Stem-like T cells are associated with the pathogenesis of ulcerative colitis in humans.

To understand the role of T cells in the pathogenesis of ulcerative colitis (UC), we analyzed colonic T cells isolated from patients with UC and controls. Here we identified colonic CD4+ and CD8+ T lymphocyte subsets with gene expression profiles resembling stem-like progenitors, previously reported in several mouse models of autoimmune disease. Stem-like T cells were increased in inflamed areas compared to non-inflamed regions from the same patients. Furthermore, TCR sequence analysis indicated stem-like T cells were clonally related to proinflammatory T cells, suggesting their involvement in sustaining effectors that drive inflammation. Using an adoptive transfer colitis model in mice, we demonstrated that CD4+ T cells deficient in either BCL-6 or TCF1, transcription factors that promote T cell stemness, had decreased colon T cells and diminished pathogenicity. Our results establish a strong association between stem-like T cell populations and UC pathogenesis, highlighting the potential of targeting this population to improve clinical outcomes.

MicroRNAs-A Promising Tool for Asthma Diagnosis and Severity Assessment: A Systematic Review.

Micro RNAs (miRNAs) are short, non-coding RNAs (Ribonucleic acids) with regulatory functions that could prove useful as biomarkers for asthma diagnosis and asthma severity-risk stratification. The objective of this systematic review is to identify panels of miRNAs that can be used to support asthma diagnosis and severity-risk assessment. Three databases (Medline, Embase, and SCOPUS) were searched up to 15 September 2020 to identify studies reporting differential expression of specific miRNAs in the tissues of adults and children with asthma. Studies reporting miRNAs associations in animal models that were also studied in humans were included in this review. We identified 75 studies that met our search criteria. Of these, 66 studies reported more than 200 miRNAs that are differentially expressed in asthma patients when compared to non-asthmatic controls. In addition, 16 studies reported 17 miRNAs that are differentially expressed with differences in asthma severity. We were able to construct two panels of miRNAs that are expressed in blood and can serve as core panels to further investigate the practicality and efficiency of using miRNAs as non-invasive biomarkers for asthma diagnosis and severity-risk assessment, respectively.

Targeting the tumor mutanome for personalized vaccination in a TMB low non-small cell lung cancer.

BACKGROUND: Cancer is characterized by an accumulation of somatic mutations, of which a significant subset can generate cancer-specific neoepitopes that are recognized by autologous T cells. Such neoepitopes are emerging as important targets for cancer immunotherapy, including personalized cancer vaccination strategies. METHODS: We used whole-exome and RNA sequencing analysis to identify potential neoantigens for a patient with non-small cell lung cancer. Thereafter, we assessed the autologous T-cell reactivity to the candidate neoantigens using a long peptide approach in a cultured interferon gamma ELISpot and tracked the neoantigen-specific T-cells in the tumor by T-cell receptor (TCR) sequencing. In parallel, identified gene variants were incorporated into a Modified Vaccinia Ankara-based vaccine, which was evaluated in the human leucocyte antigen A*0201 transgenic mouse model (HHD). RESULTS: Sequencing revealed a tumor with a low mutational burden: 2219 sequence variants were identified from the primary tumor, of which 23 were expressed in the transcriptome, involving 18 gene products. We could demonstrate spontaneous T-cell responses to 5/18 (28%) mutated gene variants, and further analysis of the TCR repertoire of neoantigen-specific CD4+ and CD8+ T cells revealed TCR clonotypes that were expanded in both blood and tumor tissue. Following vaccination of HHD mice, de novo T-cell responses were generated to 4/18 (22%) mutated gene variants; T cells reactive against two variants were also evident in the autologous setting. Subsequently, we determined the major histocompatibility complex restriction of the T-cell responses and used in silico prediction tools to determine the likely neoepitopes. CONCLUSIONS: Our study demonstrates the feasibility of efficiently identifying tumor-specific neoantigens that can be targeted by vaccination in tumors with a low mutational burden, promising successful clinical exploitation, with trials currently underway.

Intratumoral follicular regulatory T cells curtail anti-PD-1 treatment efficacy.

