Up-regulation of BTN3A1 on CD14+ cells promotes Vγ9Vδ2 T cell activation in psoriasis.

Vγ9Vδ2 T cells play an important role in the development and progression of psoriasis vulgaris (PV), but how they promote skin inflammation and the molecular mechanisms underlying Vγ9Vδ2 T cell dysfunction are poorly understood. Here, we show that circulating Vγ9Vδ2 T cells are decreased and exhibit enhanced proliferation and increased production of IFN-γ and TNF-α in PV patients. Monocytes from PV patients express higher levels of the phosphoantigen sensor butyrophilin 3A1 (BTN3A1) than monocytes from healthy controls. Blockade of BTN3A1 suppresses Vγ9Vδ2 T cell activation and abolishes the difference in Vγ9Vδ2 T cell activation between PV patients and healthy controls. The CD14+ cells in PV skin lesions highly express BTN3A1 and juxtapose to Vδ2 T cells. In addition, IFN-γ induces the up-regulation of BTN3A1 on monocytes. Collectively, our results demonstrate a crucial role of BTN3A1 on monocytes in regulating Vγ9Vδ2 T cell activation and highlight BTN3A1 as a potential therapeutic target for psoriasis.

B7H3-targeting chimeric antigen receptor modification enhances antitumor effect of Vγ9Vδ2 T cells in glioblastoma.

BACKGROUND: Glioblastoma (GBM) is a highly aggressive primary brain tumor with a poor prognosis. This study investigates the therapeutic potential of human Vγ9Vδ2 T cells in GBM treatment. The sensitivity of different glioma specimens to Vγ9Vδ2 T cell-mediated cytotoxicity is assessed using a patient-derived tumor cell clusters (PTCs) model. METHODS: The study evaluates the anti-tumor effect of Vγ9Vδ2 T cells in 26 glioma cases through the PTCs model. Protein expression of BTN2A1 and BTN3A1, along with gene expression related to lipid metabolism and glioma inflammatory response pathways, is analyzed in matched tumor tissue samples. Additionally, the study explores two strategies to re-sensitize tumors in the weak anti-tumor effect (WAT) group: utilizing a BTN3A1 agonistic antibody or employing bisphosphonates to inhibit farnesyl diphosphate synthase (FPPS). Furthermore, the study investigates the efficacy of genetically engineered Vγ9Vδ2 T cells expressing Car-B7H3 in targeting diverse GBM specimens. RESULTS: The results demonstrate that Vγ9Vδ2 T cells display a stronger anti-tumor effect (SAT) in six glioma cases, while showing a weaker effect (WAT) in twenty cases. The SAT group exhibits elevated protein expression of BTN2A1 and BTN3A1, accompanied by differential gene expression related to lipid metabolism and glioma inflammatory response pathways. Importantly, the study reveals that the WAT group GBM can enhance Vγ9Vδ2 T cell-mediated killing sensitivity by incorporating either a BTN3A1 agonistic antibody or bisphosphonates. Both approaches support TCR-BTN mediated tumor recognition, which is distinct from the conventional MHC-peptide recognition by αß T cells. Furthermore, the study explores an alternative strategy by genetically engineering Vγ9Vδ2 T cells with Car-B7H3, and both non-engineered and Car-B7H3 Vγ9Vδ2 T cells demonstrate promising efficacy in vivo, underscoring the versatile potential of Vγ9Vδ2 T cells for GBM treatment. CONCLUSIONS: Vγ9Vδ2 T cells demonstrate a robust anti-tumor effect in some glioma cases, while weaker in others. Elevated BTN2A1 and BTN3A1 expression correlates with improved response. WAT group tumors can be sensitized using a BTN3A1 agonistic antibody or bisphosphonates. Genetically engineered Vγ9Vδ2 T cells, i.e.,  Car-B7H3, show promising efficacy. These results together highlight the versatility of Vγ9Vδ2 T cells for GBM treatment.

Low plasma PD-L1 levels, early tumor onset and absence of peritoneal carcinomatosis improve prognosis of women with advanced high-grade serous ovarian cancer.

