技术资料

OBJECTIVE Tissue-resident memory T cells (TRM) are vital immune sentinels that provide protective immunity. While hepatic CD8+ TRM have been well described,little is known about the location,phenotype and function of CD4+ TRM. DESIGN We used multiparametric flow cytometry,histological assessment and novel human tissue coculture systems to interrogate the ex vivo phenotype,function and generation of the intrahepatic CD4+ T-cell compartment. We also used leukocytes isolated from human leukocyte antigen (HLA)-disparate liver allografts to assess long-term retention. RESULTS Hepatic CD4+ T cells were delineated into three distinct populations based on CD69 expression: CD69-,CD69INT and CD69HI. CD69HICD4+ cells were identified as tissue-resident CD4+ T cells on the basis of their exclusion from the circulation,phenotypical profile (CXCR6+CD49a+S1PR1-PD-1+) and long-term persistence within the pool of donor-derived leukcoocytes in HLA-disparate liver allografts. CD69HICD4+ T cells produced robust type 1 polyfunctional cytokine responses on stimulation. Conversely,CD69INTCD4+ T cells represented a more heterogenous population containing cells with a more activated phenotype,a distinct chemokine receptor profile (CX3CR1+CXCR3+CXCR1+) and a bias towards interleukin-4 production. While CD69INTCD4+ T cells could be found in the circulation and lymph nodes,these cells also formed part of the long-term resident pool,persisting in HLA-mismatched allografts. Notably,frequencies of CD69INTCD4+ T cells correlated with necroinflammatory scores in chronic hepatitis B infection. Finally,we demonstrated that interaction with hepatic epithelia was sufficient to generate CD69INTCD4+ T cells,while additional signals from the liver microenvironment were required to generate liver-resident CD69HICD4+ T cells. CONCLUSIONS High and intermediate CD69 expressions mark human hepatic CD4+ TRM and a novel functionally distinct recirculating population,respectively,both shaped by the liver microenvironment to achieve diverse immunosurveillance. View Publication

We hypothesized that combining adoptively transferred autologous T cells with a cancer vaccine strategy would enhance therapeutic efficacy by adding antimyeloma idiotype (Id)-keyhole limpet hemocyanin (KLH) vaccine to vaccine-specific costimulated T cells. In this randomized phase 2 trial,patients received either control (KLH only) or Id-KLH vaccine,autologous transplantation,vaccine-specific costimulated T cells expanded ex vivo,and 2 booster doses of assigned vaccine. In 36 patients (KLH,n = 20; Id-KLH,n = 16),no dose-limiting toxicity was seen. At last evaluation,6 (30%) and 8 patients (50%) had achieved complete remission in KLH-only and Id-KLH arms,respectively (P = .22),and no difference in 3-year progression-free survival was observed (59% and 56%,respectively; P = .32). In a 594 Nanostring nCounter gene panel analyzed for immune reconstitution (IR),compared with patients receiving KLH only,there was a greater change in IR genes in T cells in those receiving Id-KLH relative to baseline. Specifically,upregulation of genes associated with activation,effector function induction,and memory CD8+ T-cell generation after Id-KLH but not after KLH control vaccination was observed. Similarly,in responding patients across both arms,upregulation of genes associated with T-cell activation was seen. At baseline,all patients had greater expression of CD8+ T-cell exhaustion markers. These changes were associated with functional Id-specific immune responses in a subset of patients receiving Id-KLH. In conclusion,in this combination immunotherapy approach,we observed significantly more robust IR in CD4+ and CD8+ T cells in the Id-KLH arm,supporting further investigation of vaccine and adoptive immunotherapy strategies. This trial was registered at www.clinicaltrials.gov as #NCT01426828. View Publication

Persistent inflammation is a major contributor to healing impairment in diabetic chronic wounds. Paradoxically,diabetic wound environment during the acute phase of healing is completely different because it exhibits a reduced macrophage response owing to inadequate expression of CCL2 proinflammatory cytokine. What causes a reduction in CCL2 expression in diabetic wounds early after injury remains unknown. In this study,we report that in contrast to prolonged exposure to high glucose,which makes monocytes proinflammatory,short-term exposure to high glucose causes a rapid monocyte reprogramming,manifested by increased expression and secretion of IL-10,which in an autocrine/paracrine fashion reduces glucose uptake and transforms monocytes into an anti-inflammatory phenotype by dampening signaling through toll-like receptors. We show that IL-10 expression is significantly increased in diabetic wounds during the acute phase of healing,causing significant reductions in toll-like receptor signaling and proinflammatory cytokine production,delaying macrophage and leukocyte responses,and underlying healing impairment in diabetic wounds. Importantly,blocking IL-10 signaling during the acute phase of healing improves toll-like receptor signaling,increases proinflammatory cytokine production,enhances macrophage and leukocyte responses,and stimulates healing in diabetic wounds. We posit that anti-IL-10 strategies have therapeutic potential if added topically after surgical debridement,which resets chronic wounds into acute fresh wounds. View Publication

