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The impact of exposure to air pollutants, such as fine particulate matter (PM), on the immune system and its consequences on pediatric asthma are not well understood. We investigated whether the ambient levels of fine PM with aerodynamic diameter ≤ 2.5 microns (PM ) are associated with alterations in circulating monocytes in children with or without asthma. Increased exposure to ambient PM was linked to specific monocyte subtypes, particularly in children with asthma. Mechanistically, we hypothesized that innate trained immunity is evoked by a primary exposure to fine PM and accounts for an enhanced inflammatory response after secondary stimulation in vitro. We determined that the trained immunity was induced in circulating monocytes by fine particulate pollutants, and it was characterized by upregulation of proinflammatory mediators, such as TNF, IL-6, and IL-8, upon stimulation with house dust mite or LPS. This phenotype was epigenetically controlled by enhanced H3K27ac marks in circulating monocytes. The specific alterations of monocytes after ambient pollution exposure suggest a possible prognostic immune signature for pediatric asthma, and pollution-induced trained immunity may provide a potential therapeutic target for asthmatic children living in areas with increased air pollution.

Currently the clinical efficacy of colorectal cancer (CRC) which is the most common malignant tumors over the world has not reached an ideal level. Cetuximab, the monoclonal antibody targeting the extracellular domain of EGFR, has shown its great efficacy in the promotion of apoptosis and the inhibition of tumor cells-like characteristics in numerous cancers. However certain KRAS wild-type CRC patients unexpectedly show cetuximab resistance and the specific mechanism remains unclear. Circular RNAs (circRNAs) as the promising novel type of biomarkers in the cancer diagnosis and therapy, have been reported to be related with the drug resistance. In this study, with wondering the mechanism of cetuximab resistance in KRAS wild-type CRC patients, we evaluate the impact of circIFNGR2 on CRC and detect the association among circIFNGR2, miR-30b and KRAS via various experiments such as RT-qPCR, immunohistochemistry, luciferase assays, cell functional experiments and xenograft model. We conclude that circIFNGR2 induces cetuximab resistance in colorectal cancer cells by indirectly regulating target gene KRAS by sponging miR-30b at the post-transcriptional level. It is thus suggested that inhibition of circIFNGR2 can be a promising therapeutic strategy for malignant CRC patients with cetuximab resistance.

Patients with type 2 diabetes (T2D) are at an increased risk of morbidity and mortality of coronavirus disease 2019 (COVID-19). Data on the antibody response to COVID-19 vaccines in T2D patients are less studied. This study aimed to evaluate IgG antibody response to inactivated COVID-19 vaccines in hospitalized T2D patients. Hospitalized patients with no history of COVID-19 and received two doses of inactivated COVID-19 vaccines (Sinopharm or CoronaVac) were included in this study from March to October 2021. SARS-CoV-2 specific IgG antibodies were measured 14–60 days after the second vaccine dose. A total of 209 participants, 96 with T2D and 113 non-diabetes patients, were included. The positive rate and median titer of IgG antibody against receptor-binding domain (anti-RBD) of spike (S) protein of SARS-CoV-2 in T2D group were lower than in control group (67.7% vs 83.2%, p = .009; 12.93 vs 17.42 AU/ml, p = .014) respectively. Similarly, seropositivity and median titers of IgG antibody against the nucleocapsid (N) and S proteins of SARS-CoV-2 (anti-N/S) in T2D group were lower than in control group (68.8% vs 83.2%, p = .032; 18.81 vs 29.57 AU/mL, p = .012) respectively. After adjustment for age, sex, BMI, vaccine type, days after the second vaccine dose, hypertension, kidney disease, and heart disease, T2D was identified as an independent risk factor for negative anti-RBD and anti-N/S seropositivity, odd ratio 0.42 (95% confidence interval 0.19, 0.89) and 0.42 (95% CI 0.20, 0.91), respectively. T2D is associated with impaired antibody response to inactivated COVID-19 vaccine.

Inflammatory bowel disease (IBD) is a chronic gut disease with intestinal-epithelium disruption. Mast cell (MC) has been discussed in IBD studies, but its subset MCThis study was to examine the distribution of chymase-positive mast cells in the intestinal-epithelium-tissue of IBD infants, to explore the MC-chymase function on intestinal-epithelium, and to investigate the influences of DSLNT against MC-chymase-induced disruptions.The intestinal-biopsies (surgical-waste) of the infants with IBD or with intestinal-atresia (non-IBD) were paraffin-embedded for immunohistochemistry. In-situ intestinal-tissue model and in-vitro human-intestinal-epithelial-cell (Caco-2) model were established with or without the treatments of MC-chymase (50mU/mL), DSLNT (600 µM) and DSLNT + MC-chymase respectively. The tissue morphology analysis, cell proliferation assay, cell-gap-closure assessment, fluorescence-immunocytochemistry, western blot, trans-epithelial-electrical-resistance, cell-cycle and statistical analysis were applied.There was an increased number of MCChymase-positive MCs are involved in IBD progress. MC-chymase disrupts intracellular ZO-1/FAK/P38 signal pathway and cell-cell/cell-matrix contacts, while DSLNT protects intestinal-epithelium against MC-chymase to maintain the intestinal epithelium integrity.

Hyaluronic acid (HA) is the main component of the extracellular matrix (ECM) of joints, and it is important for a lubricating joint during body movement. Degradation is the main metabolic process of HA in vivo. Hyaluronidases (HAase) were known for HA degradation. The inflammation-induced HA rapid-metabolism can reduce HA viscosity and concentration in joints. Mast cells (MC) containing their specific proteases were found in synovium tissue. It is unclear if MC-proteases could be involved in HA degradation pathways. This study aims to explore the correlations between HA concentration vs mast cell proteases, or matrix metalloproteinase-2/9 (MMP-2/9) and to investigate the association of MC-specific proteases with disrupted synovial HA homeostasis in rheumatoid arthritis (RA) or collagen-induced arthritis rats. The synovial fluid samples from no-RA and RA patients were collected; the collagen-induced arthritis (CIA) rat model was established; HA concentration and the activities of MC-protease and MMP-2/9 in the samples were detected, and the correlations were analyzed. In vitro interaction experiment was carried out by mixing MC-proteases with HA to observe the degradation speed. The HA concentrations in synovial fluids were decreased in RA patients and CIA rats compared with those in no-RA subjects or normal rats respectively. The activities of mast cell proteases in synovial fluids were increased and positively correlated with MMP-9, but negatively correlated with HA concentrations. In vitro study, the addition of MC-chymase and tryptase promoted the speed in HA degradation. MC-proteases may influence HA degradation pathway.

