2.1.1 PD-1/PD-L1: Molecular Mechanisms and Therapeutic Potential in Cancer Immunotherapy

PD-1, also known as CD279, is an immune checkpoint receptor belonging to the B7-CD28 family [41]. It shares a 15% similarity in amino acid sequences to CD28 [42]. The discovery of PD-1 occurred in 1992, in a mouse T-cell hybridoma (2B4-11) cell line and in mouse hematopoietic progenitor cells (LyD9) lacking IL-3 [42, 43]. PD-1 can phosphorylate these two motifs, and they can also recruit the Src homology region 2 structural domain containing SHP-1 and SHP-2 [44]. PD-1 plays an essential function in both innate and adaptive immune reactions and is expressed on a wide range of immune cells within the TME [45], including activated T cells, B cells, NK cells, macrophages, DCs, and monocytes [43]. It is particularly strongly expressed on tumor-specific T cells [46]. PD-1 is the most extensively studied immunosuppressive factor on the surface of T cells [47].

PD-L1 is a ligand for PD-1, also known as CD274 and B7-H1, which is a member of the B7 family [42]. In 2002, research first showed that antibodies against PD-L1 could inhibit tumor growth and that PD-L1 could suppress T-cell proliferation, thereby confirming PD-L1's role in tumor immune evasion [48]. Its extracellular portion is made up of 290 amino acids [49]. PD-L1 is expressed on the surface of many immune cells [50, 51]. It is also expressed on the surface of nonhematopoietic cells, such as vascular endothelial cells, pancreatic islet cells, and placental cells [52]. Studies have confirmed that PD-L1 can also be expressed on the surface of various tumor cells, such as hematological malignancies and non-small cell lung cancer (NSCLC) [53, 54]. In addition, PD-L1 expression can be regulated by a variety of factors. For instance, For example, PD-L1 can be upregulated by TNF-α, vascular endothelial growth factor (VEGF) [55], type I and type II interferons, as well as IFN-α and IFN-β can all upregulate PD-L1 [53, 56]. TNF-α also upregulates PD-L1 expression through activation of the NF-κB pathway [57]. In addition, the JAK-STAT1-IRF1 signaling pathway, JAK-STAT3, or MEK/ERK signaling pathway all upregulated PD-L1 expression [57].

The interaction between PD-1 and PD-L1 can inhibit TCR signaling, suppress the immune response of T cells to tumors, and mediate tumor immune evasion (Figure 2A) [58, 59]. After binding to PD-1 on the surface of T cells, PD-L1 induces the phosphorylation of tyrosine in the tyrosine switch motif of PD-1's immune receptor [60]. This subsequently recruits the SHP-2 [61], causing the dephosphorylation of downstream kinases, including splenic tyrosine kinase (Syk) and PI3K [62, 63]. This inhibition affects signaling pathways such as downstream protein kinase B (PKB, also known as AKT) and extracellular regulated protein kinase (ERK) [64], ultimately inhibiting T cell activation. Furthermore, it inhibits transcription of genes and cytokines required for T-cell activation, serving a detrimental function in modulating T-cell activity [65]. Tumor cells that highly express PD-L1 bind to PD-1 on tumor-specific T cells and inhibit their activation, proliferation, and cytokine production [66, 67]. Therefore, blocking the PD-1/PD-L1 signaling pathway can restore the T cell immune cytotoxicity [68], and activate the endogenous antitumor immune response, thus exerting antitumor effects (Figure 2B).

Not only does the PD-1/PD-L1 axis have an immunosuppressive effect on T cells, but it is also essential for TAMs. PD-1 is highly expressed on TAMs, and its expression is positively correlated with tumor volume [69, 70]. Results from bone marrow transplantation experiments have shown that circulating leukocytes are the main source of PD-1 on TAMs [71, 72]. Moreover, the phagocytosis of TAMs was found to be reduced as PD-1 expression increased [73, 74]. The findings indicate that PD-1 significantly hinders the phagocytosis of TAMs [71]. Furthermore, the PD-1/PD-L1 axis suppresses macrophage activation and proliferation by blocking the mechanism of mTOR signaling in macrophages [69]. Through the Erk/Akt/mTOR regulation pathways, PD-L1 stimulates M2-type macrophage polarization and inhibits the release of pro-inflammatory cytokines and co-stimulatory molecules (CD86, MHC-II) [75]. It is noteworthy that the absence of PD-L1 has a significant impact on phagocytosis in PD-1-containing TAMs [75, 76]. PD-L1 membrane expression in these cellular populations enables engagement with macrophage-resident PD-1 receptors, thereby suppressing phagocytic functionality [77, 78]. However, it has been observed that blocking PD-L1 on the surface of extracellular cells secreted by TP53 mutant tumor cells can mitigate this inhibitory effect [69]. All of these results highlight the PD-1/PD-L1 axis's crucial function in macrophage phagocytosis. PD-L1 is expressed on DCs and can bind to PD-1 to inhibit T cell activation. It has been shown that DCs in human ovarian cancer can also express PD-1, which inhibits NF-κB activation through a SHP-2-dependent mechanism, leading to immunosuppression [79]. The combination of PD-1 and PD-L1 inhibits CD8⁺ T-cell activity and infiltration, reduces MHC I expression, and decreases cytokine secretion (e.g., IL-12, TNF-α, and IL-1), resulting in impairments of T-cell expansion and cytotoxic responses [80, 81]. In addition, PD-1 is expressed in peripheral B-cell subsets [81]. The presence of PD-1 can regulate B cell stimulation, expansion, and immune tolerance. Upon binding to PD-L1, SHP-2 is recruited and causes dephosphorylation of the BCR signaling molecule, which inhibits B cell activation, proliferation, and cytokine production [81]. Targeting the PD-1/PD-L1 axis can help restore immune cell function.

