Transient 40 °C-shock potentiates cytotoxic responses of Vδ2+ γδ T cell via HSP70 upregulation.

Vδ2+ γδ T cell, one of promising strategies for tumor immunotherapy, recognizes and kills cancer cells in a non-MHC dependent manner. Previously, we pioneeringly proved the clinical safety and efficacy of allogeneic Vδ2+ γδ T cells, in vitro expanded from healthy donors, in the treatment of late-stage cancer patients. Nevertheless, how to profoundly potentiate cytotoxic function of expanded Vδ2+ γδ T cells remains to be further explored. Here, we proposed that 40 °C-Shock could be a simple and reliable approach to in vitro boost the effector function. We found that 40 °C-shock could phosphorylate two MAPK proteins ERK and p38 through HSP70, which facilitated actyl-α-tubulin and actin augments and reorganization, elevated Ki-67 expression and cell surface adhesion, and promoted releases of cytokines IFN-γ, perforin and granzyme B, as well as downregulated LAG3 expression. We also observed 40 °C-shock induced elevations of mitochondrial metabolism. These altogether led to potentiated cytotoxic responses against cancer cells. This proof-of-concept work demonstrated that 40 °C-shock would be probably developed into an effective method to in vitro boost the cytotoxicity of Vδ2+ γδ T cell before applying it in immunotherapy, and provided scientific evidences for the view that fever can activate immune responses of innate immune cells.

Quantitative assessment of disease markers using the naked eye: point-of-care testing with gas generation-based biosensor immunochromatographic strips.

BACKGROUND: Immunochromatographic strips (ICSs) are a practical tool commonly used in point-of-care testing (POCT) applications. However, ICSs that are currently available have low sensitivity and require expensive equipment for quantitative analysis. These limitations prohibit their extensive use in areas where medical resources are scarce. METHODS: We developed a novel POCT platform by integrating a gas generation biosensor with Au@Pt Core/Shell nanoparticle (Au@PtNPs)-based ICSs (G-ICSs). The resulting G-ICSs enabled the convenient and quantitative assessment of a target protein using the naked eye, without the need for auxiliary equipment or complicated computation. To assess this platform, C-reactive protein (CRP), a biomarker commonly used for the diagnosis of acute, infectious diseases was chosen as a proof-of-concept test. RESULTS: The linear detection range (LDR) of the G-ICSs for CRP was 0.05-6.25 µg/L with a limit of detection (LOD) of 0.041 µg/L. The G-ICSs had higher sensitivity and wider LDR when compared with commonly used AuNPs and fluorescent-based ICSs. When compared with results from a chemiluminescent immunoassay, G-ICS concordance rates for CRP detection in serum samples ranged from 93.72 to 110.99%. CONCLUSIONS: These results demonstrated that G-ICSs have wide applicability in family diagnosis and community medical institutions, especially in areas with poor medical resources.

Simultaneous topographic and recognition imaging of epidermal growth factor receptor (EGFR) on single human breast cancer cells.

Epidermal growth factor receptor (EGFR) plays an important role in signaling pathway of the development of breast cancer cells. Since EGFR overexpresses in most breast cancer cells, it is regarded as a biomarker molecule of breast cancer cells. Here we demonstrated a new AFM technique-topography and recognition (TREC) imaging-to simultaneously obtain highly sensitive and specific molecular recognition images and high-resolution topographic images of EGFR on single breast cancer cells.

AFM resolves effects of ethambutol on nanomechanics and nanostructures of single dividing mycobacteria in real-time.

Dynamic nanomechanics and nanostructures of dividing and anti-mycobacterial drug treated mycobacterium remain to be fully elucidated. Atomic force microscopy (AFM) is a promising nanotechnology tool for characterization of these dynamic alterations, especially at the single cell level. In this work, single dividing mycobacterium JLS (M.JLS) before and after anti-mycobacterial drug (ethambutol, EMB) treatment was in situ quantitatively analyzed, suggesting that nanomechanics would be referred as a sensitive indicator for evaluating efficacy of anti-mycobacterial drugs. Dynamic evidence on the contractile ring and septal furrow of dividing M.JLS implied that inhibition of contractile ring formation would be a crucial process for EMB to disturb M.JLS division. These results could facilitate further explaining the regulation mechanism of the contractile ring as well as nanomechanical roles of the cell wall in the course of mycobacterial division. This work describe a new way for further elucidating the mechanisms of mycobacterial division and anti-mycobacterial drug action, as well as the drug-resistance developing mechanism of pathogenic mycobacteria.

