Identification and characterization of two related murine genes, Eat2a and Eat2b, encoding single SH2-domain adapters

Human EAT-2 (SH2D1B) and SLAM-associated protein (SAP) (SH2D1A) are single SH2-domain adapters, which bind to specific tyrosine residues in the cytoplasmic tail of six signaling lymphocytic activation molecule (SLAM) (SLAMF1)-related receptors. Here we report that, unlike in humans, the mouse and rat Eat2 genes are duplicated with an identical genomic organization. The coding regions of the mouse Eat2a and Eat2b genes share 91% identity at the nucleotide level and 84% at the protein level; similarly, segments of introns are highly conserved. Whereas expression of mouse Eat2a mRNA was detected in multiple tissues, Eat2b was only detectable in mouse natural killer cells, CD8⁺ T cells, and ovaries, suggesting a very restricted tissue expression of the latter. Both the EAT-2A and EAT-2B coimmunoprecipitated with mouse SLAM in transfected cells and augmented tyrosine phosphorylation of the cytoplasmic tail of SLAM. Both EAT-2A and EAT-2B bind to the Src-like kinases Fyn, Hck, Lyn, Lck, and Fgr, as determined by a yeast two-hybrid assay. However, unlike SAP, the EAT-2 proteins bind to their kinase domains and not to the SH3 domain of these kinases. Taken together, the data suggest that both EAT-2A and EAT-2B are adapters that recruit Src kinases to SLAM family receptors using a mechanism that is distinct from that of SAP. Electronic supplementary material Supplementary material is available for this article at and accessible for authorised users. S. Calpe and E. Erdős contributed equally to this work     

Immune regulation by SLAM family receptors and SAP-related adaptors

The signalling lymphocytic activation molecule (SLAM) family of receptors is expressed by a wide range of immune cells. Through their cytoplasmic domain, SLAM family receptors associate with SLAM-associated protein (SAP)-related molecules, a group of cytoplasmic adaptors composed almost exclusively of an SRC homology 2 domain. SAP, the prototype of the SAP family, is mutated in a human immunodeficiency named X-linked lymphoproliferative (XLP) disease. Recent observations indicate that SLAM family receptors, in association with SAP family adaptors, have crucial roles during normal immune reactions in innate and adaptive immune cells. The latest progress in this field is reviewed here.     

Identification and characterization of SF2000 and SF2001, two new members of the immune receptor SLAM/CD2 family

The SLAM family of human genes currently consists of seven related members of the immunoglobulin superfamily, membrane-associated proteins, including CD150 (SLAM), CD244 (2B4), CD84, CD229 ( Ly-9), BLAME, CD48, and 19A. These genes are expressed to varying degrees in subsets of immune cells (T, B, natural killer, and myeloid cells) and may function as ligands or receptors. This set of genes, related to CD2 and CD58 on Chromosome (Chr) 1p98, are found clustered close together in the human genome on Chr 1q22. Four of these family members (CD150, CD244, CD84, CD229) contain conserved tyrosine motifs in their cytoplasmic tails that enable them to bind intracellular signaling molecules SAP and EAT-2. SAP is mutated in human X-linked lymphoproliferative disease (XLP), and studies in XLP patients have shown that improper signaling via molecules that bind SAP contributes to the disease. We have identified two new members of the SLAM family (SF), which we term SF2000 and SF2001, which are expressed in immune cells and map in the SLAM gene cluster. SF2001 does not contain SAP-binding motifs in its short cytoplasmic tail. SF2000, which is co-expressed with SAP in T cells, binds both SAP and EAT-2. The data suggest that signaling through SF2000, together with CD150, CD244, CD84, and CD229, is controlled by SAP and therefore contributes to the pathogenesis of XLP.     

Direct and indirect interactions of the cytoplasmic region of CD244 (2B4) in mice and humans with FYN kinase

Engagement of the receptor CD244 (2B4) by its ligand CD48 has inhibitory and activating potential, and this differs depending on experimental systems in mouse and human. We show that, in both mouse and human upon engagement of its ligand CD48, CD244 can give a negative signal to natural killer cells, implying conservation of function between the two species. The signaling mechanisms used by CD244 in both human and mouse are conserved as shown by quantitative analyses of the direct molecular interactions of the SH2 domains of the adaptors SLAM-associated protein (SAP) and EAT-2 and of FYN kinase with CD244 together with the indirect interactions of the FYN SH2 domain with EAT-2. Functional experiments support the biochemical hierarchy of interactions and show that EAT-2 is not inhibitory per se. The data are consistent with a model in which the mechanism of signal transduction by CD244 is to regulate FYN kinase recruitment and/or activity and the outcome of CD48/CD244 interactions is determined by which other receptors are engaged.     

