Recombinant Macaca mulatta (Rhesus macaque) CD226 antigen (CD226)

What is CD226 and what is its significance in rhesus macaque immunology?

CD226, also known as DNAM-1 (DNAX accessory molecule-1), PTA-1 (Platelet and T-cell activation antigen 1), or TLisA1 (T lineage-specific activation antigen 1), is a member of the immunoglobulin superfamily initially identified on natural killer (NK) cells and T cells. The molecule contains two V-like domains of immunoglobulin in its membrane structure . In rhesus macaques, CD226 plays crucial roles in immune cell activation, particularly in NK cells and T cells, making it an important target for studying immune responses in this model organism. Its significance lies in its involvement in antigen-specific memory and immune cell function, which has implications for understanding responses to infections and vaccines in primates .

How is recombinant Macaca mulatta CD226 protein typically produced?

Recombinant Macaca mulatta CD226 protein can be produced using prokaryotic expression systems, typically in E. coli. The full-length mature protein (amino acids 19-336) is often expressed with an N-terminal His tag to facilitate purification. The expression construct contains the CD226 gene sequence with the appropriate tag, which is then transformed into E. coli for protein production .

The methodology involves:

• Gene synthesis or cloning of the CD226 sequence (amino acids 19-336)

• Insertion into an appropriate expression vector with a His tag

• Transformation into E. coli expression host

• Induction of protein expression

• Cell lysis and protein extraction

• Purification using affinity chromatography (His-tag binding)

• Lyophilization to produce a powder form

The purified protein typically achieves >90% purity as determined by SDS-PAGE analysis and can be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL .

How does CD226 contribute to antigen-specific NK cell memory in rhesus macaques?

CD226 plays a critical role in the development and function of antigen-specific NK cell memory in rhesus macaques. Research has demonstrated that splenic and hepatic NK cells from SHIV-SF162P3- and SIVmac251-infected macaques specifically lyse Gag- and Env-pulsed dendritic cells in an NKG2-dependent fashion . This finding challenges the traditional view of NK cells as purely innate immune cells without antigen specificity.

Methodologically, these findings were established through:

• Isolation of NK cells from infected macaques

• Functional assays measuring cytotoxicity against antigen-pulsed dendritic cells

• Blocking experiments to determine NKG2 dependency

• Longitudinal studies showing persistence of memory responses

Importantly, NK cells from Ad26-vaccinated macaques efficiently lysed antigen-matched but not antigen-mismatched targets even 5 years post-vaccination, demonstrating the durability of these memory responses . This suggests that CD226-mediated NK cell memory could be leveraged in vaccine strategies against viral pathogens like HIV-1.

Researchers investigating this phenomenon should design experiments that:

• Include appropriate controls for antigen specificity

• Measure both cytotoxicity and cytokine production

• Assess the stability of memory over time

• Consider the tissue localization of memory NK cells (particularly in spleen and liver)

What are the experimental considerations when studying the impact of CD226 polymorphisms on immune function in macaque models?

When studying CD226 polymorphisms in macaque models, researchers should consider several methodological aspects:

• Genotyping approaches: Use sequencing or SNP analysis to identify CD226 variants in your macaque cohort. While human CD226 has well-characterized polymorphisms like rs763361 associated with autoimmune diseases , macaque-specific variants may differ.

• Functional assays: Design experiments that assess:

  • Signaling pathway activation (particularly ERK1/2 and STAT4 phosphorylation)

  • Cytokine production profiles (especially IFNγ)

  • Cell-cell adhesion properties

  • Receptor-ligand interactions

• Model selection: Consider that genomic differences exist between rhesus macaques and other macaque species such as cynomolgus macaques (Macaca fascicularis) , which may affect CD226 function.

• Translational relevance: The CD226-307Ser risk variant in humans imposes immune dysregulation by increasing IFNγ signaling in CD8 T cells . When studying macaque CD226, researchers should investigate whether similar functional consequences exist for macaque variants.

• Technical considerations: Develop species-specific reagents (antibodies, primers) that accurately detect macaque CD226 variants.

For rigorous study design, include appropriate control groups, account for genetic diversity in macaque populations, and use multiple complementary assays to characterize the functional impact of CD226 variants.