Harnessing CD8+ T cell dynamics in hepatitis B virus-associated liver diseases: Insights, therapies and future directions.

Hepatitis B virus (HBV) infection playsa significant role in the etiology and progression of liver-relatedpathologies, encompassing chronic hepatitis, fibrosis, cirrhosis, and eventual hepatocellularcarcinoma (HCC). Notably, HBV infection stands as the primary etiologicalfactor driving the development of HCC. Given the significant contribution ofHBV infection to liver diseases, a comprehensive understanding of immunedynamics in the liver microenvironment, spanning chronic HBV infection,fibrosis, cirrhosis, and HCC, is essential. In this review, we focused on thefunctional alterations of CD8+ T cells within the pathogenic livermicroenvironment from HBV infection to HCC. We thoroughly reviewed the roles ofhypoxia, acidic pH, metabolic reprogramming, amino acid deficiency, inhibitory checkpointmolecules, immunosuppressive cytokines, and the gut-liver communication in shapingthe dysfunction of CD8+ T cells in the liver microenvironment. Thesefactors significantly impact the clinical prognosis. Furthermore, we comprehensivelyreviewed CD8+ T cell-based therapy strategies for liver diseases,encompassing HBV infection, fibrosis, cirrhosis, and HCC. Strategies includeimmune checkpoint blockades, metabolic T-cell targeting therapy, therapeuticT-cell vaccination, and adoptive transfer of genetically engineered CD8+ T cells, along with the combined usage of programmed cell death protein-1/programmeddeath ligand-1 (PD-1/PD-L1) inhibitors with mitochondria-targeted antioxidants.Given that targeting CD8+ T cells at various stages of hepatitis Bvirus-induced hepatocellular carcinoma (HBV + HCC) shows promise, we reviewedthe ongoing need for research to elucidate the complex interplay between CD8+ T cells and the liver microenvironment in the progression of HBV infection toHCC. We also discussed personalized treatment regimens, combining therapeuticstrategies and harnessing gut microbiota modulation, which holds potential forenhanced clinical benefits. In conclusion, this review delves into the immunedynamics of CD8+ T cells, microenvironment changes, and therapeuticstrategies within the liver during chronic HBV infection, HCC progression, andrelated liver diseases.

Investigation of free fatty acid associated recombinant membrane receptor protein expression in HEK293 cells using Raman spectroscopy, calcium imaging, and atomic force microscopy.

G-protein-coupled receptor 120 (GPR120) is a previously orphaned G-protein-coupled receptor that apparently functions as a sensor for dietary fat in the gustatory and digestive systems. In this study, a cDNA sequence encoding a doxycycline (Dox)-inducible mature peptide of GPR120 was inserted into an expression vector and transfected in HEK293 cells. We measured Raman spectra of single HEK293 cells as well as GPR120-expressing HEK293-GPR120 cells at a 48 h period following the additions of Dox at several concentrations. We found that the spectral intensity of HEK293-GPR120 cells is dependent upon the dose of Dox, which correlates with the accumulation of GPR120 protein in the cells. However, the amount of the fatty acid activated changes in intracellular calcium (Ca(2+)) as measured by ratiometric calcium imaging was not correlated with Dox concentration. Principal components analysis (PCA) of Raman spectra reveals that the spectra from different treatments of HEK293-GPR120 cells form distinct, completely separated clusters with the receiver operating characteristic (ROC) area of 1, while those spectra for the HEK293 cells form small overlap clusters with the ROC area of 0.836. It was also found that expression of GPR120 altered the physiochemical and biomechanical properties of the parental cell membrane surface, which was quantitated by atomic force microscopy (AFM). These findings demonstrate that the combination of Raman spectroscopy, calcium imaging, and AFM may provide new tools in noninvasive and quantitative monitoring of membrane receptor expression induced alterations in the biophysical and signaling properties of single living cells.

Subcellular spectroscopic markers, topography and nanomechanics of human lung cancer and breast cancer cells examined by combined confocal Raman microspectroscopy and atomic force microscopy.

