Gliomas are malignant brain tumors that originate from glial cells within the Central Nervous System (CNS) and are notorious for their rapid growth and invasiveness (1). Glioblastoma (GBM) stands out as the most prevalent and lethal subtype, accounting for approximately 57% of all gliomas and 48% of primary CNS malignant tumors. Patients with GBM face a median survival rate of merely 14–16 months (1, 2). Over the past decade, there has been a significant refinement in the World Health Organization’s classification of gliomas. The 5th edition of the WHO classification of Tumors of the Central Nervous System (WHO CNS5) has incorporated specific molecular and genetic criteria. As a result, GBMs are now delineated as grade 4 gliomas with a wild-type IDH (3). Furthermore, the classification distinguishes various histological types: adult-diffuse gliomas, pediatric low-grade diffuse gliomas, pediatric high-grade diffuse gliomas, and circumscribed astrocytic gliomas (4). Specific mutations further categorize adult diffuse gliomas into astrocytoma (with mutated IDH), oligodendroglioma (featuring mutated IDH and a 1p/19q code), and glioblastoma (with wild-type IDH) (4). It is crucial to highlight that the terms “pediatric” and “adult” types in this context do not refer to the patient’s age but rather to distinct molecular and genetic profiles (5).

This paradigmatic transition underscores the need to re-evaluate both diagnostic and therapeutic strategies. The emphasis is now on personalized medicine, particularly in the realm of immunotherapy. The CNS, contrary to previous beliefs, is an immuno-competent region (6). However, the immunosuppressive microenvironment in GBM tumors, fueled by a combination of molecular and cellular elements such as interleukin-6 (IL-6), interleukin-10 (IL-10), transforming growth factor-beta (TGF-β), M2 macrophages, microglia, and regulatory T cells (T-regs), thwarts effective anti-tumor responses (7).

In this context, numerous studies have underscored the significance of examining the role of immune checkpoint (IC) molecules and the potential benefits of inhibiting these pathways (8–13). To date, the Food and Drug Administration (FDA) has approved two immune checkpoint blockade (ICB) therapeutic strategies (14). Anti-PD1 and/or anti-CTLA-4 drugs are commonly administered to patients with advanced and recurrent glioblastoma and may be combined with temozolomide (TMZ)-based chemotherapy or radiotherapy (15, 16). While these therapies offer certain benefits, such as reinvigorating the functional state of tumor-infiltrating lymphocytes (TILs) and enhancing the expression of Interferon-Gamma (IFN-γ) and Interleukin-2 (IL-2), they have yet to demonstrate a significant improvement in overall survival (16, 17). Therefore, it might be prudent to investigate novel blockade approaches, focusing on promising immune checkpoints like VISTA, TIGIT, A2AR, and NR2F6, all of which have been linked to advanced glioma (18–21).