2.1.2 TIM-3: Surface Molecule on Immune Cells

T cell immunoglobulin (Ig) and mucin domain 3 (TIM-3) is a member of the TIM gene family [82], characterized by its presence as a cell surface molecule containing Ig and mucin domain, a type I transmembrane protein [83]. TIM-3 was mainly expressed on CD8⁺ T cells and Th1 cells [84]. In TME, TIM-3 is highly expressed on CD8⁺ tumor-infiltrating lymphocytes and CD4⁺ regulatory T cells. At the same time, TIM-3 is also expressed on innate immune cells such as DCs, macrophages, monocytes, and mast cells, and its expression is higher in M2 macrophages [85]. Functional fatigue is linked to elevated TIM-3 expression in NK cells [86]. The initial discovery of TIM-3 was made in 2001 in the course of examining genes related to asthma susceptibility in inbred mice with a congenital background. Its isoforms were later identified in 2003 [87], and since then, many studies have been conducted on TIM-3's function as an immunological checkpoint [88]. Its extracellular structural domains consist of a membrane-distal N-terminal Ig domain [89], a membrane-proximal mucin domain (which contains a site for O-linked glycosylation), and a peptide linker in the mucin and transmembrane structural domains that contains a site for N-linked sugars [84, 90]. It also has a cytoplasmic tail and a transmembrane structural domain [91]. T Five tyrosine residues are found in the cytoplasmic tail; Y256 and Y263 are essential for TIM-3 signaling because of their association with HLA-B-associated transcript 3 (Bat3) [92]. When there is no ligand binding, Bat3 can bind to the cytoplasmic tail and catalytically activate lymphocyte-specific protein tyrosine kinase (Lck), leading to the phosphorylation of the CD3ζ subunit of the TCR [93, 94]. This process promotes T cell proliferation and activation. When TIM-3 is bound to its ligand, Bat3 dissociates from the cytoplasmic tail, resulting in the phosphorylation of Y256 and Y263, Lck inactivation, and subsequent inhibition of T cell proliferation and activation [95]. TIM-3 is predominantly expressed on T helper 1 (Th1) cells rather than T helper 2 (Th2) cells among T cells [96]. Research has indicated that TIM-3 is also present on various tumor cells, including melanoma, NSCLC, hepatocellular carcinoma (HCC), and prostate cancer [97].

Current research has revealed the identification of four ligands for TIM-3. The primary ligand is galectin-9 (Gal-9) [98], which is widely expressed and considered the most significant ligand for TIM-3. High-mobility group protein B1 (HMGB1) includes carcinoembryonic antigen cell adhesion molecule 1 (Ceacam-1) and phosphatidylserine (PtdSer) is the second ligand [99, 100]. TIM-3 binds to these ligands, thereby suppressing the expansion and stimulation of adaptive immune cells (Figure 2A). In addition to these ligands, retinoic acid-inducible gene I (RIG-I) can also interact with TIM-3 [101]. Furthermore, TIM-3 attenuates RIG-I expression on macrophages in response to signal transducer and activator of transcription 1 (STAT1) [102]. It encourages RIG-I ubiquitination and destruction through RNF-122, an E3 ubiquitin ligase, leading to reduced IFN production and inhibition of antiviral activity [101, 103].

Gal-9 is a soluble protein with two tandem carbohydrate-recognition domains that recognize N-linked sugar chains of the TIM-3 immunoglobulin variable (IgV) structural domain and bind specifically to TIM-3 [83]. The activity of TIM-3-Gal-9 promotes the death of effector Th1 cells, thereby suppressing tissue inflammation and the autoimmune disease multiple sclerosis (EAE) [104]. Furthermore, the binding of TIM-3 and Gal-9 alters the production of cytokines (IL-12 and IL-23) [105], which in turn affects the Th1 response. Additionally, TIM-3 mediates T cell dysfunction and depletion by binding to Gal-9 [106]. In order for nucleic acids to translocate to endosomal cells, activated DCs release HMGB1, a damage-associated molecular pattern (DAMP) protein [107]. The interaction between TIM-3 and HMGB1 prevents nucleic acid translocation to nuclear endosomes, leading to suppression of immune responses mediated by pattern recognition receptors (PRRs) to nucleic acids from tumors. [99]. Therefore, blocking the inhibition of the nucleic acid sensing system induced by the binding of TIM-3 and HMGB1 contributes to DNA vaccine research and cytotoxic chemotherapy [99]. PtdSer functions as a non-protein ligand for TIM-3, and its binding to TIM-3 is linked to the uptake of apoptotic cells [100]. Furthermore, the co-expression of the cytokine IL-10 with T cells that express TIM-3 results in the induction of IL-10 in T cells when combined with PtdSer [108]. There are structural similarities between the membrane-distal IgV domains of TIM-3 and Ceacam-1 [109]. Co-expression of Ceacam-1 is crucial for the glycosylation and protein stability of TIM-3. Defective expression of Ceacam-1 impedes the inhibitory function of TIM-3 [100, 109]. Additionally, Ceacam1-TIM-3 transregulatory inhibition of effector T cell function plays a role in maintaining T cell tolerance.

2.1.3 V-Domain Immunoglobulin Suppressor of T Cell Activation (VISTA): A Checkpoint With Emerging Roles in Antigen Presentation and Tumor Immune Evasion

VISTA is currently a focal point in immune checkpoint research. VISTA [110], additionally referred to as VSIR, SISP1, B7H5, PD-1H, DD1α, Gi24, and Dies1, is a member of the B7 family and exhibits strong similarities to PD-L1 [111, 112]. Unlike other members, VISTA proteins contain only one IgV-like structural domain [113]. In contrast to this singular domain structure of VISTA proteins, other B7 family members possess an additional IgC-like structural domain alongside the IgV-like structural domain [114, 115]. This unique feature renders VISTA more receptor-like in nature compared to other molecular structures within the B7 family, which generally act as ligands [116]. The IgV structural domain of VISTA is characterized by a canonical disulfide bond between the putative B and F domains [117, 118]. In contrast to the B7 family, VISTA does not contain the ITIM/ITAM motifs typical of the B7 family in its cytoplasmic structural domains [119]. Instead, it contains potential latent protein kinase C-binding sites and proline-rich motifs that serve as platforms for interactions with other complexes [112, 117]. Within leukocytes [120], monocytes, macrophages, DCs, and bone marrow cells (neutrophils and microglia) all express the VISTA protein [117, 121]. In the T lymphocyte compartment, VISTA protein expression levels were highest on naive CD4⁺ and Foxp3⁺ regulatory T cells [121].