Structural basis for the interaction of the free SH2 domain EAT-2 with SLAM receptors in hematopoietic cells.

The T and natural killer (NK) cell-specific gene SAP (SH2D1A) encodes a 'free SH2 domain' that binds a specific tyrosine motif in the cytoplasmic tail of SLAM (CD150) and related cell surface proteins. Mutations in SH2D1A cause the X-linked lymphoproliferative disease, a primary immunodeficiency. Here we report that a second gene encoding a free SH2 domain, EAT-2, is expressed in macrophages and B lympho cytes. The EAT-2 structure in complex with a phosphotyrosine peptide containing a sequence motif with Tyr281 of the cytoplasmic tail of CD150 is very similar to the structure of SH2D1A complexed with the same peptide. This explains the high affinity of EAT-2 for the pTyr motif in the cytoplasmic tail of CD150 but, unlike SH2D1A, EAT-2 does not bind to non-phosphorylated CD150. EAT-2 binds to the phosphorylated receptors CD84, CD150, CD229 and CD244, and acts as a natural inhibitor, which interferes with the recruitment of the tyrosine phosphatase SHP-2. We conclude that EAT-2 plays a role in controlling signal transduction through at least four receptors expressed on the surface of professional antigen-presenting cells.     

SLAM-Family Receptors: Immune Regulators with or without SAP-Family Adaptors

The signaling lymphocytic activation molecule (SLAM) family of receptors and the SLAM-associated protein (SAP) family of intracellular adaptors are expressed in immune cells. By way of their cytoplasmic domain, SLAM-related receptors physically associate with SAP-related adaptors. Evidence is accumulating that the SLAM and SAP families play crucial roles in multiple immune cell types. Moreover, the prototype of the SAP family, that is SAP, is mutated in a human immunodeficiency, X-linked lymphoproliferative (XLP) disease. In the presence of SAP-family adaptors, the SLAM family usually mediates stimulatory signals that promote immune cell activation or differentiation. In the absence of SAP-family adaptors, though, the SLAM family undergoes a “switch-of-function,” thereby mediating inhibitory signals that suppress immune cell functions. The molecular basis and significance of this mechanism are discussed herein.Immune cells undergo differentiation and, once mature, are activated through the integrated actions of many molecules, including cell surface receptors and intracellular signaling effectors. Whereas some of these molecules have “primary” roles in the immune response, others have secondary, albeit still critical, functions in this process. For example, differentiation and activation of B cells are strictly dependent on the function of the B-cell receptor (BCR) and its intracellular effectors. Other receptors present on B cells, such as CD19 and CD40, influence B-cell functions in critical ways, by modulating BCR-triggered signals (Cambier et al. 1994).There is accumulating evidence that the signaling lymphocytic activation molecule (SLAM) family of receptors plays important roles in immunity (Schwartzberg et al. 2009; Ma et al. 2007; Veillette et al. 2007; Veillette 2006b; Veillette 2006a). This class of receptors provides key effects in multiple immune cell types. Recent data indicate that SLAM-family receptors can either promote or inhibit the functions of primary activating receptors (Cruz-Munoz et al. 2009; Dong et al. 2009). These alternative activities are controlled by whether or not SLAM-related receptors are coexpressed with members of the SLAM-associated protein (SAP) family of intracellular adaptor molecules. The functions and mechanisms of action of the SLAM and SAP families are reviewed herein.     

SAP couples Fyn to SLAM immune receptors

SAP (SLAM-associated protein) is a small lymphocyte-specific signalling molecule that is defective or absent in patients with X-linked lymphoproliferative syndrome (XLP). Consistent with its single src homology 2 (SH2) domain architecture and unusually high affinity for SLAM (also called CD150), SAP has been suggested to function by blocking binding of SHP-2 or other SH2-containing signalling proteins to SLAM receptors. Additionally, SAP has recently been shown to be required for recruitment and activation of the Src-family kinase FynT after SLAM ligation. This signalling 'adaptor' function has been difficult to conceptualize, because unlike typical SH2-adaptor proteins, SAP contains only a single SH2 domain and lacks other recognized protein interaction domains or motifs. Here, we show that the SAP SH2 domain binds to the SH3 domain of FynT and directly couples FynT to SLAM. The crystal structure of a ternary SLAM-SAP-Fyn-SH3 complex reveals that SAP binds the FynT SH3 domain through a surface-surface interaction that does not involve canonical SH3 or SH2 binding interactions. The observed mode of binding to the Fyn-SH3 domain is expected to preclude the auto-inhibited conformation of Fyn, thereby promoting activation of the kinase after recruitment. These findings broaden our understanding of the functional repertoire of SH3 and SH2 domains.     

