BTLA levels vary during T cell differentiation and activation processes. Specifically, BTLA is expressed in naive T cells, and the expression transiently increases upon activation, but is decreased in activated T cells (15–18). Moreover, the interaction manners between BTLA and HVEM also changes. Under homeostatic conditions, HVEM and BTLA interact in cis to provide intrinsic inhibitory signals. Upon T cell activation, HVEM is internalized, which allows BTLA to interact with HVEM in trans (6, 10). BTLA-deficient mice exhibit no defects in lymphocyte development, suggesting that BTLA is not necessary for T cell development (19). However, BTLA can significantly inhibit T cell activation and proliferation. BTLA-deficient T cells show increased proliferation (4) and are hyperresponsive to TCR-mediated activation (19). BTLA agonistic monoclonal antibody (mAb) suppresses the proliferation of T cells and the secretion of interleukin (IL)-10 and interferon γ (IFN-γ) upon anti-CD3 stimulation (20). Antibodies targeting BTLA enhance the proliferation of CD8⁺ T cells and blocking BTLA in combination with PD-1 is found to be effective in enhancing the exhausted human T cell response (21). Krieg et al. found that BTLA^-/- mice showed an elevated memory CD8⁺ T cell count and proposed that the increased T cell proliferation mediated by BTLA deficiency was associated with alterations in T cell memory subsets but not co-inhibition (22). However, Deppong et al. discovered that T cells with BTLA deficiency exhibited no enhanced proliferation, but showed decreased death (23). In addition, BTLA contributes to the induction of peripheral tolerance in CD4⁺ and CD8⁺ T cells (24). In naive T-cells, BTLA and HVEM form a cis-heterodimeric complex, which blocks other co-signaling molecules from binding to HVEM and stimulating the NF-κB signaling pathway, thus maintaining the tolerance of T cells (6). BTLA engagement has been suggested to facilitate SHP-1 and SHP-2 recruitment (3, 4). However, Chemnitz et al. reported that SHP-1 recruitment was not related to the function of BTLA, as blocking SHP-1 recruitment did not affect the function of BTLA (25). Paradoxically, it was recently shown that BTLA preferentially recruits SHP-1 to suppress T cell signaling more efficiently (26, 27). γδT cells can quickly produce inflammatory cytokines at sites of barrier to protect against pathogens. BTLA can limit γδ T cell numbers and control proliferation and cytokine secretion in mature lymph node γδ T cells (28). Vγ9Vδ2 cells are the main subset of γδ T cells in peripheral blood that are reactive to tumors and microbial agents, and BTLA was found to be strongly expressed in resting Vγ9Vδ2 T cells. BTLA engagement inhibited Vγ9Vδ2 T cell proliferation, while targeting BTLA resulted in the enhancement of Vγ9Vδ2 TCR-mediated signaling (29).

BTLA in B cells has rarely been studied compared to that in T cells. In human B cell subsets, mature peripheral B cells display the most significant BTLA level, whereas a uniform BTLA expression is observed in naive, transitional, and memory peripheral B cells, and the lowest level is detected in bone marrow-derived precursor B cells (30). In addition, BTLA levels are downregulated in B cells of the aged, which is related to the decreased reactivity to the trivalent influenza vaccine (31). In mouse spleen, compared to T cells, B cells display higher surface BTLA level (12, 32). Consistent with its effect on the development of T cells, BTLA does not significantly affect the development of B cells, because normal growth of lymphocytes is detected in mice with BTLA deficiency (4, 19). Moreover, it was found that BTLA/HVEM ligation can suppress the functions of B cells (such as proliferation, cytokine secretion, and co-stimulatory molecule upregulation) (30, 33). Engagement of BTLA recruits SHP-1, leading to reduced activation of BCR downstream signaling molecules (30). However, Zhang et al. reported that BTLA antibodies had no effect on lipopolysaccharide (LPS)- or anti-IgM antibody-induced B cell proliferation in vitro (34). Regarding helper T (Th) cells, follicular Th (Tfh) cells represent the dominant subset that promotes antibody secretion by B cells. Mintz et al. discovered that HVEM engagement of BTLA on Tfh cells reduced T cell receptor (TCR) signaling and CD40 ligand mobilization to the synapse, thereby reducing the help to B cells and inhibiting B cell proliferation (35). Additionally, it was found that BTLA could inhibit IL-21 production by Tfh cells to suppress the development of germinal center B cells and subsequent IgG responses (36).