What are the optimal protocols for assessing CD226-dependent NK cell responses in rhesus macaque samples?

To effectively assess CD226-dependent NK cell responses in rhesus macaque samples, researchers should follow these methodological guidelines:

• Sample preparation:

  • Isolate peripheral blood mononuclear cells (PBMCs) from whole blood using density gradient centrifugation

  • For tissue-resident NK cells, prepare single-cell suspensions from spleen or liver samples

  • Use negative selection to isolate untouched NK cells to prevent activation during isolation

• Flow cytometry phenotyping:

  • Use antibody panels including macaque-reactive anti-CD226 antibodies

  • Include markers for NK cells (CD3-CD56+/CD16+) and activation markers

  • Consider intracellular staining for cytokines (IFNγ, TNFα) and cytotoxic molecules (perforin, granzymes)

• Functional assays:

  • Cytotoxicity assays using CD226 ligand-expressing target cells

  • Redirected killing assays with anti-CD226 antibodies

  • CD226 blocking experiments to demonstrate CD226-dependence

  • NKG2D blocking to distinguish between CD226 and NKG2D-mediated effects

• Antigen-specific memory assessment:

  • Challenge with antigen-pulsed dendritic cells

  • Measure specific lysis of antigen-matched versus mismatched targets

  • Longitudinal studies to assess durability of memory responses

• Signaling studies:

  • Assess phosphorylation of ERK1/2 and STAT4 following CD226 engagement

  • Evaluate Ca2+ flux after CD226 cross-linking

For reliable results, always include appropriate positive and negative controls, validate antibody cross-reactivity with rhesus macaque CD226, and carefully interpret data in the context of other activating and inhibitory receptors that may influence NK cell function.

How can recombinant Macaca mulatta CD226 protein be effectively used in immunological assays?

Recombinant Macaca mulatta CD226 protein can be employed in various immunological assays with the following methodological approaches:

• Antibody development and validation:

  • Use as an immunogen for generating anti-CD226 antibodies

  • Employ in ELISA as a coating antigen for antibody screening

  • Create standard curves for quantitative assays

• Binding and interaction studies:

  • Surface Plasmon Resonance (SPR) to measure binding kinetics with ligands

  • Pull-down assays to identify novel interaction partners

  • Competition binding assays with known ligands (CD112/Nectin-2, CD155/PVR)

• Functional studies:

  • T cell stimulation assays using plate-bound recombinant CD226

  • NK cell modulation experiments

  • Blocking experiments to evaluate the role of CD226 in specific immune functions

• Structural studies:

  • Crystallography of CD226 alone or in complex with ligands

  • Epitope mapping using fragments or mutants of the recombinant protein

When working with the recombinant protein, follow these technical considerations:

• Store at -20°C/-80°C upon receipt

• Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

• Add 5-50% glycerol for long-term storage

• Avoid repeated freeze-thaw cycles

• Working aliquots can be stored at 4°C for up to one week

What are the key considerations when studying CD226 in the context of autoimmune disease models using rhesus macaques?

When investigating CD226 in autoimmune disease models using rhesus macaques, researchers should address these critical considerations:

• Disease model selection and validation:

  • Choose appropriate autoimmune disease models that are well-established in macaques

  • Consider that CD226 has been implicated in multiple human autoimmune conditions including MS, T1D, RA, AITD, and SSc

  • Validate the relevance of CD226 in your specific model through preliminary studies

• Genetic considerations:

  • Screen for CD226 polymorphisms in your macaque cohort

  • Consider that the rs763361/Gly307Ser variant in humans affects T cell function

  • Identify macaque-equivalent polymorphisms that might influence disease susceptibility

• Cellular and molecular analyses:

  • Evaluate CD226 expression across immune cell subsets (NK cells, T cells, platelets)

  • Assess CD226-dependent signaling pathways, particularly focusing on:

    • ERK1/2 activation

    • STAT4 phosphorylation

    • IFNγ production

  • Study CD226 interactions with its ligands CD112 and CD155

• Therapeutic targeting strategies:

  • Develop CD226-blocking antibodies specific for macaque CD226

  • Test the effect of CD226 blockade on disease progression

  • Consider the balance between CD226 and competing inhibitory receptors like TIGIT

• Translational considerations:

  • Design experiments that can inform human therapeutic development

  • Address how findings in macaques may translate to human autoimmune diseases

  • Consider differences in CD226 expression and function between species

How does CD226 function differ between tissue-resident and circulating NK cells in rhesus macaques?