BackgroundAlthough the gut microbiota may be involved in obesity onset and progression, the exact association of the gut microbiota in metabolically healthy obesity (MHO) remains largely unknown.MethodsAn integrated paired-sample metagenomic analysis was conducted to investigate the gut microbial network and biomarkers of microbial species from the MHO and healthy non-obese subjects in the GMrepo database. Further explorations were performed in the MHO mice model using a multiomics analysis to detect changes in the composition and function of the intestinal microbiome and associated metabolites.ResultsIn the human study, 314 matched metagenomic data were qualified for the final analysis. We identified seven significantly changed species possibly involved in MHO pathogenesis (MHO-enriched: Bacteroides vulgatus, Megamonas sp; MHO-depleted: Butyrivibrio crossotus, Faecalibacterium prausnitzii, Bacteroides cellulosilyticus; Eubacterium siraeum; Bacteroides massiliensis). In the murine study, we found 79 significantly-changed species which may have possible associations with the MHO phenotype. The depletion of Bacteroides cellulosilyticus was commonly recognized in the human and murine MHO phenotype. Consistent with the metagenomic data, liquid chromatography-mass spectrometry (LC/MS) revealed significantly changed gut metabolites, which may promote MHO pathogenesis by altering the amino acids and lipid metabolic pathways. In the microbe-metabolites interaction analysis, we identified certain fatty acids (Dodecanedioic acid, Arachidic Acid, Mevalonic acid, etc.) that were significantly correlated with the MHO-enriched or depleted species.ConclusionThis study provides insights into identifying specific microbes and metabolites that may involve in the development of obesity without metabolic disorders. Future modalities for MHO intervention may be further validated by targeting these bacteria and metabolites.

Background and AimsThis study aimed to develop an interpretable random forest model for predicting severe acute pancreatitis (SAP).MethodsClinical and laboratory data of 648 patients with acute pancreatitis were retrospectively reviewed and randomly assigned to the training set and test set in a 3:1 ratio. Univariate analysis was used to select candidate predictors for the SAP. Random forest (RF) and logistic regression (LR) models were developed on the training sample. The prediction models were then applied to the test sample. The performance of the risk models was measured by calculating the area under the receiver operating characteristic (ROC) curves (AUC) and area under precision recall curve. We provide visualized interpretation by using local interpretable model-agnostic explanations (LIME).ResultsThe LR model was developed to predict SAP as the following function: -1.10-0.13×albumin (g/L) + 0.016 × serum creatinine (μmol/L) + 0.14 × glucose (mmol/L) + 1.63 × pleural effusion (0/1)(No/Yes). The coefficients of this formula were utilized to build a nomogram. The RF model consists of 16 variables identified by univariate analysis. It was developed and validated by a tenfold cross-validation on the training sample. Variables importance analysis suggested that blood urea nitrogen, serum creatinine, albumin, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, calcium, and glucose were the most important seven predictors of SAP. The AUCs of RF model in tenfold cross-validation of the training set and the test set was 0.89 and 0.96, respectively. Both the area under precision recall curve and the diagnostic accuracy of the RF model were higher than that of both the LR model and the BISAP score. LIME plots were used to explain individualized prediction of the RF model.ConclusionsAn interpretable RF model exhibited the highest discriminatory performance in predicting SAP. Interpretation with LIME plots could be useful for individualized prediction in a clinical setting. A nomogram consisting of albumin, serum creatinine, glucose, and pleural effusion was useful for prediction of SAP.

Previous researches have emphasized a trypsin-centered theory of acute pancreatitis (AP) for more than a century. With additional studies into the pathogenesis of AP, new mechanisms have been explored. Among them, the role of immune response bears great importance. Pro-inflammatory substances, especially damage-associated molecular patterns (DAMPs), play an essential role in activating, signaling, and steering inflammation. Meanwhile, activated neutrophils attach great importance to the immune defense by forming neutrophil extracellular traps (NETs), which cause ductal obstruction, premature trypsinogen activation, and modulate inflammation. In this review, we discuss the latest advances in understanding the pathological role of DAMPs and NETs in AP and shed light on the flexible crosstalk between these vital inflammatory mediators. We, then highlight the potentially promising treatment for AP targeting DAMPs and NETs, with a focus on novel insights into the mechanism, diagnosis, and management of AP.

Ellipticine, a natural product from Ochrosia elliptica, has been broadly investigated for its anticancer effects. Although inflammation has been clearly identified as a key factor in the onset and progression of cancer, the relationship between ellipticine and inflammation remains unknown. Hence, the aims of the present study were to assess the effects of ellipticine on the inflammatory responses to lipopolysaccharide (LPS)-induced macrophages and to potentially identify the underlying mechanisms involved. Viability testing showed that ellipticine was not significantly toxic to Raw264.7 cells and actually conveyed protective effects to LPS-stimulated Raw264.7 cells and human peripheral blood monocytes by decreasing the secretion of inflammatory factors (TNF-α and IL-6). The results of western blot analysis and electrophoretic mobility shift assays showed that ellipticine markedly suppressed LPS-induced activation of the JNK/AP-1 (c-Fos and c-Jun) signaling pathway, but not ERK/p38/NF-κB pathway (p65 and p50) activation. Furthermore, ellipticine reduced the inflammatory response and mortality in a mouse model of LPS-induced endotoxic shock. Collectively, these data indicate that ellipticine may be a potential therapeutic agent for the treatment of inflammation-associated diseases.

Atopic dermatitis (AD) in early childhood is often the initial manifestation of allergic disease associated with high IgE. Accumulating evidences show that follicular helper T (Tfh) cells play a critical role in promoting B cell differentiation and IgE production, human regulatory B (Breg) cells participate in immunomodulatory processes and inhibition of allergic inflammation. However, the roles and interactions between IL-10-producing Breg cells and Tfh cells in childhood AD are unclear. In this study, we found that the percentage of CD19+IL-10+ Breg cells in children with extrinsic AD was significantly lower than that in age-matched healthy controls, and that it correlated negatively with enhanced CD4+CXCR5+PD-1+ICOS+ circulating Tfh cell responses and increased disease activity; however, there was no significant correlation with serum total IgE levels. A co-culture system revealed that Breg cells from patients with extrinsic AD cannot effectively inhibit differentiation of Tfh cells in an IL-10 dependent manner. Abnormal pSTAT3 signaling induced via Toll-like receptors (TLR), but not the B-cell receptor (BCR) signaling, might contribute to the defect of Breg cells in AD. Taken together, these observations demonstrate an important role for IL-10-producing Breg cells in inhibiting Tfh cell differentiation, and suggest that they may participate in the pathogenesis of AD.