As its name implies, BTLA shows preferential expression in T and B cells, yet its expression can also be detected in additional immune cells, such as DCs. Immature DCs express lower levels of BTLA, and BTLA expression increases with maturation (37). The number of DCs in BTLA^-/- mice spleen was found to be similar to that in wild-type mice, suggesting that BTLA is not essential for DC development (38). The HVEM-BTLA pathway plays an important role in regulating DC homeostasis. The lymphotoxin β receptor signaling pathway triggers the proliferation of DCs, while the HVEM-BTLA signaling pathway suppresses DC proliferation, suggesting that the HVEM-BTLA pathway provides an inhibitory checkpoint for DC homeostasis (39). Xin et al. reported that BTLA overexpression suppressed DC maturation and enhanced the immune tolerance of immature DCs (40). BTLA can inhibit toll-like receptor 4 signaling and proinflammatory cytokine production in DCs, thereby inhibiting LPS-induced endotoxic shock (38). Jones et al. discovered that BTLA⁺ DCs governed the conversion of peripheral Treg cells by upregulating CD5, thus enhancing the tolerance of peripheral Treg cells (41). Moreover, active pulmonary tuberculosis drives BTLA expression in DCs, thereby inhibiting Th17 and Th22 responses induced by DCs and promoting Treg and Th2 differentiation (42). Further, urothelial cancer induces the overexpression of BTLA in DCs, resulting in reduced secretion of effector cytokines (43).

BTLA is found to be expressed in tumor-infiltrating lymphocytes (TILs) and is often associated with impaired anti-tumor immune response. Upregulated BTLA expression in gallbladder cancer (GBC) plays a role in inhibiting anticancer immunity, and an increased proportion of BTLA⁺CD8⁺ cells is related to the unfavorable outcome of GBC patients (44). In hepatocellular carcinoma (HCC) cases, the expression level of BTLA markedly increases in circulating CD4⁺ cells rather than in CD8⁺ T cells (45, 46). Besides, blocking the BTLA/HVEM signaling pathway can promote IFN-γ secretion by circulating CD4⁺ and CD8⁺ T cells (45). BTLA⁺ T cells show increased levels of additional checkpoint molecules, such as PD-1, lymphocyte-activation gene-3, T cell immunoglobulin and mucin-do-main-containing molecule-3 (TIM-3). In addition, these cells exhibit a poorly differentiated phenotype, reduced cytolysis, and increased proliferation potential among patients with diffuse large B-cell lymphoma (DLBCL). Further, increased BTLA levels are related to an advanced disease stage in DLBCL patients (47). BTLA expression is elevated in T cells from patients with melanoma (48, 49). Furthermore, PD-1⁺TIM-3^-CD8⁺ T cells expressing BTLA are the largest tumor-specific CD8⁺ T cell subset among melanoma cases that show partial dysfunction with decreased IFN-γ production compared with BTLA^- T cells (49). BTLA is highly expressed in type I NKT cells in murine autochthonous mammary tumors, and BTLA-neutralizing antibodies can inhibit tumor proliferation and pulmonary metastasis (50). BTLA expression was also found to be upregulated in T cells from patients with lung cancer (51). In addition, soluble BTLA (sBTLA) in plasma is significantly associated with the risk of death in patients with clear cell renal cell cancer, suggesting that sBTLA has a similar role to membranous BTLA in inhibiting T cell response (52). However, some studies have reported contradictory results. For instance, as suggested by Song et al., BTLA levels decreased in colorectal cancer tissues in comparison with levels in matched non-carcinoma tissues. In addition, decreased BTLA levels predicted poor overall survival and less LNM (53). Notably, BTLA is suggested to trigger both inhibitory and survival signaling, and may have a context-specific function in TILs (7). Although BTLA is a co-inhibitory receptor, higher frequencies and numbers of BTLA-expressing CD8⁺ TILs are markedly associated with positive adoptive cell therapy responses among melanoma cases (54). This team further found that BTLA marks a less-differentiated TIL subset, which displays enhanced resistance to apoptosis and improved