CD226 Human

What is the basic structure of human CD226 and where is it expressed?

Human CD226 (DNAM-1) is a 65 kDa type I transmembrane glycoprotein belonging to the immunoglobulin superfamily. The mature protein contains a 236 amino acid extracellular domain (ECD) with two Ig-like C2-set domains and a 61 amino acid cytoplasmic region with motifs for binding PDZ domains and band 4.1 family proteins. CD226 is primarily expressed on multiple lymphoid and myeloid cells, including natural killer (NK) cells, T cells, monocytes, and platelets. The protein plays a critical role in immune cell activation through interactions with its ligands CD155/PVR and Nectin-2/CD112 .

How is the CD226 gene regulated at the transcriptional level?

The human CD226 gene contains two distinct promoters, designated P1 and P2, located at -810 to -287 bp and +33 to +213 bp, respectively. A negative regulatory element exists between these promoters. Both P1 and P2 can be regulated by phorbol ester (12-O-tetradecanoylphorbol-13-acetate) and calcium ionophore (A23187). Transcription factor analysis reveals putative binding sites for AP-1, Sp1, PEA3, and Ets-1 within the regulatory regions. Interestingly, the transcription factor Ets-1 has opposing effects on the two promoters: it promotes AP-1-induced P2 activity while inhibiting AP-1-induced P1 activity. This differential regulation appears to involve a 10-bp AP-1/Ets-1 composite site (CCTTCCTTCC) in the P1 promoter .

How conserved is CD226 across species and what implications does this have for research models?

CD226 exhibits moderate sequence conservation between species. Human CD226 shares approximately 50% and 52% amino acid sequence identity with mouse and rat CD226, respectively, within the extracellular domain. This conservation level allows for cross-species research while necessitating caution when extrapolating findings between models. The human and mouse CD226 genes are located on chromosomes 18q22.3 and 18E4, respectively, suggesting conserved genomic context. When designing research models, investigators should account for these species-specific differences, particularly when testing therapeutic approaches targeting CD226 or its signaling pathways .

What are the primary cellular functions of CD226 in immune responses?

CD226 functions as a key costimulatory receptor across multiple immune cell types. In NK cells, CD226 ligation promotes activation, cytotoxicity, and memory-like NK cell (mlNK) proliferation. For T cells, CD226 contributes to differentiation, activation, and cytotoxic functions. CD226 also enhances dendritic cell maturation, facilitates megakaryocyte and activated platelet adhesion to vascular endothelial cells, and promotes monocyte extravasation. Additionally, CD226 inhibits osteoclast formation. These diverse functions position CD226 as a central regulator of both innate and adaptive immune responses. The functionality of CD226 depends on its interactions with ligands CD155/PVR and Nectin-2/CD112, which are widely expressed on various cell types .

How does CD226 signaling affect memory-like NK cell responses in latent tuberculosis infection?

CD226 signaling critically regulates memory-like NK cell (mlNK) proliferation and function in individuals with latent tuberculosis infection (LTBI). Research shows that CD226 expression increases on mlNKs (CD56+CD27+) from LTBI+ donors following stimulation with γ-irradiated Mycobacterium tuberculosis (γ-Mtb). Blockade of CD226 signaling, either through antibody treatment or CRISPR/Cas9-mediated deletion of the CD226 gene, significantly reduces mlNK proliferation. Furthermore, CD226 blockade decreases IFN-γ production, degranulation, and activation markers (CD69 and HLA-DR) in mlNKs from LTBI+ individuals. Mechanistically, CD226 signaling promotes mlNK proliferation by enhancing FOXO1 phosphorylation and increasing cMyc expression, resulting in elevated glycolytic activity. These findings suggest that CD226 signaling is essential for maintaining protective memory NK cell responses in tuberculosis infection .

What molecular pathways are activated downstream of CD226 and how do they vary across immune cell types?

CD226 activates multiple downstream signaling pathways that vary by cell type. In NK cells from LTBI+ individuals, CD226 signaling enhances the phosphorylation of FOXO1 and increases cMyc expression, driving cell proliferation and glycolytic metabolism. CD226 blockade reduces the production of IFN-γ, TNF-α, MIP-1β, and MCP1, indicating its role in regulating cytokine and chemokine production. In T cells, particularly CD8+ T cells carrying the CD226-307Ser risk variant, CD226 engagement leads to enhanced phosphorylation of ERK1/2 (extracellular signal–regulated kinases) and STAT4, resulting in increased IFN-γ production. This suggests that CD226 couples to the mitogen-activated protein kinase pathway and JAK/STAT signaling. Additionally, CD226 contains binding motifs for PDZ domains and band 4.1 family proteins in its cytoplasmic region, indicating potential interactions with cytoskeletal components and additional signaling complexes .

How does the rs763361 variant of CD226 affect immune cell function and disease susceptibility?