Immune-checkpoint blockade (ICB) has shown remarkable clinical success in boosting antitumor immunity. However, the breadth of its cellular targets and specific mode of action remain elusive. We find that tumor-infiltrating follicular regulatory T (TFR) cells are prevalent in tumor tissues of several cancer types. They are primarily located within tertiary lymphoid structures and exhibit superior suppressive capacity and in vivo persistence as compared with regulatory T cells, with which they share a clonal and developmental relationship. In syngeneic tumor models, anti-PD-1 treatment increases the number of tumor-infiltrating TFR cells. Both TFR cell deficiency and the depletion of TFR cells with anti-CTLA-4 before anti-PD-1 treatment improve tumor control in mice. Notably, in a cohort of 271 patients with melanoma, treatment with anti-CTLA-4 followed by anti-PD-1 at progression was associated with better a survival outcome than monotherapy with anti-PD-1 or anti-CTLA-4, anti-PD-1 followed by anti-CTLA-4 at progression or concomitant combination therapy.

Circulating miRNAs-A potential tool to identify severe asthma risk?

BACKGROUND: Identifying patients at risk of severe asthma is vitally important given the disproportionate burden of disease imposed by that state. However, biomarkers to support such needs remain elusive. METHODS: In this letter, we assessed whether specific panels of circulating miRNAs (microRNAs) can differentiate between mild and severe asthma patients as well as between healthy subjects and severe asthma patients. RESULTS: To our knowledge, the miRNAs identified in our work such as miR-28-3p, miR-16-2-3p, and miR-210-3p have not been previously reported as differentially expressed in the serum of severe asthma patients. CONCLUSION: Our findings suggest that miRNA expression profiles may have the capability as potential biomarkers that signal the risk of having severe asthma. As such, these findings have significant novelty and merit wider dissemination to facilitate further work in this field.

Human Intestinal Macrophages Are Involved in the Pathology of Both Ulcerative Colitis and Crohn Disease.

BACKGROUND: Intestinal macrophages are key immune cells in the maintenance of intestinal immune homeostasis and have a role in the pathogenesis of inflammatory bowel disease (IBD). However, the mechanisms by which macrophages exert a pathological influence in both ulcerative colitis (UC) and Crohn disease (CD) are not yet well understood. METHODS: We purified intestinal macrophages from gastrointestinal mucosal biopsies (patients with UC, patients with CD, and healthy donors) and analyzed their transcriptome by RNA sequencing and bioinformatics, confirming results with quantitative polymerase chain reaction and immunohistochemistry. RESULTS: Compared with those of healthy donors, intestinal macrophages in patients with UC and with CD showed cellular reprograming of 1287 and 840 dysregulated genes, respectively (false discovery rate ≤ 0.1). The UC and CD intestinal macrophages showed an activated M1 inflammatory phenotype and the downregulation of genes engaged in drug/xenobiotic metabolism. Only macrophages from CD showed, concomitant to an M1 phenotype, a significant enrichment in the expression of M2 and fibrotic and granuloma-related genes. For the first time, we showed (and validated by quantitative polymerase chain reaction and immunohistochemistry) that intestinal macrophages in patients with IBD present both M1 and M2 features, as recently described for tumor-associated macrophages, that affect key pathways for IBD pathology, represented by key markers such as MMP12 (fibrosis), CXCL9 (T-cell attraction), and CD40 (T-cell activation). CONCLUSIONS: Our data support the therapeutic targeting of macrophages to maintain remission in IBD but also indicate that a shift toward an M2 program-as proposed by some reports-may not limit the recruitment and activation of T cells because M2 features do not preclude M1 activation in patients with UC or CD and could exacerbate M2-related CD-specific features such as fibrosis and the formation of granulomas.

M1hot tumor-associated macrophages boost tissue-resident memory T cells infiltration and survival in human lung cancer.