survival after tumor killing (55, 56). Besides, growing evidence suggests that polymorphisms of BTLA increase susceptibility to a wide range of cancers. Rs1982809 is a functional single nucleotide polymorphism (SNP) that affects BTLA 3′-UTR activity and BTLA expression, and has been found to be associated with a higher incidence of renal cell carcinoma (57), chronic lymphocytic leukemia (58) and esophagogastric junction adenocarcinoma (59). Five BTLA gene SNPs were found to be related to progesterone receptor, estrogen receptor, tumor size, p53, and C-erbB-2 statuses among Chinese females from the northeast Heilongjiang Province (60). However, Cao et al. found no significant relationship between BTLA polymorphisms and esophageal squamous cell carcinoma (61).

BTLA negatively regulates T cell-mediated inflammation. BTLA^-/- mice experience a prolonged duration of allergic airway inflammation and enhanced recruitment of inflammatory cells relative to those in wild-type mice (62). Moreover, DNFB-mediated contact hypersensitivity, CD8⁺ T cell proliferation, and IFN-γ generation increased in BTLA^-/- mice, whereas these effects were reversed by an agonistic anti-BTLA antibody (63). The BTLA⁺ T cell frequency is elevated in the mucosa and blood of mice with colitis, and BTLA can control inflammatory responses by upregulating Foxp3 expression (64). In a Con A challenge model of acute hepatitis, BTLA^-/- mice showed reduced survival and increased early secretion of serum cytokines, which was partly due to the negative regulation of NKT cells by BTLA (65). Moreover, Shi et al. discovered that BTLA could induce the self-tolerance of CD8⁺BTLA⁺ T cells to reduce the attack on hepatocytes, thus regulating hepatic homeostasis in a Con A-induced hepatitis model in zebrafish (66).

Notably, a growing body of research suggests that BTLA plays an important role in sepsis. Sepsis mortality is prevented in mice with BTLA deficiency, which presents with reduced leukocyte recruitment into the peritoneum, reduced IL-10 secretion, increased bacterial clearance, and suppressed neutrophil activation (67). Besides, it is further found that critically ill sepsis patients show a markedly increased proportion of BTLA⁺ CD4⁺ lymphocytes in peripheral blood, which is associated with a subsequent infection (68). The BTLA level in Tregs (69) and plasma concentration of sBTLA (70, 71) are elevated in sepsis cases, and they are related to sepsis severity. However, some studies have reported different results. Specifically, relative to that in normal subjects, the number of BTLA⁺CD4⁺ T cells decreased among patients with severe sepsis, and a lower percentage of BTLA⁺CD4⁺ T cells during the early stage of sepsis was associated with the severity and mortality of sepsis patients (72). Furthermore, Spec et al. reported that differences in BTLA levels in CD4⁺ and CD8⁺ T cells were not statistically significant between patients with Candida sepsis and controls (73). Interestingly, different clones of BTLA antibodies have two contradictory effects, including neutralizing and potentiating effects on BTLA-mediated signaling (74, 75). Cheng et al. reported that BTLA antibody treatment increased the levels of cytokines and chemokines and promoted the recruitment of inflammatory cells into the peritoneal cavity, leading to deteriorated organ damage and increased mortality in mice with hemorrhagic shock/sepsis (76). However, Kobayashi et al. found that a BTLA antibody rescued mice from LPS-induced endotoxic shock (38). The authors also suggested that mice with BTLA deficiency showed high susceptibility to LPS-induced endotoxic shock and that LPS-mediated production of IL-12 and tumor necrosis factor α significantly increased in macrophages and DCs of mice with BTLA deficiency (38). These discrepant results from different studies may be related to the different functions of the antibodies used. Moreover, genetic variations in BTLA are associated with sepsis morbidity. In addition to increasing cancer susceptibility, Rs1982809 is also associated with the incidence of sepsis and multiple organ dysfunction scores (77).