Increased expression of SLAM receptors SLAMF3 and SLAMF6 in systemic lupus erythematosus T lymphocytes promotes Th17 differentiation

Altered T cell function in systemic lupus erythematosus (SLE) is determined by various molecular and cellular abnormalities, including increased IL-17 production. Recent evidence suggests a crucial role for signaling lymphocyte activation molecules (SLAMs) in the expression of autoimmunity. In this study, we demonstrate that SLAMF3 and SLAMF6 expression is increased on the surface of SLE T cells compared with normal cells. SLAM coengagement with CD3 under Th17 polarizing conditions results in increased IL-17 production. SLAMF3 and SLAMF6 T cell surface expression and IL-17 levels significantly correlate with disease activity in SLE patients. Both naive and memory CD4(+) T cells produce more IL-17 in response to SLAM costimulation as compared with CD28 costimulation. In naive CD4(+) cells, IL-17 production after CD28 costimulation peaks on day 3, whereas costimulation with anti-SLAMF3 and anti-SLAMF6 Abs results in a prolonged and yet increasing production during 6 d. Unlike costimulation with anti-CD28, SLAM costimulation requires the presence of the adaptor molecule SLAM-associated protein. Thus, engagement of SLAMF3 and SLAMF6 along with Ag-mediated CD3/TCR stimulation represents an important source of IL-17 production, and disruption of this interaction with decoy receptors or blocking Abs should mitigate disease expression in SLE and other autoimmune conditions.     

Functional requirements for interactions between CD84 and Src homology 2 domain-containing proteins and their contribution to human T cell activation

Cell surface receptors belonging to the CD2 subset of the Ig superfamily of molecules include CD2, CD48, CD58, 2B4, signaling lymphocytic activation molecule (SLAM), Ly9, CD84, and the recently identified molecules NTB-A/Ly108/SLAM family (SF) 2000, CD84H-1/SF2001, B lymphocyte activator macrophage expressed (BLAME), and CRACC (CD2-like receptor-activating cytotoxic cells)/CS-1. Some of these receptors, such as CD2, SLAM, 2B4, CRACC, and NTB-A, contribute to the activation and effector function of T cells and NK cells. Signaling pathways elicited via some of these receptors are believed to involve the Src homology 2 (SH2) domain-containing cytoplasmic adaptor protein SLAM-associated protein (SAP), as it is recruited to SLAM, 2B4, CD84, NTB-A, and Ly-9. Importantly, mutations in SAP cause the inherited human immunodeficiency X-linked lymphoproliferative syndrome (XLP), suggesting that XLP may result from perturbed signaling via one or more of these SAP-associating receptors. We have now studied the requirements for SAP recruitment to CD84 and lymphocyte activation elicited following ligation of CD84 on primary and transformed human T cells. CD84 was found to be rapidly tyrosine phosphorylated following receptor ligation on activated T cells, an event that involved the Src kinase Lck. Phosphorylation of CD84 was indispensable for the recruitment of SAP, which was mediated by Y(262) within the cytoplasmic domain of CD84 and by R(32) within the SH2 domain of SAP. Furthermore, ligating CD84 enhanced the proliferation of anti-CD3 mAb-stimulated human T cells. Strikingly, this effect was also apparent in SAP-deficient T cells obtained from patients with XLP. These results reveal a novel function of CD84 on human lymphocytes and suggest that CD84 can activate human T cells via a SAP-independent mechanism.     