Consistent with this approach, the present study seeks to ascertain the potential role of Butyrophilin Subfamily 3 Member A1 (BTN3A1 or CD277) in advanced gliomas. BTN3A molecules are members of the immunoglobulin family and exist in three distinct isoforms: BTN3A1, BTN3A2, and BTN3A3, which exhibit 95% of structural homology (22). These paralogous genes encode transmembrane proteins featuring both IgV and IgC extracellular domains, as well as a B30.2 intracellular region pivotal for downstream signaling (23). BTN3A1 appears to be the only one that can efficiently elicit signal transduction, either after stimulation by binding to its putative ligand on its extracellular domain, or by the direct interaction of a phosphoantigen with the B30.2 intracellular domain. Studies have highlighted the significant role of the intracellular domain B30.2 in cell signaling (24). Unlike BTN3A1 and BTN3A3, BTN3A2 lacks it, which limits its involvement in signaling processes (24). Regarding BTN3A3, despite the presence of the intracellular domain B30.2, it would appear to be less efficient than BTN3A1 in cell signaling due to slight changes in amino acid residues in the B30.2 structure (25) The BTN3A1 molecule is expressed on a variety of cells, including T cells, B cells, monocytes, dendritic cells, natural killer (NK) cells, and even tumor cells (26). Most studies on BTN3A1 focus mainly on how it activates γδ T cells, whereas BTN3A molecules are also involved in αβ T cell immunomodulation (27). As previously demonstrated, BTN3A1 is implicated in CD4+/CD8+ αβ T-cells inhibition. Using genetically engineered APCs expressing BTN3A1, Kyle K. Payne et al. demonstrated reduced IFNγ release and CD4+/CD8+ T-cell proliferation (27). Jinghua. W and coworkers highlighted the interaction between BTN3A1 on T cells and LSECtin on melanoma cells, resulting in decreased production of IFN-γ, IL-2, and TNF-α (28). The ability of BTN3A2 and BTN3A3 to induce these inhibitions on CD4+/CD8+ αβ T-cells has not yet been fully demonstrated. While some studies have identified LSECtin as a ligand for BTN3A1, the definitive binding partner for this molecule remains inadequately characterized (28, 29). As such, the prognostic value of BTN3A1 is intricately tied to the specific cancer type and the signaling pathways activated upon binding with its presumptive ligand on APC/tumor cells (26). For instance, overexpression of BTN3A1 in ovarian cancer (OC) has been linked to suppressed αβ T cell proliferation and reduced T helper 1 (Th1) cytokine production, culminating in unfavorable patient outcomes (30). Additionally, the correlation of BTN3A expression with immune evasion strategies of pancreatic tumor (PC) cells reinforces the role of BTN3A in facilitating tumor progression (31). A notable recent study elucidated that BTN3A1 overexpression in esophageal squamous cell carcinoma (ESCC) augments radioresistance by modulating the expression and phosphorylation of UNC-51-like autophagy-activating kinase (ULK1), subsequently correlating with adverse prognoses (32).

In our study, we endeavored to probe and underscore the significance of BTN3A1 in gliomas. By leveraging the TCGA database in tandem with our in-house cohort, we scrutinized the relationship between BTN3A1 expression, and clinicopathological as well as molecular factors. Our findings indicated that BTN3A1 could play a part in promoting tumor progression, and its overexpression correlates with poor prognosis. We are optimistic that our findings will pave the way for devising potential anti-BTN3A1 therapeutic avenues for addressing advanced gliomas.

The central nervous system is an intricate and meticulously organized structure responsible for many crucial biological functions. Pathological disturbances within this system can lead to significant lifestyle implications (36). Glioblastoma (GBM), which accounts for over 50% of gliomas, is classified as an IDH-wildtype and grade 4 tumor according to the latest WHO classification of the CNS (4). The aggressive and invasive nature of GBM can compromise the integrity of the blood-brain barrier (BBB) and hampers the CNS’s proper functioning, resulting in a markedly low survival rate (37). Immunotherapy emerges as a promising therapeutic strategy that extends patient care beyond conventional treatments (38). While CTLA-4 and PD-1/PD-L1 immune checkpoint blockades have yielded significant results in various cancers, such as melanoma, non-small cell lung cancer (NSCLC), and bladder cancer (BC), their survival benefits have not yet been established in gliomas (39–41). Consequently, the exploration of new immune checkpoint candidate molecules might yield better outcomes for glioma patients. The BTN3A1 (CD277) molecule, a member of the immunoglobulin superfamily receptors, has been shown to play a role in Vγ9Vδ2 T-cell activation through the sensitivity of its B30.2 domain to phosphoantigens (pAg). Recently, there has been growing interest in investigating the relationship between BTN3A1 and cancer, with prior results indicating its dual role in tumor progression (26). In this study, we assessed the expression pattern and potential role of BTN3A1 using both TCGA datasets and our own Moroccan glioma cohort.

Transcriptomic and protein expression analyses of BTN3A1 expression in our Moroccan cohort revealed a significant upregulation in grade 4 gliomas when compared to grades 2 and 3. This was corroborated by the TCGA dataset, which displayed a heightened expression of BTN3A1 in grade 4 gliomas relative to grades 2 and 3, and also in grade 3 compared to grade 2. Following the World Health Organization CNS5 guidelines, glioblastomas are now categorized as IDH-WILD type/grade 4 gliomas (5). Our findings suggest that an increased expression of BTN3A1 is associated with more aggressive glioma subtypes. This suggests that BTN3A1 could serve as a potential biomarker for glioma diagnosis and grading. In clinical scenarios, elevated BTN3A1 expression could raise suspicion for grade 4 gliomas, leading to a more precise classification and facilitating timely and appropriate clinical management. Previous research has demonstrated that soluble plasma levels of BTN3A1 (sBTN3A1) may predict the efficacy of nivolumab therapy in patients with metastatic renal cell carcinoma (mRCC) (42). Therefore, recognizing elevated BTN3A1 expression as a marker for grade 4 gliomas could augment the molecular characteristics delineated in the fifth edition of the WHO classification of central nervous system tumors (WHO CNS5).