Understanding the differential expression and function of CD226 in tissue-resident versus circulating NK cells in rhesus macaques requires specific methodological approaches:

• Tissue sample processing:

  • For circulating NK cells: Isolate PBMCs from peripheral blood

  • For tissue-resident NK cells: Process spleen, liver, lymph nodes, or other tissues of interest

  • Use gentle enzymatic digestion methods that preserve surface receptor expression

  • Isolate NK cells through magnetic separation or FACS sorting

• Phenotypic characterization:

  • Develop comprehensive flow cytometry panels to identify tissue-resident versus circulating NK cell populations

  • Include markers of tissue residency (CD69, CD103) alongside CD226

  • Quantify CD226 expression levels across different NK cell subsets

• Functional assessments:

  • Compare CD226-dependent cytotoxicity between tissue-resident and circulating NK cells

  • Evaluate antigen-specific memory capabilities in both populations

  • Research suggests that splenic and hepatic NK cells may have enhanced antigen-specific memory capacity compared to circulating NK cells

• Transcriptomic and proteomic profiling:

  • Perform RNA-seq on sorted NK cell populations to identify differences in CD226-related gene expression

  • Use proteomic approaches to identify differential signaling pathway activation

• In situ analysis:

  • Employ multiplex immunohistochemistry to visualize CD226+ NK cells in tissues

  • Assess the spatial relationship between CD226+ NK cells and other immune cells or CD226 ligand-expressing cells

Research has demonstrated that splenic and hepatic NK cells from SHIV-infected and vaccinated macaques show robust antigen-specific memory responses , suggesting that tissue-resident NK cells may have unique CD226-dependent functions. Furthermore, these tissue-resident memory NK cells showed durable responses even 5 years post-vaccination, highlighting their potential importance in long-term immunity.

What are common technical challenges when working with recombinant Macaca mulatta CD226 protein and how can they be addressed?

Researchers working with recombinant Macaca mulatta CD226 protein may encounter several technical challenges that can be addressed through specific methodological approaches:

• Protein stability issues:

  • Challenge: CD226 protein degradation during storage or handling

  • Solution: Store as lyophilized powder; after reconstitution, add 5-50% glycerol and store at -20°C/-80°C

  • Solution: Aliquot to avoid repeated freeze-thaw cycles; working aliquots can be kept at 4°C for up to one week

• Solubility problems:

  • Challenge: Poor solubility or precipitation after reconstitution

  • Solution: Reconstitute in appropriate buffer (Tris/PBS-based buffer, pH 8.0)

  • Solution: Add low concentrations of non-ionic detergents if needed for specific applications

• Functional activity assessment:

  • Challenge: Determining if the recombinant protein retains native conformation and function

  • Solution: Validate using binding assays with known ligands (CD112/Nectin-2, CD155/PVR)

  • Solution: Compare activity with commercially available standards when possible

• Cross-reactivity considerations:

  • Challenge: Limited cross-reactivity between human and macaque-specific reagents

  • Solution: Validate antibodies specifically against the macaque protein

  • Solution: Develop macaque-specific detection reagents when necessary

• Post-translational modification differences:

  • Challenge: E. coli-expressed proteins lack glycosylation present in native CD226

  • Solution: For applications where glycosylation is critical, consider using mammalian or insect cell expression systems

  • Solution: Validate function through multiple complementary assays

How can researchers overcome limitations in studying CD226 signaling pathways in primary rhesus macaque cells?