Immune Cell Inhibition by SLAMF7 Is Mediated by a Mechanism Requiring Src Kinases,CD45, and SHIP-1 That Is Defective in Multiple Myeloma Cells

Signaling lymphocytic activation molecule F7 (SLAMF7) is a receptor present on immune cells, including natural killer (NK) cells. It is also expressed on multiple myeloma (MM) cells. This led to development of an anti-SLAMF7 antibody, elotuzumab, showing efficacy against MM. SLAMF7 mediates activating or inhibitory effects in NK cells, depending on whether cells express or do not express the adaptor EAT-2. Since MM cells lack EAT-2, we elucidated the inhibitory effectors of SLAMF7 in EAT-2-negative NK cells and tested whether these effectors were triggered in MM cells. SLAMF7-mediated inhibition in NK cells lacking EAT-2 was mediated by SH2 domain-containing inositol phosphatase 1 (SHIP-1), which was recruited via tyrosine 261 of SLAMF7. Coupling of SLAMF7 to SHIP-1 required Src kinases, which phosphorylated SLAMF7. Although MM cells lack EAT-2, elotuzumab did not induce inhibitory signals in these cells. This was at least partly due to a lack of CD45, a phosphatase required for Src kinase activation. A defect in SLAMF7 function was also observed in CD45-deficient NK cells. Hence, SLAMF7-triggered inhibition is mediated by a mechanism involving Src kinases, CD45, and SHIP-1 that is defective in MM cells. This defect might explain why elotuzumab eliminates MM cells by an indirect mechanism involving the activation of NK cells.     

The Adaptor Protein SAP Directly Associates with CD3ζ Chain and Regulates T Cell Receptor Signaling

Mutations altering the gene encoding the SLAM associated protein (SAP) are responsible for the X-linked lymphoproliferative disease or XLP1. Its absence is correlated with a defective NKT cells development, a decrease in B cell functions and a reduced T cells and NK cells cytotoxic activities, thus leading to an immunodeficiency syndrome. SAP is a small 128 amino-acid long protein that is almost exclusively composed of an SH2 domain. It has been shown to interact with the CD150/SLAM family of receptors, and in a non-canonical manner with SH3 containing proteins such as Fyn, βPIX, PKCθ and Nck1. It would thus play the role of a minimal adaptor protein. It has been shown that SAP plays an important function in the activation of T cells through its interaction with the SLAM family of receptors. Therefore SAP defective T cells display a reduced activation of signaling events downstream of the TCR-CD3 complex triggering. In the present work, we evidence that SAP is a direct interactor of the CD3ζ chain. This direct interaction occurs through the first ITAM of CD3ζ, proximal to the membrane. Additionally, we show that, in the context of the TCR-CD3 signaling, an Sh-RNA mediated silencing of SAP is responsible for a decrease of several canonical T cell signaling pathways including Erk, Akt and PLCγ1 and to a reduced induction of IL-2 and IL-4 mRNA. Altogether, we show that SAP plays a central function in the T cell activation processes through a direct association with the CD3 complex.     

Dual functional roles for the X-linked lymphoproliferative syndrome gene product SAP/SH2D1A in signaling through the signaling lymphocyte activation molecule (SLAM) family of immune receptors

The X-linked lymphoproliferative (XLP) syndrome gene encodes a protein named SAP or SH2D1A that is composed of a single Src homology 2 (SH2) domain. Two models have been proposed for its function in lymphocyte signaling. One postulates that it acts as an inhibitor of interactions between the phosphatase SHP-2 and the immune receptor SLAM. The other suggests that it functions as an adaptor to promote the recruitment of a kinase, FynT, to SLAM. Here, we provide evidence in support of both roles for SAP. Using an array of peptides derived from the SLAM family of receptors, we demonstrate that SAP binds with comparable affinities to the same sites in these receptors as do the SH2 domains of SHP-2 and SHIP, suggesting that these three proteins may compete against one another in binding to a given SLAM family receptor. Furthermore, in vitro and in vivo binding studies indicate that SAP is capable of binding directly to FynT, an interaction mediated by the FynT SH3 domain. In cells, FynT was shown to be indispensable for SLAM tyrosine phosphorylation, which, in turn, was drastically enhanced by SAP. Because SAP also blocked the recruitment of SHP-2 to SLAM in these cells, we propose a dual functional role for SAP in SLAM signaling by acting both as an adaptor for FynT and an inhibitor to SHP-2 binding. The physiological relevance of the dual functional role for SAP is underscored by the observation that disease-causing SAP mutants exhibited significantly reduced affinities to both FynT and SLAM.     