Our single-cell analysis indicated that BTN3A1 is expressed in both immune and glioma cells. Interestingly, Recent findings have delineated four distinct glioblastoma cell states: Neural-Progenitor-like (NPC-like), Oligodendrocyte-Progenitor-like (OPC-like), Astrocyte-like (AC-like), and Mesenchymal-like (MES-like). Each of these states mirrors the neurodevelopmental differentiation and damage responses observed in the normal neural lineage (33). While the reaction of normal neural cells to tumor cells plays a role in shaping the tumor microenvironment, altered responses from glioblastoma progenitor cells lead to overlapping effects that significantly influence the propagation and persistence of glioblastoma (33). Our results revealed an upregulation of BTN3A1 in the AC-like cell state. Notably, the AC-Like state is characterized by aggressive and proliferative cells (33). Furthermore, adult diffuse gliomas exhibit a higher proportion of AC-Like cells compared to pediatric diffuse gliomas. This distinction could partially account for the more aggressive nature of adult gliomas (33, 43). Given these findings, the pronounced expression of BTN3A1 in AC-Like cells supports our initial observations, underscoring BTN3A1’s potential as a promising marker for glioblastomas.

With respect to the profile of immune infiltration, we observed that BTN3A1 expression directly correlated with the infiltration levels of B cells, CD8+ T cells, general CD4+ T cell populations, and M2 macrophages. Conversely, there was a negative correlation with Th1 and M0 macrophages. Recent research posits the Central Nervous System (CNS) as an immune-competent region. Moreover, evidence indicates that the glioma environment can impair and render the Blood-Brain Barrier (BBB) permeable, thus promoting the migration of immune effectors (6, 44).

In light of our findings, BTN3A1 seems to drive a pro-tumor immune response by bolstering M2 macrophage activities. Studies have demonstrated that M2 macrophages foster tumor progression and engender an immunosuppressive microenvironment within gliomas, notably through the secretion of cytokines IL-6, TGF-β, and IL-10, which in turn recruit Treg cells (45, 46). This assertion aligns seamlessly with our observations where elevated BTN3A1 levels were associated with heightened IL-10 and TGF-β expression. However, there wasn’t any significant difference in the expression of IFN-γ and Granzyme B between “high-BTN3A1” and “low-BTN3A1” groups. Notably, BTN3A1 exhibited a positive correlation with IL-10 and FoxP3, and single-cell analysis indicated that exhausted CD8+ T cells expressed BTN3A1.

Drawing from the data, we hypothesize that (1) BTN3A1 propels its pro-tumor effect in glioblastoma by inciting M2 macrophages to produce IL-10. This, in turn, activates and attracts Treg cells, culminating in CD8+ T-cell exhaustion. Earlier research also identified an association between BTN3A1 upregulation in ovarian cancer and the suppression of αβ T cell activity, ostensibly due to BTN3A1’s interference with CD45-mediated signaling, thereby obstructing TCR-mediated T cell activation (27). Adding to this, Wang J. et al. presented evidence that BTN3A1, when expressed on T-cells, binds to LSECtin on melanoma cells, resulting in the suppression of T cell proliferation and the production of cytokines like (28). A holistic analysis spanning various omics data sets revealed a positive association between BTN3A1 and immune cells such as B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells in conditions like Breast Cancer (BRCA), Lung Adenocarcinoma (LUAD), and Lung Squamous Cell Carcinoma (LUSC) (47).