VISTA has a complex function in immunomodulation, with studies demonstrating that it functions not only as a co-inhibitory receptor for T cells but also as a ligand expressed on APCs [111, 122]. The majority of current research has focused on VISTA's function as an inhibitory receptor on T cells, where it serves to negatively modulate the immune response [123]. The recombinant VISTA Ig fusion protein was shown to suppress CD4⁺ T cell proliferation and the cytokine production of IL-2 and IFN-γ in vitro [124]. Additionally, it promoted the conversion of naive T cells into FoxP3⁺ regulatory T cells [125]. Furthermore, elevated expression of VISTA on DCs from bone marrow led to decreased cell proliferation and cytokine production [111, 112, 123]. Additionally, the treatment of CD4⁺ T cells with VISTA-specific agonistic antibodies has been shown to inhibit CD4⁺ T cell activation, both in vitro and in vivo [126]. This demonstrates that VISTA functions as a CD4⁺ T cell inhibitory receptor [127, 128]. The ligand for VISTA, immunoglobulin superfamily (IgSF) member 11 (IGSF11), can also be expressed on tumor cells [129]. The interaction between VISTA and IGSF11 inhibits human T cell proliferation and cytokine production [130, 131]. Under acidic conditions, VISTA expressed on TAMs, MDSC, or tumor epithelial cells may interact with T cell-expressed PSGL-1 (P-selectin glycoprotein ligand-1) [132]. This interaction enables VISTA to specifically inhibit immune activation in acidic microenvironments (Figure 2A) [133, 134].

2.2.1 MHC-I: From Classic Antigen Presentation to Novel Regulatory Functions in Immune Homeostasis

The MHC-I plays a crucial role in antigen presentation and the regulation of macrophage phagocytosis [138]. MHC is a term used to describe a group of highly polymorphic gene clusters that encode major histocompatibility antigens in animals. In humans, the MHC is referred to as human leukocyte antigen (HLA) [139], which includes classical HLA-I, HLA-II, and nonclassical HLA-III molecules [140, 141]. In mice, the MHC is known as H-2 [142]. The human HLA is located on the short arm of chromosome 6, while the mouse H-2 is located on chromosome 17. MHC-I is found on the surface of APCs and represents the first human leukocyte antigenic product involved in tumor immune response [143, 144]. It consists of a heterodimer containing two structural domains: an alpha heavy chain and a fully extracellular light chain β2m [145]. The α heavy chain consists of three extracellular structural domains (α1, α2, and α3) as well as one transmembrane structural domain [146]. The α3 structural region is located on the surface of T cells and is responsible for binding to CD8 [147]. The antigen-binding site for MHC-I is formed by the interaction of the α1 and α2 structural domains [147, 148]. β2m is a soluble protein that does not cross cell membranes but instead binds noncovalently to the extracellular portion of the α heavy chain [149].

Binding of MHC-I to leukocyte immunoglobulin-like receptor b1 (LILRB1) isoforms promotes immune evasion, serving as a phagocytic checkpoint in cancer immunotherapy [150-152]. LILRB1 belongs to the class B LIR receptor subfamily and contains multiple cytoplasmic ITIMs [153]. It is also known as CD85J and LIR-1 [154]. LILRB1 is widely distributed and expressed on macrophages, NK cells, DCs, B cells, and T cells [155-157]. LILRB1 is composed of an extracellular Ig-like domain, a transmembrane domain, and a cytoplasmic domain containing ITIM sequences [158]. It triggers inhibitory signals primarily through the ITIM sequences in the cytoplasmic domain, which can also recruit SHIP [154]. The binding of LILRB1 to the α3 structural domain and β2m subunit of MHC-I results in ITIM phosphorylation [159]. Phosphorylated ITIM is activated by Src-family protein tyrosine kinases, which phosphorylate tyrosine residues and then recruit the phosphatase SHIP [160]. The recruitment of SHIP inhibits ITAM tyrosine kinase activity, prompting the engagement of ITAM from the Syk/ZAP70 kinase family [161]. Consequently, this triggers the PI3K/AKT pathway to promote tumor cell proliferation and suppress immune cell function [162]. Compared to CD8⁺ T cells, MHC-I exhibits a higher affinity for LILRB1. The binding of MHC-I to LILRB1 not only triggers inhibitory signals but also hinders MHC-I from presenting antigens to CD8⁺ T cells and stimulates adaptive immune responses (Figure 3A) [163, 164], thereby negatively regulating immune function and promoting tumor immune escape.

2.2.2 CD47-SIRPα: The “Don't Eat Me” Signaling Pathway in Immune Regulation

CD47, commonly referred to as the integrin-associated protein, is a highly glycosylated integral membrane protein with an approximate molecular weight of 50 kDa [165]. It is a member of the Ig family and is expressed across various tissues. CD47 contains a single IgV-like domain at its N-terminus [166]. Within the immune system, CD47 stands out as the sole 5-transmembrane (5-TM) receptor [167]. In 1987, CD47 was initially identified on the surface of red blood cells [168]. Until 2000, CD47 was considered a “self-marker” on murine red blood cells, which interacts with signal regulatory protein alpha (SIRPα) on phagocytes [169]. In 2019, it was confirmed that CD47 acts as a tumor phagocytosis checkpoint by delivering a “don't eat me” signal [170]. In cancer immunotherapy, research on CD47 has primarily focused on the inhibitory role of the CD47-SIRPα axis in phagocytosis [165]. SIRPα was initially identified as a receptor tyrosine kinase that is associated with the inhibitory phosphatases SHP-1 and SHP-2. It consists of three extracellular IgSF structural domains, with the furthest domain being the Ig-variable structural domain [167, 171, 172]. The intracellular structural domains of SIRPα contain immunoreceptor tyrosine motifs, which confer inhibitory receptor properties upon it. The n-terminus of SIRPα and the single lg-V structural domain of CD47 are the key mediators of the mutual engagement of SIRPα and CD47 [169, 173]. Ligation of CD47 to SIRPα promotes the phosphorylation of intracellular ITIM and activates the inhibitory phosphatases SHP-1 and SHP-2 [174]. The dephosphorylation of proteins with tyrosine-activating motifs present on immune receptors is one of the many ways that SHP phosphatases mediate the suppression of immune cell activation [169]. In contrast to the T cell-associated immune checkpoint pathway, which is targeted in cancer immunotherapy, the CD47/SIRP pathway serves as a natural immune checkpoint that inhibits the phagocytic activity and antigen presentation ability of macrophages [169, 175]. Macrophages operate as key effectors in innate immunity as specialized phagocytes that engulf senescent and dead cells within the body [176]. Within tumor tissues, macrophages have the ability to remove tumor cells through phagocytosis. When CD47 binds to SIRPα, it induces phosphorylation of the ITIM tyrosine [177, 178]. Activation of the ITIM through phosphorylation leads to the recruitment and stimulation of SHP-1 and SHP-2, which in turn dephosphorylates several intracellular proteins, leading to the loss of their biological functions [174]. In the end, this results in the suppression of macrophage phagocytosis and antigen presentation functions (Figure 3B) [179]. This function can be utilized in the normal human environment to distinguish between “self” and “non-self” to prevent unintentional injury [180]. When tumor cells that have been phagocytosed bind to IgG through in vivo conditioning, the Fc domain of IgG attaches to Fc receptor (FcR) on the surface of the macrophage, thereby activating the macrophage [181, 182]. Activation of the FcR signaling pathway leads to the formation of a “phagocytic synapse” between tumor cells and phagocytes. This process involves three sequential events: initial contact between tumor cells and phagocytes [183], followed by encirclement of tumor cells by macrophage pseudopods, and ultimately the phagocytosis of tumor cells. The current findings suggest that CD47-SIRPα functions as an immune checkpoint by inhibiting the formation of macrophage pseudopods [178, 184].