BACKGROUND: The most common subtype of ovarian cancer (OC) showing immunogenic potential is represented by the high-grade serous ovarian cancer (HGSOC), which is characterized by the presence of tumor-infiltrating immune cells able to modulate immune response. Because several studies showed a close correlation between OC patient's clinical outcome and expression of programmed cell death protein-1 or its ligand (PD-1/PD-L1), the aim of our study was to investigate if plasma levels of immunomodulatory proteins may predict prognosis of advanced HGSOC women. PATIENTS AND METHODS: Through specific ELISA tests, we analyzed plasma concentrations of PD-L1, PD-1, butyrophilin sub-family 3A/CD277 receptor (BTN3A1), pan-BTN3As, butyrophilin sub-family 2 member A1 (BTN2A1), and B- and T-lymphocyte attenuator (BTLA) in one hundred patients affected by advanced HGSOC, before surgery and therapy. The Kaplan-Meier method was used to generate the survival curves, while univariate and multivariate analysis were performed using Cox proportional hazard regression models. RESULTS: For each analyzed circulating biomarker, advanced HGSOC women were discriminated based on long (≥ 30 months) versus short progression-free survival (PFS < 30 months). The concentration cut-offs, obtained by receiver operating characteristic (ROC) analysis, allowed to observe that poor clinical outcome and median PFS ranging between 6 and 16 months were associated with higher baseline levels of PD-L1 (> 0.42 ng/mL), PD-1 (> 2.48 ng/mL), BTN3A1 (> 4.75 ng/mL), pan-BTN3As (> 13.06 ng/mL), BTN2A1 (> 5.59 ng/mL) and BTLA (> 2.78 ng/mL). Furthermore, a lower median PFS was associated with peritoneal carcinomatosis, age at diagnosis > 60 years or Body Mass Index (BMI) > 25. A multivariate analysis also suggested that plasma concentrations of PD-L1 ≤ 0.42 ng/mL (HR: 2.23; 95% CI: 1.34 to 3.73; p = 0.002), age at diagnosis ≤ 60 years (HR: 1.70; 95% CI: 1.07 to 2.70; p = 0.024) and absence of peritoneal carcinomatosis (HR: 1.87; 95% CI: 1.23 to 2.85; p = 0.003) were significant prognostic marker for a longer PFS in advanced HGSOC patients. CONCLUSIONS: The identification of high-risk HGSOC women could be improved through determination of the plasma PD-L1, PD-1, BTN3A1, pan-BTN3As, BTN2A1 and BTLA levels.

Expression level of BTN3A1 on the surface of CD14+ monocytes is a potential predictor of γδ T cell expansion efficiency.

Human γδ T cells expressing Vγ9Vδ2 T cell receptors exert a robust response to pathogens and malignant cells. These cells are activated by BTN3A1, which is expressed by pathogen-derived phosphoantigens (pAgs) or host-derived pAgs that accumulate in transformed cells or in cells exposed to aminobisphosphonates. Activated Vδ2 (+) T cells exert multiple effector functions; therefore, they are a promising candidate for immunotherapy. However, not all donors have γδ T cells with adequate proliferative activity. Here, we performed ex vivo culture of γδ T cells from 20 healthy donors and explored factors that may affect their expansion efficiency. Consistent with previous studies, we found that amplification of γδ T cells requires CD14+ monocytes to act as accessory cells. We also show here that surface expression of BTN3A1 by monocytes correlates positively with γδ T cell expansion. Moreover, treatment with BTN3A1-Fc increased the expansion efficiency of peripheral blood mononuclear cells (PBMCs) from donors harboring γδ T cells with poor expansion capacity. Taken together, the data suggest that the level of BTN3A1 expressed on the surface of monocytes is a useful biomarker for predicting the degree of expansion of γδ T cells.

Incorporation of a FRET pair within a phosphonate diester.