The rs763361 nonsynonymous variant in the CD226 gene results in a glycine-to-serine substitution at position 307 of the CD226 protein (CD226-307Ser) and has been identified as a risk factor for multiple immune-mediated diseases, including multiple sclerosis (MS). Functional studies reveal that this risk variant significantly alters immune cell function. In CD8+ T cells, the CD226-307Ser variant enhances T-cell receptor (TCR) signaling, with transcriptomic analyses showing enrichment in TCR, JAK/STAT, and IFNγ signaling pathways. At the molecular level, this variant leads to increased phosphorylation of ERK1/2 and STAT4, resulting in enhanced IFNγ production. Previous studies have also shown that this variant decreases the immune-regulatory capacity of Treg cells while increasing the proinflammatory potential of effector CD4+ T cells. These functional alterations collectively contribute to immune dysregulation and increased susceptibility to autoimmune conditions .

What methodologies are most effective for studying CD226 variants in human populations?

Studying CD226 variants in human populations requires a multi-faceted approach combining genomic, transcriptomic, and functional analyses. For genomic characterization, next-generation sequencing and genotyping arrays are effective for identifying variants such as rs763361. To assess functional consequences, researchers should isolate immune cells from donors based on their CD226 genotype (e.g., homozygous for either protective or risk alleles). High-parameter flow cytometry enables detailed phenotypic characterization of ex vivo cells, while bulk or single-cell RNA sequencing identifies transcriptomic signatures associated with specific variants. Pathway enrichment analysis can then reveal affected signaling networks. For functional validation, researchers should examine canonical signaling pathways (e.g., phosphorylation of ERK1/2, STAT4) and cytokine production (particularly IFNγ) in response to receptor engagement. These methodologies, as demonstrated in recent CD226 research, provide comprehensive insights into how genetic variants influence immune cell function and disease susceptibility .

Beyond multiple sclerosis, what other autoimmune or inflammatory conditions are associated with CD226 variants?

CD226 variants, particularly rs763361 (Gly307Ser), have been implicated in multiple autoimmune and inflammatory conditions beyond multiple sclerosis. These include type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosus, autoimmune thyroid disease, and psoriasis. The broad association with various immune-mediated conditions suggests that CD226 variants affect fundamental immune regulatory mechanisms common to multiple pathologies. The risk variant appears to promote a proinflammatory phenotype across different immune cell populations, potentially through enhanced TCR signaling, increased cytokine production (particularly IFNγ), and reduced regulatory T cell function. Understanding the shared pathogenic mechanisms across these conditions may provide insights for developing targeted therapies that modulate CD226 signaling in multiple autoimmune diseases .

What flow cytometry protocols are optimal for detecting CD226 expression on different immune cell subsets?

For optimal flow cytometric detection of CD226 on various immune cell subsets, a multiparametric approach is recommended. Begin with freshly isolated peripheral blood mononuclear cells (PBMCs) and use appropriate fixation methods that preserve CD226 epitopes. A recommended antibody panel includes anti-CD226 PE-conjugated monoclonal antibody (such as clone 102511) combined with lineage markers: CD3 and CD56 for NK cells; CD3, CD4, and CD8 for T cell subsets; CD14 for monocytes; and CD19 for B cells. For more detailed characterization, include markers CD27 (important for identifying memory-like NK cells), activation markers (CD69, HLA-DR), and functional markers (IFN-γ, granzyme B) following stimulation. Set quadrant markers based on appropriate isotype control antibodies. For preservation of intracellular phospho-epitopes when examining CD226 signaling, use rapid fixation with paraformaldehyde followed by methanol permeabilization. This methodology allows accurate detection of CD226 expression and its correlation with phenotypic and functional parameters across immune cell populations .

How can researchers effectively manipulate CD226 signaling in experimental systems?

Researchers can manipulate CD226 signaling through several complementary approaches. For acute blockade, anti-CD226 blocking antibodies provide a straightforward method, as demonstrated in studies of memory-like NK cells from LTBI+ individuals. For genetic manipulation, CRISPR/Cas9-mediated deletion of the CD226 gene offers more permanent functional disruption. When studying specific signaling pathways downstream of CD226, selective inhibitors of key molecules (such as cMyc inhibitors or glycolysis inhibitors) can help dissect the relative contribution of each pathway. To enhance CD226 signaling, researchers can use plate-bound or soluble recombinant CD155 or CD112 (CD226 ligands). For in vivo studies, conditional knockout models or administration of blocking antibodies can be employed. Each approach has advantages and limitations that should be considered based on the specific research question. Importantly, validation experiments should confirm the efficacy of these manipulations, such as verifying reduced CD226 protein expression or diminished downstream signaling events .

What advanced techniques are available for studying CD226-mediated signaling networks?