In addition to SIRPα, CD47 also binds with significantly lower affinity to SIRPγ. SIRPγ is expressed on human T cells and is believed to play a role in transendothelial migration [185, 186]. Other proteins that interact with CD47 include extracellular matrix proteins, platelet reactive protein-1, and various integrins. These integrins include integrin αVβ3, integrin α2bβ3, integrin α2β1, and integrin α4β1, with which CD47 binds and interacts in cis [187]. CD47 is frequently overexpressed in various hematological and solid tumors, and it is often associated with a poor prognosis [188]. This suggests that tumor cells can avoid being destroyed by phagocytes because of the CD47-SIRPα pathway [189, 190]. Studies in mouse tumor models have suggested that the blockade of SIRPα or CD47, in combination with other ICIs targeting PD-1 or CTLA-4, may potentially have synergistic antitumor effects [191].

2.2.3 Cluster of Differentiation 24 (CD24)-Siglec-10: A Key Player in Immune Regulation and Tumor Escape Mechanisms

In 1978, scientists first discovered CD24 [192], a short peptide containing only 31 amino acids [192, 193]. CD24 has been found to be strongly expressed in specific types of tumor cells, such as those found in ovarian cancer and triple-negative breast cancer [194]. This suggests a potential role for CD24 in the development and progression of these malignancies [195]. A 2019 study revealed CD24's role in suppressing innate immune phagocytic activity, functioning as a tumor-protective signaling marker through macrophage evasion mechanisms. CD24 acts as a mediator of intermolecular interactions at cellular junction sites, facilitating intercellular and matrix adhesion. It is essential for mediating cellular identification, triggering activation pathways, transducing signals, and regulating both multiplication and specialization processes in cells, as well as cellular stretching and motility [196, 197]. CD24, a GPI-anchored surface protein, chiefly resides within plasma membranes across healthy and malignant cellular populations [198]. The function of CD24 in the cell membrane is dependent on its binding protein. Siglec-10, which serves as the ligand for CD24, is a sialic acid-binding immunoglobulin-type lectin (Siglecs) [197]. CD24 evades macrophage phagocytosis by engaging with siglec-10. Siglec-10 is a lectin that specifically binds to α-2, 3- or α-2, 6-linked sialic acid [197]. It is a member of the Siglecs family that functions to inhibit the activation of immune cells. This protein is typically expressed on cells of the innate immune system. Its main function is to regulate the signaling of immune cells [17, 199]. Siglec-10 features a singular transmembrane segment and consists of an extracellular region with a V-set Ig-like domain accompanied by three C2-set domains, along with an intracellular region that possesses an ITIM [200]. Following tyrosine residue modification, the structural motif engages Src homology 2 (SH2)-containing phosphatase effectors, including SHP-1/PTPN6, effectively terminating intracellular communication cascades [201, 202]. CD24, CD52 (target of the therapeutic monoclonal antibody alemtuzumab), and vascular adhesion protein-1 have been described as ligands for Siglec-10 [197, 203]. Studies demonstrate that CD24 engagement with Siglec-10 on innate immune effector cells inhibits detrimental inflammatory activation during pathogenic challenges, including infection, sepsis, hepatic damage, and chronic graft-versus-host disease [204]. Many researchers have characterized CD24-Siglec-10 interaction as a natural immune checkpoint crucial for mediating antitumor immunity and as a potential target for future immunotherapy [203, 205].

CD24 is significantly overexpressed in various tumors compared to normal tissue, while Siglec-10 shows higher expression in TAMs [203]. The CD24-Siglec-10 axis functions by releasing a “don't-eat-me” signal that helps the tumor cells evade the immune system and avoid phagocytosis [206]. When CD24 binds to Siglec-10, it triggers a suppressive signaling pathway that involves the activation of phosphatases SHP-1 and SHP-2 through the Src homology region 2 domain (Figure 3C) [207]. Siglec-10 recruits dual ITIM-bound phosphatases that suppress TLR-induced inflammatory signaling and disrupt actin-dependent phagocytic machinery in macrophages. [208]. Phosphorylation of Src kinases is triggered through ITIM-related signaling modules following ligand engagement between Siglec-10's Ig variable region and CD24's ectodomain-bound sialic acid residues [209, 210]. Subsequently, the phosphorylated ITIM tyrosine in the cytoplasm recruit's tyrosine phosphatases, leading to a reduction in signaling within the CD24-Siglec-10 axis [207, 210]. This process suppresses the immunoreactivity of macrophages, diminishes their phagocytic activity, and weakens their ability to surveil tumor cells.