Traumatic injuries,such as burn,are often complicated by ethanol intoxication at the time of injury. This leads to a myriad of complications and post-burn pathologies exacerbated by aberrant immune responses. Recent findings suggest that immune cell dysfunction in the gastrointestinal system is particularly important in deleterious outcomes associated with burn injuries. In particular,intoxication at the time of burn injury leads to compromised intestinal T cell responses,which can diminish intestinal immunity and promote bacterial translocation,allowing for increased secondary infections in the injured host and associated sequelae,such as multiple organ failure and sepsis. Regulatory T cells (Treg) have been identified as important mediators of suppressing effector T cell function. Therefore,the goal of this study was to assess the effects of ethanol intoxication and burn injury on Treg populations in small intestinal immune organs. We also evaluated the suppressive capability of Tregs isolated from injured animals. Male C57BL/6 mice were gavaged with 2.9?Šg/kg ethanol before receiving a ˆ¼12.5% total body surface area scald burn. One day after injury,we identified a significant increase in Tregs number in small intestine Peyer's patches (ˆ¼?—1.5) and lamina propria (ˆ¼?—2). Tregs-producing cytokine IL-10 were also increased in both tissues. Finally,Tregs isolated from ethanol and burn-injured mice were able to suppress proliferation of effector T cells to a greater degree than sham vehicle Tregs. This was accompanied by increased levels of IL-10 and decreased levels of pro-proliferative cytokine IL-2 in cultures containing ethanol + burn Tregs compared with sham Tregs. These findings suggest that Treg populations are increased in intestinal tissues 1 day following ethanol intoxication and burn injury. Tregs isolated from ethanol and burn-injured animals also exhibit a greater suppression of effector T cell proliferation,which may contribute to altered T cell responses following injury. View Publication

Resting CD4+ T cells are primary targets of early HIV infection events in vivo,but do not readily support HIV replication in vitro. This barrier to infection can be overcome by exposing resting CD4+ T cells to endothelial cells (ECs). ECs line blood vessels and direct T cell trafficking into inflamed tissues. Cell trafficking pathways have been shown to have overlapping roles in facilitating HIV replication,but their relevance to EC-mediated enhancement of HIV susceptibility in resting CD4+ T cells has not previously been examined. We characterized the phenotype of primary human resting CD4+ T cells that became productively infected with HIV when cocultured with primary human blood and lymphatic ECs. The infected CD4+ T cells were primarily central memory cells enriched for high expression of the integrins LFA-1 and VLA-4. ICAM-1 and VCAM-1,the cognate ligands for LFA-1 and VLA-4,respectively,were expressed by the ECs in the coculture. Blocking LFA-1 and VLA-4 on resting CD4+ T cells inhibited infection by 65.4%-96.9%,indicating that engagement of these integrins facilitates EC-mediated enhancement of productive HIV infection in resting CD4+ T cells. The demonstration that ECs influence cellular HIV susceptibility of resting memory CD4+ T cells through cell trafficking pathways engaged during the transmigration of T cells into tissues highlights the physiological relevance of these findings for HIV acquisition and opportunities for intervention. View Publication

