CD226 is a transmembrane glycoprotein with a well-defined structural organization, which is primarily expressed on natural killer (NK) cells, cytotoxic T cells, and some subsets of B cells [1,2]. Its primary structure consists of an extracellular domain, a transmembrane domain, and a cytoplasmic tail [1]. Each of these domains contributes to the protein's overall function. The extracellular domain of CD226 is the region responsible for interacting with its ligands, CD155 (PVR) and CD112 (Nectin-2). It comprises eight N-linked glycoprotein sites and two immunoglobulin V-like domains [1]. The IgV domain is particularly significant for ligand binding and recognition, as it contains specific amino acid residues that form the interface with ligands, contributing to the specificity and strength of the interaction and initiating downstream signaling events [3]. The transmembrane segment comprises a sequence of 28 amino acids. The cytoplasmic tail of CD226 is relatively short but essential for signaling. It contains tyrosine-based signaling motifs, including immunoreceptor tyrosine-based activation motifs (ITAMs) [4], which play a crucial role in downstream signaling pathways upon ligand binding. For example, tyrosine residues within the cytoplasmic tail, like Y319 and Y322, are notable for their role in signaling cascades. Phosphorylation of these residues is associated with downstream signaling events crucial for cellular responses [2]. CD226's cytoplasmic tail is involved in activating cytotoxic responses in immune cells and regulating immune checkpoint pathways [2,5].

The regulation of CD226 is a complex and tightly controlled process. Various mechanisms and factors that regulate the expression and function of CD226, including transcriptional regulation, protein-level regulation, environmental factors, and tumor microenvironment. Transcription factors such as activating protein-1 (AP-1) are known to positively regulate CD226 expression [6]. Conversely, certain transcription factors like FoxP3, which is highly expressed in regulatory T cells (Tregs), can inhibit CD226 expression [7-9]. Tregs suppress immune responses, and reduced CD226 expression on their surface may contribute to immune tolerance. In addition, exhausted CD8⁺ T cells elevate the transcriptional regulator EOMES, which interacts directly with a binding site in the CD226 gene, resulting in the inhibition of CD226 expression [10]. CD226 can undergo various protein-level modifications. For example, CD226 can be glycosylated, which can affect its ligand-binding affinity and cellular localization [1]. Phosphorylation of CD226 upon ligand binding is another key regulatory mechanism, initiating subsequent signaling cascades that bolster immune cell activation, under the influence of regulatory factors such as PD-1 and TIGIT [5,11-13]. The activation of the PD-1 pathway leads to the dephosphorylation of CD226, facilitated by the PD-1-SHP2 complex [11,13]. TIGIT outcompetes and replaces CD226 owing to its higher affinity for shared ligands [5]. Additionally, TIGIT hinders the formation of CD226 homodimers via engaging in cis interactions with CD226 [12]. This interaction impedes CD226's ability to induce activation signals. The distinctive mechanisms through which PD-1 and TIGIT regulate CD226 dephosphorylation underscore the imperative need to combine PD-1/PD-L1 blockade with anti-TIGIT to fully activate CD226. Additionally, the interaction between CD226 and PVR initiates the phosphorylation of a critical tyrosine residue (Y319) in CD226. This phosphorylation event render CD226 susceptible to ubiquitination, internalization, and degradation by Cbl-b in CD8⁺ T cells [14]. The presence of various cytokines and inflammatory mediators in the microenvironment can also influence CD226 expression and function. For instance, interferon-gamma (IFN-γ) might inhibit CD226 expression on CD8⁺ TILs cells [15]. In the context of cancer, the tumor microenvironment has a significant impact on CD226 regulation. Some tumors actively downregulate CD226 ligands (CD155 and CD112) on their surface, limiting CD226-mediated immune recognition [16,17]. Tumor-secreted factors and immune suppressive cells in the tumor microenvironment may also affect CD226 signaling.

CD226 is a crucial component of immune cell recognition and cytotoxicity against tumor cells. It binds to its ligands, CD155 and CD112, which are often expressed on the surface of tumor cells [18]. CD226 engagement with its ligands on tumor cells enhances the cytotoxic activity of NK cells and CD8⁺ T cells. This engagement initiates the activation of pathways associated with immune cell activation and effector functions, leading to increased tumor cell killing, thereby limiting tumor growth and progression. CD226 interactions can also influence immune checkpoint regulation, affecting the balance between activating and inhibitory signals in the tumor microenvironment. Additionally, CD226 has been associated with the regulation of immune cell infiltration into the tumor microenvironment. Its engagement with ligands may contribute to the recruitment of immune cells within tumors [19]. These effects of CD226 work together to inhibit tumor progression. However, tumor cells have developed several strategies to evade CD226-mediated immune responses. This includes downregulation or shedding of CD226 ligands, rendering the tumor cells less recognizable by immune cells [5,12]. Furthermore, the activation of inhibitory signaling like PD-1 pathway can suppress CD226-mediated immune responses [11,13].