BACKGROUND: The role of tumor-associated macrophages (TAMs) in determining the outcome between the antitumor effects of the adaptive immune system and the tumor's anti-immunity stratagems, is controversial. Macrophages modulate their activities and phenotypes by integration of signals in the tumor microenvironment. Depending on how macrophages are activated, they may adopt so-called M1-like, antitumor or M2-like, protumor profiles. In many solid tumors, a dominance of M2-like macrophages is associated with poor outcomes but in some tumor types, strong M1-like profiles are linked to better outcomes. We aimed to investigate the interrelationship of these TAM populations to establish how they modulate the efficacy of the adaptive immune system in early lung cancer. METHODS: Macrophages from matched lung (non-tumor-associated macrophages (NTAMs)) and tumor samples (TAMs) from resected lung cancers were assessed by bulk and single-cell transcriptomic analysis. Protein expression of genes characteristic of M1-like (chemokine (C-X-C motif) ligand 9) or M2-like (matrix metallopeptidase 12) functions was confirmed by confocal microscopy. Immunohistochemistry related the distribution of TAM transcriptomic signatures to density of CD8+ tissue-resident memory T cells (TRM) in tumors and survival data from an independent cohort of 393 patients with lung cancer. RESULTS: TAMs have significantly different transcriptomic profiles from NTAMs with >1000 differentially expressed genes. TAMs displayed a strong M2-like signature with no significant variation between patients. However, single-cell RNA-sequencing supported by immuno-stained cells revealed that additionally, in 25% of patients the M2-like TAMs also co-expressed a strong/hot M1-like signature (M1hot). Importantly, there was a strong association between the density of M1hot TAMs and TRM cells in tumors that was in turn linked to better survival. Our data suggest a mechanism by which M1hot TAMs may recruit TRM cells via CXCL9 expression and sustain them by making available more of the essential fatty acids on which TRM depend. CONCLUSIONS: We showed that in early lung cancer, expression of M1-like and M2-like gene signatures are not mutually exclusive since the same TAMs can simultaneously display both gene-expression profiles. The presence of M1hot TAMs was associated with a strong TRM tumor-infiltrate and better outcomes. Thus, therapeutic approaches to re-program TAMs to an M1hot phenotype are likely to augment the adaptive antitumor responses.

Back to Normal: An Old Physics Route to Reduce SARS-CoV-2 Transmission in Indoor Spaces.

We advocate the widespread use of UV-C light as a short-term, easily deployable, and affordable way to limit virus spread in the current SARS-CoV-2 pandemic. Radical social distancing with the associated shutdown of schools, restaurants, sport clubs, workplaces, and traveling has been shown to be effective in reducing virus spread, but its economic and social costs are unsustainable in the medium term. Simple measures like frequent handwashing, facial masks, and other physical barriers are being commonly adopted to prevent virus transmission. However, their efficacy may be limited, particularly in shared indoor spaces, where, in addition to airborne transmission, elements with small surface areas such as elevator buttons, door handles, and handrails are frequently used and can also mediate transmission. We argue that additional measures are necessary to reduce virus transmission when people resume attending schools and jobs that require proximity or some degree of physical contact. Among the available alternatives, UV-C light satisfies the requirements of rapid, widespread, and economically viable deployment. Its implementation is only limited by current production capacities, an increase of which requires swift intervention by industry and authorities.

MicroRNA23a Overexpression in Crohn's Disease Targets Tumour Necrosis Factor Alpha Inhibitor Protein 3, Increasing Sensitivity to TNF and Modifying the Epithelial Barrier.

BACKGROUND AND AIMS: Mucosal healing is important in Crohn's disease therapies. Epithelial homeostasis becomes dysregulated in Crohn's, with increased permeability, inflammation, and diarrhoea. MicroRNAs are small non-coding RNAs that regulate gene expression and show changes in inflammatory bowel disease. Tumour necrosis factor alpha [TNFα] inhibitor protein 3 is raised in Crohn's and regulates TNFα-mediated activation of NFκB. We investigated TNFα regulation by microRNA in Crohn's disease [CD], and studied effects on epithelial permeability and inflammation. METHODS: Colonic epithelium from CD and healthy donor biopsies was isolated using laser capture microdissection, and microRNA was quantified. Tumour necrosis factor alpha inhibitor protein 3 was characterised immunohistochemically on serial sections. Expression effect of microRNA was confirmed with luciferase reporter assays. Functional barrier permeability studies and innate cytokine release were investigated with cell and explant culture studies. RESULTS: MicroRNA23a levels significantly increased in colonic Crohn's epithelium compared with healthy epithelium. Luciferase reporter assays in transfected epithelial cells confirmed that microRNA23a repressed expression via the 3' untranslated region of tumour necrosis factor alpha inhibitor protein 3 mRNA, coinciding with increased NFκB-mediated transcription. Immunohistochemical staining of TNFAIP3 protein in colonic biopsies was reduced or absent in adjacent Crohn's sections, correlating inversely with microRNA23a levels and encompassing some intercohort variation. Overexpression of microRNA23a increased epithelial barrier permeability in a colonic epithelial model and increased inflammatory cytokine release in cultured explant biopsies, mimicking Crohn's disease characteristics. CONCLUSIONS: MicroRNA23a overexpression in colonic Crohn's epithelium represses tumour necrosis factor alpha inhibitor protein 3, enhancing sensitivity to TNFα, with increased intestinal permeability and cytokine release.