The clinical evolution of patients with chronic Chagas disease (CCD) is mainly associated with an excessive inflammation and a defective immunomodulatory profile caused by the interaction between T. cruzi and the host. Regulatory B (Breg) cells exert immune suppression mostly through IL-10 production (B10 cells),but also through IL-10-independent mechanisms. Previously,we demonstrated that CCD patients with cardiomyopathy show changes in the ex vivo Breg cell phenotypic distribution although maintain IL-10 production capacity. Here,we sought to identify potential alterations on Breg cells upon in vitro stimulation. Isolated B cells from CCD patients with or without cardiomyopathy and non-infected (NI) donors were stimulated with T. cruzi lysate or CpG + CD40L,and characterized by flow cytometry based on the expression of CD24,CD27,CD38,and the regulatory molecules IL-10 and PD-L1. IL-10 and IL-17 secretion in the supernatant of B cells was evaluated by ELISA. Data showed that T. cruzi stimulation diminished the expression of CD24 and CD38 on CD27- B cells while reducing the percentage of CD24high inside CD27+ B cells. Furthermore,T. cruzi induced a regulatory B cell phenotype by increasing B10 cells and IL-10 secretion in all the groups. The innate-like B10 cells expansion observed in patients with cardiomyopathy would be associated with CD27- B10 cell subsets,while no predominant phenotype was found in the other groups. Patients with cardiomyopathy also displayed higher IL-17 secretion levels in T. cruzi-activated B cells. CpG + CD40L stimulation revealed that B cells from CCD patients and NI donors had the same ability to differentiate into B10 cells and secrete IL-10 in vitro. Additionally,CCD patients showed an increased frequency of CD24-CD27- B cells and a reduction in the percentage of CD24highCD27+ Breg cells,which appeared to be inversely correlated with the presence of T. cruzi DNA in blood. Finally,CCD patients exhibited a higher frequency of PD-L1+ B cells in T. cruzi-stimulated samples,suggesting that IL-10-independent mechanisms could also be tangled in the control of inflammation. Altogether,our results provide evidence about the potential role of Breg cells in the immune response developed against T. cruzi and its contribution to chronic Chagas cardiomyopathy. View Publication

RNA-targeting CRISPR-Cas13 proteins have recently emerged as a powerful platform to modulate gene expression outcomes. However,protein and CRISPR RNA (crRNA) delivery in human cells can be challenging with rapid crRNA degradation yielding transient knockdown. Here we compare several chemical RNA modifications at different positions to identify synthetic crRNAs that improve RNA targeting efficiency and half-life in human cells. We show that co-delivery of modified crRNAs and recombinant Cas13 enzyme in ribonucleoprotein (RNP) complexes can alter gene expression in primary CD4+ and CD8+ T cells. This system represents a robust and efficient method to modulate transcripts without genetic manipulation. View Publication

Neutrophils (PMNs) contain and release a powerful arsenal of mediators,including several granular enzymes,reactive oxygen species (ROS) and neutrophil extracellular traps (NETs). Although airway neutrophilia is associated with severity,poor response to glucocorticoids and exacerbations,the pathophysiological role of neutrophils in asthma remains poorly understood. Twenty-four patients with asthma and 22 healthy controls (HCs) were prospectively recruited. Highly purified peripheral blood neutrophils (> 99%) were evaluated for ROS production and activation status upon stimulation with lipopolysaccharide (LPS),N-formylmethionyl-leucyl-phenylalanine (fMLP) and phorbol 12-myristate 13-acetate (PMA). Plasma levels of myeloperoxidase (MPO),CXCL8,matrix metalloproteinase-9 (MMP-9),granulocyte-monocyte colony-stimulating factor (GM-CSF) and vascular endothelial growth factor (VEGF-A) were measured by ELISA. Plasma concentrations of citrullinated histone H3 (CitH3) and circulating free DNA (dsDNA) were evaluated as NET biomarkers. Activated PMNs from asthmatics displayed reduced ROS production and activation status compared to HCs. Plasma levels of MPO,MMP-9 and CXCL8 were increased in asthmatics compared to HCs. CitH3 and dsDNA plasma levels were increased in asthmatics compared to controls and the CitH3 concentrations were inversely correlated to the % decrease in FEV1/FVC in asthmatics. These findings indicate that neutrophils and their mediators could have an active role in asthma pathophysiology. View Publication

Understanding human hematopoietic stem cell fate control is important for its improved therapeutic manipulation. Asymmetric cell division,the asymmetric inheritance of factors during division instructing future daughter cell fates,was recently described in mouse blood stem cells. In human blood stem cells,the possible existence of asymmetric cell division remained unclear because of technical challenges in its direct observation. Here,we use long-term quantitative single-cell imaging to show that lysosomes and active mitochondria are asymmetrically inherited in human blood stem cells and that their inheritance is a coordinated,nonrandom process. Furthermore,multiple additional organelles,including autophagosomes,mitophagosomes,autolysosomes,and recycling endosomes,show preferential asymmetric cosegregation with lysosomes. Importantly,asymmetric lysosomal inheritance predicts future asymmetric daughter cell-cycle length,differentiation,and stem cell marker expression,whereas asymmetric inheritance of active mitochondria correlates with daughter metabolic activity. Hence,human hematopoietic stem cell fates are regulated by asymmetric cell division,with both mechanistic evolutionary conservation and differences to the mouse system. View Publication