The nanostructures and hydrophobic properties of cancer cell membranes are important for membrane fusion and cell adhesion. They are directly related to cancer cell biophysical properties, including aggressive growth and migration. Additionally, chemical component analysis of the cancer cell membrane could potentially be applied in clinical diagnosis of cancer by identification of specific biomarker receptors expressed on cancer cell surfaces. In the present work, a combined Raman microspectroscopy (RM) and atomic force microscopy (AFM) technique was applied to detect the difference in membrane chemical components and nanomechanics of three cancer cell lines: human lung adenocarcinoma epithelial cells (A549), and human breast cancer cells (MDA-MB-435 with and without BRMS1 metastasis suppressor). Raman spectral analysis indicated similar bands between the A549, 435 and 435/BRMS1 including ~720 cm(-1) (guanine band of DNA), 940 cm(-1) (skeletal mode polysaccharide), 1006 cm(-1) (symmetric ring breathing phenylalanine), and 1451 cm(-1) (CH deformation). The membrane surface adhesion forces for these cancer cells were measured by AFM in culture medium: 0.478 ± 0.091 nN for A549 cells, 0.253 ± 0.070 nN for 435 cells, and 1.114 ± 0.281 nN for 435/BRMS1 cells, and the cell spring constant was measured at 2.62 ± 0.682 mN m(-1) for A549 cells, 2.105 ± 0.691 mN m(-1) for 435 cells, and 5.448 ± 1.081 mN m(-1) for 435/BRMS1 cells.

Characterization and analysis of mycobacteria and Gram-negative bacteria and co-culture mixtures by Raman microspectroscopy, FTIR, and atomic force microscopy.

The molecular composition of mycobacteria and Gram-negative bacteria cell walls is structurally different. In this work, Raman microspectroscopy was applied to discriminate mycobacteria and Gram-negative bacteria by assessing specific characteristic spectral features. Analysis of Raman spectra indicated that mycobacteria and Gram-negative bacteria exhibit different spectral patterns under our experimental conditions due to their different biochemical components. Fourier transform infrared (FTIR) spectroscopy, as a supplementary vibrational spectroscopy, was also applied to analyze the biochemical composition of the representative bacterial strains. As for co-cultured bacterial mixtures, the distribution of individual cell types was obtained by quantitative analysis of Raman and FTIR spectral images and the spectral contribution from each cell type was distinguished by direct classical least squares analysis. Coupled atomic force microscopy (AFM) and Raman microspectroscopy realized simultaneous measurements of topography and spectral images for the same sampled surface. This work demonstrated the feasibility of utilizing a combined Raman microspectroscopy, FTIR, and AFM techniques to effectively characterize spectroscopic fingerprints from bacterial Gram types and mixtures.

γδ T cells: origin and fate, subsets, diseases and immunotherapy.

The intricacy of diseases, shaped by intrinsic processes like immune system exhaustion and hyperactivation, highlights the potential of immune renormalization as a promising strategy in disease treatment. In recent years, our primary focus has centered on γδ T cell-based immunotherapy, particularly pioneering the use of allogeneic Vδ2+ γδ T cells for treating late-stage solid tumors and tuberculosis patients. However, we recognize untapped potential and optimization opportunities to fully harness γδ T cell effector functions in immunotherapy. This review aims to thoroughly examine γδ T cell immunology and its role in diseases. Initially, we elucidate functional differences between γδ T cells and their αß T cell counterparts. We also provide an overview of major milestones in γδ T cell research since their discovery in 1984. Furthermore, we delve into the intricate biological processes governing their origin, development, fate decisions, and T cell receptor (TCR) rearrangement within the thymus. By examining the mechanisms underlying the anti-tumor functions of distinct γδ T cell subtypes based on γδTCR structure or cytokine release, we emphasize the importance of accurate subtyping in understanding γδ T cell function. We also explore the microenvironment-dependent functions of γδ T cell subsets, particularly in infectious diseases, autoimmune conditions, hematological malignancies, and solid tumors. Finally, we propose future strategies for utilizing allogeneic γδ T cells in tumor immunotherapy. Through this comprehensive review, we aim to provide readers with a holistic understanding of the molecular fundamentals and translational research frontiers of γδ T cells, ultimately contributing to further advancements in harnessing the therapeutic potential of γδ T cells.

Programmed Cell Death Tunes Tumor Immunity.