Small RNA Species and microRNA Profiles are Altered in Severe Asthma Nanovesicles from Broncho Alveolar Lavage and Associate with Impaired Lung Function and Inflammation.

MicroRNAs are known to regulate important pathways in asthma pathology including the IL-6 and IFN pathways. MicroRNAs have been found not only within cells but also within extracellular vesicles such as exosomes. In this study, we particularly focused on microRNA cargo of nanovesicles in bronchoalveolar lavage of severe asthmatic patients. We extracted nanovesicle RNA using a serial filtration method. RNA content was analyzed with small RNA sequencing and mapped to pathways affected using WebGestalt 2017 Software. We report that severe asthma patients have deficient loading of microRNAs into their airway luminal nanovesicles and an altered profile of small RNA nanovesicle content (i.e., ribosomal RNA and broken transcripts, etc.). This decrease in microRNA cargo is predicted to increase the expression of genes by promoting inflammation and remodeling. Consistently, a network of microRNAs was associated with decreased FEV1 and increased eosinophilic and neutrophilic inflammation in severe asthma. MicroRNAs in airway nanovesicles may, thus, be valid biomarkers to define abnormal biological disease processes in severe asthma and monitor the impact of interventional therapies.

Single-cell transcriptomic analysis of tissue-resident memory T cells in human lung cancer.

High numbers of tissue-resident memory T (TRM) cells are associated with better clinical outcomes in cancer patients. However, the molecular characteristics that drive their efficient immune response to tumors are poorly understood. Here, single-cell and bulk transcriptomic analysis of TRM and non-TRM cells present in tumor and normal lung tissue from patients with lung cancer revealed that PD-1-expressing TRM cells in tumors were clonally expanded and enriched for transcripts linked to cell proliferation and cytotoxicity when compared with PD-1-expressing non-TRM cells. This feature was more prominent in the TRM cell subset coexpressing PD-1 and TIM-3, and it was validated by functional assays ex vivo and also reflected in their chromatin accessibility profile. This PD-1+TIM-3+ TRM cell subset was enriched in responders to PD-1 inhibitors and in tumors with a greater magnitude of CTL responses. These data highlight that not all CTLs expressing PD-1 are dysfunctional; on the contrary, TRM cells with PD-1 expression were enriched for features suggestive of superior functionality.

Immunological biomarkers as indicators for outcome after discontinuation of nucleos(t)ide analogue therapy in patients with HBeAg-negative chronic hepatitis B.

The optimal duration of treatment with nucleos(t)ide analogues (NAs) for patients with HBeAg-negative chronic hepatitis B (CHB) is unknown. The aim of this study was to identify an immune signature associated with off-treatment remission to NA therapy. We performed microarray analysis of peripheral blood mononuclear cell (PBMCs) from six patients with chronic hepatitis B who stopped NA therapy (three with off-treatment remission, three with relapse) and five patients with chronic HBV infection (previously termed 'inactive carriers') served as controls. Results were validated using qRT-PCR on a second group of 21 individuals (17 patients who stopped treatment and four controls). PBMCs from 38 patients on long-term NA treatment were analysed for potential to stop treatment. Microarray analysis indicated that patients with off-treatment remission segregated as a distinct out-group. Twenty-one genes were selected for subsequent validation. Ten of these were expressed at significantly lower levels in the patients with off-treatment remission compared to the patients with relapse and predicted remission with AUC of 0.78-0.92. IFNγ, IL-8, FASLG and CCL4 were the most significant by logistic regression. Twelve (31.6%) of 38 patients on long-term NA therapy had expression levels of all these four genes below cut-off values and hence were candidates for stopping treatment. Our data suggest that patients with HBeAg-negative CHB who remain in off-treatment remission 3 years after NA cessation have a distinct immune signature and that PBMC RNA levels of IFNγ, IL-8, FASLG and CCL4 may serve as potential biomarkers for stopping NA therapy.