2.2.4 SLAMF 3/4: Key Inhibitory Receptors in Immune Regulation

The signaling lymphocyte activation molecule (SLAM) family of immune cell surface receptors belongs to the CD2 subfamily of the IgSF and is comprised of nine members, namely SLAMF1-9 [211]. SLAMs play a crucial role as immunomodulatory receptors [69]. SLAM family receptors (SFRs) belong to the IgSF and are widely expressed on hematopoietic cells, including macrophages [212]. They play a crucial role in regulating the activation and cytotoxicity of these cells [22, 69]. LAM family receptors serve as critical regulators orchestrating immunoregulatory functions across both innate and adaptive immune cellular networks [213]. They typically recruit downstream SAP family proteins to deliver activation signals that regulate a variety of immune responses, such as NK cell activation, NK-T cell development, and antibody production [214]. The SLAM family of receptors, often utilized as markers to distinguish hematopoietic stem cells from other hematopoietic precursor cells, has been demonstrated to bear similarity to CD47 and may serve as a novel “self” molecule that specifically inhibits macrophage phagocytosis of its own blood and maintains macrophage tolerance [215]. Therefore, the SLAM family of receptors may serve as blood tissue-specific “do not eat me” receptors [216]. As a result, SFRs are anticipated to function as novel immune checkpoint molecules, offering new targets for tumor immunotherapy [217]. Specifically, SFR members, particularly SLAMF3 and SLAMF4 (receptors for SLAMF2), work together to inhibit the phagocytosis of hematopoietic cells by macrophages (Figure 3D). They achieve this by suppressing “eat-me” signaling in macrophages through SH2 domain-containing phosphatases [211, 218].

2.2.5 Stanniocalcin 1 (STC-1): Key Regulators of Tumor Invasion, Metastasis, and Immune Escape

STC-1 is a homodimeric glycoprotein initially discovered in the corpuscles of Stannius in teleost fish [219]. Functions as a pivotal regulator maintaining calcium-phosphate equilibrium while exhibiting multifaceted roles spanning reproductive biology through gestation and lactation, developmental processes including vascular patterning and tissue morphogenesis, cellular fate determination via proliferation-apoptosis balance, and pathological pathways such as ischemic injury and oncogenic progression [220, 221]. There is emerging evidence suggesting that STC1 is present in various human cancer cells [69]. It can promote the invasiveness of breast cancer by activating the JNK/c-Jun pathway. STC1 induces activation of the JNK pathway and modulates the expression of key molecular targets, such as p-JNK and p-c-Jun [222], thereby enhancing the metastatic potential of HCC [223]. STC-1 orchestrates pro-tumorigenic vascularization and metastatic dissemination through VEGF transactivation mediated by PKCβII-dependent ERK1/2 phosphorylation cascades in neoplastic cells [69]. STC1 can also upregulate its expression through the PI-3K/Akt/NF-κB-dependent signaling pathway to promote TNBC cell metastasis. Abnormal STC1 expression has been observed to be associated with metastasis or advanced tumor stage in tumor tissues compared with corresponding normal tissue controls, suggesting that STC1 may play a role in promoting tumor progression [224].

STC-1 has been shown to be an intracellular “eat-me” signaling inhibitor and an immune checkpoint. Mechanistically, tumor STC-1 interacts with calreticulin as an “eat-me” signal to block targeted phagocytosis of membrane calreticulin by APCs (Figure 4A) [225]. Such disruption compromises the antigen presentation process between APCs and T lymphocytes while suppressing macrophage-mediated clearance of malignant cellular targets [226]. Targeting the interaction between STC-1 and calmodulin represents a promising approach to enhance the efficacy of cancer immunotherapy in patients. Tumor-derived STC-1 plays a critical role in evading immune responses within the TME, exerting suppressive effects on anticancer immunity [227]. STC1 suppresses intracellular second messenger signaling through calcium, leading to a reduction in macrophage mobility [228]. Specifically, it inhibits the expression of uncoupling protein-2 (UCP2) in response to chemical inducers, resulting in decreased mitochondrial membrane potential and superoxide production in macrophages [229]. This ultimately contributes to the modulation of cellular functions and inflammatory responses [229, 230]. UCP2 plays a crucial role in regulating the production of superoxide in macrophages, which is a key factor in controlling macrophage function and viability [231]. Therefore, STC-1 reduces superoxide production in macrophages by promoting UCP2 expression, thereby establishing its significance in tumor immunotherapy [226, 230]. The targeting of STC-1-expressing tumor cells demonstrates potent antitumor effects.

2.2.6 Fc Gamma Receptors: FcγRIIB Was Used as a Key Inhibitory Regulator

FcRs are specific receptors for Igs that bind the Fc portion of antibodies [232]. They are predominantly expressed on innate immune cells and serve as receptors for antigen-antibody immune complexes, thereby providing a cellular effector arm of the humoral immune system that links the adaptive and innate immune systems [233, 234]. The cross-linking of IgG immune complexes by FcγRs induces phosphorylation of their ITAMs, leading to the activation of SYK, SRC, and PKC pathway kinases (Figure 4B) [235]. This kinase activation subsequently triggers actin remodeling, which is crucial for the phagocytosis of IgG immune complexes [236]. FcRs have the ability to induce a variety of biological functions, including the modulation of macrophage phagocytosis, cytolysis, and transcriptional activation of cytokine genes. This can trigger an inflammatory cascade response [237]. FcRs consist of FcγRIIB (CD32B), FcγRI, FcγRIIIA (CD16), and FcγRIIA (CD32A), all of which are expressed on macrophages [238]. However, FcγRIIB stands out as the sole inhibitor of phagocytosis among the human Fcγ receptor family, while the remaining members serve to enhance this process [69]. Activated FcRs related to FcR common γ-chain all contain an ITAM [239]. In contrast, the FcγRIIB, a single-chain molecule belonging to the inhibitory FcγR subclass, serves as a potent negative regulator by containing an ITIM in its cytoplasmic structural domain [232]. Activation of ITIM through phosphorylation brings in the SHP1 and SHP2 phosphatases that work to suppress the subsequent phagocytosis process [234, 237]. FcγRIIB is commonly expressed in various types of immune cells, such as B cells, mast cells, DCs, neutrophils, and macrophages [240]. It plays a vital role in negatively regulating immune cell functions, including the proliferation of B cells and DCs, as well as the degranulation of mast cells [241]. Additionally, FcγRIIB has been implicated in the development of numerous autoimmune and neoplastic diseases, such as arthritis, systemic lupus erythematosus, inflammatory bowel disease, colorectal cancer, and melanoma [242]. Autoimmune symptoms are exacerbated in FcγRIIB-deficient mice, and normal expression of FcγRIIB prevents mice from developing an SLE-like phenotype [243]. Currently, commercially available monoclonal antibodies specific for FcγRIIB, such as mAb2B6, have been humanized and are capable of directly inducing myeloid cytotoxicity in B cells [244].