The demise of cells in various ways enables the body to clear unwanted cells. Studies over the years revealed distinctive molecular mechanisms and functional consequences of several key cell death pathways. Currently, the most intensively investigated programmed cell death (PCD) includes apoptosis, necroptosis, pyroptosis, ferroptosis, PANoptosis, and autophagy, which has been discovered to play crucial roles in modulating the immunosuppressive tumor microenvironment (TME) and determining clinical outcomes of the cancer therapeutic approaches. PCD can play dual roles, either pro-tumor or anti-tumor, partly depending on the intracellular contents released during the process. PCD also regulates the enrichment of effector or regulatory immune cells, thus participating in fine-tuning the anti-tumor immunity in the TME. In this review, we focused primarily on apoptosis, necroptosis, pyroptosis, ferroptosis, PANoptosis, and autophagy, discussed the released molecular messengers participating in regulating their intricate crosstalk with the immune response in the TME, and explored the immunological consequence of PCD and its implications in future cancer therapy developments.

Apoptosis, Pyroptosis, and Ferroptosis Conspiringly Induce Immunosuppressive Hepatocellular Carcinoma Microenvironment and γδ T-Cell Imbalance.

Hepatocellular carcinoma (HCC) is highly malignant and prone to metastasize due to the heterogeneous and immunosuppressive tumor microenvironment (TME). Programmed cell deaths (PCDs) including apoptosis, ferroptosis, and pyroptosis routinely occur in the HCC TME and participate in tumorigenesis. However, how apoptosis, ferroptosis, and pyroptosis are involved in constructions of the immunosuppressive TME and their underlying cross-talk remains to be further unveiled. In this work, we deciphered the immunosuppressive landscape of HCC TME, which demonstrated high expressions of inhibitory checkpoint molecules and infiltration of protumor immune cells but low infiltration of antitumor effector immune cells. Further investigations unequivocally revealed that marker genes of apoptosis, ferroptosis, and pyroptosis are closely correlated with expressions and infiltrations of inhibitory checkpoint molecules and immune cells and that higher "-optosis" links to poorer patient prognosis. Notably, such three types of "-optosis" interact with each other at both the gene and protein levels, suggesting that they conspiringly induce the establishment of the immunosuppressive HCC TME. Interestingly, examinations of circulating γδ T cells in HCC patients revealed a noticeable dysfunction phenotype. The strikingly elevated ratio of the Vδ1+ versus the Vδ2+ subset suggested that the Vδ1+/Vδ2+ ratio would be a potential biomarker for the diagnosis and prognosis in HCC patients. Altogether, this work thoroughly decrypted the underlying correlations between apoptosis, ferroptosis, and pyroptosis and the formation of immunosuppressive HCC TME and, meanwhile, indicated that allogeneic Vδ2+ γδ T-cell transfer would be a promising adjuvant strategy for renormalizing circulating γδ T cell and thus achieving sound clinical efficacy against HCC.

Hepatocellular carcinoma-infiltrating γδ T cells are functionally defected and allogenic Vδ2+ γδ T cell can be a promising complement.

In hepatocellular carcinoma (HCC), γδ T cells participate in mediating the anti-tumour response and are linked with a positive prognosis. However, these cells can become pro-tumoural in the tumour microenvironment (TME). We aimed to decipher the immune landscape and functional states of HCC-infiltrating γδ T cells to provide fundamental evidence for the adoptive transfer of allogeneic Vδ2+ γδ T cells in HCC immunotherapy. We performed single-cell RNA sequencing (scRNA-seq) on γδ T cells derived from HCC tumours and healthy donor livers. Confocal microscopy, flow cytometry and a Luminex assay were applied to validate the scRNA-seq findings. The γδ T cells in the HCC TME entered G2/M cell cycle arrest, and expressed cytotoxic molecules such as interferon-gamma and granzyme B, but were functionally exhausted as indicated by upregulated gene and protein LAG3 expression. The γδ T cells in the HCC TME were dominated by the LAG3+ Vδ1+ population, whereas the Vδ2+ γδ T population was greatly depleted. Moreover, glutamine metabolism of γδ T cells was markedly upregulated in the glutamine-deficient TME. Both in vitro and in vivo experiments showed that glutamine deficiency upregulated LAG3 expression. Finally, our results indicated that ex vivo-expanded Vδ2+ γδ T cells from healthy donor could complement the loss of T cell receptor clonality and effector functions of HCC-derived γδ T cells. This work deciphered the dysfunctional signatures of HCC-infiltrating γδ T cells in the HCC TME, providing scientific support for the use of allogeneic Vδ2+ γδ T cells in HCC cellular therapy.

Metabolites in the Tumor Microenvironment Reprogram Functions of Immune Effector Cells Through Epigenetic Modifications.