Our research underscored the presence of BTN3A1 in both immune and glioblastoma cells. Its expression was clearly associated with immune cell infiltration and immunosuppressive activity. We highlighted the potential role of BTN3A1 in the establishment of an immunosuppressive microenvironment, thus contributing to the inhibition of αβ T cell activity in gliomas. In a recent study, Wang Y and coworkers demonstrated that high BTN3A1 protein expression could predict a favorable Vγ9Vδ2 T-cell-mediated cytotoxic response in glioblastoma (48). Increased levels of BTN3A1 may enhance phosphoantigen presentation to γδ T cells, improving their anti-tumor responses. In such a glioma microenvironment, the higher the expression of BTN3A1, the more effective the cytotoxicity of γδ T cells should be, leading to tumour regression and improved prognosis. Although this hypothesis is fully cogent, it does, have some limitations. γδ T cells are uncommon, representing less than 5% of all T lymphocytes (49). In addition, as mentioned in the related study, the cytotoxicity of the γδ T cells against GBM cells was limited (≈30%) (48). High levels of BTN3A1 will tip the balance in favor of tumour progression and αβ T cell inhibition, despite the low compensation of γδ T cell activation. By prioritizing the reversal of αβ T cell inhibition over γδ T cell activation, our results suggest that blocking BTN3A1 may be an effective strategy and will represent a promising avenue for immunotherapy in glioblastoma. Alternatively, a strategy based on preventing BTN3A1-induced inhibition of αβ T cells, combined with stimulation of γδ T cells, will contribute meaningfully to improving anti-tumor response. In their study, Kyle K. Payne et al. demonstrated that CTX-2026, an anti-BTN3A1 antibody, can both hinder BTN3A1-mediated inhibition of αβ T-cells and activate γδ T cells (27). To summarize, the tumor microenvironment specificity is pivotal and governs how some therapeutic strategies can be implemented to foster relevant outcomes. Our findings suggest targeting BTN3A1 may present a promising avenue for immunotherapy in glioblastoma.

Combined therapies have consistently demonstrated superior efficacy compared to monotherapies. Notably, while the efficacy of immunotherapies for glioblastoma remains constrained, there’s compelling evidence supporting enhanced survival outcomes through combination immune checkpoint blockade strategies (50). In our investigation into the relationship between BTN3A1 and various checkpoints such as PD-1, CTLA-4, VISTA, TIGIT, and TIM-3, we discerned a significant association. Elevated BTN3A1 expression correlated positively with heightened TIM-3 expression in glioblastoma. Previous research has highlighted TIM-3’s role in advancing glioblastoma progression by curtailing the effector functions of CD8+ T cells and Th1 cells and attenuating IFNγ production (51). Additionally, TIM-3 plays a pivotal role in the migration and polarization of pro-tumoral M2 macrophages (52). Our findings, juxtaposed with prior research, suggest that (2) BTN3A1 might expedite glioma progression through the activation of the IL-6/JAK/STAT3 pathway (as depicted in Figure 6A ). This leads to the upregulation of TIM-3, potentially inhibiting T cell activities while stimulating M2 macrophages. Furthermore, existing studies have characterized a pro-tumoral glioma phenotype distinctively marked by the TIM-3/interleukin 6 interaction (51, 52). It should also be noted that in addition to the IL-6/JAK/STAT3 pathway, Tim-3 seems to promote tumor progression through the NF-κB signaling pathway (53). Herein, we found that high expression of BTN3A1 was associated with the activation of the NF-κB via the TNF-α signaling pathway. The involvement of NF-κB in oncogenesis is well established (54, 55) Thus, co-expression of BTN3A1 with TIM-3 may result in a synergistic pro-tumor effect by enhancing the IL-6/JAK/STAT3 and TNF-α/NF-κB pathways. Previous studies demonstrated that the combination of anti-TIM-3 with anti-PD1 has aroused more encouraging results than with anti-PDL1 in melanoma, NSCLC, and ESCC (53). Combined immune checkpoint blockade is a fairly recent strategy in the treatment of gliomas, as evidenced by the ongoing clinical trial which will assess the combination of Anti-Tim-3 with Anti-PD-1 (56). This positive correlation between BTN3A1 and TIM-3 holds promising implications for potential combined immune-checkpoint blockade strategies in glioblastoma.

