Beyond PD-1/CTLA-4! The Emerging Immune Checkpoint CD160 is Transforming the Landscape of Tumor Immunotherapy

1. What is CD160?

CD160 is a membrane molecule closely associated with immune regulation, primarily distributed on immune cells such as NK cells, certain T cells, and intraepithelial lymphocytes. In recent years, driven by continuous advancements in tumor immunology, chronic infection research, and immune checkpoint studies, CD160 has gradually emerged as a notable research target. Its core characteristic lies in the fact that CD160 is not a molecule with a single function; rather, it exhibits diverse biological functions, such as activation or inhibition, depending on the cell type, tissue environment, and ligand conditions.

Currently, immune checkpoint research is no longer confined to classic molecules like PD-1/PD-L1 or CTLA-4. A growing body of evidence suggests that immune cell dysfunction is typically not determined by a single molecule but results from the combined involvement of multiple inhibitory receptors. To address this, researchers established OMIP-037, a 16-color flow cytometry panel for analyzing human peripheral blood and tumor-infiltrating lymphocytes. This panel incorporates multiple inhibitory receptors, including PD-1, TIM-3, CD160, LAG-3, and TIGIT, into a single detection system, enabling the identification of up to 32 inhibitory receptor combinations at the single-cell level and further distinguishing subsets such as CD4+, CD8+, NK, iNKT, and γδ T cells. This provides a crucial tool for systematically investigating the expression characteristics of CD160 across different immune cell populations ^[1].

This research framework highlights two key issues. First, immune exhaustion is essentially a complex process involving multiple molecules, making a single marker insufficient to fully reflect functional status. Second, CD160 is not merely a redundant co-inhibitory molecule. Current evidence indicates that it can promote metabolic activation and cytokine secretion in NK cells, yet it may also be associated with inhibitory phenotypes in certain T cell environments and participate in negative regulation through binding with HVEM. Therefore, CD160 likely represents an immunoregulatory pathway with independent biological significance ^[1].

Pan-cancer analyses across various solid tumors further support the importance of CD160 as an emerging immune checkpoint. Integrated TCGA studies reveal that in multiple tumors, such as lung cancer, breast cancer, and colorectal cancer, CD160, along with checkpoint molecules like LAG-3 and TIGIT, is commonly highly expressed on CD8+ T cells, with no particularly significant differences observed among different tumors, suggesting its potential involvement in widespread tumor immune suppression mechanisms. Furthermore, the cellular sources of different checkpoint ligands within the tumor microenvironment vary; for instance, certain ligands are preferentially expressed by macrophages, while others originate more from epithelial cells. This implies that CD160-related signaling depends not only on the molecule itself but also on the spatial distribution of cells and the local microenvironment ^[2].

However, current research on CD160 still has notable limitations. In the existing literature, many findings focus on expression profiling and correlational analyses, while the downstream signaling mechanisms in different cells, isoform differences, membrane environment influences, and functional division of labor with other checkpoint molecules remain incompletely elucidated. Therefore, although CD160 has become a hotspot molecule in tumor immunology and immunoregulation research, its mechanistic studies and clinical translation are still in a phase of continuous advancement ^{[1, 2]}.

2. Molecular Structure and Basic Features of CD160

2.1 Discovery History of CD160

CD160 was initially discovered as a GPI-anchored immunoglobulin-like receptor, identified on peripheral blood CD56dim NK cells and TCRγδ lymphocytes using the BY55 monoclonal antibody. Studies also found that although the number of CD4+ or CD8+ T cells expressing CD160 in peripheral blood is limited, its expression is more pronounced in tissues such as the intestine and skin, suggesting its potential involvement in tissue immune surveillance and local effector functions ^[3].