Background and AimsThe aim of this study was to apply machine learning models and a nomogram to differentiate critically ill from non-critically ill COVID-19 pneumonia patients.MethodsClinical symptoms and signs, laboratory parameters, cytokine profile, and immune cellular data of 63 COVID-19 pneumonia patients were retrospectively reviewed. Outcomes were followed up until Mar 12, 2020. A logistic regression function (LR model), Random Forest, and XGBoost models were developed. The performance of these models was measured by area under receiver operating characteristic curve (AUC) analysis.ResultsUnivariate analysis revealed that there was a difference between critically and non-critically ill patients with respect to levels of interleukin-6, interleukin-10, T cells, CD4+ T, and CD8+ T cells. Interleukin-10 with an AUC of 0.86 was most useful predictor of critically ill patients with COVID-19 pneumonia. Ten variables (respiratory rate, neutrophil counts, aspartate transaminase, albumin, serum procalcitonin, D-dimer and B-type natriuretic peptide, CD4+ T cells, interleukin-6 and interleukin-10) were used as candidate predictors for LR model, Random Forest (RF) and XGBoost model application. The coefficients from LR model were utilized to build a nomogram. RF and XGBoost methods suggested that Interleukin-10 and interleukin-6 were the most important variables for severity of illness prediction. The mean AUC for LR, RF, and XGBoost model were 0.91, 0.89, and 0.93 respectively (in two-fold cross-validation). Individualized prediction by XGBoost model was explained by local interpretable model-agnostic explanations (LIME) plot.ConclusionsXGBoost exhibited the highest discriminatory performance for prediction of critically ill patients with COVID-19 pneumonia. It is inferred that the nomogram and visualized interpretation with LIME plot could be useful in the clinical setting. Additionally, interleukin-10 could serve as a useful predictor of critically ill patients with COVID-19 pneumonia.

Activated-mast cells (MCs) within gingival-tissue of chronic-periodontitis (CP) patients, release various inflammatory-factors. Bradykinin is a nine-amino-acid peptide and pro-inflammatory mediator, produced through factor-XII-cascade or tryptase-cascade. The ability of MC-chymase in bradykinin generation has not been discussed yet. This study investigated the salivary levels of MC-chymase, high molecular weight kininogen (HMWK) and bradykinin of CP patients; examined the potential of MC-proteases in bradykinin production using biochemistry-models; and explored the effects of bradykinin on gingival fibroblasts (GFs). Saliva-samples were collected; MC-protease activities were detected; HMWK cleavage was assessed by western-blot and SDS-PAGE; bradykinin levels were measured using immunoassay. Primary GFs were extracted and cultured with or without bradykinin; cell-viability, gelatine-zymography and flow-cytometry were applied. Immunocytochemistry and western-blot were used to detect intracellular protein expressions of bradykinin-stimulated GFs. The data showed that the salivary-levels of MC-proteases, bradykinin, HMWK, and lactoferrin of CP-patients were increased. HMWK was cleaved by MC-chymase in-vitro, resulting in bradykinin generation. Bradykinin promoted cell proliferation, cell cycle and matrix-metalloproteinase-2(MMP-2) activity, and increased intracellular expressions of nuclear-factor-kappa-B(NF-κB), focal-adhesion-kinase(FAK), transforming-growth-factor-β(TGF-β), P38, P53 of GFs. MC-chymase promotes bradykinin production to stimulate GFs and to continue inflammation during CP development. A new BK-generation cascade found in this study provides a new basis for the pathogenesis of CP and the mechanism of continuous inflammation. The activation of MC-chymase/bradykinin-generation cascade depends on HMWK level and MC-chymase activity under inflammatory condition. MC-chymase contributes to bradykinin production, mediating the cross-talks between MCs and GFs. MC-chymase can be used as a therapeutic target and a salivary biomarker in this case.

CD8+ T cells are seldom considered in IgE mediated food allergy; we show that peanut specific CD8+ T cells are increased in peanut allergic human subjects.

Rheumatoid arthritis (RA) is an autoimmune disease and characterized by the excessive cell proliferation, abnormal cell cycle of lymphocytes and synovial cells. The therapeutic effects of curcumin in active RA patients were reported, but limited by its insolubility and rapid systemic elimination. Dimethyl curcumin (DiMC) is a metabolically stable analogue of curcum with anti-inflammatory property. In this study, liposomes encapsulated dimethyl curcumin (Lipo-DiMC) was prepared to improve the bioavailability and metabolic-stability; collagen induced arthritis (CIA) rat model was employed to investigate the effects of Lipo-DiMC treatments during CIA progress. Physical assessments and routine-blood-test were performed. Fresh spleen lymphocytes were isolated from normal, CIA and Lipo-DiMC-treated CIA rats; flow-cytometry for cell-cycle analysis, western-blotting for intracellular signal pathway protein expressions, gelatin-zymography for matrix-metalloproteases 2/9 (MMP-2/9) and GF-AFC for dipeptidyl-peptidase I (DPPI) activity assay. Compared with untreated CIA rats, Lipo-DiMC treatments relieved paw-swellings, suppressed the increments of immunocytes numbers and inhibited DPPI and MMP-2/9 over-activity in blood. Lipo-DiMC adjusted CIA-induced cell cycle dysfunction at G0/G1-phase and S-phase of spleen lymphocytes for CIA rats. The intracellular expression-trends of P38, P21, Bcl-2, JNK-1 and DPPI of spleen lymphocytes were observed during CIA progress with and without Lipo-DiMC administrations. Lipo-DiMC exhibited its therapeutic functions by attenuating CIA development in rats, associated with down-regulating CIA-induced lymphocytes numbers, inhibiting over-expressed of DPPI and MMP-2/9, and adjusting cell cycles. These findings provide a new insight into the mechanism of Lipo-DiMC treatment in CIA rat model and suggest that Lipo-DiMC could be considered as a potential drug for RA treatment.