2.2.7 TIM-1: Mediates B-Cell Immunosuppression

T cell Ig and mucin domain 1 (TIM-1) encoded by Havcr1 gene is an important immune checkpoint molecule on B cells [39]. Its expression and action mechanism on B cells are of great significance for the regulation of immune response [39]. In B cells, TIM-1 is mainly expressed in specific subsets of B cells, especially the subsets of B cells that expand in TME [40]. The mechanism of TIM-1 on B cells mainly includes the following aspects: first, TIM-1 binds to phosphatidylserine on the surface of apoptotic cells to enhance tissue tolerance [245]. Second, TIM-1 expression limits the type I interferon (IFN-I) response of B cells, thereby inhibiting B cell activation and antigen presentation ability(Figure 4C) [40]. The suppressive impact not only hinders the growth of T cells but also undermines the body's ability to combat tumors through immune mechanisms. In addition, TIM-1 interacts with a variety of co-inhibitory molecules, such as PD-1, TIM-3, to form an immunosuppressive microenvironment and promote tumor immune escape [40].

TIM-1 expression is regulated by a variety of factors. Both IFN-I and IFN-II regulate TIM-1 expression, and the loss of TIM-1 enhances the ability of B cells to respond to these interferons [246]. Because of the immunosuppressive effect of TIM-1 on B cells, therapeutic strategies targeting TIM-1 have shown great potential. For example, the use of a high-affinity anti-TIM-1 antibody can significantly inhibit tumor growth and enhance the antitumor immune response. Furthermore, PD-1 blockade and anti-TIM-1 antibodies together can enhance the therapeutic outcome. These discoveries offer fresh concepts and targets for immunotherapy against cancer.

2.3.1 Role of CTLA-4 in Immune Regulation

CTLA-4, also known as CD152, is a leukocyte differentiation antigen [251]. It is classified under the IgSF of functionally important Igs and serves as a transmembrane receptor on T-cells [252]. CTLA-4 is broadly expressed, primarily on activated Tregs [253]. The CTLA-4 molecule contains a lead peptide and three structural domains that are associated with exons 2, 3, and 4, respectively [254]. And the first structural domain functions to bind ligands, while the second and third structural domains are transmembrane and cytoplasmic structural domains [255]. The first identification of CTLA-4 was obtained by screening a cDNA library derived from mouse CTL, which revealed a high degree of homology between the mouse and human CTLA-4 genes [256, 257]. In vivo, CTLA-4 serves as an important immune checkpoint receptor for the co-stimulatory ligands B7-1 (CD80) and B7-2 (CD86) on T-cells, exerting a negative regulatory effect on T-cell activation in immunotherapy [258].

Pioneering murine genetic ablation models in the mid-1990s first established CTLA-4's critical inhibitory role in modulating T lymphocyte activation cascades [259]. Research established CTLA-4 as a potent CD80/CD86-binding receptor, functioning as a critical inhibitory checkpoint that suppresses adaptive immunity through competitive ligand interactions [260]. This molecule has been extensively studied in immune response therapy and has been successfully utilized in clinical therapy, playing a pivotal role in impeding anticancer immunity [261].

CTLA-4 exhibits significant structural homology with the ectodomain of CD28, which is another co-stimulatory receptor for CD80/CD86 [260]. CTLA-4 demonstrates a higher binding affinity for the ligand compared to CD28. In fact, its affinity for the CD80/CD86 ligand is 10 to 100 times higher than that of CD28 [256, 262]. CTLA-4 is initially present in intracellular vesicles of inactivated T cells and can only be detected 24 h after activation [263]. Its expression reaches a maximum at 36 to 48 h but can also be constitutively expressed on Tregs (Figure 5) [262, 264]. Furthermore, it was shown that CTLA-4 is closely associated with the differential expression of T cell subsets and that a portion of its biological function is also reliant on the stage of T cell differentiation [256, 265]. In addition, the expression of CTLA-4 serves as a mechanism to maintain the balance of T-cell activation intensity [266]. This not only ensures the normal functioning of the body's immune response but also facilitates the survival of CTLA-4 expressing cells within the body [267, 268]. The interaction between CD28 on T-cells and CD80/CD86 on APCs initiates a co-stimulatory signal essential for T-cell activation. This, together with the first signal generated by the recognition of MHC by the antigen-specific T-cell receptor, constitutes a dual signal for T-cell activation [269]. Due to the high degree of homology between the extracellular domains of CTLA-4 and CD28, CTLA-4 can antagonize the interaction of CD28 with its ligands. This action reduces the production of co-stimulatory signals and further hinders the promotional function of CD28 molecules on T-cell activation [270]. Ultimately, this leads to T-cell suppression, reduced cytokine production, and proliferation restriction [264, 271]. This type of inhibition of T cell activation is a cell-intrinsic mechanism.

CTLA-4 can transmit inhibitory signals into APCs via CD80/CD86 ligands, leading to the inhibition of the production of the tryptophan-depleting enzyme indoleamine-2, 3-dioxygenase and T-cell effector responses [251, 272]. In addition, binding of CTLA-4 to CD80/CD86 ligands can also result in transcytosis of APC ligands [273]. Furthermore, CTLA-4 expressing T cells have the ability to deplete ligands from APCs, thereby decreasing the level of ligands on the surface of APCs and subsequently inhibiting co-stimulation of opposing T cells [274]. This type of inhibition represents an extracellular mechanism for regulating T cell activation. In contrast, CTLA-4 also enhances T cell/APC adhesion through an LFA1-mediated pathway and leads to attenuated T cell activation by reducing the duration of APC/T cell interactions [275].