2.2 Main Isoforms of CD160

Subsequent studies have shown that CD160 exists in at least two main forms: the classic GPI-anchored CD160-GPI and the transmembrane CD160-TM. Both isoforms can be detected in primary CD4+ and CD8+ T cells, but their functions differ. Experiments showed that CD160-GPI, upon stimulation with HVEM-Fc or anti-CD160 antibodies, could enhance Jurkat cell activation, suggesting it possesses certain positive co-stimulatory potential. In contrast, CD160-TM responded less robustly under the same conditions and exhibited lower binding affinity for HVEM ^[4].

This phenomenon indicates that CD160's function is determined not only by extracellular recognition but may also be influenced by membrane anchoring mode, membrane localization, and receptor clustering ability. In other words, the different isoforms do not merely represent structural variations but may dictate that CD160 plays entirely distinct biological roles in different cellular environments ^[4].

2.3 Binding Mechanism of CD160 with HVEM

Structural biology studies have provided deeper insights into the binding mode between CD160 and HVEM. Based on co-expression in insect cells and in-situ purification, the native, non-ligated CD160-HVEM complex forms a 3:3 trimer in solution, a conclusion supported by relevant crystallographic analyses ^[5]. This result is not entirely consistent with earlier studies that considered CD160 a monomer, suggesting that different experimental systems, expression systems, glycosylation states, and recombinant strategies can all affect the determination of its oligomeric form ^[5].

From a functional perspective, it is plausible that the GPI-anchored form facilitates lateral movement and clustering of CD160 in lipid rafts on the membrane, thereby enhancing its ability to form stable complexes with HVEM. Conversely, the transmembrane CD160-TM may exhibit weaker signaling activity due to differences in spatial orientation or local membrane environment. However, this inference currently lacks direct high-resolution evidence within the native cellular membrane environment and requires further validation ^{[4, 5]}.

3. Expression Patterns and Ligand Recognition of CD160

3.1 Cellular Expression Distribution of CD160

CD160 was initially identified as an NK cell-associated molecule, primarily expressed on CD56dim NK cells, TCRγδ lymphocytes, and a subset of cytotoxic CD8+ T cells, and is also highly expressed in intestinal intraepithelial lymphocytes (IELs). With further research, scientists discovered that CD160 expression is not confined to these classic cell populations. For instance, in the skin, a subset of effector memory CD4+ T cells also expresses CD160, along with skin-homing molecules such as CLA and CCR4 and cytotoxic molecules like perforin, suggesting CD160 might also serve as a marker for tissue-resident or terminally differentiated effector cells ^[6].

Within the tumor microenvironment, HVEM and CD160 often form an interconnected regulatory network with molecules like BTLA. Studies show that BTLA axis-related genes frequently exhibit co-expression patterns in tumor tissues, indicating the potential involvement of the HVEM-CD160/BTLA pathway in tumor immunosuppression and tumor microenvironment remodeling ^[7]. Furthermore, in tumor antigen-specific CD8+ T cells, CD160 is often co-expressed with molecules such as PD-1, TIM-3, and LAG-3, a phenomenon typically associated with dysfunction or exhaustion, though variations exist among different tumors and tissue sites ^[8].

3.2 Structural Recognition Mechanism of CD160 for HVEM

At the structural level, the extracellular region of human CD160 possesses a unique immunoglobulin-like fold and can form a 1:1 stoichiometric complex with HVEM. Its binding interface shares overall similarity with the BTLA-HVEM complex but retains its own distinct characteristics ^[9]. More broadly, HVEM itself serves as a multi-ligand platform; besides binding CD160 and BTLA, it can also recognize the TNF family ligand LIGHT, and these binding sites are not entirely overlapping. In certain cases, HVEM can even simultaneously participate in forming higher-order complexes, thereby integrating multiple immune signals ^[10].

Early studies using HDX-MS and molecular modeling, despite having limited resolution, provided descriptions of key contact regions and conformational changes that are largely consistent with subsequent crystallographic findings ^[11]. Therefore, current evidence generally supports the view that the interaction between CD160 and HVEM has a clear structural basis and is not merely a simple binary interaction but is embedded within a more complex HVEM ligand network ^{[9, 10, 11]}.