DNA methylation (DNAm) has been shown to play a role in mediating food allergy; however, the mechanism by which it does so is poorly understood. In this study, we used targeted next-generation bisulfite sequencing to evaluate DNAm levels in 125 targeted highly informative genomic regions containing 602 CpG sites on 70 immune-related genes to understand whether DNAm can differentiate peanut allergy (PA) versus nonallergy (NA). We found PA-associated DNAm signatures associated with 12 genes (7 potentially novel to food allergy, 3 associated with Th1/Th2, and 2 associated with innate immunity), as well as DNAm signature combinations with superior diagnostic potential compared with serum peanut-specific IgE for PA versus NA. Furthermore, we found that, following peanut protein stimulation, peripheral blood mononuclear cell (PBMCs) from PA participants showed increased production of cognate cytokines compared with NA participants. The varying responses between PA and NA participants may be associated with the interaction between the modification of DNAm and the interference of environment. Using Euclidean distance analysis, we found that the distances of methylation profile comprising 12 DNAm signatures between PA and NA pairs in monozygotic (MZ) twins were smaller than those in randomly paired genetically unrelated individuals, suggesting that PA-related DNAm signatures may be associated with genetic factors.

Ambient air pollution exposure is associated with cardiovascular dysregulation and immune system alterations, yet no study has investigated both simultaneously in children. Understanding the multifaceted impacts may provide early clues for clinical intervention prior to actual disease presentation. We therefore determined the associations between exposure to multiple air pollutants and both immunological outcomes (methylation and protein expression of immune cell types associated with immune regulation) and cardiovascular outcomes (blood pressure) in a cohort of school-aged children (6–8 years; n = 221) living in a city with known elevated pollution levels. Exposure to fine particular matter (PM2.5), carbon monoxide (CO), and ozone (O3) was linked to altered methylation of most CpG sites for genes Foxp3, IL-4, IL-10 and IFN-g, all involved in immune regulation (e.g. higher PM2.5 exposure 1 month prior to the study visit was independently associated with methylation of the IL-4 CpG24 site (est = 0.16; P = 0.0095). Also, immune T helper cell types (Th1, Th2 and Th17) were associated with short-term exposure to PM2.5, O3 and CO (e.g. Th1 cells associated with PM2.5 at 30 days: est = − 0.34, P 456, NOx and/or NO2 (P ≤ 0.038 for all). Finally, diastolic BP (DBP) was inversely associated with long-term exposures to both CO and PAH456, and both systolic and pulse pressure were associated with short-term NO2 and chronic NOx exposure. Our findings demonstrate links between air pollution exposure and methylation of immunoregulatory genes, immune cell profiles and blood pressure, suggesting that even at a young age, the immune and cardiovascular systems are negatively impacted by exposure to air pollution.

Necrotizing enterocolitis (NEC) remains a fatal gastrointestinal disorder in neonates. Disialyllacto-N-tetraose (DSLNT), a function-unclear human milk-derived hexasaccharide, shows anti-NEC potential in previous animal studies. This study is aimed to explore the role of mast cell (MC), a fundamental cell type of mucosal immune system and protective DSLNT in regulating pathological process of NEC. For this purpose, infantile intestinal-tissues were collected from NEC neonates for examination of MCs and its proteases-positive cells. MC accumulation and MC-specific proteases (chymase, tryptase and dipeptidyl peptidase I) were firstly found in lesioned area of NEC infants in-vivo. Subsequent in-situ experiments on neonatal ileum segments showed that purified MC-chymase induced a destructive epithelial layer shedding from basement and microvascular endothelium damage in infantile intestinal segments. Human foreskin MC-activation model was established and DSLNT were applied; MC products (histamine and MC-proteases) were used as MC activation/degranulation indicators. In this in-vitro model, DSLNT pretreatment suppressed release of histamine, chymase and tryptase by MC to the tissue supernatants during lipopolysaccharide or complement C5a stimulation. Newborn rats were formula-hand-fed with or without DSLNT supplement and exposed to hypoxia/cold-stress to induce experimental-NEC-model. In NEC rats, DSLNT supplementation reduced the incidence and pathological scores of NEC, inhibited local accumulation of MC and reduced cytokines (IL-1β, IL-6 and TNF-α) levels in the ileum of rats. In conclusion, MC was causally implicated in epithelium barrier failure in pathogenesis of NEC. DSLNT favorably modulated MC homeostasis by regulating MC degranulation/accumulation, contributing to attenuated NEC. This indicated novel pathomechanisms and potential targets of NEC.

BACKGROUND: There has been a striking generational increase in the prevalence of food allergies. We have proposed that this increase can be explained, in part, by alterations in the commensal microbiome. METHODS: To identify bacterial signatures and metabolic pathways that may influence the expression of this disease, we collected fecal samples from a unique, well-controlled cohort of twins concordant or discordant for food allergy. Samples were analyzed by integrating 16S rRNA gene amplicon sequencing and liquid chromatography–tandem mass spectrometry metabolite profiling. RESULTS: A bacterial signature of 64 operational taxonomic units (OTUs) distinguished healthy from allergic twins; the OTUs enriched in the healthy twins were largely taxa from the Clostridia class. We detected significant enrichment in distinct metabolite pathways in each group. The enrichment of diacylglycerol in healthy twins is of particular interest for its potential as a readily measurable fecal biomarker of health. In addition, an integrated microbial-metabolomic analysis identified a significant association between healthy twins and Phascolarctobacterium faecium and Ruminococcus bromii, suggesting new possibilities for the development of live microbiome-modulating biotherapeutics. CONCLUSION: Twin pairs exhibited significant differences in their fecal microbiomes and metabolomes through adulthood, suggesting that the gut microbiota may play a protective role in patients with food allergies beyond the infant stage. TRIAL REGISTRATION: Participants in this study were recruited as part of an observational study (ClinicalTrials.gov NCT01613885) at multiple sites from 2014 to 2018. FUNDING: This work was supported by the Sunshine Charitable Foundation; the Moss Family Foundation; the National Institute of Allergy and Infectious Diseases (NIAID) (R56AI134923 and R01AI 140134); the Sean N. Parker Center for Allergy and Asthma Research; the National Heart, Lung, and Blood Institute (R01 HL 118612); the Orsak family; the Kepner family; and the Stanford Institute for Immunity, Transplant and Infection.