Cell-cleavable protecting groups are an effective tactic for construction of biological probes because such compounds can improve problems with instability, solubility, and cellular uptake. Incorporation of fluorescent groups in the protecting groups may afford useful probes of cellular functions, especially for payloads containing phosphonates that would be highly charged if not protected, but little is known about the steric or electronic factors that impede release of the payload. In this report we present a strategy for the synthesis of a coumarin fluorophore and a 4-((4-(dimethylamino)phenyl)diazenyl)benzoic acid (DABCYL) ester chromophore incorporated as a FRET pair within a single phosphonate. Such compounds were designed to deliver a BTN3A1 ligand payload to its intracellular receptor. Both final products and some synthetic intermediates were evaluated for their ability to undergo metabolic activation in γδ T cell functional assays, and for their photophysical properties by spectrophotometry. One phosphonate bearing a DABCYL acyloxyester and a novel tyramine-linked coumarin fluorophore exhibited strong, rapid, and potent cellular activity for γδ T cell stimulation and also showed FRET interactions. This strategy demonstrates that bioactivatable phosphonates containing FRET pairs can be utilized to develop probes to monitor cellular uptake of otherwise charged payloads.

Potent double prodrug forms of synthetic phosphoantigens.

Phosphoantigens are ligands of BTN3A1 that stimulate anti-cancer functions of γδ T cells, yet the potency of natural phosphoantigens is limited by low cell permeability and low metabolic stability. Derivatives of BTN3A1 ligand prodrugs were synthesized that contain an acetate-protected allylic alcohol and act as doubly protected prodrugs. A novel set of phosphonates, phosphoramidates, and phosphonamidates has been prepared through a new route that simplifies synthesis and postpones the point of divergence into different prodrug forms. One of the new prodrugs, compound 11, potently stimulates γδ T cell proliferation (72 h EC50 = 0.12 nM) and interferon γ response to loaded leukemia cells (4 h EC50 = 19 nM). This phosphonamidate form was > 900x more potent than the corresponding phosphoramidate, and the phosphonamidate form was also significantly more stable in plasma following acetate hydrolysis. Therefore, prodrug modification of phosphonate butyrophilin ligands at the allylic alcohol can both facilitate chemical synthesis and improve potency of γδ T cell stimulation.

MAP4-regulated dynein-dependent trafficking of BTN3A1 controls the TBK1-IRF3 signaling axis.

The innate immune system detects viral nucleic acids and induces type I interferon (IFN) responses. The RNA- and DNA-sensing pathways converge on the protein kinase TANK-binding kinase 1 (TBK1) and the transcription factor IFN-regulatory factor 3 (IRF3). Activation of the IFN signaling pathway is known to trigger the redistribution of key signaling molecules to punctate perinuclear structures, but the mediators of this spatiotemporal regulation have yet to be defined. Here we identify butyrophilin 3A1 (BTN3A1) as a positive regulator of nucleic acid-mediated type I IFN signaling. Depletion of BTN3A1 inhibits the cytoplasmic nucleic acid- or virus-triggered activation of IFN-ß production. In the resting state, BTN3A1 is constitutively associated with TBK1. Stimulation with nucleic acids induces the redistribution of the BTN3A1-TBK1 complex to the perinuclear region, where BTN3A1 mediates the interaction between TBK1 and IRF3, leading to the phosphorylation of IRF3. Furthermore, we show that microtubule-associated protein 4 (MAP4) controls the dynein-dependent transport of BTN3A1 in response to nucleic acid stimulation, thereby identifying MAP4 as an upstream regulator of BTN3A1. Thus, the depletion of either MAP4 or BTN3A1 impairs cytosolic DNA- or RNA-mediated type I IFN responses. Our findings demonstrate a critical role for MAP4 and BTN3A1 in the spatiotemporal regulation of TBK1, a central player in the intracellular nucleic acid-sensing pathways involved in antiviral signaling.

BTN3A1-antibodies and phosphoantigens: TCRVγ9Vδ2 "see" the difference.