Studying CD226 signaling in primary rhesus macaque cells presents specific challenges that can be addressed through these methodological approaches:

• Limited cell numbers:

  • Challenge: Restricted access to primary macaque samples

  • Solution: Optimize miniaturized assays requiring fewer cells

  • Solution: Expand primary cells using appropriate cytokines while monitoring for phenotypic changes

  • Solution: Consider immortalization of primary cells for specific applications

• Signaling pathway analysis:

  • Challenge: Detecting phosphorylation events in rare cell populations

  • Solution: Use phospho-flow cytometry to analyze signaling at single-cell resolution

  • Solution: Employ multiplexed phosphoprotein detection systems

  • Solution: Focus on key pathways implicated in CD226 signaling, particularly ERK1/2 and STAT4 phosphorylation

• Cross-reactivity of reagents:

  • Challenge: Many antibodies developed for human studies have limited reactivity with macaque proteins

  • Solution: Validate commercial antibodies against macaque samples

  • Solution: Use conserved epitope-targeting antibodies when possible

  • Solution: Develop macaque-specific antibodies for critical signaling molecules

• Functional validation:

  • Challenge: Connecting signaling events to functional outcomes

  • Solution: Couple signaling analysis with functional readouts (cytotoxicity, cytokine production)

  • Solution: Use specific inhibitors of signaling pathways to establish causality

  • Solution: Employ genetic approaches (siRNA, CRISPR) when feasible

• System integration:

  • Challenge: Understanding how CD226 signaling interacts with other pathways

  • Solution: Conduct comprehensive phosphoproteomic analysis

  • Solution: Use network analysis to identify pathway crosstalk

  • Solution: Consider the balance between activating (CD226) and inhibitory (TIGIT) signals

When studying CD226-307Ser variants (equivalent to human rs763361), researchers should particularly focus on IFNγ signaling pathways and production, as these have been implicated in the risk variant's contribution to immune dysregulation .

What strategies can be employed to study the co-expression and functional relationship between CD226 and TIGIT in rhesus macaque immune cells?

The balance between CD226 (activating) and TIGIT (inhibitory) signaling is critical for immune regulation. To effectively study their co-expression and functional relationship in rhesus macaque immune cells, researchers should consider these methodological approaches:

• Co-expression analysis:

  • Develop multi-parameter flow cytometry panels that simultaneously detect CD226 and TIGIT

  • Include markers for relevant cell subsets (T cells, NK cells, Tregs)

  • Quantify expression ratios across different activation states and disease conditions

  • Consider that the CD226:TIGIT ratio may be more informative than absolute expression levels

• Competitive binding studies:

  • Examine competition for shared ligands (CD112/Nectin-2, CD155/PVR)

  • Use recombinant proteins to quantify binding affinities

  • Develop blocking strategies that selectively interrupt specific receptor-ligand pairs

• Functional assessments:

  • Compare effects of selective CD226 or TIGIT blockade versus combined blockade

  • Assess how changing the CD226:TIGIT balance affects:

    • Cytokine production

    • Cytotoxicity

    • Proliferation

    • Regulatory function

• Signaling interaction analysis:

  • Investigate how CD226 and TIGIT signaling pathways interact

  • Determine whether one receptor can modulate signaling from the other

  • Examine downstream convergence points in signaling cascades

• In vivo models:

  • Develop approaches to selectively modulate CD226 or TIGIT in vivo

  • Study disease outcomes in models where CD226:TIGIT balance is perturbed

What are promising areas for further investigation of CD226 function in rhesus macaque models of human disease?

Several high-priority research directions for CD226 in rhesus macaque models present opportunities for significant scientific advancement:

• Vaccine development applications:

  • Investigate how CD226-dependent NK cell memory can be harnessed for vaccine design

  • Explore adjuvants that specifically enhance CD226-mediated responses

  • Develop strategies to induce durable tissue-resident memory NK cells

  • Build on findings that NK cells from Ad26-vaccinated macaques show antigen-specific memory for at least 5 years

• Autoimmune disease modeling:

  • Establish macaque models that recapitulate CD226 polymorphism effects seen in human autoimmune diseases

  • Investigate whether CD226-307Ser equivalent variants in macaques increase susceptibility to autoimmune conditions

  • Explore the CD226 signaling mechanisms that promote autoimmunity, particularly focused on IFNγ pathway enhancement

• Therapeutic targeting approaches:

  • Develop and test CD226-targeting biologics in macaque models prior to human translation

  • Investigate cell-type specific targeting strategies

  • Compare CD226 blockade versus TIGIT enhancement approaches

  • Explore combination approaches targeting multiple co-stimulatory pathways

• Tissue-specific immunity:

  • Map tissue distribution and function of CD226+ immune cells across different tissues

  • Investigate how tissue environments influence CD226 expression and function

  • Develop strategies to modulate tissue-resident CD226+ cells for therapeutic purposes

• Integrative multiomics analysis:

  • Combine genomic, transcriptomic, and proteomic approaches to understand CD226 regulation

  • Investigate epigenetic control of CD226 expression in different immune contexts

  • Apply systems biology approaches to position CD226 within broader immune networks

These directions build upon findings that CD226 plays roles in antigen-specific memory , is implicated in autoimmune disease susceptibility through polymorphisms , and functions within a complex network of activating and inhibitory receptors.

How might emerging technologies enhance our understanding of CD226 biology in rhesus macaques?

Emerging technologies offer unprecedented opportunities to advance our understanding of CD226 biology in rhesus macaques:

• Single-cell technologies:

  • Single-cell RNA sequencing to identify CD226-expressing cell populations with high resolution

  • Single-cell ATAC-seq to understand chromatin accessibility patterns around the CD226 locus

  • Spatial transcriptomics to map CD226+ cells within tissue microenvironments

  • Single-cell proteomics to understand protein co-expression patterns with CD226

• Advanced imaging approaches:

  • Multiplex imaging platforms (CODEX, Imaging Mass Cytometry) to visualize CD226+ cells in tissues

  • Intravital microscopy to study CD226-dependent interactions in live animals

  • Super-resolution microscopy to examine CD226 clustering and organization at the cell membrane

• Genetic manipulation tools:

  • CRISPR/Cas9 gene editing in primary macaque cells to study CD226 function

  • AAV-delivered gene modification approaches for in vivo CD226 manipulation

  • Development of rhesus macaque iPSCs with defined CD226 genotypes

• Protein interaction technologies:

  • Proximity labeling approaches (BioID, APEX) to identify novel CD226 interaction partners

  • Hydrogen-deuterium exchange mass spectrometry to map structural dynamics of CD226

  • AlphaFold or other structural prediction tools to model CD226 interactions

• Systems biology approaches:

  • Network analysis to position CD226 within immune signaling networks

  • Multi-omics integration to understand CD226 regulation across biological scales

  • Machine learning approaches to predict CD226-dependent outcomes from complex datasets

What approaches can integrate findings from rhesus macaque CD226 studies with human translational research?

To maximize the translational value of rhesus macaque CD226 research for human applications, researchers should implement these integrative approaches:

• Comparative genomics and proteomics:

  • Perform detailed sequence and structural comparisons between macaque and human CD226

  • Identify conserved functional domains and species-specific differences

  • Map polymorphisms in macaque CD226 that correspond to human disease-associated variants

  • Create a comprehensive database of cross-species CD226 variants and their functional implications

• Parallel experimental design:

  • Conduct matched experiments in both macaque and human samples when possible

  • Use identical protocols, reagents, and analytical approaches for cross-species comparisons

  • Develop dual-specificity reagents that work equally well in both species

  • Establish standardized reporting formats to facilitate data integration

• Translational model validation:

  • Validate that CD226-dependent mechanisms discovered in macaques are conserved in humans

  • Use humanized mouse models as an intermediate validation step

  • Develop ex vivo human systems to test predictions from macaque studies

  • Build computational models that account for species differences

• Biomarker development pipeline:

  • Identify CD226-related biomarkers in macaque models that have potential clinical relevance

  • Validate candidate biomarkers in human samples

  • Develop assays that can be used in both preclinical macaque studies and human clinical trials

  • Create reference standards for CD226-related measurements across species

• Therapeutic development strategies:

  • Design biologics targeting CD226 with cross-species reactivity

  • Use macaque studies to inform optimal dosing, timing, and combinations for human trials

  • Develop companion diagnostics for CD226-targeted therapies

  • Establish predictive markers of response based on macaque findings