Advanced techniques for studying CD226-mediated signaling networks include phospho-flow cytometry, which allows simultaneous detection of multiple phosphorylated proteins at the single-cell level in heterogeneous populations. This technique has been used to identify enhanced ERK1/2 and STAT4 phosphorylation in CD8+ T cells with the CD226-307Ser variant. Mass cytometry (CyTOF) offers even higher dimensionality for comprehensive signaling network analysis. For temporal dynamics of signaling, live-cell imaging with fluorescent biosensors can track pathway activation in real-time. Bulk and single-cell RNA sequencing provide transcriptomic profiles associated with CD226 signaling, as demonstrated in studies showing enrichment of TCR, JAK/STAT, and IFNγ pathways in CD226-307Ser variant cells. Proteomics approaches, including proximity labeling techniques and immunoprecipitation followed by mass spectrometry, can identify novel interaction partners of CD226. Systems biology approaches integrating these multiomics data help construct comprehensive signaling networks. For functional validation, CRISPR screens targeting components of these networks can establish their contribution to CD226-mediated cellular responses .

How does CD226 contribute to tuberculosis immunity and what are the implications for vaccine development?

CD226 plays a crucial role in tuberculosis immunity by regulating memory-like NK cell (mlNK) responses. Research on individuals with latent tuberculosis infection (LTBI) shows that CD226 signaling enhances the proliferation, cytokine production, and degranulation of CD56+CD27+ mlNKs in response to Mycobacterium tuberculosis antigens. Mechanistically, CD226 promotes FOXO1 phosphorylation and increases cMyc expression, driving metabolic reprogramming toward glycolysis to support mlNK expansion. Blocking CD226 signaling reduces the production of protective cytokines including IFN-γ and TNF-α, which are critical for controlling mycobacterial infection. These findings suggest that CD226-dependent NK cell memory may be an important component of protective immunity against tuberculosis. For vaccine development, strategies that enhance CD226 signaling or expression could potentially boost protective NK cell responses. Conversely, defects in CD226 signaling might contribute to inadequate immune control of Mtb infection, representing a potential target for host-directed therapies to enhance TB immunity .

What is the current understanding of CD226's role in multiple sclerosis pathogenesis and progression?

CD226 contributes to multiple sclerosis (MS) pathogenesis primarily through the rs763361 variant (CD226-307Ser), which has been identified as a genetic risk factor for MS. This variant enhances proinflammatory responses in both CD4+ and CD8+ T cells. In CD8+ T cells, the CD226-307Ser variant leads to enhanced TCR signaling, increased phosphorylation of ERK1/2 and STAT4, and elevated IFNγ production. Transcriptomic analyses of CD8+ T cells from individuals with this variant show enrichment in pathways related to TCR, JAK/STAT, and IFNγ signaling. Earlier studies have also demonstrated that this variant decreases the immune-regulatory capacity of Treg cells while increasing the proinflammatory potential of effector CD4+ T cells. Together, these effects create an immune environment favoring autoreactive T cell activation and inflammatory demyelination. The broad expression of CD226 across multiple immune cell types suggests it may influence various aspects of MS immunopathology, from initial T cell activation to effector functions at sites of CNS inflammation. These findings position CD226 as both a genetic risk factor and a functional contributor to MS disease processes .

How do interactions between CD226 and its competing receptors (like CD96) shape immune responses?

The immune response is finely tuned by the balance between CD226 and competing receptors like CD96, which both bind to CD155 but produce different functional outcomes. CD96 competes with CD226 for binding to CD155 and blocks CD226-mediated NK cell activation, creating a regulatory checkpoint system. This competition involves both binding affinity differences and dynamic regulation of receptor expression. Future research should investigate how these receptors are co-regulated during immune responses, including whether they form heterotypic complexes and how their expression is modulated in different disease states. Advanced imaging techniques like super-resolution microscopy could reveal spatial organization of these receptors in immune synapses. Understanding this regulatory network could identify novel therapeutic targets that selectively modify specific receptor interactions rather than globally blocking CD226 function. Additionally, studying how these competing receptor systems differ between individuals with protective versus risk variants of CD226 could provide insights into disease susceptibility mechanisms .

What is the role of CD226 in metabolic reprogramming of immune cells and how does this influence functional outcomes?

CD226 signaling significantly influences immune cell metabolism, particularly in memory-like NK cells where it enhances glycolysis to support proliferation. Research in LTBI+ individuals demonstrates that CD226 blockade reduces glycolysis in NK cells, and inhibiting glycolysis diminishes memory-like NK cell expansion in response to Mycobacterium tuberculosis stimulation. This metabolic regulation appears to operate through CD226's effects on cMyc expression, a key regulator of metabolic pathways in immune cells. Future research should comprehensively characterize how CD226 signaling affects various metabolic pathways (glycolysis, oxidative phosphorylation, fatty acid metabolism) in different immune cell types and activation states. Metabolomic profiling combined with seahorse analysis could provide detailed insights into these metabolic changes. Understanding the metabolic consequences of CD226 variants, particularly the CD226-307Ser risk variant, might explain their functional impact on immune responses. This knowledge could lead to metabolic interventions that selectively target CD226-mediated immune dysregulation in autoimmune diseases or enhance protective responses in infectious contexts .