However, it is also important to note that most structural studies have been conducted using recombinant extracellular fragments and have not fully incorporated factors such as glycosylation, membrane orientation, lipid raft localization, and intercellular spatial configuration. Consequently, how the CD160-HVEM interaction is dynamically regulated in the authentic physiological environment remains an important question for future research ^{[9, 10, 11]}.

4. Signaling Pathways and Immunoregulatory Mechanisms of CD160

4.1 Signal Transduction Mechanisms of the CD160/HVEM/BTLA Axis

HVEM is not a molecule with a fixed output of either activation or inhibition signals; it functions more as a "signaling hub," with its effect depending on the ligand bound and the spatial context of the interaction. Research shows that in the human system, binding of HVEM to CD160 can selectively co-stimulate NK cells. HVEM on tumor cell surfaces can enhance the activation of CD56dim NK cells, promote IFN-γ and TNF-α secretion induced by type I IFN and IL-2, trigger rapid phosphorylation of ERK1/2 and AKT, and enhance cytotoxicity ^[12]. This indicates that CD160-HVEM interaction can engage classic effector pathways like MAPK and PI3K-AKT, exerting pro-activation effects in NK cells ^[12].

However, when HVEM binds to BTLA, the outcome may shift towards inhibition. Studies have found that BTLA can inhibit cytotoxic activity, suggesting that the signal output of HVEM depends on the identity of the ligand and is not inherently biased towards activation ^[12]. Furthermore, BTLA and HVEM can form cis-complexes on the same cell membrane; this configuration blocks exogenous HVEM co-stimulation while retaining BTLA's inhibitory function, thus favoring an overall inhibitory signal ^[13]. This also explains why HVEM does not exhibit a strong co-stimulatory effect in some primary T cells.

In certain viral infection models, more complex bidirectional regulatory relationships can form between HVEM and BTLA. For instance, in a vaccinia virus model, deficiency in either HVEM or BTLA impaired the survival and memory formation of effector CD8+ T cells, suggesting that BTLA can, under specific circumstances, act as a reverse ligand for HVEM, delivering pro-survival signals to T cells in trans ^[14].

On the other hand, the expression of CD160 itself is regulated by upstream induction programs. In a model where HIV-1-exposed dendritic cells activate T cells, the p38 MAPK/STAT3 pathway can promote the upregulation of inhibitory receptors including CD160, while blocking this pathway reduces the expression of these molecules and restores T cell proliferation ^[15]. This implies that CD160-related signaling is not merely a result of receptor-ligand binding but is also influenced by intracellular transcriptional regulatory networks ^[15].

4.2 Functional Regulation of Different Immune Cells by CD160

The role of CD160 in NK cells is relatively well-defined. Studies in gene-knockout mice show that CD160 deficiency does not affect NK cell development or basal cytotoxic capacity but impairs the body's ability to control NK-sensitive tumors and significantly reduces IFN-γ secretion by NK cells. Bone marrow chimera and adoptive transfer experiments further demonstrate that CD160-positive NK cells play a critical role in early tumor immune surveillance ^[16]. Thus, the primary value of CD160 in NK cells may lie more in promoting cytokine output and early anti-tumor effects rather than merely influencing degranulation capacity ^[16].

This function is closely linked to metabolic status. In HIV-infected individuals, decreased CD160 expression on NK cells correlates negatively with disease progression. CD160-positive NK cells typically exhibit higher GLUT1 expression and glucose uptake capacity; their activation is accompanied by enhanced PI3K/AKT/mTOR/S6K metabolic axis signaling, while elevated plasma TGF-β1 levels are associated with CD160 downregulation ^[17]. Similar findings are observed in hepatocellular carcinoma: reduced CD160 levels on intratumoral NK cells correlate with poorer prognosis and higher recurrence risk; high TGF-β1 levels inhibit IFN-γ production by CD160+ NK cells, while blocking TGF-β1 partially restores this function ^[18]. These studies indicate that CD160 is not just a cell surface marker on immune cells but is also closely related to NK cell metabolic fitness and effector status ^{[17, 18]}.