Association between SAP and FynT: Inducible SH3 domain-mediated interaction controlled by engagement of the SLAM receptor

SAP is an intracellular adaptor molecule composed almost exclusively of an SH2 domain. It is mutated in patients with X-linked lymphoproliferative disease, a human immunodeficiency. Several immune abnormalities were also identified in SAP-deficient mice. By way of its SH2 domain, SAP interacts with tyrosine-based motifs in the cytoplasmic domain of SLAM family receptors. SAP promotes SLAM family receptor-induced protein tyrosine phosphorylation, due to its capacity to recruit the Src-related kinase FynT. This unusual property relies on the existence of a second binding surface in the SAP SH2 domain, centered on arginine 78 of SAP, that binds directly to the FynT SH3 domain. Herein, we wanted to further understand the mechanisms controlling the interaction between SLAM-SAP and FynT. Our experiments showed that, unlike conventional associations mediated by SH3 domains, the interaction of the FynT SH3 domain with SLAM-SAP was strictly inducible. It was absolutely dependent on engagement of SLAM by extracellular ligands. We obtained evidence that this inducibility was not due to increased binding of SLAM to SAP following SLAM engagement. Furthermore, it could occur independently of any appreciable SLAM-dependent biochemical signal. In fact, our data indicated that the induced association of the FynT SH3 domain with SLAM-SAP was triggered by a change in the conformation of SLAM-associated SAP caused by SLAM engagement. Together, these data elucidate further the events initiating SLAM-SAP signaling in immune cells. Moreover, they identify a strictly inducible interaction mediated by an SH3 domain.     

Crystal structures of the XLP protein SAP reveal a class of SH2 domains with extended, phosphotyrosine-independent sequence recognition.

SAP, the product of the gene mutated in X-linked lymphoproliferative syndrome (XLP), consists of a single SH2 domain that has been shown to bind the cytoplasmic tail of the lymphocyte coreceptor SLAM. Here we describe structures that show that SAP binds phosphorylated and nonphosphorylated SLAM peptides in a similar mode, with the tyrosine or phosphotyrosine residue inserted into the phosphotyrosine-binding pocket. We find that specific interactions with residues N-terminal to the tyrosine, in addition to more characteristic C-terminal interactions, stabilize the complexes. A phosphopeptide library screen and analysis of mutations identified in XLP patients confirm that these extended interactions are required for SAP function. Further, we show that SAP and the similar protein EAT-2 recognize the sequence motif TIpYXX(V/I).     

The SAP and SLAM families in immune responses and X-linked lymphoproliferative disease

SAP (signalling lymphocytic activation molecule (SLAM)-associated protein) is a T- and natural killer (NK)-cell-specific protein containing a single SH2 domain encoded by a gene that is defective or absent in patients with X-linked lymphoproliferative syndrome (XLP). The SH2 domain of SAP binds with high affinity to the cytoplasmic tail of the haematopoietic cell-surface glycoprotein SLAM and five related receptors. SAP regulates signal transduction of the SLAM-family receptors by recruiting SRC kinases. Similarly, the SAP-related proteins EAT2A and EAT2B are thought to control signal transduction that is initiated by SLAM-related receptors in professional antigen-presenting cells. In this review, we discuss recent findings on the structure and function of proteins of the SAP and SLAM families.     

The isolation, characterization, and molecular cloning of a 75-kDa gelatinase B-like enzyme, a member of the matrix metalloproteinase (MMP) family. An avian enzyme that is MMP-9-like in its cell expression pattern but diverges from mammalian gelatinase B in sequence and biochemical properties