Human blood γδ T lymphocytes express TCRVγ9Vδ2 and respond to nonpeptide phosphoantigens (PAgs) by a mysterious mechanism involving the BTN3A1 (CD277) molecule . BTN3A1 is a butyrophilin-like protein related to CD80, PD-L1, and MHC, and is either a presenting or a co-stimulatory molecule for PAgs. Although the precise roles and molecular interactions with the TCRVγ9Vδ2 are currently not determined, it is commonly thought that all TCRVγ9Vδ2 lymphocytes 'see' PAg and BTN3A1 together, presumably in a single molecular recognition event. But whether this recognition event could be reproduced in a simplified model was not addressed in previous studies. In this issue, Starick et al. (Eur. J. Immunol. 2017. 47: 982-992) compared the response of three TCRVγ9Vδ2 pairs of murine and human cell transfectants to PAg and anti-BTN3A1 antibodies using IL-2 release as a readout. The authors found that although the two murine transfectants responded similarly to either stimuli, one murine TCRVγ9Vδ2 transfectant reacted to PAgs but not to anti-BTN3A1 (mAb 20.1). Human transductants behave in a similar fashion, demonstrating that TCRVγ9Vδ2 lymphocytes differentiate PAg and BTN3A1 signals, while species of the transductants unmask this differential sensitivity. Indeed, understanding the puzzling mode of antigen recognition by γδ T lymphocytes will be essential for developing γδ T-cell-based immunotherapies, and the authors of this study now demonstrate that TCRVγ9Vδ2 lymphocytes are able to differentiate the PAg and BTN3A1 stimuli.

A Photo-Crosslinkable Biotin Derivative of the Phosphoantigen (E)-4-Hydroxy-3-Methylbut-2-Enyl Diphosphate (HMBPP) Activates Vγ9Vδ2 T Cells and Binds to the HMBPP Site of BTN3A1.

Vγ9Vδ2 T cells play an important role in the cross talk of the innate and adaptive immune system. For their activation by phosphoantigens (PAgs), both cell surface receptors, the eponymous Vγ9Vδ2 T cell antigen receptors (Vγ9Vδ2 TCRs) on Vγ9Vδ2 T cells and butyrophilin 3A1 (BTN3A1) on the phosphoantigen-"presenting" cell, are mandatory. To find yet undetected but further contributing proteins, a biotinylated, photo-crosslinkable benzophenone probe BioBP-HMBPP (2) was synthesized from a known allyl alcohol in nine steps and overall 16 % yield. 2 is based on the picomolar PAg (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP, 1). Laser irradiation of 2 at 308 nm initiated the photo-crosslinking reaction with proteins. When the B30.2 domain of BTN3A1, which contains a positively charged PAg-binding pocket, was exposed to increasing amounts of HMBPP (1), labeling by BioBP-HMBPP (2) was reduced significantly. Because BSA labeling was not impaired, 2 clearly binds to the same site as natural ligand 1. Thus, BioBP-HMBPP (2) is a suitable tool to identify co-ligands or receptors involved in PAg-mediated T cell activation.

Synergistic effects of BTN3A1, SHP2, CD274, and STAT3 gene polymorphisms on the risk of systemic lupus erythematosus: a multifactorial dimensional reduction analysis.