In addition, CTLA-4 can also disrupt TCR signaling by interacting with PP2A (protein phosphatase 2) and SHP2 [276]. This interaction is believed to downregulate TCR signaling on T cells upon ligand binding between the TCR and the MHC and antigenic peptide on DCs [277, 278]. The inhibitory effect of CTLA-4 on T cells is further demonstrated by the physiological function of CTLA-4-deficient mice [279]. These mice develop normally before birth but are born with various lymphoproliferative disorders of the thymus, spleen, and lymph nodes [280]. These lymphoid organs enlarge 5 to 10 times larger than those of normal mice and usually die by the third or fourth week [281]. Studies showed that T cells in these mice were in a highly activated state and would proliferate more compared to normal mice when triggered by TCR/CD3, leading to a significant increase in CD4 and CD8 numbers [282]. The aforementioned examples further illustrate that the deficiency of CTLA-4 leads to uncontrolled activation of T cells, highlighting the pivotal role of CTLA-4 in inhibiting T cell activation. Additionally, the disease observed in CTLA-4-deficient mice is not attributed to active T-cell defects [283]; rather, normal T cells triggered by CTLA-4 produce factors that inhibit disease induced by CTLA-4 deficiency [284]. CTLA-4 serves as the target gene for Forkhead box protein P3 (Foxp3), a master regulator of Tregs. Consequently, inactivation of the gene encoding Foxp3 in CTLA-4-deficient mice results in fatal autoimmune disease [251], underscoring the essential role of CTLA-4 in enabling Treg cells to suppress immune responses and maintain immune self-tolerance [284].

2.3.2 Diversity, Structural Characteristics of Killer Immunoglobulin-Like Receptor (KIR) and Its Function in Immune Regulation

KIR is an important class of receptors expressed on the surface of NK cells and T cells, which play a key role in regulating the activity of NK cells [285]. The KIR gene family is highly polymorphic, showing random, diverse, single allele expression patterns, and this diversity may lead to different KIR expression patterns and functions [286, 287]. KIRs are categorized as activating KIRs (aKIRs) and inhibitory KIRs (iKIRs), and their family members are classified into two types: the KIR2D and KIR3D [288-290]. The naming of KIRs is based on the structural characteristics of their extracellular regions (“2D” and “3D” represent the number of extracellular Ig-like domains) and the length of their cytoplasmic tails (“L” for long and “S” for short). These structural domains endow them with specificity for particular HLA molecules (Figure 6) [291]. The function of KIR can be deduced based on the extent of the cytoplasmic region; L receptors are usually inhibitory, and S receptors are usually activating [292, 293]. Currently, iKIRs play a dominant role in controlling NK cell function by interacting with specific MHC-I molecules (human HLA-I molecules) and modulating immune activity by regulating either killing or inhibitory signals in the context of recognizing different HLA-like molecules and other ligands [288].

iKIRs are type I transmembrane receptors targeting polymorphic HLA-A, B, and C molecules [294]. For example, KIR2DL1 primarily recognizes the HLA-C C2 population with high affinity, whereas KIR2DL2 and KIR2DL3 recognize the HLA-C C1 allotype [295-297]. KIR3DL1 recognizes HLA-B and some HLA-A carrying Bw4 [298, 299]. When iKIRs bind to their ligands, they transmit inhibitory signals through ITIMs in cytoplasmic tails [300, 301]. These ITIMs are phosphorylated by specific tyrosine kinases (e.g., Src family kinases) upon binding [162, 302, 303]. Phosphorylated ITIMs recruit and activate phosphatases containing the SH2 structural domain, such as SHP-1 and SHP-2 [304, 305]. These phosphatases dephosphorylate and inhibit signaling molecules associated with NK cell activation, such as Lck, Fyn, and Syk, which downregulate PI3K, MAPK, and NF-κB signaling [303, 306, 307]. The above mechanisms lead to downregulation of NK cell-mediated cytotoxicity, attenuation of cell proliferation, and significant reduction in cytokine production [302, 304]. In addition, NK cells do not mount an attack on cells expressing normal HLA-class I molecules. Under normal conditions, iKIRs help NK cells distinguish between self and non-self-cells, thus playing an important role in immune surveillance [303]. If cells (e.g., tumor cells or virus-infected cells) reduce the expression of HLA-class I molecules, the inhibitory signals of iKIRs are weakened, and NK cells may then be activated and kill these abnormal cells.

2.3.3 NKG2A/CD94: A Key Inhibitory Receptor Complex in NK Cell and T Cell Regulation

NKG2A (CD159) is a heterodimeric inhibitory receptor of the C-type lectin family expressed predominantly on the surface of NK cells and also present in certain T cell subsets [308]. NKG2A is expressed in several organ tissues of the human body, and especially the highest expression is found in peripheral blood cells. CD94 is a 70 kDa type II transmembrane glycoprotein belonging to the C-type lectin superfamily, which, like NKG2A, is mainly expressed on the surface of NK cells and some T cells [309, 310]. The NKG2A/CD94 molecule, after being linked by disulfide bonds to form a heterodimeric complex, is recognized by HLA-E, a nonclassical MHC-I molecule on the target cell, which induces cascading inhibitory signals and suppresses cytotoxic activity and cytokine secretion from NK cells(Figure 6) [37, 311]. The N-terminus of NKG2A is located in the cytoplasm, and like KIR, it contains ITIMs [312]. After NKG2A binds to CD94 to form a heterodimer, it can recognize the target cell-specific ligand HLA-E and transmit inhibitory signals to NK cells by recruiting SHP-1 and SHP-2 [312-314]. In addition, during the activation of CD8⁺ T cells, the NKG2A on the surface of CD8⁺ T cells can bind to the HLA-E molecule, thereby inhibiting the activation process of CD8⁺ T cells [315, 316].

In the tumor-infiltrated areas, the expression of HLA-E and NKG2A on the surface of NK and CD8⁺ T cells increases, and the ability of local NKG2A to bind to HLA-E in the tumor increases, thereby inhibiting the function of NK and CD8⁺ T cells, which helps tumor cells evade immune surveillance [317, 318]. Therefore, NKG2A targeting agents are being studied for the intervention of various cancer types, including, but not limited to, colorectal, head and neck, ovarian, Hodgkin's lymphoma, and lung cancers [319-321]. Investigating the inhibitors of NKG2A holds significant clinical potential.

