways

  • CD86 expression levels may predict response to checkpoint inhibitor treatments

• Therapeutic targeting strategies:

  • Direct modulation of CD86 expression or function

  • Targeting the CD86-CTLA-4 axis to enhance anti-tumor immunity

  • Combination approaches targeting multiple costimulatory pathways

Researchers identified CD86 as a potential immunotherapy target through a multi-step process: constructing a co-expression network based on immune-related genes, building protein-protein interaction networks, and validating findings through experimental assays .

How does the combined expression pattern of CD86 and BTLA influence B cell activation thresholds?

The interplay between CD86 and BTLA creates a dynamic regulation system that determines B cell activation thresholds :

Subtype-Specific Expression Patterns:

This "activation permissive" versus "controlled" balance determines several critical B cell functions:

• Antigen sensitivity: The more "activation permissive" state of SM cells (high CD86, low BTLA) may lower the threshold for antigen activation, requiring less antigen concentration or stimulation for response.

• Differentiation dynamics: The expression pattern likely influences rapid differentiation of SM cells into plasma cells upon reactivation, working in conjunction with BCR affinity and immunoglobulin tail tyrosine (ITT) motifs.

• Temporal regulation: CD86 shows quicker adjustment compared to BTLA during B cell development and activation, suggesting different kinetics in their regulatory functions.

• Functional plasticity: The differential expression may allow memory B cells to maintain distinct functional capabilities despite sharing memory status .

How do CD86+ regulatory T cells influence immune response dynamics?

The unexpected discovery of CD86 expression on a subset of activated regulatory T cells (Tregs) has revealed new dimensions of immune regulation :

Key Findings:
Using advanced transcriptomic and proteomic hybrid technology, researchers detected CD80 and CD86 (normally associated with antigen-presenting cells) on a subset of activated Tregs . This observation challenges conventional understanding of Treg function and suggests several novel regulatory mechanisms:

• Bi-directional immune modulation:

  • CD86+ Tregs may directly interact with CD28/CTLA-4 on other T cells

  • This creates a T cell-to-T cell regulatory circuit independent of APCs

  • May provide more localized and specific immune suppression

• Functional implications:

  • In patients with active coeliac disease, these CD86+ Tregs could modulate local immune responses

  • The expression may enable Tregs to compete with APCs for interaction with effector T cells

  • Could represent a negative feedback mechanism during intense immune activation

• Methodological significance:

  • The finding highlights the limitations of conventional cell characterization approaches

  • Demonstrates the value of multi-omics approaches for discovering unexpected cell populations

  • Reveals the frequently low correlation between mRNA and protein expression in primary CD4+ T cells

This discovery has significant implications for understanding Treg plasticity and function in various disease contexts, particularly autoimmune conditions .

What are the emerging technologies for studying CD86 dynamics in complex immune environments?

Advanced technologies are revolutionizing our ability to study CD86 dynamics in complex immune environments :

• Single-cell multi-omics approaches:

  • Single-cell RNA sequencing (scRNA-seq) combined with protein detection (AbSeq)

  • Enables simultaneous analysis of transcriptome and surface protein expression

  • Revealed unexpected CD86 expression on activated Tregs

  • Cost-effective solution for dissecting immune cell heterogeneity at high resolution

• Systems biology computational methods:

  • Weighted Gene Co-Expression Network Analysis (WGCNA)

  • Identifies modules of highly correlated genes associated with clinical features

  • Successfully identified CD86 as a key immune-related gene in bladder cancer

  • Enables integration of multiple datasets for comprehensive analysis

• Advanced flow cytometry techniques:

  • Mass cytometry (CyTOF) for simultaneous analysis of dozens of markers

  • Spectral flow cytometry for improved resolution of co-expressed markers

  • High-dimensional analysis approaches (tSNE, UMAP) for visualizing complex relationships

• In vivo imaging technologies:

  • Intravital microscopy for tracking CD86-expressing cells in living tissues

  • CRISPR-based reporters for real-time monitoring of CD86 expression

  • Enables temporal and spatial analysis of CD86 dynamics during immune responses

• Functional genomics approaches:

  • CRISPR-Cas9 screening to identify regulators of CD86 expression

  • Epigenetic profiling to understand transcriptional control mechanisms

  • DNA methylation analysis reveals regulatory mechanisms of CD86 expression

These technologies collectively provide unprecedented insights into CD86 biology, enabling researchers to study its dynamics at cellular, molecular, and systems levels in complex immune environments .

What are the unresolved questions about CD86 expression and function?

Despite significant advances, several critical questions about CD86 remain unresolved and represent important areas for future research:

• Sex-dependent expression differences:
  Research has unexpectedly revealed significantly higher CD86 expression on plasmablasts, switched memory and non-switched memory B cells in men compared to women . The biological significance and mechanisms underlying these sex differences remain poorly understood.

• Temporal dynamics of CD86/BTLA regulation:
  While CD86 appears to have quicker adjustment capabilities compared to BTLA during B cell development, the precise molecular mechanisms orchestrating this differential regulation and its functional consequences need further investigation .

• CD86 expression on unconventional cell types:
  The discovery of CD86 on activated regulatory T cells challenges conventional understanding and raises questions about whether other lymphocyte populations might express CD86 under specific conditions .

• Epigenetic regulation mechanisms:
  CD86 expression negatively correlates with DNA methylation , but the detailed epigenetic regulatory mechanisms controlling CD86 expression across different cell types and activation states remain unclear.

• CD86 polymorphism functional consequences:
  While the Ile179Val polymorphism has been associated with asthma and allergic disorders , the precise molecular mechanisms by which this variant alters CD86 function and influences disease susceptibility need further characterization.

Addressing these questions will provide deeper insights into CD86 biology and potentially reveal new therapeutic opportunities.

How might therapeutic targeting of CD86 evolve in future immunomodulatory approaches?

The future of CD86-targeted therapies holds promise for more precise immunomodulation across multiple disease contexts:

• Beyond CTLA-4-based approaches:

  • Direct CD86 modulators rather than targeting receptor interactions

  • Subtype-selective targeting to affect specific CD86-expressing cells

  • Temporal control systems to modulate CD86 function at specific disease stages

• Precision medicine applications:

  • CD86 polymorphism-based patient stratification for personalized therapy

  • Sex-specific dosing regimens based on differential CD86 expression

  • Combination therapies targeting multiple costimulatory pathways

• Advanced delivery technologies:

  • Cell type-specific delivery of CD86 modulators

  • Tissue-targeted approaches for localized immunomodulation

  • Responsive systems that activate only under specific immune conditions

• Expanded disease applications:

  • Autoimmune conditions beyond current indications

  • Infectious disease interventions targeting CD86-mediated responses

  • Metabolic disorders with immune components

• Monitoring approaches:

  • Real-time assessment of CD86 expression for therapy optimization

  • Biomarker panels including CD86 expression patterns

  • Functional assays measuring CD86-dependent T cell responses