SUMMARY Clinical definitions of asthma fail to capture the heterogeneity of immune dysfunction in severe, treatment-refractory disease. Applying mass cytometry and machine learning to bronchoalveolar lavage (BAL) cells, we find that corticosteroid-resistant asthma patients cluster largely into two groups: one enriched in interleukin (IL)-4+ innate immune cells and another dominated by interferon (IFN)-γ+ T cells, including tissue-resident memory cells. In contrast, BAL cells of a healthier population are enriched in IL-10+ macrophages. To better understand cellular mediators of severe asthma, we developed the Immune Cell Linkage through Exploratory Matrices (ICLite) algorithm to perform deconvolution of bulk RNA sequencing of mixed-cell populations. Signatures of mitosis and IL-7 signaling in CD206−FcεRI+CD127+IL-4+ innate cells in one patient group, contrasting with adaptive immune response in T cells in the other, are preserved across technologies. Transcriptional signatures uncovered by ICLite identify T-cell-high and T-cell-poor severe asthma patients in an independent cohort, suggesting broad applicability of our findings., Graphical abstract, In brief Asthma is an immune-mediated heterogeneous disease. Current type-2 biomarkers provide limited understanding of underlying pathobiology or therapeutic guidance. Using a multi-omics approach, Camiolo et al. identify two clusters of severe asthma patients with similar type-2 biomarkers but with strikingly distinct immune profiles and associated biological pathways.

Vaccination against hepatitis B is the most effective strategy to control HBV infection. The first licensed hepatitis B vaccine was developed by the purification of hepatitis B surface antigen (HBsAg) from plasma of asymptomatic HBsAg carriers. Then, the recombinant DNA technology enabled the development of recombinant hepatitis B vaccine. A series of three doses vaccine can elicit long-term protection more than 30 y. Concurrent use of hepatitis B immunoglobulin and hepatitis B vaccine has substantially reduced the mother-to-child transmission of HBV, nearly zero infection in children of carrier mother with negative hepatitis B e antigen (HBeAg) and 5–10% infection in children of HBeAg-positive mothers. By the end of 2018, 189 countries adopted universal hepatitis B vaccination program, which has dramatically reduced the global prevalence of HBsAg in children

BackgroundDNA methylation of CpG sites on genetic loci has been linked to increased risk of asthma in children exposed to elevated ambient air pollutants (AAPs). Further identification of specific CpG sites and the pollutants that are associated with methylation of these CpG sites in immune cells could impact our understanding of asthma pathophysiology. In this study, we sought to identify some CpG sites in specific genes that could be associated with asthma regulation (Foxp3 and IL10) and to identify the different AAPs for which exposure prior to the blood draw is linked to methylation levels at these sites. We recruited subjects from Fresno, California, an area known for high levels of AAPs. Blood samples and responses to questionnaires were obtained (n = 188), and in a subset of subjects (n = 33), repeat samples were collected 2years later. Average measures of AAPs were obtained for 1, 15, 30, 90, 180, and 365days prior to each blood draw to estimate the short-term vs. long-term effects of the AAP exposures.ResultsAsthma was significantly associated with higher differentially methylated regions (DMRs) of the Foxp3 promoter region (p = 0.030) and the IL10 intronic region (p = 0.026). Additionally, at the 90-day time period (90days prior to the blood draw), Foxp3 methylation was positively associated with NO2, CO, and PM2.5 exposures (p = 0.001, p = 0.001, and p = 0.012, respectively). In the subset of subjects retested 2years later (n = 33), a positive association between AAP exposure and methylation was sustained. There was also a negative correlation between the average Foxp3 methylation of the promoter region and activated Treg levels (p = 0.039) and a positive correlation between the average IL10 methylation of region 3 of intron 4 and IL10 cytokine expression (p = 0.030).ConclusionsShort-term and long-term exposures to high levels of CO, NO2, and PM2.5 were associated with alterations in differentially methylated regions of Foxp3. IL10 methylation showed a similar trend. For any given individual, these changes tend to be sustained over time. In addition, asthma was associated with higher differentially methylated regions of Foxp3 and IL10.

Diffuse large B-cell lymphoma (DLBCL), the most common type of malignant lymphoma, accounts for 30% of adult non-Hodgkin lymphomas. Epstein-Barr virus (EBV) -positive DLBCL of the elderly is a newly recognized subtype that accounts for 8-10% of DLBCLs in Asian countries, but is less common in Western populations. Five DLBCL-derived cell lines were employed to characterize patterns of EBV latent gene expression, as well as response to cytokines and chemotaxis. Interleukin-4 and interleukin-21 modified LMP1, EBNA1 and EBNA2 expression depending on cell phenotype and type of EBV latent programme (type I, II or III). These cytokines also affected CXCR4- or CCR7-mediated chemotaxis in two of the cell lines, Farage (type III) and Val (type II). Further, we investigated the effect of EBV by using dominant-negative EBV nuclear antigen 1(dnEBNA1) to eliminate EBV genomes. This resulted in decreased chemotaxis. By employing an alternative way to eliminate EBV genomes, Roscovitine, we show an increase of apoptosis in the EBV-positive lines. These results show that EBV plays an important role in EBV-positive DLBCL lines with regard to survival and chemotactic response. Our findings provide evidence for the impact of microenvironment on EBV-carrying DLBCL cells and might have therapeutic implications.

Background X-linked inhibitor of apoptosis (XIAP) deficiency is a rare primary immunodeficiency disease characterized by haemophagocytic lymphohistiocytosis, recurrent splenomegaly and inflammatory bowel disease (IBD). The only curative treatment is haematopoietic stem cell transplant (HSCT). Case presentation Here, we report the case of a 22-month-old male with a long history of abdominal distension and anaemia. Clinical and laboratory findings were consistent with eosinophilic colitis. To identify the underlying disease, we performed exome sequencing, which showed an unreported frameshift mutation in the XIAP gene. Conclusion We present eosinophilic colitis as the initial manifestation of XIAP deficiency for the first time in this article, which expands the mutation spectrum and phenotype of this disease.