Expression and localisation of thymosin beta-4 in the developing human early fetal heart.

BACKGROUND: The objective of this study was to investigate the expression and localisation of thymosin ß4 (Tß4) in the developing human heart. Tß4 is a cardioprotective protein which may have therapeutic potential. While Tß4 is an endogenously produced protein with known importance during development, its role within the developing human heart is not fully understood. Elucidating the localisation of Tß4 within the developing heart will help in understanding its role during cardiac development and is crucial for understanding its potential for cardioprotection and repair in the adult heart. METHODS: Expression of Tß4 mRNA in the early fetal human heart was assessed by PCR using both ventricular and atrial tissue. Fluorescence immunohistochemistry was used to assess the localisation of Tß4 in sections of early fetal human heart. Co-staining with CD31, an endothelial cell marker, and with myosin heavy chain, a cardiomyocyte marker, was used to determine whether Tß4 is localised to these cell types within the early fetal human heart. RESULTS: Tß4 mRNA was found to be expressed in both the atria and the ventricles of the early fetal human heart. Tß4 protein was found to be primarily localised to CD31-expressing endothelial cells and the endocardium as well as being present in the epicardium. Tß4-associated fluorescence was greater in the compact layer of the myocardial wall and the interventricular septum than in the trabecular layer of the myocardium. CONCLUSIONS: The data presented illustrates expression of Tß4 in the developing human heart and demonstrates for the first time that Tß4 in the human heart is primarily localised to endothelial cells of the cardiac microvasculature and coronary vessels as-well as to the endothelial-like cells of the endocardium and to the epicardium.

MicroRNA-31 Targets Thymic Stromal Lymphopoietin in Mucosal Infiltrated CD4+ T Cells: A Role in Achieving Mucosal Healing in Ulcerative Colitis?

Background: Ulcerative colitis (UC) is characterized by disruption of the mucosal intestinal barrier. MicroRNAs, single-stranded noncoding RNAs of approximately 22nt, are dysregulated in UC. MicroRNAs targeting thymic stromal lymphopoietin (TSLP), a cytokine involved in T-cell maturation and polarization, may be involved in regulating UC inflammation and mucosal healing. Methods: Biopsy samples from non-UC (n = 38), inactive UC (n = 18), and active UC (n = 23) patients were analyzed for mRNA (real-time quantitative polymerase chain reaction) or TSLP protein expression (enzyme-linked immunosorbent assay). Flow cytometry was used to isolate CD4+ T cells from biopsies. The functional mechanism was shown using luciferase assays and antago-miR transfections. The TSLP/miR-31 association was analyzed on 196 subjects from a previous clinical trial that tested the anti-IL-13 drug tralokinumab, whereas mucosal healing effects were studied on a subset of patients (n = 13) from this trial. Results: We found that TSLP is reduced at both mRNA and protein levels in inflamed UC patients when compared with healthy subjects, in both whole biopsies and biopsy-isolated CD4+ CD25+ T cells. The expression of miR-31, predicted to target TSLP, inversely co-related to the levels of TSLP mRNA in T cells. Blocking miR-31 in vitro in T cells increased both TSLP mRNA expression and protein secretion. Luciferase assays showed that miR-31 directly targeted TSLP mRNA, suggesting a direct mechanistic link. We also found that TSLP is increased in patients who achieve mucosal healing, comparing biopsies before and after treatment from the tralokinumab trial. Conclusions: Our data suggest a role for TSLP in promoting mucosal healing and regulating inflammation in UC, whereas miR-31 can directly block this effect.

Genome-Wide Posttranscriptional Dysregulation by MicroRNAs in Human Asthma as Revealed by Frac-seq.

