Natural killer cells (NK cells), a unique subset of cytotoxic lymphocytes, play pivotal roles in immune defense through directly recognizing and eliminating pathogen-infected cells or cancer cells [1]. Currently, NK cells have been identified in humans, mice, and teleost fishes [2-5]. However, the existence, identification and functional characterization of NK-like cells in invertebrates remain poorly understood. Studies in Drosophila [6] and shrimp [7] had revealed that their undefined type of hemocytes analogous to vertebrate NK cells had direct cytotoxic activity against invading pathogens or abnormal cells. The identification of NK cells primarily relies on the surface marker neural cell adhesion molecule 1 (NCAM1/CD56) [8] and their distinct cytotoxic function [9]. NCAM1 is mainly expressed in immune cells, particularly NK cells, and has been used as a marker for NK cell identification. Here, our study identified the NCAM1 homolog (CgNCAM1) from the Pacific oyster Crassostrea gigas and explored its mediated immune functions [10]. We obtained the nucleotide and amino acid sequence of CgNCAM1 from oyster C. gigas genome [11]. Its structure is similar to that of the classical NCAM1, but its intracellular domain contains immunoreceptor tyrosine-based inhibition motif (ITIMs). This is the first reported instance of ITIMs in an invertebrate NCAM1 homolog, suggesting a potential immunosuppressive role. Moreover, CgNCAM1 was also mainly expressed in immune cells (hemocytes) and localized in the cytoplasm and cell membrane. After Vibrio splendidus stimulation, CgNCAM1 was translocated from the hemocyte cytoplasm to cell membrane. These findings suggested that there were significant structural, distributional, and functional differences between molluscan NCAM1 and classical NCAM1. Below, we present the key findings of the study and outline potential directions for future research.

NCAMs, initially identified in chicken embryonic nerve tissue, are termed as "neural cell adhesion molecules" and belong to the immunoglobulin (Ig) superfamily. NCAM1, a cell adhesion molecule, is located on the cell membrane surface and mediates both homophilic and heterophilic cell interactions [12]. In humans, NCAM1 exists as three isoforms, NCAM-120, NCAM-140, and NCAM-180, which are named according to their molecular weights [25]. In teleosts, NCAM1 homologs (NCAM1a/b) had been identified in the mandarin fish (Synchiropus splendidus), barbel chub (Squaliobarbus curriculus), and zebrafish (Danio rerio) [13-15]. In vertebrates, the orthologs of NCAM1 typically contain two or more immunoglobulin (Ig)-like domains, two fibronectin type III (FN3) domains, a transmembrane (TM) region, and a cytoplasmic tail. Similarly, the amino acid sequence of CgNCAM1 identified from C. gigas also contained an extracellular region with one signal peptide, three Ig domains, one FN3 domain, and a TM domain [10].

In humans, mice and other vertebrates, the intracellular domain of NCAM1 lacks typical structural features, and thus the molecular mechanisms in signal transduction remain unclear. In our study, the intracellular region of CgNCAM1 in C. gigas contains two ITIMs. ITIMs are typically found in inhibitory receptors, such as TIGIT, TIM-3, etc in NK cells [16-20]. The amino acid sequences of NCAM1s, T cell immunoreceptor with Ig and ITIM domains (TIGITs), T cell immunoglobulin and mucin domains (TIMs) and killer immunoglobulin-like receptors (KIRs) from various species were retrieved from NCBI database. The amino acid sequences of CgNCAM1 and other retrieved sequences were used for constructing a phylogenetic tree with neighbor-joining (NJ) method with 1000 bootstrap replicates. Phylogenetic analysis showed that CgNCAM1 belonged to the NCAM1 family, while it exhibited higher homology to immunoglobulin superfamily (IgSF) inhibitory receptors TIGIT from species of vertebrates. Some inhibitory receptors had also been identified in both lower vertebrates and invertebrates [21-25]. The unique protein structure suggests that CgNCAM1 exhibits a functional divergence in that ITIM-dependent signaling enables its negative regulation for innate immune activation.