OBJECTIVE: Systemic lupus erythematosus is a complex autoimmune disorder, and evidence supports the significance of genetic polymorphisms in SLE genetic susceptibility. The aim of this study was to assess the effects of BTN3A1 (butyrophilin 3A1), SHP2 (Src homology-2 containing protein tyrosine phosphatase), CD274 (programmed cell death 1 ligand 1), and STAT3 (signal transducer-activator of transcription 3) gene interactions on SLE risk. MATERIALS AND METHODS: Two hundred and ninety patients diagnosed with SLE and 370 healthy controls were recruited. A multifactor dimensionality reduction (MDR) approach was used to determine the epistasis among single nucleotide polymorphisms (SNPs) on the BTN3A1 (rs742090), SHP2 (rs58116261), CD174 (rs702275), and STAT3 (rs8078731) genes. The best risk prediction model was identified in terms of precision and cross-validation consistency. RESULTS: Allele A and genotype AA were negatively related to genetic susceptibility of SLE for BTN3A1 rs742090 (OR = 0.788 (0.625-0.993), P = 0.044; OR = 0.604 (0.372-0.981), P = 0.040). For STAT3 rs8078731, allele A and genotype AA were positively related to the risk of SLE (OR = 1.307 (1.032-1.654), P = 0.026; OR = 1.752 (1.020-3.010), P = 0.041). MDR analysis revealed the most significant interaction between BTN3A1 rs742090 and SHP2 rs58116261. The best risk prediction model was a combination of BTN3A1 rs742090, SHP2 rs58116261, and STAT3 rs8078731 (accuracy = 0.5866, consistency = 10/10, OR = 1.9870 (1.5964-2.4731), P = 0.001). CONCLUSION: These data indicate that risk prediction models formed by gene interactions (BTN3A1, SHP2, STAT3) can identify susceptible populations of SLE. Key Points • BTN3A1 rs742090 polymorphism was a protective factor for systemic lupus erythematosus, while STAT3 rs8078731 polymorphism was a risk factor. • There was a strong synergistic effect of BTN3A1 rs742090 and SHP2 rs58116261, and interaction among BTN3A1 rs742090, SHP2 rs58116261, and STAT3 rs8078731 constructed the best model to show association with SLE risk.

High expression of BTN3A1 is associated with clinical and immunological characteristics and predicts a poor prognosis in advanced human gliomas.

Introduction: Gliomas represent the most prevalent and aggressive tumors within the central nervous system. Despite the current standard treatments, the median survival time for glioblastoma patients remains dismal, hovering around 14 months. While attempts have been made to inhibit the PD-1/PD-L1 and CTLA-4/CD80-CD86 axes through immunotherapy, the outcomes have yet to demonstrate significant efficacy. The immune checkpoint Butyrophilin 3A1 (BTN3A1) can either be involved in advantageous or detrimental function depending on the cancer type. Methods: In our study, we utilized a Moroccan cohort to delve into the role of BTN3A1 in gliomas. A transcriptomic analysis was conducted on 34 patients, which was then corroborated through a protein analysis in 27 patients and validated using the TCGA database (n = 667). Results: Our results revealed an elevated expression of BTN3A1 in glioblastoma (grade 4), as evidenced in both the TCGA database and our cohort of Moroccan glioma patients. Within the TCGA cohort, BTN3A1 expression was notably higher in patients with wild-type IDH. We observed a positive correlation between BTN3A1 expression and immune infiltration of B cells, CD8+ T cells, naive CD4+ T cells, and M2 macrophages. Patients exhibiting increased BTN3A1 expression also presented elevated levels of TGF-ß, IL-10, and TIM-3 compared to those with reduced BTN3A1 expression. Notably, patients with high BTN3A1 expression were associated with a poorer prognosis than their counterparts with lower expression. Conclussion: Our findings suggest that BTN3A1 might promote the establishment of an immunosuppressive microenvironment. Consequently, targeting BTN3A1 could offer novel therapeutic avenues for the management of advanced gliomas.

Division of labor and cooperation between different butyrophilin proteins controls phosphoantigen-mediated activation of human γδ T cells.

Butyrophilin (BTN)-3A and BTN2A1 molecules control TCR-mediated activation of human Vγ9Vδ2 T-cells triggered by phosphoantigens (PAg) from microbes and tumors, but the molecular rules governing antigen sensing are unknown. Here we establish three mechanistic principles of PAg-action. Firstly, in humans, following PAg binding to the BTN3A1-B30.2 domain, Vγ9Vδ2 TCR triggering involves the V-domain of BTN3A2/BTN3A3. Moreover, PAg/B30.2 interaction, and the critical γδ-T-cell-activating V-domain, localize to different molecules. Secondly, this distinct topology as well as intracellular trafficking and conformation of BTN3A heteromers or ancestral-like BTN3A homomers are controlled by molecular interactions of the BTN3 juxtamembrane region. Finally, the ability of PAg not simply to bind BTN3A-B30.2, but to promote its subsequent interaction with the BTN2A1-B30.2 domain, is essential for T-cell activation. Defining these determinants of cooperation and division of labor in BTN proteins deepens understanding of PAg sensing and elucidates a mode of action potentially applicable to other BTN/BTNL family members.