AIMS Whereas intravenous administration of Toll-like receptor 4 ligand lipopolysaccharide (LPS) to human volunteers is frequently used in clinical pharmacology studies,systemic use of LPS has practical limitations. We aimed to characterize the intradermal LPS response in healthy volunteers,and as such qualify the method as local inflammation model for clinical pharmacology studies. METHODS Eighteen healthy male volunteers received 2 or 4 intradermal 5 ng LPS injections and 1 saline injection on the forearms. The LPS response was evaluated by noninvasive (perfusion,skin temperature and erythema) and invasive assessments (cellular and cytokine responses) in skin biopsy and blister exudate. RESULTS LPS elicited a visible response and returned to baseline at 48??hours. Erythema,perfusion and temperature were statistically significant (P  View Publication

Despite the growing knowledge of T cell responses in COVID-19 patients,there is a lack of detailed characterizations for T cell-antigen interactions and T cell functions. Here,with a predicted peptide library from SARS-CoV-2 S and N proteins,we found that specific CD8+ T cell responses were identified in over 75% of COVID-19 convalescent patients (15/20) and an epitope from the N protein,N361-369 (KTFPPTEPK),was the most dominant epitope from our selected peptide library. Importantly,we discovered 2 N361-369-specific T cell receptors (TCRs) with high functional avidity that were independent of the CD8 co-receptor. These TCRs exhibited complementary cross-reactivity to several presently reported N361-369 mutant variants,as to the wild-type epitope. Further,the natural functions of these TCRs in the cytotoxic immunity against SARS-CoV-2 were determined with dendritic cells (DCs) and the lung organoid model. We found that the N361-369 epitope could be normally processed and endogenously presented by these different types of antigen presenting cells,to elicit successful activation and effective cytotoxicity of CD8+ T cells ex vivo. Our study evidenced potential mechanisms of cellular immunity to SARS-CoV-2,and illuminated potential ways of viral clearance in COVID-19 patients. These results indicate that utilizing CD8-independent TCRs against SARS-CoV-2-associated antigens may provide functional superiority that is beneficial for the adoptive cell immunotherapies based on natural or genetically engineered T cells. Additionally,this information is highly relevant for the development of the next-generation vaccines with protections against continuously emerged SARS-CoV-2 mutant strains. View Publication

Acute coronary syndrome (ACS) is the most severe clinical manifestation of coronary heart disease.We performed an epigenome-wide analysis of circulating CD4+ and CD8+ T cells isolated from ACS patients and healthy subjects (HS),enrolled in the DIANA clinical trial,by reduced-representation bisulphite sequencing (RRBS). In CD4+ T cells,we identified 61 differentially methylated regions (DMRs) associated with 57 annotated genes (53% hyper- and 47% hypo-methylated) by comparing ACS patients vs HS. In CD8+ T cells,we identified 613 DMRs associated with 569 annotated genes (28% hyper- and 72% hypo-methylated) in ACS patients as compared to HS. In CD4+vs CD8+ T cells of ACS patients we identified 175 statistically significant DMRs associated with 157 annotated genes (41% hyper- and 59% hypo-methylated). From pathway analyses,we selected six differentially methylated hub genes (NFATC1,TCF7,PDGFA,PRKCB,PRKCZ,ABCA1) and assessed their expression levels by q-RT-PCR. We found an up-regulation of selected genes in ACS patients vs HS (P < 0.001). ABCA1,TCF7,PDGFA,and PRKCZ gene expression was positively associated with CK-MB serum concentrations (r = 0.75,P = 0.03; r = 0.760,P = 0.029; r = 0.72,P = 0.044; r = 0.74,P = 0.035,respectively).This pilot study is the first single-base resolution map of DNA methylome by RRBS in CD4+ and CD8+ T cells and provides specific methylation signatures to clarify the role of aberrant methylation in ACS pathogenesis,thus supporting future research for novel epigenetic-sensitive biomarkers in the prevention and early diagnosis of this pathology. View Publication