Cellular metabolism of both cancer and immune cells in the acidic, hypoxic, and nutrient-depleted tumor microenvironment (TME) has attracted increasing attention in recent years. Accumulating evidence has shown that cancer cells in TME could outcompete immune cells for nutrients and at the same time, producing inhibitory products that suppress immune effector cell functions. Recent progress revealed that metabolites in the TME could dysregulate gene expression patterns in the differentiation, proliferation, and activation of immune effector cells by interfering with the epigenetic programs and signal transduction networks. Nevertheless, encouraging studies indicated that metabolic plasticity and heterogeneity between cancer and immune effector cells could provide us the opportunity to discover and target the metabolic vulnerabilities of cancer cells while potentiating the anti-tumor functions of immune effector cells. In this review, we will discuss the metabolic impacts on the immune effector cells in TME and explore the therapeutic opportunities for metabolically enhanced immunotherapy.

Circulating PD1+Vδ1+γδ T Cell Predicts Fertility in Endometrial Polyp Patients of Reproductive-Age.

Clinically, immune cell function is correlated with pathogenesis of endometrial polyp (EP) and infertility of women of reproductive-age. However, the underlying immune cell hallmark in EP patients remains unclear. Here, we focused on analyzing circulating immune cells, and attempted to reveal the correlation between peripheral immune cell functional phenotypes and fertility in EP patients. Through comparison of circulating CD4+/CD8+ T cells, NK cells, and γδ T cells between 64 EP patients and 68 healthy females, we found that γδ T cells, but not CD4+/CD8+ T cells and NK cells, were immunologically correlated with conception rate and conception interval time. Specifically, total γδ T cells and the Vδ1+PD1+ γδ T subpopulation decreased whereas the Vδ1/Vδ2 ratio increased in EP patients compared to healthy controls. Moreover, the patients with the higher Vδ1/Vδ2 ratio (median value equals 1.04) had a poorer fertility and longer interval time of conception (210 days versus 158 days for control). Meanwhile, higher Vδ1+PD1+ γδ T cell proportion (median equals 15.7) was positively correlative with both higher conception rate and shortened median conception interval time (130 days for Vδ1+PD1high group versus 194 days for Vδ1+PD1low group). Notably, in healthy controls, both Vδ1/Vδ2 ratio and Vδ1+PD1+ γδ T cell proportion correlated with pregnancy rate oppositely, comparing to EP patients. Together, our results suggested that imbalanced γδ T cell population occurred in EP patients, and that Vδ1/Vδ2 ratio and PD-1 expression of Vδ1+ γδ T cells could be potentially developed into valuable predictors for fertility in EP patients.

Human mesenchymal stem cells promote ischemic repairment and angiogenesis of diabetic foot through exosome miRNA-21-5p.

BACKGROUND: Diabetic foot is caused by ischemic disease of lower extremities of diabetic patients, and the effective therapy is very limited. Mesenchymal stem cells (MSCs) based cell therapy had been developed into a new treatment strategy for diabetic foot clinically. However, the underlying molecular mechanism remains to be fully addressed. Exosomes (extracellular vesicles) secreted by MSCs may play crucial role in the processes of MSCs mediated inhibition of inflammatory microenvironment as well as pro-angiogenesis of ischemic tissue of diabetic foot. METHODS: Exosomes were isolated from MSCs using ultracentrifugation, and further characterized by the nanoparticle tracking analyzer and flow cytometry. Moreover, RNA sequencing, Western Blot, in vitro cell proliferation, in vivo pro-angiogenesis, as well as ischemic repairment of diabetic foot through rat model were performed to evaluate exosome physiological functions. RESULTS: We found that inflammatory cytokines (tumor necrosis factor α and interleukin-6) and vascularcelladhesion molecule-1 induced MSCs to secrete exosomes heterogeneously, including exosome size and quantity. Through RNA sequencing, we defined a new proangiogenic miRNA, miRNA-21-5p. Further knockdown and overexpression of miRNA-21-5p by manipulating MSCs validated the biological activity of exosome miRNA-21-5p, including in vitro cell proliferation, in vivo pro-angiogenesis in Chick Chorioallantoic Membrane (CAM) assay, and in vivo pro-angiogenesis experiments (tissue injury and repair) in diabetic rat models. Furthermore, we discovered that exosomemiRNA-21-5p promoted angiogenesis through upregulations of vascular endothelial growth factor receptor (VEGFR) as well as activations of serine/threonine kinase (AKT) and mitogen-activated protein kinase (MAPK). Together, our work suggested miRNA-21-5p could be a novel mechanism by which exosomes promote ischemic tissue repair and angiogenesis. Meanwhile, miRNA-21-5p could be potentially developed into a new biomarker for exosomes of MSCs to treat diabetic foot. CONCLUSIONS: miRNA-21-5p is a new biomarker and a novel mechanism by which exosomes promote ischemic tissue repair and angiogenesis of diabetic foot. Our work could not only provide new scientific evidences for revealing pro-angiogenesis mechanism of MSCs, but also eventually benefit MSCs-based clinical therapy for diabetic foot of diabetes patients.