MicroRNAs are small noncoding RNAs that inhibit gene expression posttranscriptionally, implicated in virtually all biological processes. Although the effect of individual microRNAs is generally studied, the genome-wide role of multiple microRNAs is less investigated. We assessed paired genome-wide expression of microRNAs with total (cytoplasmic) and translational (polyribosome-bound) mRNA levels employing subcellular fractionation and RNA sequencing (Frac-seq) in human primary bronchoepithelium from healthy controls and severe asthmatics. Severe asthma is a chronic inflammatory disease of the airways characterized by poor response to therapy. We found genes (i.e., isoforms of a gene) and mRNA isoforms differentially expressed in asthma, with novel inflammatory and structural pathophysiological mechanisms related to bronchoepithelium disclosed solely by polyribosome-bound mRNAs (e.g., IL1A and LTB genes or ITGA6 and ITGA2 alternatively spliced isoforms). Gene expression (i.e., isoforms of a gene) and mRNA expression analysis revealed different molecular candidates and biological pathways, with differentially expressed polyribosome-bound and total mRNAs also showing little overlap. We reveal a hub of six dysregulated microRNAs accounting for ∼90% of all microRNA targeting, displaying preference for polyribosome-bound mRNAs. Transfection of this hub in bronchial epithelial cells from healthy donors mimicked asthma characteristics. Our work demonstrates extensive posttranscriptional gene dysregulation in human asthma, in which microRNAs play a central role, illustrating the feasibility and importance of assessing posttranscriptional gene expression when investigating human disease.

MicroRNA-31 and MicroRNA-155 Are Overexpressed in Ulcerative Colitis and Regulate IL-13 Signaling by Targeting Interleukin 13 Receptor α-1.

Interleukin-13 (IL-13) is an important Type 2 T helper (Th2) cytokine, controlling biological functions in epithelium and has been linked to asthma, atopic dermatitis and ulcerative colitis (UC). Interleukin-13 signals through IL-13 receptor α-1 (IL13RA1 (gene) and IL13Rα1 (protein)), a receptor that can be regulated by microRNAs (miRs). MicroRNAs are small non-coding single-stranded RNAs with a role in several pathologies. However, their relevance in the pathophysiology of UC, a chronic inflammatory condition of the colonic mucosa, is poorly characterised. Here, we determined the expression of IL13Rα1 in UC, its potential regulation by miRs and the subsequent effect on IL-13 signalling. Inflamed mucosa of UC patients showed decreased mRNA and protein expression of IL13RA1 when compared to healthy controls. We show that miR-31 and miR-155 are upregulated in inflamed UC mucosa and that both directly target the 3' untranslated region of IL13RA1 mRNA. Transfection of miR-31 and miR-155 mimics reduced the expression of IL13RA1 mRNA and protein, and blocked IL-13-dependent phosphorylation of signal transducer and activator of transcription 6 (STAT6) in HT-29 cells, a gut epithelium cell line. Interleukin-13 activation of suppressor of cytokine signaling 1 (SOCS1) and eotaxin-3 (CCL26) expression was also diminished. MicroRNA-31/microRNA-155 mimics also downregulated IL13RA1 in ex vivo human inflamed UC biopsies. We propose that miR-31 and miR-155 have an important role in limiting IL-13 signalling in UC disease.

Mycobacterium tuberculosis subverts negative regulatory pathways in human macrophages to drive immunopathology.

Tuberculosis remains a global pandemic and drives lung matrix destruction to transmit. Whilst pathways driving inflammatory responses in macrophages have been relatively well described, negative regulatory pathways are less well defined. We hypothesised that Mycobacterium tuberculosis (Mtb) specifically targets negative regulatory pathways to augment immunopathology. Inhibition of signalling through the PI3K/AKT/mTORC1 pathway increased matrix metalloproteinase-1 (MMP-1) gene expression and secretion, a collagenase central to TB pathogenesis, and multiple pro-inflammatory cytokines. In patients with confirmed pulmonary TB, PI3Kδ expression was absent within granulomas. Furthermore, Mtb infection suppressed PI3Kδ gene expression in macrophages. Interestingly, inhibition of the MNK pathway, downstream of pro-inflammatory p38 and ERK MAPKs, also increased MMP-1 secretion, whilst suppressing secretion of TH1 cytokines. Cross-talk between the PI3K and MNK pathways was demonstrated at the level of eIF4E phosphorylation. Mtb globally suppressed the MMP-inhibitory pathways in macrophages, reducing levels of mRNAs encoding PI3Kδ, mTORC-1 and MNK-1 via upregulation of miRNAs. Therefore, Mtb disrupts negative regulatory pathways at multiple levels in macrophages to drive a tissue-destructive phenotype that facilitates transmission.