Compared to NK cells, the role of CD160 in T cells is more complex. In TCR-engineered T cell studies, persistent graft T cells upregulate multiple co-inhibitory molecules, including CD160, accompanied by functional decline ^[19]. In patients with chronic lymphocytic leukemia (CLL), CD160 is one of the significantly upregulated co-inhibitory receptors on CD8+ T cells; its high expression correlates with an exhaustion-like phenotype and is regulated by extracellular vesicles and inflammatory factors ^[20]. Additionally, some studies suggest CD160 may play a negative regulatory role in certain T cell environments ^[21]. Since CD160 itself is a GPI-anchored molecule lacking a typical intracellular signaling domain, the precise mechanisms by which it exerts inhibitory effects in T cells---whether through membrane microdomains, co-receptors, or reverse signaling---require further elucidation ^{[19, 20, 21]}.

Beyond tumor and infection contexts, CD160 also plays a physiological role in tissue repair. Research shows that in a chemotherapy-induced intestinal injury model, intestinal intraepithelial lymphocytes can interact with HVEM on epithelial cells via CD160, activating epithelial NF-κB signaling and promoting transit-amplifying cell proliferation and mucosal repair. Deficiency in this pathway leads to impaired intestinal regeneration and increased mortality, while adoptive transfer of CD160-positive IELs ameliorates this phenotype ^[22]. This demonstrates that CD160 is not merely an "inhibitory checkpoint" but also participates in immune-tissue crosstalk and damage repair in specific tissue environments ^[22].

5. CD160 in Diseases: Tumors, Infections, Autoimmunity, and Inflammation

5.1 Dual Role of CD160 in Tumors

In tumor research, CD160 exhibits a clear dual nature: on one hand, it may mark effective anti-tumor immune cells; on the other, it may participate in immune evasion or dysfunction.

In a study of neoadjuvant immunotherapy for mismatch repair-deficient/microsatellite instability-high (dMMR/MSI-H) colorectal cancer, a subset of intratumoral PD-1^lo CD8+ T cells that highly expressed CD160, TRGC2, and KLRB1, while lowly expressing typical proliferation/exhaustion genes, was significantly associated with pathological complete response, suggesting that CD160 might mark a subset of functional effector cells responsive to PD-1 blockade ^[23]. In lung adenocarcinoma, a plasma extracellular vesicle transcriptome study found that baseline EV-CD160 levels positively correlated with response to immunochemotherapy, progression-free survival, and overall survival, and its dynamic changes could be used for efficacy monitoring ^[24].

On the other hand, CD160 may also be utilized by tumor cells themselves. In triple-negative breast cancer, researchers found that tumor cells could express the transmembrane CD160-TM isoform and subsequently developed a specific antibody, 22B12, which induced antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) in vitro and demonstrated anti-tumor activity in mouse models ^[25]. This indicates that CD160 is not merely an immune cell marker but may also serve as a direct therapeutic target in certain tumors ^[25].

Furthermore, results regarding CD160 vary across different tumor types. In esophageal squamous cell carcinoma, high expression of XCL1 and CD160 by tumor cells may correlate with immune escape ^[26]; an epidemiological proteomics study suggested an association between plasma CD160 levels and breast cancer risk ^[27]; in hematological malignancies, CLL-derived exosomes induced upregulation of PD-1 and CD160 in recipient cells, whereas in acute myeloid leukemia, although CD8+ T cells upregulated CD160 and PD-1, they did not necessarily exhibit a classic terminally exhausted state, possibly resembling an activation-associated or "pseudo-exhausted" status ^{[28, 29]}.

Therefore, CD160 can be understood as an emerging molecule possessing properties of both a tumor immune marker and a potential therapeutic target. However, its true application value remains dependent on distinguishing cellular origins, identifying isoforms, and analyzing specific tumor types ^{[23, 24, 25]}.