2.3.4 Lymphocyte Activation Gene-3 (LAG-3): Structure, Function, and Its Critical Role in the Immune System

LAG-3, also referred to as CD223, was first identified in 1990 from a screen of CD3-negative IL-2-dependent NK cell line F5 cells [322]. LAG-3, a protein classified within the IgSF, is specifically identified as a mature type I transmembrane glycoprotein. It is located on human chromosome 12 and serves as a critical regulator of the immune system [91, 323]. This biomolecule features three distinct topological segments: a membrane-spanning segment, an ectodomain, and an intracellular module containing functional motifs [324]. The extracellular structure is composed of four IgSF structural domains (D1–D4). The significance of the extracellular domain in binding ligands to LAG-3 is primarily attributed to the presence of a proline-rich ring structure and an intrachain disulfide bond within the D1 domain [325]. This domain belongs to the IgSF and exhibits species-specific characteristics [91]. In the transmembrane-cytoplasmic fraction, LAG-3 undergoes proteolytic shedding from the plasma membrane, mediated predominantly by family metalloproteases (ADAM10 and ADAM17). This, in turn, regulates the function of LAG-3 [325]. The intracellular domain contains three structural elements: (1) a phosphorylatable serine residue at position S454 (substrate of protein kinase C), (2) a conserved KIEELE amino acid sequence, and repeating glutamic acid-proline dipeptide units [326, 327]. The highly conserved “KIEELE” motif is crucial for the function of LAG-3, and mutation of KIEELE can result in complete loss of LAG-3 function [328]. LAG-3 can co-localize with CD4, CD8, and CD3 molecules. However, structurally, LAG-3 is not identical to CD3 and CD8 but shares approximately 20% amino acid sequence homology with CD4 in the extracellular region [329]. LAG-3 is predominantly expressed on the surface of effector T cells and regulatory Tregs, NK cells, B cells, and DCs. Similar to CTLA-4, LAG-3 is not expressed on naive T cells but is induced by CD8⁺ and CD4⁺ T cells in response to sustained antigenic stimulation [330].

Studies have indicated that the LAG-3 ligands identified in the TME consist of MHC-II molecules, galactose lectin-3 (Gal-3) [331], fibrinogen-like protein 1 (FGL1), and liver sinusoidal endothelial cell lectin (LSECtin) [323]. While LAG-3 and CD4 both engage MHC-II molecules,346 LAG-3 demonstrates superior binding affinity over CD4, competitively impairing CD4-MHC-II complex formation [332]. Moreover, LAG-3 selectively binds to the MHC-II/peptide (pMHC-II) complex on the surface of APCs [333]. leading to signaling of MHC-II molecules in DCs [334]. Upon binding of LAG-3 to MHC-II, inhibitory signals can be transmitted through the cytoplasmic structural domains, leading to the inhibition of CD4 T cell activation and cytokine production [335, 336]. This ultimately impacts the immune response. Furthermore, the interaction between LAG-3 and MHC-II promotes the recruitment of tumor-specific CD4T cells and facilitates tumor escape [337]. Gal-3 is a soluble molecule secreted by tumor-associated stromal cells. It is predominantly expressed on the surface of tumor cells and activated T cells [338]. It is binding to LAG-3, which results in the inhibition of IFN-γ production by CD8⁺ T cells and plays a critical role in suppressing the expansion of plasmacytoid DCs [339]. FGL1 is produced and released by both tumor cells and hepatocytes, functioning to suppress IL-2 LSECtin production. This cytokine, primarily located in the liver, has also been detected on tumor cell surfaces [333]. Upon binding to LAG-3, both FGL1 and LSECtin have demonstrated significant inhibition of antitumor responses [340, 341]. To date, LAG-3 has been identified as a key factor in various pathological conditions and is pivotal in the progression of malignancies. The relationship between LAG-3 expression and clinical outcomes differs significantly among cancer types.

2.3.5 T Cell Immunoreceptor With Immunoglobulin and ITIM Domains (TIGIT): Emerging Immunotherapy Targets in the IgSF

TIGIT is an IgSF receptor that has become a target for cancer immunotherapy [342, 343]. It was discovered in 2009 through genome-wide analysis of genes expressed on immune cells [344], revealing that these genes contain structural domains commonly found in inhibitory receptors, thus identifying TIGIT as an inhibitory receptor [345, 346]. The human TIGIT gene is located on chromosome 3 at q13. 31 and has a cDNA length of 2, 926 bp, which can encode 244 amino acids with 6 TIGIT isoforms [345]. TIGIT is structurally organized into three distinct regions: an extracellular IgV domain, a type I transmembrane topology, and a cytoplasmic segment housing both an ITIM and an immunoglobulin tyrosine tail [347]. TIGIT functions as an inhibitory checkpoint modulator for cytotoxic immune responses, with preferential expression observed across innate lymphoid populations (NK cells/γδ T cells) and adaptive subsets including CD4⁺/CD8⁺ T lymphocytes, regulatory T cells, and Th cell populations [348]. However, its expression is weak when CD4⁺ and CD8⁺ T cells are in the naive state but can be highly expressed when they are activated [349].

TIGIT has three primary ligands: CD155 (also known as poliovirus receptor, PVR), CD112, and CD113, all of which are part of the connexin and NECL families [342]. CD155 contains Ig domains, C1-like and C2-like domains in its extracellular regions, a long C-terminal domain, and ITIM in its transmembrane region [350]. Demonstrating potent TIGIT-binding specificity, this molecule modulates multifaceted immunoregulatory pathways within TME [351]. TIGIT binds to CD155 on DCs and induces phosphorylation of CD155, initiating a biochemical transduction chain that reduces IL-12 production and suppresses T cell function [352]. The binding of TIGIT to its ligands inhibits the activity of T cells and NK cells, as well as reduces antigen presentation and anti-inflammatory cytokine secretion [353]. In addition to binding to these ligands, CD226 also plays a crucial role in the mechanism of action of TIGIT [354]. CD226 is a co-stimulatory receptor expressed on T cells and NK cells, which binds to LFA-1 and mediates the activation of immune cell function [355]. The binding of TIGIT to CD155 interferes with CD226 activation signaling and suppresses tumor immune response [356, 357]. Notably, highly TIGIT-expressing Tregs have a potent inhibitory effect on the immune response. They can upregulate various Tregs gene markers such as CTLA-4, LAG-3, PD-1, Foxp3, and so forth, and attenuate Th1 and Th17 functions [358]. Furthermore, these highly TIGIT-expressing Tregs have also been found to attenuate Th1 and Th17 functions [359]. TIGIT inhibits NK cell degranulation and cytokine production. Additionally, the binding of TIGIT⁺NK cells to CD155⁺ MDSCs inhibits the phosphorylation of ZAP70/SYK and ERK1/2, ultimately attenuating NK cell cytolysis [306, 360]. TIGIT is widely expressed on lymphocytes and is currently a popular immune checkpoint, particularly when combined with PD-1/PD-L1 [361, 362]. This combination has demonstrated improved therapeutic effects in clinical settings.