Our GSEA analysis probed the influence of BTN3A1 in orchestrating both immunological and tumor pathways. Overexpression of BTN3A1 was associated with hallmark genes implicated in inflammatory responses, encompassing INF-α, INF-γ, TNF-α, and the complement system. Distinct research underscores the ambivalent role of these inflammatory agents in glioblastoma (57). For instance, in addition to INF-α, INF-γ, and TNF-α’s pro-inflammatory functions, INF-γ has been shown to amplify PD-L1 expression and foster T-reg differentiation in glioblastoma, thereby abetting glioma cells in evading immune surveillance (7, 58). Preliminary transcriptomic analyses aimed at deriving a risk score grounded on INF-γ expression posited that elevated INF-γ levels correlated with a more adverse clinicopathological and molecular prognosis in glioblastoma (59, 60). Simultaneously, TNF-α exhibited heightened expression in GBM and was implicated in bolstering tumor proliferation (61). Current literature remains scant regarding the roles of INF-α and the complement system in glioblastoma. Our research suggests that BTN3A1 is potentially instrumental in triggering inflammatory cascades, as evidenced by its association with hallmark genes of inflammatory responses. Subsequently, an immune-suppressive microenvironment seems to be established, steered by increased expression of BTN3A1. Furthermore, our data reveals an association of heightened BTN3A1 expression in glioblastoma with signature genes of pathways such as IL6/JAK/STAT3, TGF-β, KRAS, P53, and PI3K/AKT/MTOR. It’s intriguing that BTN3A1’s role in tumorigenesis can oscillate between pro-tumoral and anti-tumoral, contingent on the pathological milieu and the specific downstream signaling cascades activated by specific interactions between the IgC extracellular domain of BTN3A1 and its putative ligand (26, 29). Prior investigations affirm the profound implications of pathways like KRAS, P53, and PI3K/AKT/MTOR in the onset, evolution, and metastasis of glioblastoma (62–64). We postulate that (3) BTN3A1 may champion glioblastoma establishment and expansion by actuating the KRAS, P53, and PI3K/AKT/MTOR signaling conduits. BTN3A1’s correlation with hallmark genes of the IL6/JAK/STAT3 and TGF-β pathways reaffirms and validates our preceding observations. These observations indicated that (1) BTN3A1 mediates a pro-tumoral influence in glioblastoma, principally through the induction of M2 macrophages to synthesize IL-10 and TGF-β. This cascade eventually leads to CD8+ T-cell exhaustion and (2) accentuates glioma development via the IL-6/JAK/STAT3 pathway.

The analysis of BTN3A1’s prognostic significance revealed an association between elevated BTN3A1 expression and poorer prognosis in glioma patients. Elevated BTN3A1 expression has also been linked to unfavorable outcomes in esophageal squamous cell carcinoma (ESCC), metastatic gastrointestinal stromal tumors (mGISTs), and ovarian cancer (OC) (27, 32, 65).

The present study suggests that BTN3A1 expression can serve as a prognostic indicator for worse outcomes in advanced human gliomas.

In summary, our study posits that elevated BTN3A1 expression correlates with an immunosuppressive microenvironment in gliomas, precipitating a heightened aggressive pathological state. BTN3A1 seems to foster a pro-tumor response, operating in association with TIM3, M2 macrophages, Tregs, and other pro-tumor pathways. Consequently, BTN3A1 expression may serve as a pioneering immunological indicator to foresee unfavorable outcomes in advanced human gliomas.

However, our study remains limited due to the limited size of our Moroccan patient cohort. Further exploration is essential to validate the mechanistic and functional significance of BTN3A1 in the modulation of immune cells, paving the way for potential anti-BTN3A1 immunotherapies.

The original contributions presented in the study are included in the article/ Supplementary Materials , further inquiries can be directed to the corresponding author/s.

The present study received approval from the Ethical Board of Ibn Rochd University Hospital, Casablanca, with the approval code 28/15. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants’ legal guardians/next of kin.

AK: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. AG: Data curation, Formal analysis, Visualization, Writing – review & editing. AQ: Data curation, Formal analysis, Software, Writing – review & editing. NS: Formal analysis, Investigation, Writing – review & editing. YB: Formal analysis, Writing – review & editing. AL: Data curation, Writing – review & editing. MK: Data curation, Formal analysis, Writing – review & editing. AB: Funding acquisition, Methodology, Supervision, Validation, Visualization, Writing – review & editing.