The molecular mechanism responsible for Helicobacter pylori infection-mediated gastritis and carcinogenesis is not yet clear. Increased evidence suggests that chronic gastritis and elevated gastric epithelial cell (GEC) apoptosis are crucial events during stomach carcinoma transformation. PUMA is a potent proapoptotic Bcl-2 protein and mediates acute tissue injury. In this study, we aimed to investigate the role of PUMA in GEC apoptosis and inflammation induced by H. pylori infection. As a result, we found that PUMA expression was elevated in gastritis tissues compared with uninvolved tissues, and it was correlated with the severity of apoptosis and gastritis. In mice, PUMA mRNA and protein were markedly induced in GECs upon induction of gastritis by H. pylori. PUMA-deficient mice were highly resistant to apoptosis and gastritis induced by H. pylori. Furthermore, the transcription factor NF-κB p65 binds to PUMA promoter to activate PUMA transcription after H. pylori infection. In addition, NF-κB inhibitor could rescue H. pylori-induced apoptosis and gastritis. Finally, H. pylori-induced activation of p-p65 and PUMA was mediated via Toll-like receptor 2 (TLR2) and blocked in TLR2 knockout mice. Taken together, these results verified the pro-inflammatory effect of PUMA in H. pylori-infected gastric tissue. Moreover, TLR2/NF-κB-mediated transcriptional regulation of PUMA contributes to the pathogenesis of H. pylori-infected gastritis.

// Xiaoying Zhou 1, 2, * , Han Chen 1, 2, * , Li Zhu 3, * , Bo Hao 1 , Weifeng Zhang 1 , Jie Hua 1 , Huiyuan Gu 4 , Wujuan Jin 1, 2 , Guoxin Zhang 1, 2 1 Department of Gastroenterology, First Affiliated Hospital of Nanjing Medical University, Nanjing, China 2 First Clinical Medical College of Nanjing Medical University, Nanjing, China 3 Department of Gastroenterology, People’s Hospital of Jingjiang, Taizhou, China 4 Department of Gastroenterology, First Affiliated Hospital of Soochow University, Suzhou, China * These authors contributed equally to this work Correspondence to: Guoxin Zhang, email: guoxinz@njmu.edu.cn Keywords: helicobacter pylori infection, lncRNA, gastric cancer, MUC2 Received: April 27, 2016 Accepted: October 10, 2016 Published: November 07, 2016 ABSTRACT Long non-coding RNAs (lncRNAs) were shown to play critical roles in cancer biology. We investigated whether H. pylori infection could promote gastric cancer by regulating lncRNAs expression. Differentially expressed lncRNAs between H. pylori positive and negative tissues were identified by microarray and validated by qRT-PCR. Our results indicated that H. pylori positive tissues have a specific profile of lncRNAs. Cell biological assays with siRNA-mediated knockdown or lentivirus vector-mediated over-expression were performed to probe the functional relevance of the lncRNAs. We identified an lncRNA-AF147447 decreased expressed by H. pylori infection, which can inhibit GC proliferation and invasion in vitro and in vivo , act as a tumor suppressor in the development of H. pylori induced GC. LncRNA AF147447 could repress MUC2 expression by direct binding or increasing miR-34c expression. We also found that transcription factor E2F1 could be recruited to lncRNA AF147447 promoter by RNA immunoprecipatation and RNA pull down assays. These findings support a role of lncRNA AF147447 in tumor suppression. This discovery contributes to a better understanding of the importance of the deregulated lncRNAs by H. pylori infection and provides a rationale for the potential development of lncRNA-based targeted approaches for the treatment of H. pylori -related gastric cancer.

Glutaredoxin 3 (GLRX3) is antioxidant enzyme, maintaining a low level of ROS, thus contributing to the survival and metastasis of several types of cancer. However, the expression and functions of GLRX3 have not been addressed in nasopharyngeal carcinoma (NPC). In this study, we found that GLRX3 was overexpressed in NPC. Knockdown of GLRX3 in NPC cell lines inhibited proliferation in vitro, tumorignesis in vivo, and colony formation. In addition, GLRX3 knockdown decreased the migration and invasion capacity of NPC cells by reversing the epithelial-mesenchymal transition (EMT). Furthermore, stabilization of GLRX3 was positively related to with epidermal growth factor receptor (EGFR) expression and negatively with ROS generation. Phosphorylation of Akt, a key downstream effector, was induced by EGFR signaling but did not rely on increasing ROS level in NPC cells. GLRX3 might be an oncoprotein in NPC, playing important roles in increasing redox reaction and activating EGFR/ Akt signals, so it may be a therapeutic target for NPC.

Necrotizing enterocolitis (NEC), a devastating infant disease characterized by severe intestinal necrosis, its pathogenesis is poorly understood, but appears to be multifactorial and highly associated with immaturity of gastrointestinal tract and immature innate-immune system. Breast-milk is effective strategy to protect infants against NEC. This study is using a NEC rat model to investigate the pathological mechanism of NEC involved intestinal-damages, and the therapeutic mechanism of sialylated human milk oligosaccharides (SHMOs) on NEC rats; also using cell model to investigate the effects of SHMOs on colon-epithelial cells (Caco-2) in-vitro. Extraction and characterization of SHMOs from breast milk, establishment of a NEC rat model, histopathological analysis and mast cell accounting of the terminal ileum were taken; The levels of DPPI, TLR4, IL-6, TNF-α, MMP-2/9 and glutathione were measured using various methods. Caco-2 cells were pre-treated with SHMOs and cultured with LPS, histamine, chymase or DPPI, cell viabilities and mitochondrial membrane potential were examined; flow cytometry was used to detect cell cycle. The accumulation of mast cells was found in the ileum of NEC rats, but prohibited by SHMOs treatment; the increased levels of TLR4, DPPI, IL-6, TNF-α, MMP-2/9 in NEC ileum were suppressed by SHMOs in-vivo. SHMOs prevented Caco-2 cells from LPS, histamine, chymase induced damages by surviving cell viability, regulating G0/G1 and S phase in cell cycles, and increasing mitochondrial membrane potential. These findings provide a new insight into the pharmacological mechanism of SHMOs treatment for NEC and suggest that SHMOs needs well attention for therapeutic aims.

The aryl hydrocarbon receptor (AhR) is an important immune regulator with a role in inflammatory response. However, the role of AhR in IL-10 production by inflammatory macrophages is currently unknown. In this study, we investigated LPS-induced IL-10 expression in macrophages from AhR-KO mice and AhR-overexpressing RAW264.7 cells. AhR was highly expressed after LPS stimulation through NF-κB pathway. Loss of AhR resulted in reduced IL-10 expression in LPS-induced macrophages. Moreover, the IL-10 expression was elevated in LPS-induced AhR-overexpressing RAW264.7 cells. Maximal IL-10 expression was dependent on an AhR non-genomic pathway closely related to Src and STAT3. Furthermore, AhR-associated Src activity was responsible for tyrosine phosphorylation of STAT3 and IL-10 expression by inflammatory macrophages. Adoptive transfer of AhR-expressing macrophages protected mice against LPS-induced peritonitis associated with high IL-10 production. In conclusion, we identified the AhR-Src-STAT3-IL-10 signaling pathway as a critical pathway in the immune regulation of inflammatory macrophages, It suggests that AhR may be a potential therapeutic target in immune response.