BTLA is suggested to play a vital role in the protection from autoimmunity. Mice with BTLA deficiency exhibit increased antigen-specific IgG responses and are sensitive to experimental autoimmune encephalomyelitis (EAE) (4, 78). BTLA deficiency results in spontaneous development of autoimmune hepatitis-like disease, which is characterized by elevated transaminase levels, interface hepatitis, and hepatic spotty necrosis (79). The number of BTLA-expressing B cells and CD19⁺/BTLA⁺/IL-10⁺ cells obviously decreased in multiple sclerosis (MS) cases, while the remission of fingolimod-induced relapsed-remitted MS is related to a significantly increased number of CD19⁺/BTLA⁺/IL-10⁺ B lymphocytes (80). In patients with active systemic lupus erythematosus (SLE), the proportion of CD4⁺BTLA⁺ T cells is markedly decreased (81). Sawaf et al. found that in SLE cases, BTLA had a lower ability to suppress the activation of T cells, and such impaired BTLA function in lupus CD4⁺ T cells was found to be related to disease activity (82). BTLA deficiency accelerates the lupus-like phenotype in autoimmune-prone MRL-lpr/lpr mice, and severe lymphocytic infiltration is detected in the lungs, kidneys, salivary glands, joints, and pancreas (83). However, a study conducted in the Japanese population showed that there was no significant difference in haplotypes, genotypes, and alleles of the BTLA gene between SLE and healthy groups (84). Anti-BTLA mAb treatment can delay the onset of autoimmune diabetes in non-obese diabetic (NOD) mice, increase the proportion of Tregs, and direct the cytokine milieu away from autoimmunity (85). Transferring transgenic BTLA-expressing DCs into NOD mice markedly triggers the tolerance of CD8⁺ T cells, while attenuating the autoimmune diabetes severity (86). In rheumatoid arthritis (RA) patients, BTLA-expressing CD3⁺/CD4⁺/CD8⁺ T cell proportions are remarkably increased, and the swollen joint count is negatively correlated with the percentage of BTLA⁺CD8⁺ T cells (87). Lin et al. found that the C+800T SNP in the BTLA gene was associated with RA susceptibility (88). Besides, the 590C SNP, a functional polymorphism of the BTLA gene, is markedly associated with RA susceptibility in the Japanese population (89).