Butyrophilins: γδ T Cell Receptor Ligands, Immunomodulators and More.

Butyrophilins (BTN) are relatives of the B7 family (e.g., CD80, PD-L1). They fulfill a wide range of functions including immunomodulation and bind to various receptors such as the γδ T cell receptor (γδTCR) and small molecules. One intensively studied molecule is BTN3A1, which binds via its cytoplasmic B30.2 domain, metabolites of isoprenoid synthesis, designated as phosphoantigen (PAg), The enrichment of PAgs in tumors or infected cells is sensed by Vγ9Vδ2 T cells, leading to the proliferation and execution of effector functions to remove these cells. This article discusses the contribution of BTNs, the related BTNL molecules and SKINT1 to the development, activation, and homeostasis of γδ T cells and their immunomodulatory potential, which makes them interesting targets for therapeutic intervention.

Ligand-induced interactions between butyrophilin 2A1 and 3A1 internal domains in the HMBPP receptor complex.

The ligand-bound (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) receptor (BTN3A1 and BTN2A1) is detectable by the T cell receptor (TCR) of Vγ9Vδ2 T cells. Although BTN3A1 binds to phosphoantigens (pAgs), the mechanisms resulting in receptor activation are not clear. We used CRISPR-Cas9, ELISA, nano-bioluminescence resonance energy transfer (BRET), and isothermal titration calorimetry (ITC) to evaluate the role of BTN2A1. Depletion of BTN2A1 and rescue experiments demonstrate that its internal domain is essential for pAg detection. Internal hetero-BRET signals are observed between BTN2A1 and BTN3A1 that are increased by pAg. ITC detects a direct interaction between the intracellular domains of BTN3A1 and BTN2A1 only in the presence of pAg. This interaction is abrogated by removal of the BTN2A1 juxtamembrane (JM) region but not by removal of the BTN3A1 JM region. Regional mutations between BTN2A1 316-326 clearly affect the interferon γ (IFNγ) response and hetero-BRET signal. Mutations to amino acids L318, W320, and L325 indicate that these amino acids are crucial. This study demonstrates a pAg-inducible interaction between BTN2A1 and BTN3A1 internal domains.

Long noncoding RNA HOXA-AS2 accelerates cervical cancer by the miR-509-3p/BTN3A1 axis.

OBJECTIVES: Cervical cancer is an aggressive malignant tumour and causes high mortality in women. LncRNA HOXA-AS2 is a tumour promoter in many cancers. The current work was designed to elucidate the functions of HOXA-AS2 in cervical cancer and the underlying regulatory mechanism. METHODS: qRT-PCR was conducted to reveal RNA levels. A FISH assay was conducted for the identification of the subcellular location of HOXA-AS2. MTT, EdU, Transwell and tube formation were used for detection of cell growth, migration and angiogenesis, respectively. In-vivo studies were conducted to reveal the role of HOXA-AS2 on transplanted tumour growth in mice. KEY FINDINGS: The HOXA-AS2 level was found high in tissues and cells of cervical cancer. Silencing of HOXA-AS2 restrained cell proliferation, migration and invasion. Angiogenesis of HUVECs was restrained after silencing HOXA-AS2. Additionally, HOXA-AS2 upregulated the BTN3A1 by interaction with miR-509-3p. BTN3A1 overexpression rescues the inhibitory effect of silenced HOXA-AS2 on cell phenotypes in cervical cancer. Moreover, xenograft tumour growth in mice was suppressed by HOXA-AS2 depletion and was facilitated by BTN3A1 overexpression. CONCLUSIONS: HOXA-AS2 accelerates cellular progression in cervical cancer by the miR-509-3p/BTN3A1 axis.