Background: tryptase, the most abundant protease of the human mast cell, has been implicated as a key mediator of allergic inflammation that acts through activation of PAR2.Objectives: to investigate the contribution of PAR2 in the pro-inflammatory actions mediated by tryptase in a mice model.Methods: we have injected recombinant human βII-tryptase into the peritoneum of PAR2-deficient and wild-type C57BL/6 mice. After 6, 12 and 24 hours mice were euthanized, peritoneal lavage performed and inflammatory changes investigated.Results: tryptase stimulated an increase in neutrophil numbers in the peritoneum, but responses did not differ between PAR2-deficient and wild-type mice. Heat-inactivation of tryptase or pre-incubation with a selective tryptase inhibitor reduced neutrophilia, but neutrophil accumulation was not elicited with a peptide agonist of PAR2 (SLIGRL-NH2 ). Zymography indicated that tryptase stimulated the release of matrix metalloproteinases (MMP) 2 and 9 in the peritoneum of both mouse strains. Studies involving immunomagnetic isolation of neutrophils suggested that neutrophils represent the major cellular source of tryptase-induced MMP2 and MMP9. At 24 h after tryptase injection there was increased microvascular leakage as indicated by high levels of albumin in peritoneal lavage fluid, and this appeared to be partially abolished by heat-inactivating tryptase or addition of a protease inhibitor. There was no corresponding increase in levels of histamine or total protein. The extent of tryptase-induced microvascular leakage or gelatinase release into the peritoneum did not differ between PAR2-deficient and wild-type mice.Conclusions: our findings indicate that tryptase is a potent stimulus for neutrophil accumulation, MMP release and microvascular leakage. Though these actions required an intact catalytic site, the primary mechanism of tryptase in vivo would appear to involve processes independent of PAR2. This article is protected by copyright. All rights reserved.

Activated hepatic stellate cells (aHSCs) are now established as a central driver of fibrosis in human liver injury. In the presence of chronic or repeated injury, fibrosis, cirrhosis, and hepatocellular carcinoma (HCC) can occur, so there is interest in down-regulating aHSCs activity in order to treat these diseases. Here, we report that Vγ9Vδ2 T cells are reduced in patients with liver cirrhosis, stimulating us to investigate possible interactions between Vγ9Vδ2 T cells and aHSCs. We find that Vγ9Vδ2 T cells kill aHSCs and killing is enhanced when aHSCs are pretreated with BPH-1236, a lipophilic analog of the bone resorption drug zoledronate. Cytotoxicity is mediated by direct cell-to-cell contact as shown by Transwell experiments and atomic force microscopy, with BPH-1236 increasing the adhesion between aHSCs and Vγ9Vδ2 T cells. Mechanistically, BPH-1236 functions by inhibiting farnesyl diphosphate synthase, leading to accumulation of the phosphoantigen isopentenyl diphosphate and recognition by Vγ9Vδ2 T cells. The cytolytic process is largely dependent on the perforin/granzyme B pathway. In a Rag2−/−γc−/− immune-deficient mouse model, we find that Vγ9Vδ2 T cells home-in to the liver, and when accompanied by BPH-1236, kill not only orthotopic aHSCs but also orthotopic HCC tumors. Collectively, our results provide the first proof-of-concept of a novel immunotherapeutic strategy for the treatment of fibrosis–cirrhosis–HCC diseases using adoptively transferred Vγ9Vδ2 T cells, combined with a lipophilic bisphosphonate.

Background Recent studies found that gastric cancer patients with Helicobacter pylori infection had a better response to chemotherapy and had an improved overall prognosis compared with those without. However, the underlying mechanism remains unknown. Methods Quantitative real-time PCR (qRT-PCR) was utilized to determine the expression profile of miR-141 in H. pylori infected cells and tissues and their respective controls. qRT-PCR and Western blot were used to determine the expression level of KEAP-1. Luciferase reporter assays were used to determine whether KEAP-1 was a direct target of miR-141 in the gastric cancer cells. MTT and apoptosis assay were performed to detect the survival of cells under cisplatin treatment. Result We found that H. pylori infection can significantly down-regulate miR-141 expression. Knockdown miR-141 expression in 7901/DDP and 7901 cells could significantly improve cisplatin sensitivity. Over-expression of miR-141 resulted in enhanced resistance to cisplatin in both gastric cancer cells. We also demonstrated that miR-141 directly targets KEAP1 by luciferase reporter assay, and that down-regulation of KEAP1 induces cisplatin resistance. Conversely, over-expression of KEAP1 significantly enhanced cisplatin sensitivity. Our 75 pairs of tissues also showed that KEAP1 was significantly up-regulated in H. pylori-positive tissues. Conclusion Altogether, these findings demonstrated that the H. pylori infection could modulate cisplatin sensitivity through miR-141-mediated regulation of KEAP1.

Backgroud Dipeptidyl peptidase I (DPPI), a lysosomal cysteine protease is derived from granule immune cells including mast cell, neutrophils, and toxicity T cells. DPPI can activate serine proteases by removal of dipeptides from N-termini of the pro-proteases, resulting in granule immune cells activation which involved in physiological or pathological responses. Triperygium Wilfordii Polyglucoside (TWP) is one of the traditional Chinese medicines, and commonly used in rheumatoid arthritis (RA) treatment. The present study intended to evaluate the effects of TWP on DPPI activity. Methods In vivo and in vitro studies were carried out to investigate the functions of TWP or triptolide (TP) on DPPI activities in serum, tissues of CIA rats. Rats were divided into five groups randomly: normal group, untreated CIA rat group, TWP treatment CIA groups (the low dose 2.5 mg/100 g body-weight and high dose 5 mg/100 g body-weight), and TP treatment CIA group (4 μg/100 g body-weight). Arthritis development was monitored visually, and joint pathology was examined radiologically. Total protein concentrations in synovial fluids (SFs) were determined by BCA method. Serums and tissue homogenates from CIA rats were collected and DPPI activities were detected using fluorescence substrate GF-AFC. The in vitro interactions between DPPI in serums or in tissue homogenates and TWP or TP were assessed. Results TWP-treated CIA rats showed a significant improvement in bone erosion. TWP significantly suppressed paw swelling and total protein concentration in the SFs of CIA rats compared with untreated CIA rats. The elevated activities of DPPI in serums or tissues of CIA rats were significantly inhibited by TWP, but not by TP in vivo . The inhibitory effects of TWP on DPPI activities were also confirm by in vitro study. Conclusion One of the therapeutic functions of TWP in RA treatment could be inhibiting DPPI activity in serums and synovial tissue produced during RA development, and then reducing inflammatory serine proteases activities and further recovering CIA rats from RA symptoms.