CD226 signaling plays a pivotal role in activating and enhancing the cytotoxicity of NK cells and CD8⁺ T cells. It promotes the release of cytotoxic granules, such as perforin and granzyme, inducing apoptosis in tumor cells. Additionally, it stimulates the release of cytokines, such as IFN-γ and TNF-α, contributing to anti-tumor immune responses [10]. CD226 is not only involved in tumor recognition but also functions as a co-stimulatory molecule. It synergizes with other activating receptors on immune cells, such as NKG2D and T-cell receptors, to strengthen the immune response against tumors [20]. In addition, as mentioned above, CD226 also influences immune checkpoint regulation and the recruitment of immune cells, exerting its intricate role in the tumor microenvironment.

There is growing interest in targeting CD226 as a part of cancer immunotherapy. To date, the development and investigation of monoclonal antibodies that activate CD226 signaling have been limited. A notable example is LY3435151 (Eli Lilly), which entered a phase 1 clinical trial; however, this trial was abruptly halted shortly after its initiation. The reasons for its termination are unclear; however, it is crucial to explore strategies for overcoming the limitations associated with using CD226 as a target for cancer treatment.

Research into the role of CD226 in cancer is an active and evolving field, and there are still several unanswered questions and areas of ongoing investigation.

Mechanisms of immune evasion

While CD226 plays a crucial role in tumor surveillance and cytotoxicity, many cancers can evade immune responses. Understanding the mechanisms by which tumors suppress or downregulate CD226 expression on immune cells or its ligands on tumor cells is an important area of research. What are the specific mechanisms that allow tumors to escape CD226-mediated immune recognition?

Context-specific effects

CD226's role in cancer appears to be context-specific, with different outcomes in various cancer types and stages [19]. It is essential to delineate the factors that determine whether CD226 activation enhances or inhibits tumor growth. Are there specific tumor microenvironment factors that modulate CD226 function?

Patient stratification

CD226-targeted therapies have the potential for personalized medicine. However, identifying which patients are most likely to benefit from CD226-based immunotherapies remains a challenge. Researchers are working to define biomarkers or criteria that can predict a patient's response to CD226-targeted treatments. What are the best predictive markers for CD226-based therapies, and how can they be applied in clinical practice?

Long-term safety

As with any immunotherapy, the long-term safety of CD226-targeted treatments is a critical concern. Multiple studies have revealed that CD226 expression levels are associated with several autoimmune disorders, including autoimmune thyroid disease (AITD), rheumatoid arthritis (RA), systemic sclerosis (SSc), type 1 diabetes (T1D), and multiple sclerosis (MS) [1,20-25]. Ongoing research is needed to assess potential side effects, immune-related adverse events, and the durability of responses. What are the long-term safety profiles and outcomes associated with CD226-based therapies?

CD226 in combination with other immune checkpoints

CD226 interacts with various other immune checkpoint molecules, such as PD-1 and TIGIT [19]. Understanding the complex crosstalk between these checkpoints and how they collectively influence the immune response in cancer is an ongoing area of investigation. How can the combined targeting of multiple immune checkpoints, including CD226, be optimized for maximal therapeutic benefit?

Ideal modification of CD226

While CD226 agonist approaches for bolstering anti-tumor responses hold promise, the presence of CD226 on platelets, and its association with their activation and adhesion, introduces potential complexities in its intended pharmacological role in anti-tumor therapy. It is imperative to devise strategies to surmount the limitations associated with employing CD226 as a target for cancer treatment.

Combinatorial immunotherapies

Combining multiple immunotherapeutic approaches has shown promise in cancer treatment. However, the optimal combinations and sequencing of therapies involving CD226 modulation are not fully understood. What are the most effective combination strategies involving CD226-targeted therapies and immune checkpoint inhibitors?

Addressing these unanswered questions will contribute to a more comprehensive understanding of CD226's role in cancer and pave the way for the development of innovative therapeutic strategies for improving cancer treatment outcomes.

The author declares no conflict of interest.