Since BTLA can provide both inhibitory and pro-survival signals to T cells, it plays a dual role during infection. BTLA expression increases in various infectious diseases, which is generally related to an impaired immune response against infection. BTLA is strongly upregulated in both CD4⁺ and CD8⁺ T cells from COVID-19 patients (90). Similarly, BTLA levels increase in CD4⁺ and CD8⁺ T cells from patients with pulmonary tuberculosis, and this is related to disease progression (91). During infection with primary cytomegalovirus (CMV), BTLA is highly induced in CD8⁺ T cells, and BTLA blockade enhances CMV-specific CD8⁺ T cell proliferation (92). Increased BTLA level in intrahepatic lymphocytes is related to impaired T cell responses in the process of chronic hepatitis B virus (HBV) infection, whereas blocking BTLA promotes cytokine production and T cell proliferation (93). In patients with chronic HBV infection, a subset of antigen-specific CD8⁺ T cells with low IFN-γ production express high levels of BTLA, and these cells play a vital role in the regulation of CD8⁺ T cell responses via BTLA signaling (94). In patients with severe community-acquired pneumonia and in mice with acute lung inflammation, circulating BTLA⁺CD4⁺ lymphocyte proportions markedly increased, whereas agonistic anti-BTLA antibody reduced the activation of the NF-κB pathway and attenuated the inflammatory responses (95). In mice infected with the parasitic nematode Strongyloides ratti, BTLA expression in CD4⁺ T cells was upregulated. Additionally, deficiency of either BTLA or HVEM leads to decreased amounts of adult parasites in the small intestine, while reducing the larval output in the process of infection (96). Moreover, BTLA^-/- mice have also shown resistance to infection in several other disease models. During infection with murine hepatitis virus strain-3 (MHV-3), BTLA^-/- mice show markedly improved spleen and liver injuries and reduced mortality (97). BTLA suppresses both the innate and T/B cell-dependent adaptive immune responses during experimental malaria, whereas BTLA^-/- mice show markedly decreased parasitemia, and early clearance of infections (98). Furthermore, BTLA^-/- mice are more resistant to listeriosis infection (99), whereas targeting BTLA promotes primary and memory T cell responses (100) and accelerates early bacterial clearance (99). Interestingly, BTLA^-/- mice have a decreased number of Listeria-specific CD8⁺ T cells, which reveals the dual effects of BTLA in regulating anti-intracellular pathogen responses in the host (100, 101). Furthermore, HVEM expression in CD8⁺ T lymphocytes and BTLA expression in other cell types are necessary for the optimal survival of effector and memory T cells, suggesting that the BTLA/HVEM pathway enhances activated CD8⁺ T cell survival in the process of Listeria infection (101). During vaccinia virus infection, BTLA or HVEM deficiency markedly damages the survival of effector CD8⁺ T cells and protective immune memory (102). BTLA⁺ αβ T cells display a central memory phenotype to combat Mycobacterium tuberculosis (Mtb) infection, as manifested by increased cell proliferation and cytokine secretion, suggesting that BTLA expression in αβT cells is involved in protective immune memory against Mtb infection among patients with active pulmonary tuberculosis (103).

The inhibitory role of BTLA on T cells has also been supported by transplantation studies. BTLA can inhibit donor anti-host T cell responses and ameliorate graft-versus-host disease (GVHD) resulting from allogeneic bone marrow transplantation (104, 105). In patients with acute renal allograft rejection, BTLA levels in peripheral CD3⁺ T lymphocytes are markedly lower than those in normal controls who show stable functions of the transplanted kidney (106, 107). Agonistic anti-BTLA antibody treatment prolongs cardiac allograft survival in mice with increased CD4⁺CD25⁺Foxp3⁺ cells and reduced IL-2 and IFN-γ production (108). Furthermore, BTLA overexpression in a rat model can markedly suppress acute kidney rejection and extend allograft survival (106). Partially MHC-mismatched allografts can potently induce BTLA expression, while targeting BTLA prompts rapid rejection (109). Agonistic anti­BTLA antibody combined with CTLA-4 immunoglobulin treatment can prolong the survival of islet allografts, while promoting indefinite graft acceptance (110, 111). Moreover, according to the latest research, overexpression of BTLA combined with CTLA-4 in kidney transplant recipients reduced IL-2 production, suppressed T cell proliferation, improved graft function, attenuated acute T cell-mediated rejection, and extended graft survival (112). However, there have been some contradictory reports. For example, Del Rio et al. reported that BTLA blockade alleviated acute GVHD reaction in an F1 transfer semiallogeneic murine model (113, 114). Rodriguez-Barbosa et al. found that targeting BTLA/HVEM showed no effect on modulating graft rejection across a fully MHC mismatched barrier and that the donor-specific allogeneic immune response was independent of the HVEM/BTLA signaling pathway (115). Besides, Wang et al. explored the role of BTLA and HVEM polymorphisms in antibody-mediated rejection in renal transplant recipients and found that none of the polymorphisms identified was associated with antibody-mediated rejection (116).