How does epigenetic regulation of CD226 influence its expression and function across different immune cell subsets?

The epigenetic regulation of CD226 across immune cell subsets remains largely unexplored but represents a critical area for future research. The presence of two distinct promoters (P1 and P2) in the human CD226 gene suggests complex regulatory mechanisms that may be differentially controlled by epigenetic modifications. Research should investigate how DNA methylation, histone modifications, and chromatin accessibility patterns at these promoters vary across immune cell types and activation states. Additionally, understanding how microRNAs and long non-coding RNAs regulate CD226 expression could reveal post-transcriptional regulatory mechanisms. Epigenetic profiling of CD226 in individuals with autoimmune diseases compared to healthy controls might identify disease-associated epigenetic signatures. Similarly, examining how environmental factors (infections, inflammation, metabolic changes) influence the epigenetic landscape of CD226 could explain variability in immune responses. Technologies like ATAC-seq, ChIP-seq, and bisulfite sequencing, combined with single-cell approaches, would enable comprehensive mapping of CD226's epigenetic regulation. These insights could lead to targeted epigenetic therapies that normalize CD226 expression in disease states .

What are the principal challenges in developing specific and effective antibodies against CD226 and its ligands?

Developing specific and effective antibodies against CD226 and its ligands presents several challenges. First, the structural similarity between CD226's ligands (CD155/PVR and Nectin-2/CD112) and their overlapping binding sites can lead to cross-reactivity issues. Second, the conformational changes that CD226 undergoes upon ligand binding make it difficult to develop antibodies that specifically block these interactions without affecting other functions. Third, species differences in CD226 structure (human CD226 shares only 50-52% sequence identity with mouse and rat homologs) complicate the development of cross-reactive antibodies for translational research. To overcome these challenges, researchers should employ detailed epitope mapping, generate recombinant protein fragments as immunogens, and use multiple screening approaches including functional assays to verify specificity and activity. Humanized antibody development may be necessary for therapeutic applications, requiring additional validation steps. Furthermore, considering the diverse expression of CD226 across immune cell types, antibodies should be validated across multiple cellular contexts to ensure consistent specificity and effectiveness .

How can researchers reconcile contradictory findings about CD226 function across different experimental systems?

Reconciling contradictory findings about CD226 function requires systematic analysis of multiple variables that differ between experimental systems. First, researchers should carefully document the specific cell types studied, as CD226 functions vary between NK cells, CD4+ T cells, CD8+ T cells, and other immune populations. Second, the activation state of cells significantly influences CD226 expression and function; comparing resting versus activated cells may explain discrepancies. Third, genetic background matters—particularly CD226 variants like rs763361 that alter function—so genotyping study subjects is crucial. Fourth, differences in experimental approaches (antibody blockade versus genetic deletion, acute versus chronic manipulation) can yield different outcomes. To address these issues, researchers should perform parallel experiments using multiple methodologies on the same biological samples, incorporate appropriate controls for each variable, conduct meta-analyses of published data to identify patterns, and utilize systems biology approaches to model contextual differences in CD226 signaling networks. Collaborative efforts sharing standardized protocols across laboratories would also help resolve contradictions and establish consensus on CD226 functions in different contexts .

What considerations are important when designing clinical studies to evaluate CD226-targeting therapeutics?

Designing clinical studies for CD226-targeting therapeutics requires careful consideration of multiple factors. First, patient stratification based on CD226 genotype (particularly rs763361 status) is essential, as therapeutic responses may differ between those with protective versus risk variants. Second, given CD226's roles in anti-tumor immunity and infection control, comprehensive safety monitoring must include surveillance for increased susceptibility to infections and malignancies. Third, selecting appropriate biomarkers that reflect CD226 pathway modulation is crucial—these might include phosphorylation of downstream signaling molecules (ERK1/2, STAT4), cytokine production patterns (especially IFNγ), and immune cell phenotyping with emphasis on regulatory versus effector balance. Fourth, the timing of intervention is important; CD226-targeting may be more effective during disease initiation rather than established disease. Lastly, considering combination therapies that address multiple pathways simultaneously may yield better outcomes than CD226 blockade alone. Trial designs should include dose-finding studies with careful pharmacokinetic/pharmacodynamic assessments and potentially adaptive designs that allow modification based on early response indicators. Long-term follow-up will be necessary to evaluate both sustained efficacy and delayed adverse effects .