5.2 Role of CD160 in Chronic Infections

In infectious disease research, CD160 is most frequently associated with T cell exhaustion. HIV studies show that HIV-specific CD8+ T cells expressing either CD160 or PD-1 alone retain some functionality, whereas cells co-expressing CD160 and PD-1 exhibit more pronounced impaired proliferation and reduced cytokine production, accompanied by downregulation of NFκB-related nodes and upregulation of multiple inhibitory molecules. Blocking the CD160-HVEM interaction could restore the proliferation and cytokine secretion of these cells in vitro, indicating that this axis plays a functional role in maintaining or reinforcing the exhausted state ^[30].

Further research revealed that the T-bet^dimEomes^hi phenotype in chronic HIV infection is closely associated with high expression of PD-1, CD160, and 2B4; such cells typically exist in a transitional memory/exhausted state and can persist after long-term suppression by antiretroviral therapy (ART) ^[31]. Concurrently, high-avidity HIV-specific T cells also tend to enrich within the PD-1/2B4/CD160 co-expressing population, exhibiting stronger dysfunction and clonal turnover upon viral rebound ^[32]. These results suggest that CD160-associated exhaustion is not a transient phenomenon but may be embedded within stable transcriptional and differentiation programs ^{[30, 31, 32]}.

However, high CD160 expression does not always equate to inhibition. For example, in sepsis, the 2B4^hiPD-1^lowCD160^hi phenotype was associated with stronger cytokine production and poorer prognosis, suggesting it might also represent a hyperactivated but dysregulated immune state ^[33]. In diseases such as acute hepatitis E virus (HEV) infection and malaria, CD160 similarly exhibits complex roles related to the pathogen type and immunopathological processes ^{[34, 35]}. Thus, in infection contexts, CD160 is better characterized as a context-dependent indicator of immune status, rather than simply being classified as an "inhibitory marker" ^{[30, 33, 34, 35]}.

5.3 Role of CD160 in Autoimmunity, Inflammation, and Metabolic Diseases

Beyond tumors and infections, CD160 appears in research on various autoimmune and inflammatory diseases. In patients with primary Sjögren's syndrome, the expression and co-expression frequencies of BTLA, HVEM, and CD160 in peripheral blood are decreased, suggesting potential impairment of the BTLA-HVEM-CD160 network in maintaining immune homeostasis ^[36]. In systemic lupus erythematosus, CD160 levels on CD8+ T cells are reduced and correlate with disease activity ^[37]. This contrasts with the upregulation of CD160 as an exhaustion-associated molecule in chronic infections, again underscoring that the significance of CD160 must be interpreted within specific disease contexts ^{[38, 39]}.

At the genetic level, a study on autoimmune thyroid disease identified an association between the CD160-related polymorphism rs744877 and Graves' disease risk, but no significant association with Hashimoto's thyroiditis, suggesting that the CD160 pathway may contribute to susceptibility in some autoimmune diseases ^[40].

In inflammatory bowel disease models, single-cell studies identified a population of IL-23R-dependent Th1-like pathogenic cells expressing CD160; interfering with this molecule inhibited transplant colitis, indicating that CD160 may participate in maintaining pathogenic programs in certain pro-inflammatory T cell subsets ^[41]. In non-alcoholic fatty liver disease (NAFLD) research, CD160 was incorporated into a NAFLD-associated tryptophan metabolism-immune interaction network, demonstrating high diagnostic discrimination and correlation with M2 macrophage infiltration ^[42]. However, such evidence currently remains largely correlative and insufficient to prove CD160 is a direct driving factor ^[42].

Overall, a common feature of CD160 in these diseases is that it may both reflect disruption of immune homeostasis and directly participate in inflammatory or pathogenic processes. Therefore, whether CD160 is suitable as a therapeutic target or biomarker requires further support from cell type-specific and longitudinal follow-up studies ^{[36, 37, 40, 41, 42]}.