Comprehensive analysis of BTN3A1 in cancers: mining of omics data and validation in patient samples and cellular models.

Butyrophilin 3A1 (BTN3A1), a major histocompatibility complex-associated gene that encodes a membrane protein with two extracellular immunoglobulin domains and an intracellular B30.2 domain, is critical in T-cell activation and adaptive immune response. Here, the expression of BTN3A1 in cancers was analyzed in eight databases comprising 86 733 patients of 33 cancers, and the findings were validated in patient samples and cell models. We showed that BTN3A1 was expressed in most cancers, and its expression level was strongly correlated with clinical outcome of 13 cancers. Mutations of BTN3A1 were detected, and the mutations were distributed throughout the entire gene. Gene set enrichment analysis showed that BTN3A1 co-expression genes and interacting proteins were enriched in immune regulation-related pathways. BTN3A1 was associated with tumor-infiltrating immune cells and was co-expressed with multiple immune checkpoints in patients with breast cancer (BRCA) and non-small cell lung cancer (NSCLC). We reported that BTN3A1 was downregulated in 46 of 65 (70.8%) NSCLCs, and its expression level was inversely associated with clinical outcome of the patients. BTN3A1 in tumor samples was lower than in counterpart normal tissues in 31 of 38 (81.6%) BRCAs. Bioinformatics analyses showed that BTN3A1 could be a target gene of transcription factor Spi-1 proto-oncogene (SPI1), and our 'wet' experiments showed that ectopic expression of SPI1 upregulated, whereas silencing of SPI1 downregulated, BTN3A1 expression in cells. These results suggest that BTN3A1 may function as a tumor suppressor and may serve as a potential prognostic biomarker in NSCLCs and BRCAs.

Prognostic Role of Plasma PD-1, PD-L1, pan-BTN3As and BTN3A1 in Patients Affected by Metastatic Gastrointestinal Stromal Tumors: Can Immune Checkpoints Act as a Sentinel for Short-Term Survival?

Gastrointestinal stromal tumors (GISTs) represent 1% of all primary gastrointestinal tumors. Immune surveillance is often overcome by cancer cells due to the activation of immunoregulatory molecules such as programmed death protein (PD-1) and its ligand PD-L1, and butyrophilin sub-family 3A/CD277 receptors (BTN3A). Because several studies demonstrated that tumor PD-1 and PD-L1 expression may have a prominent prognostic function, this investigation aimed to discover if soluble forms of these molecules may be useful in predicting survival of metastatic GIST (mGIST) patients. Through specific ad hoc developed ELISA assays not yet available on the market, the circulating PD-1, PD-L1, BTN3A1, and pan-BTN3As levels were examined in 30 c-KIT exon 11-mutated mGIST patients, prior to imatinib therapy. Using specific thresholds derived by ROC analysis, we found that high baseline levels of sPD-1 (>8.1 ng/mL), sPD-L1 (>0.7 ng/mL), sBTN3A1 (>7.0 ng/mL), and pan-BTN3As (>5.0 ng/mL) were correlated with shorter progression-free survival (PFS) and poor prognosis. Contrariwise, subjects with lower plasma concentrations exhibited a median PFS about 20 months longer than to the earlier. Finally, an additional multivariate analysis revealed that circulating levels of sPD-L1 ≤ 0.7 ng/mL and pan-sBTN3As ≤ 5.0 ng/mL, and the absence of KIT exon 11 deletions or delins at codons 557 and/or 558 were associated with a longer PFS in mGIST patients. Our investigation, for the first time, revealed that evaluating the plasma concentration of some immune checkpoints may help prognosticate survival in mGIST patients, suggesting their potential use as prognostic biomarkers beyond the presence of KIT exon 11 Del or Delins at codons 557/558.

High Abundance of Intratumoral γδ T Cells Favors a Better Prognosis in Head and Neck Squamous Cell Carcinoma: A Bioinformatic Analysis.