We have isolated a novel 75-kDa gelatinase from a chicken macrophage cell line, HD11. Biochemical and immunological characterization of the purified enzyme demonstrated that it is distinct from the chicken 72-kDa gelatinase A (MMP-2). The enzyme is capable of specific gelatin binding and rapid gelatin cleavage. Incubation with an organomercurial compound (p-aminophenylmercuric acetate) induces proteolytic processing and activation of this enzyme, and the resultant gelatinolytic activity is sensitive to both zinc chelators and tissue inhibitors of metalloproteinases. A full-length cDNA for the enzyme has been cloned, and sequence analysis demonstrated that the enzyme possesses the characteristic multidomain structure of an MMP gelatinase including a cysteine switch prodomain, three fibronectin type II repeats, a catalytic zinc binding region, and a hemopexin-like domain. The 75-kDa gelatinase is produced by phorbol ester-treated chicken bone marrow cells, monocytes, and polymorphonuclear leukocytes, cell types that charac- teristically produce the 92-kDa mammalian gelatinase B (MMP-9). The absence of a 90-110-kDa gelatinase in these cell types indicates that the 75-kDa gelatinase is likely the avian counterpart of gelatinase B. However, the protein is only 59% identical to human gelatinase B, whereas all previously cloned chicken MMP homologues are 75-90% identical to their human counterparts. In addition, the new 75-kDa chicken gelatinase lacks the type V collagen domain that is found in all mammalian gelatinase Bs. Furthermore, the secreted enzyme appears structurally distinct from known gelatinase Bs and the activated enzyme can cleave fibronectin, which is not a substrate for mammalian gelatinase B. Thus the results of this study indicate that a second MMP gelatinase exists in chickens, and although it is MMP-9/gelatinase B-like in its overall domain structure and expression pattern, it appears to be biochemically divergent from mammalian gelatinase B.     

Molecular pathogenesis of EBV susceptibility in XLP as revealed by analysis of female carriers with heterozygous expression of SAP

X-linked lymphoproliferative disease (XLP) is a primary immunodeficiency caused by mutations in SH2D1A which encodes SAP. SAP functions in signalling pathways elicited by the SLAM family of leukocyte receptors. A defining feature of XLP is exquisite sensitivity to infection with EBV, a B-lymphotropic virus, but not other viruses. Although previous studies have identified defects in lymphocytes from XLP patients, the unique role of SAP in controlling EBV infection remains unresolved. We describe a novel approach to this question using female XLP carriers who, due to random X-inactivation, contain both SAP(+) and SAP(-) cells. This represents the human equivalent of a mixed bone marrow chimera in mice. While memory CD8(+) T cells specific for CMV and influenza were distributed across SAP(+) and SAP(-) populations, EBV-specific cells were exclusively SAP(+). The preferential recruitment of SAP(+) cells by EBV reflected the tropism of EBV for B cells, and the requirement for SAP expression in CD8(+) T cells for them to respond to Ag-presentation by B cells, but not other cell types. The inability of SAP(-) clones to respond to Ag-presenting B cells was overcome by blocking the SLAM receptors NTB-A and 2B4, while ectopic expression of NTB-A on fibroblasts inhibited cytotoxicity of SAP(-) CD8(+) T cells, thereby demonstrating that SLAM receptors acquire inhibitory function in the absence of SAP. The innovative XLP carrier model allowed us to unravel the mechanisms underlying the unique susceptibility of XLP patients to EBV infection in the absence of a relevant animal model. We found that this reflected the nature of the Ag-presenting cell, rather than EBV itself. Our data also identified a pathological signalling pathway that could be targeted to treat patients with severe EBV infection. This system may allow the study of other human diseases where heterozygous gene expression from random X-chromosome inactivation can be exploited.     

SLAMF8, a potential new immune checkpoint molecule,is associated with the prognosis of colorectal cancer

Recently, immune checkpoint inhibitors (ICIs), such as programmed cell death 1 (PD-1) monoclonal antibodies (mAbs), have revolutionized the treatment of malignant tumors. Therefore, the number of studies aiming to screen and identify new immune checkpoint molecules for antitumor immunotherapy is increasing. Signaling lymphocytic activation molecule (SLAM) family members are mainly expressed by and regulate the functions of immune cells. Recent studies have shown that several SLAM family members are involved in the regulation of the tumor immune microenvironment and are promising targets for antitumor immunotherapy. Signaling lymphocytic activation molecule family member 8 (SLAMF8) is a type I cell surface glycoprotein and is encoded on chromosome 1q21. To further illustrate the clinical value of SLAMF8 in colorectal cancer (CRC), we retrospectively analyzed the relationship between SLAMF8 expression and the prognosis of CRC patients and the associations between SLAMF8 expression and the expression levels of other SLAM family members and other classic immune checkpoint molecules using The Cancer Genome Atlas (TCGA) data, RNA sequencing data, tissue immunohistochemistry staining, and systematic follow-up analysis. Here, high SLAMF8 expression was associated with poor overall survival (OS) in CRC. The mRNA expression level of SLAMF8 was positively correlated with the expression levels of multiple classic immune checkpoints and other SLAM family members. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis suggested that the pathways enriched in CRC tissues with high SLAMF8 expression were associated with the regulation of the tumor immune microenvironment.