Allogeneic Vγ9Vδ2 T-cell immunotherapy exhibits promising clinical safety and prolongs the survival of patients with late-stage lung or liver cancer.

Vγ9Vδ2 T cells are promising candidates for cellular tumor immunotherapy. Due to their HLA-independent mode of action, allogeneic Vγ9Vδ2 T cells can be considered for clinical application. To apply allogeneic Vγ9Vδ2 T cells in adoptive immunotherapy, the methodology used to obtain adequate cell numbers with optimal effector function in vitro needs to be optimized, and clinical safety and efficacy also need to be proven. Therefore, we developed a novel formula to improve the expansion of peripheral γδ T cells from healthy donors. Then, we used a humanized mouse model to validate the therapeutic efficacy of expanded γδ T cells in vivo; furthermore, the expanded γδ T cells were adoptively transferred into late-stage liver and lung cancer patients. We found that the expanded cells possessed significantly improved immune effector functions, including proliferation, differentiation, and cancer cell killing, both in vitro and in the humanized mouse model. Furthermore, a phase I clinical trial in 132 late-stage cancer patients with a total of 414 cell infusions unequivocally validated the clinical safety of allogeneic Vγ9Vδ2 T cells. Among these 132 patients, 8 liver cancer patients and 10 lung cancer patients who received ≥5 cell infusions showed greatly prolonged survival, which preliminarily verified the efficacy of allogeneic Vγ9Vδ2 T-cell therapy. Our clinical studies underscore the safety and efficacy of allogeneic Vγ9Vδ2 T-cell immunotherapy, which will inspire further clinical investigations and eventually benefit cancer patients.

Allogeneic Vγ9Vδ2 T-Cell Therapy Promotes Pulmonary Lesion Repair: An Open-Label, Single-Arm Pilot Study in Patients With Multidrug-Resistant Tuberculosis.

The WHO's "Global tuberculosis report 2020" lists tuberculosis (TB) as one of the leading causes of death globally. Existing anti-TB therapy strategies are far from adequate to meet the End TB Strategy goals set for 2035. Therefore, novel anti-TB therapy protocols are urgently needed. Here, we proposed an allogeneic Vγ9Vδ2 T-cell-based immunotherapy strategy and clinically evaluated its safety and efficacy in patients with multidrug-resistant TB (MDR-TB). Eight patients with MDR-TB were recruited in this open-label, single-arm pilot clinical study. Seven of these patients received allogeneic Vγ9Vδ2 T-cell therapy adjunct with anti-TB drugs in all therapy courses. Cells (1 × 108) were infused per treatment every 2 weeks, with 12 courses of cell therapy conducted for each patient, who were then followed up for 6 months to evaluate the safety and efficacy of cell therapy. The eighth patient initially received four courses of cell infusions, followed by eight courses of cell therapy plus anti-MDR-TB drugs. Clinical examinations, including clinical response, routine blood tests and biochemical indicators, chest CT imaging, immune cell surface markers, body weight, and sputum Mycobacterium tuberculosis testing, were conducted. Our study revealed that allogeneic Vγ9Vδ2 T cells are clinically safe for TB therapy. These cells exhibited clinical efficacy in multiple aspects, including promoting the repair of pulmonary lesions, partially improving host immunity, and alleviating M. tuberculosis load in vivo, regardless of their application in the presence or absence of anti-TB drugs. This pilot study opens a new avenue for anti-TB treatment and exhibits allogeneic Vγ9Vδ2 T cells as promising candidates for developing a novel cell drug for TB immunotherapy. Clinical Trial Registration: (https://clinicaltrials.gov/ct2/results?cond=&term=NCT03575299&cntry=&state=&city=&dist=) ( NCT03575299).