Tissue-resident memory features are linked to the magnitude of cytotoxic T cell responses in human lung cancer.

Therapies that boost the anti-tumor responses of cytotoxic T lymphocytes (CTLs) have shown promise; however, clinical responses to the immunotherapeutic agents currently available vary considerably, and the molecular basis of this is unclear. We performed transcriptomic profiling of tumor-infiltrating CTLs from treatment-naive patients with lung cancer to define the molecular features associated with the robustness of anti-tumor immune responses. We observed considerable heterogeneity in the expression of molecules associated with activation of the T cell antigen receptor (TCR) and of immunological-checkpoint molecules such as 4-1BB, PD-1 and TIM-3. Tumors with a high density of CTLs showed enrichment for transcripts linked to tissue-resident memory cells (TRM cells), such as CD103, and CTLs from CD103hi tumors displayed features of enhanced cytotoxicity. A greater density of TRM cells in tumors was predictive of a better survival outcome in lung cancer, and this effect was independent of that conferred by CTL density. Here we define the 'molecular fingerprint' of tumor-infiltrating CTLs and identify potentially new targets for immunotherapy.

IFN-γ Influences Epithelial Antiviral Responses via Histone Methylation of the RIG-I Promoter.

The asthmatic lung is prone to respiratory viral infections that exacerbate the symptoms of the underlying disease. Recent work has suggested that a deficient T-helper cell type 1 response in early life may lead to these aberrant antiviral responses. To study the development of long-term dysregulation of innate responses, which is a hallmark of asthma, we investigated whether the inflammatory environment of the airway epithelium can modulate antiviral gene expression via epigenetic mechanisms. We primed AALEB cells, a human bronchial epithelial cell line, with IFN-γ and IL-13, and subsequently infected the cells with respiratory syncytial virus (RSV). We then analyzed the expression of innate antiviral genes and their epigenetic markers. Priming epithelial cells with IFN-γ reduced the RSV viral load. Microarray analysis identified that IFN-γ priming enhanced retinoic acid-inducible gene (RIG)-I mRNA expression, and this expression correlated with epigenetic changes at the RIG-I promoter that influenced its transcription. Using chromatin immunoprecipitation, we observed a reduction of trimethylated histone 3 lysine 9 at the RIG-I promoter. Addition of inhibitor BIX-01294 to this model indicated an involvement of lysine methyltransferase G9a in RIG-I epigenetic regulation. These data suggest that prior exposure to IFN-γ may leave an epigenetic mark on the chromatin that enhances airway cells' ability to resist infection, possibly via epigenetic upregulation of RIG-I. These observations provide further evidence for a crucial role of IFN-γ in the development of mature antiviral responses within a model of respiratory infection. Further clinical validation is required to determine whether this effect in early life leads to changes in antiviral responses associated with asthma.

MiR-146b is down-regulated during the chondrogenic differentiation of human bone marrow derived skeletal stem cells and up-regulated in osteoarthritis.

Articular cartilage injury can result in chondrocyte loss and diminishment of specialised extracellular matrix, which can progress to an osteoarthritic (OA) phenotype. Stem cells have emerged as a favourable approach for articular cartilage regeneration. Identification of miRNAs which influence stem cell fate offers new approaches for application of miRNAs to regenerate articular cartilage. Skeletal stem cells (SSCs) isolated from human bone marrow were cultured as high density micromass' using TGF-ß3 to induce chondrogenesis. qPCR and TaqMan qPCR were used to assess chondrogenic gene and miRNA expression. Target prediction algorithms identified potential targets of miR-146b. Transient transfection with miR-146b mimic and western blotting was used to analyse SOX5. Human OA articular chondrocytes were examined for miR-146b expression. Chondrogenic differentiation of human bone marrow derived SSCs resulted in significant down-regulation of miR-146b. Gain of miR-146b function resulted in down-regulation of SOX5. MiR-146b expression was up-regulated in OA chondrocytes. These findings demonstrate the functional role of miR-146b in the chondrogenic differentiation of human bone marrow derived SSCs. MiR-146b may play a role in the pathophysiology of OA. Application of miR-146b combined with stem cell therapy could enhance regeneration of cartilaginous tissue and serve as a potential therapeutic target in the treatment of OA.