Vertebrate NCAM1s primarily function in cell adhesion, neurite fasciculation, and outgrowth [26]. In humans, NCAM1 regulates the interaction between T cells and antigen-presenting cells [27] and mediates human NK cell migration [28]. NCAM1 can also promote multiple myeloma (MM)-cell growth, survival and adhesion to stromal cells [29]. In teleosts, NCAM1a, NCAM1b, and their polysialic acid (polySia) modifications played functional roles in axon guidance [14]. Emerging studies reported that NCAM1 could respond to immune stimuli and directly mediate NK cell recognition for Aspergillus fumigatus [30,31]. In the mandarin fish, the expression levels of mfNCAM1a and mfNCAM1b were significantly upregulated after LPS or poly (I:C) stimulation, while their binding capacity to multiple pathogen-associated molecular patterns (PAMPs) remained unverified. In the present study, CgNCAM1 could bind to various microbes (V. splendidus, Escherichia coli, Staphylococcus aureus, Micrococcus luteus, and Pichia pastoris) as well as PAMPs (LPS, Mannan, Peptidoglycan, and Poly(I:C)). As the concentration of rCgNCAM1-3×Ig-FN3 increased from 9.86 μg mL⁻¹ to 315.53 μg mL⁻¹, the P/N values of its binding to LPS, PGN, MAN and Poly (I: C) increased from 3.84 to 12.59, 4.43 to 11.40, 3.79 to 8.52 and 3.70 to 8.94, respectively. These results indicated that CgNCAM1 could function as a pattern recognition receptor (PRR). In vertebrates, NCAM1 expressions are upregulated after viral or bacterial stimulation. The increase of NCAM1 primarily regulates cellular processes such as proliferation, differentiation migration and adhesion, thereby influencing the efficiency and efficacy of immune responses [32,33]. In multiple myeloma (MM)-cell lines, NCAM1 activates p90 ribosomal S6 kinase 2 (RSK2) and induces phosphorylation of cAMP-responsive element-binding protein 1 (CREB1), triggering the transcription of anti-apoptotic genes (such as BCL2, MCL1, etc.) [29]. However, the detailed molecular mechanism by which NCAM1 regulates downstream signaling remains unclear. Our study demonstrated that CgNCAM1 expression was significantly upregulated in hemocytes and translocated from hemocyte cytoplasm to cell membrane after V. splendidus stimulation. After binding to ligands, CgNCAM1 recruited CgSHIP2 to inhibit the P38-H3K4me and P38-NF-κB pathways, reducing the mRNA expressions of CgIL17-2/3/6 and CgTNF-2, and ultimately suppressing inflammation. Both NCAM1 and TIGIT act as inhibitory receptors. However, TIGIT's ligands (e.g., CD155, CD112, and CD113 [34]) have been better characterized. Beyond the differences in downstream pathways, TIGIT was phosphorylated at Tyr225 via its intracellular ITT-like motif (distinct from an ITIM motif). The phosphorylated TIGIT then recruited the adapter Grb2 and SHIP1 to terminate phosphatidylinositol 3-kinase (PI3K) and MAPK signaling, thereby suppressing NK cell activation [35]. Overall, both pathways suppress downstream signaling via phosphatase activity. These findings provide evidence that primitive molluscs have evolved NCAM1-mediated immunosuppressive pathways. It also suggests that there has evolved dual functionality of pathogen sensing coupled with inhibitory control to balance protective immunity and inflammation control in invertebrate immune system.

In vertebrates, NCAM1 is also known as CD56, a well-established surface marker for NK cells. NCAM1/CD56, a well-characterized glycoprotein of IgSF, exhibits expression levels that directly correlate with NK cell activation status. According to CD56 expression levels, human NK cells are classified into two subsets: immature CD56^bright NK cells and mature CD56^dim NK cells [36]. The CD56^bright subset constitutes approximately 10% of circulating NK cells in blood and is primarily localized in lymph nodes. These cells possess immunoregulatory functions and produce cytokines such as tumor necrosis factor-α (TNF-α), TNF-β, interleukin-10 (IL-10), and interferon-γ (IFN-γ) [37]. In contrast, CD56^dim NK cells primarily exert direct cytotoxic effects against both pathogen-infected and tumor cells. These cells express high levels of cytotoxic effector molecules including KIRs, perforin, and granzymes [38]. Furthermore, CD56^bright NK cells can differentiate into CD56^dim NK cells. However, conclusive evidence for NCAM1 as a surface marker of NK/NK-like cells in lower vertebrates and invertebrates is still lacking. The cytotoxic activity of NK cells is co-regulated by surface-expressed killer cell inhibitory receptors (KIRs) [39,40] and killer activation receptors (KARs) [41,42], enabling selective elimination of abnormal cells while protecting normal cells. Both CD56 and these receptors belong to the transmembrane glycoprotein family, and they exhibit pathogen recognition capabilities through ligand binding [30,31]. KIRs include IgSF inhibitory receptors, such as TIGIT, TIM-3, and PD-1 [16-20]. And these inhibitory receptors contain ITIMs in their cytoplasmic tails. Interestingly, CgNCAM1 shares similar structures, possessing extracellular Ig domains and intracellular ITIMs. This discovery suggests that during the early evolution of NK cells, NCAM1 may have served as both a marker molecule for NK-like cells and a functional regulator for their activation in early metazoan immune systems. However, the role of NCAM1 in regulating the cytotoxic function of NK-like cells remains unclear and still needs further to be investigated in future.

The oyster, an important mariculture species in China, faces growing disease-related challenges that threaten the sustainable development of shellfish aquaculture. As essential effectors of the oyster's innate immune system, hemocytes play vital roles in both tissue repair and immune defense. Unlike adaptive immune responses, NK cells can initiate immune defense without antigen-specific stimulation. In mammals, the identification, morphological characteristics, and functions of NK cells are well-characterized, whereas the knowledge on NK cells in invertebrates is still very limited. In this study, we identify CgNCAM1 as the first NCAM1 homolog in invertebrates with ITIM-dependent immunosuppressive function. These findings provide a critical basis for defining and classifying NK-like hemocytes in oysters. Furthermore, this study provides new insights into the activation and regulatory mechanisms of NK cells during early evolution, offers a theoretical basis for understanding the differences between invertebrate NK-like cells and vertebrate NK cells, and provides a new perspective for understanding the origin and functional evolution of immune cells. Based on the established immunoregulatory roles of CgNCAM1, we will investigate CgNCAM1⁺ hemocytes by: (i) developing a high-purity sorting system based on specific surface markers, (ii) quantifying their cytotoxicity against pathogen-infected cells and tumor cells, and (iii) clarifying its evolutionary conservation with vertebrate NK cells. In addition, although we have initially confirmed that CgNCAM1 exerts anti-inflammatory effects through its ITIM, the spatiotemporal characteristics of ITIM phosphorylation and the regulatory details of its recruitment of effector molecules such as SHIP2 still need to be systematically analyzed through methods like site-directed mutagenesis and phosphoproteomics.

This research was supported by National Natural Science Foundation of China (32222086), Liaoning Revitalization Talents Program (XLYC2203087).