γδ T cells are a small subset of unconventional T cells that are enriched in the mucosal areas, and are responsible for pathogen clearance and maintaining integrity. However, the role of γδ T cells in head and neck squamous cell carcinoma (HNSCC) is largely unknown. Here, by using RNA-seq data from The Cancer Genome Atlas (TCGA), we discovered that HNSCC patients with higher levels of γδ T cells were positively associated with lower clinical stages and better overall survival, and high abundance of γδ T cells was positively correlated with CD8+/CD4+ T cell infiltration. Gene ontology and pathway analyses showed that genes associated with T cell activation, proliferation, effector functions, cytotoxicity, and chemokine production were enriched in the group with a higher γδ T cell abundance. Furthermore, we found that the abundance of γδ T cells was positively associated with the expression of the butyrophilin (BTN) family proteins BTN3A1/BTN3A2/BTN3A3 and BTN2A1, but only MICB, one of the ligands of NKG2D, was involved in the activation of γδ T cells, indicating that the BTN family proteins might be involved in the activation and proliferation of γδ T cells in the tumor microenvironment of HNSCC. Our results indicated that γδ T cells, along with their ligands, are promising targets in HNSCC with great prognostic values and treatment potentials.

Baseline plasma levels of soluble PD-1, PD-L1, and BTN3A1 predict response to nivolumab treatment in patients with metastatic renal cell carcinoma: a step toward a biomarker for therapeutic decisions.

Despite a proportion of renal cancer patients can experiment marked and durable responses to immune-checkpoint inhibitors, the treatment efficacy is widely variable and identifying the patient who will benefit from immunotherapy remains an issue. We performed a prospective study to investigate if soluble forms of the immune-checkpoints PD-1 (sPD-1), PD-L1 (sPD-L1), pan-BTN3As, BTN3A1, and BTN2A1, could be candidate to predict the response to immune-checkpoint blockade therapy. We evaluated the plasma levels in a learning cohort of metastatic clear cell renal carcinoma (mccRCC) patients treated with the anti-PD-1 agent nivolumab by ad hoc developed ELISA's. Using specific cut-offs determined through ROC curves, we showed that high baseline levels of sPD-1 (>2.11 ng/ml), sPD-L1 (>0.66 ng/ml), and sBTN3A1 (>6.84 ng/ml) were associated with a longer progression-free survival (PFS) to nivolumab treatment [median PFS, levels above thresholds: sPD-1, 20.7 months (p < .0001); sPD-L1, 19 months (p < .0001); sBTN3A1, 17.5 months (p = .002)]. High sPD-1 and sBTN3A1 levels were also associated with best overall response by RECIST and objective response of >20%. The results were confirmed in a validation cohort of 20 mccRCC patients. The analysis of plasma dynamic changes after nivolumab showed a statistically significant decrease of sPD-1 after 2 cycles (Day 28) in the long-responder patients. Our study revealed that the plasma levels of sPD-1, sPD-L1, and sBTN3A1 can predict response to nivolumab, discriminating responders from non-responders already at therapy baseline, with the advantages of non-invasive sample collection and real-time monitoring that allow to evaluate the dynamic changes during cancer evolution and treatment.

The TRIM14 PRYSPRY domain mediates protein interaction via its basic interface.

Tripartite motif (TRIM)14 was recently shown to be an important molecule against pathogens. Its PRYSPRY domain acts as a protein-protein interaction module. TRIM14 exerts distinct functions via its PRYSPRY domain by interacting with different partners. However, the structural basis for its binding specificity remains unknown. Here we solved the crystal structure of the TRIM14 PRYSPRY domain, and found a positively charged surface that may mediate its partner specificity. Isothermal titration calorimetry reveals that the TRIM14 PRYSPRY domain binds to acidic peptides, and the analysis of the reported partners of TRIM14 is consistent with our assumption. Therefore, we demonstrate that the PRYSPRY domain of TRIM14 harbors a putative basic interface that may favorably bind to acidic amino acid residues.