Vitamin C promotes the proliferation and effector functions of human γδ T cells.

γδ T cells are of interest as effector cells for cellular immunotherapy due to their HLA-non-restricted lysis of many different tumor cell types. Potential applications include the adoptive transfer of in vitro-expanded γδ T cells. Therefore, it is important to optimize the culture conditions to enable maximal proliferative and functional activity. Vitamin C (L-ascorbic acid) is an essential vitamin with multiple effects on immune cells. It is a cofactor for several enzymes, has antioxidant activity, and is an epigenetic modifier. Here, we investigated the effects of vitamin C (VC) and its more stable derivative, L-ascorbic acid 2-phosphate (pVC), on the proliferation and effector function of human γδ T cells stimulated with zoledronate (ZOL) or synthetic phosphoantigens (pAgs). VC and pVC did not increase γδ T-cell expansion within ZOL- or pAg-stimulated PBMCs, but increased the proliferation of purified γδ T cells and 14-day-expanded γδ T-cell lines in response to γδ T-cell-specific pAgs. VC reduced the apoptosis of γδ T cells during primary stimulation. While pVC did not prevent activation-induced death of pAg-restimulated γδ T cells, it enhanced the cell cycle progression and cellular expansion. Furthermore, VC and pVC enhanced cytokine production during primary activation, as well as upon pAg restimulation of 14-day-expanded γδ T cells. VC and pVC also increased the oxidative respiration and glycolysis of γδ T cells, but stimulus-dependent differences were observed. The modulatory activity of VC and pVC might help to increase the efficacy of γδ T-cell expansion for adoptive immunotherapy.

Bibliometrics Analysis of Butyrophilins as Immune Regulators [1992-2019] and Implications for Cancer Prognosis.

The butyrophilins (BTNs) represent a unique family of immunoglobulin. They were considered to be involved in milk lactation after their discovery in 1981. With the development of research, an increasing number of research revealed that BTNs play important roles in immune regulation [1992-2019]. Our research aimed to summarize the BTN research status and their relationship with lung cancers and breast cancers by bibliometrics and bioinformatics methods. Our results indicate that the researches on immune-regulatory functions of BTNs gradually developed from 1992 to 2006, whereas they increased quickly after 2007. There are international cooperations among 56 countries, of which the United States is the most active one with the highest number of studies as well as highest citations. By coauthorship and cocitation analysis, we showed that Adrian Hayday, who is active in γδ T-cell field, was an active author in BTN publications with average year of 2015 and led a subfield. By keywords co-occurrence analysis, we found that γδ T cell, which is an important cancer immune regulator, is one important hotspot. Finally, we found that several BTN members' expression levels were significantly correlated with prognosis of lung cancer and breast cancer patients. Thus, these BTNs might play immune regulatory effects and could serve as potential biomarkers for cancer.

Roles of mTORC1 and mTORC2 in controlling γδ T1 and γδ T17 differentiation and function.

The metabolism-controlled differentiation of αß T cells has been well documented; however, the role of a metabolism program in γδ T cell differentiation and function has not been clarified. Here, using CD2-cre; mTORC1 Raptor-f/f, and mTORC2 Rictor-f/f mice (KO mice), we found that mTORC1, but not mTORC2, was required for the proliferation and survival of peripheral γδ T cells, especially Vγ4 γδ T cells. Moreover, mTORC1 was essential for both γδ T1 and γδ Τ17 differentiation, whereas mTORC2 was required for γδ T17, but not for γδ Τ1, differentiation. We further studied the underlying molecular mechanisms and found that depletion of mTORC1 resulted in the increased expression of SOCS1, which in turn suppressed the key transcription factor Eomes, consequentially reducing IFN-γ production. Whereas the reduced glycolysis resulted in impaired γδ Τ17 differentiation in Raptor KO γδ T cells. In contrast, mTORC2 potentiated γδ Τ17 induction by suppressing mitochondrial ROS (mitoROS) production. Consistent with their cytokine production profiles, the Raptor KO γδ T cells lost their anti-tumor function both in vitro and in vivo, whereas both Raptor and Rictor KO mice were resistant to imiquimod (IMQ)-induced psoriasis-like skin pathogenesis. In summary, we identified previously unknown functions of mTORC1 and mTORC2 in γδ T cell differentiation and clarified their divergent roles in mediating the activity of γδ T cells in tumors and autoimmunity.