Summary Introduction Helicobacter pylori (H. pylori) is regarded as a major pathogen in gastrointestinal diseases and the infection rate is still at a high level in children. Therefore, the diagnosis is of great clinical importance. The objective was to perform a meta-analysis on H. pylori stool antigen test (SAT) for the diagnosis of the infection in children. Methods Published information on the sensitivity and specificity of stool antigen test in each study was collected to assess the accuracy of the test for the diagnosis of H. pylori infection. Forty-five eligible studies were selected for analysis. Data on the publication year, H. pylori prevalence, eradication rate and gold standard of each study were summarized. Result Forty-five studies, including 5931 patients, evaluated the accurancy of H. pylori SAT. Pooled sensitivity, specificity, LR+ and LR– were: 92.1%, 94.1%, 17.01, 0.085, respectively. Subgroup analyses were conducted to evaluate the sensitivity and specificity of H. pylori SAT in different situations and found that sensitivity and specificity were significantly higher when monoclonal H. pylori SAT and two or more reference methods were used. Conclusions Detection of H. pylori antigen in stools with ELISA monoclonal antibodies is a non-invasive efficient test for the diagnosis of infection in children. However, the available one-step and polyclonal SAT tests are still unreliable.

Background: The association between Helicobacter pylori infection and gastric cancer has been identified recently. However, the molecular mechanism remained largely unknown. Methods and Results: We found that miR-141 was decreased in Helicobacter pylori positive specimens (n=75) compared with negative tissues (n=75). The knockdown of miR-141 enhanced the invasion ability of gastric cancer cells; meanwhile, over-expression of miR-141 could inhibit the abilities of gastric cancer cells in vitro. A luciferase assay revealed that miR-141 was directly bound to the 3'-untranslated regions (3'-UTR) of STAT4. STAT4 was found up-regulated at mRNA and protein levels, as shown by qRT-PCR and western blot. Over-expression of STAT4 was used to mimic miR-141 action in the invasion of gastric cancer. Conclusion: MiR-141 may play a pivotal role in controlling gastric cancer invasion through regulating STAT4 and maybe a potential target to treat gastric cancer.

Mesenchymal stem cells (MSCs), as well as osteoblastic cells derived from these MSCs, have been shown to be key components of the hematopoietic stem cell (HSC) niche. In this study, we wished to examine whether other cell types that are known to differentiate from MSCs similarly regulate the stem cell niche, namely cells of the adipocyte lineage. Recent studies have examined the role that adipocytes play in the biology of the HSCs in different bone locations and in transplantation settings; however, none have examined their role under homeostatic conditions. We compared the ability of adipocytic and nonadipocytic cell lines to support primitive hematopoietic cells in vitro. Preadipocytic cell lines demonstrated enhanced support of hematopoietic cells. Similarly, primary bone marrow (BM) cells treated with troglitazone, a drug that enhances adipogenesis, also demonstrated augmented support over control-treated stromal cells. We further examined the effects of increased adipocyte number in vivo under homeostatic conditions using troglitazone treatment and found that these alterations had no effect on HSC frequency. Taken together, we demonstrate that cells of the adipocyte lineage promote the ability of stromal cells to support primitive hematopoietic cells in vitro, yet alterations of adipocyte number and volume in vivo have no effect. These data suggest that adipocytes are not a component of the adult BM HSC niche under homeostatic conditions.

Asthma constitutes a spectrum of conditions that affect a substantial proportion of the population in developed countries. A large segment of asthmatics are characterized by an immunologic type 2 allergic profile. A preponderance of conventional and biologically-based therapies are available to treat these subjects. However, a small minority of individuals clinically classified as severe asthmatics (SA), are highly refractory to treatment and account for a significantly disproportionate share of the clinical and economic impact of the asthma condition as a whole. In order to better define the immune response in SA with the ultimate goal of developing novel therapeutic modalities, we analyzed bronchoalveolar lavage (BAL) cells from a cohort of SA, mild to moderate asthmatics (M/M), and healthy controls by mass cytometry (CyTOF). Unbiased bioinformatic assessment revealed 34 clusters of BAL cells with assignment of subjects to one of four groupings by principal component analysis (PCA). The majority of SA (78.9%) fell into two of these groups and constituted 65.2% of their makeup, with M/M representing the remaining 34.8%. Similarly, analysis of airway bronchial epithelial cells (BECs) by RNA sequencing (RNASeq) revealed three subject groups with SA falling largely into one of these. Synthesis of CyTOF and RNASeq data in the context of relevant clinical parameters resulted in a combined profile for each subject, which was able to distinguish those with the most severe disease. Our approach demonstrates that an unbiased, multifactorial assessment of immune and epithelial profiles can predict asthma severity, but may also reveal mechanism-based clues for targeted, personalized therapeutic intervention in severe asthmatics.

Summary Human Vγ9Vδ2 T cells respond to microbial infections and malignancy by sensing diphosphate-containing metabolites called phosphoantigens, which bind to the intracellular domain of butyrophilin 3A1, triggering extracellular interactions with the Vγ9Vδ2 T cell receptor (TCR). Here, we examined the molecular basis of this “inside-out” triggering mechanism. Crystal structures of intracellular butyrophilin 3A proteins alone or in complex with the potent microbial phosphoantigen HMBPP or a synthetic analog revealed key features of phosphoantigens and butyrophilins required for γδ T cell activation. Analyses with chemical probes and molecular dynamic simulations demonstrated that dimerized intracellular proteins cooperate in sensing HMBPP to enhance the efficiency of γδ T cell activation. HMBPP binding to butyrophilin doubled the binding force between a γδ T cell and a target cell during “outside” signaling, as measured by single-cell force microscopy. Our findings provide insight into the “inside-out” triggering of Vγ9Vδ2 T cell activation by phosphoantigen-bound butyrophilin, facilitating immunotherapeutic drug design.