COL4A3 expression in asthmatic epithelium depends on intronic methylation and ZNF263 binding

BackgroundReduction of COL4A3 in asthmatic airways, one of the six isoforms of collagen 4 results in increased inflammation and angiogenesis implicating it as a central part of asthma pathogenesis. However, the path underlying these diminished COL4A3 levels has been elusive to date. This study investigated a possible mechanism underlying the reduction of COL4A3 expression.MethodsBronchial biopsies of n=76 asthmatics and n=83 controls were subjected to RNA-sequencing and DNA methylation bead arrays to identify expression and methylation changes. The binding of ZNF263 was analysed by ChiP-Seq coupled with qPCR. Effects of ZNF263 silencing, using siRNA, on the COL4A3 expression were studied by qPCR.ResultsCOL4A3 expression was significantly reduced in bronchial biopsies compared to healthy controls whereas DNA methylation levels at cg11797365 were increased. COL4A3 expression levels were significantly low in asthmatics without ICS use whereas the expression was not statistically different between asthmatics using ICS and controls. Methylation levels at cg11797365 in vitro were increased upon consecutive rhinovirus infections.ConclusionOur data indicates an epigenetic modification as a contributing factor for the loss of COL4A3 expression in asthmatic airway epithelium.

Inflammatory Determinants of Differential Tuberculosis Risk in Pre-Adolescent Children and Young Adults

The risk of progression from Mycobacterium tuberculosis (M.tb) infection to active tuberculosis (TB) disease varies markedly with age. TB disease is significantly less likely in pre-adolescent children above 4 years of age than in very young children or post-pubescent adolescents and young adults. We hypothesized that pro-inflammatory responses to M.tb in pre-adolescent children are either less pronounced or more regulated, than in young adults. Inflammatory and antimicrobial mediators, measured by microfluidic RT-qPCR and protein bead arrays, or by analyzing published microarray data from TB patients and controls, were compared in pre-adolescent children and adults. Multivariate analysis revealed that M.tb-uninfected 8-year-old children had lower levels of myeloid-associated pro-inflammatory mediators than uninfected 18-year-old young adults. Relative to uninfected children, those with M.tb-infection had higher levels of similar myeloid inflammatory responses. These inflammatory mediators were also expressed after in vitro stimulation of whole blood from uninfected children with live M.tb. Our findings suggest that myeloid inflammation is intrinsically lower in pre-pubescent children than in young adults. The lower or more regulated pro-inflammatory responses may play a role in the lower risk of TB disease in this age group.

Exploring autoantibody signatures in brain tissue from patients with severe mental illness

In recent years, studies have shown higher prevalence of autoantibodies in patients with schizophrenia compared to healthy individuals. This study applies an untargeted and a targeted affinity proteomics approach to explore and characterize the autoantibody repertoire in brain tissues from 73 subjects diagnosed with schizophrenia and 52 control subjects with no psychiatric or neurological disorders. Selected brain tissue lysates were first explored for IgG reactivity on planar microarrays composed of 11,520 protein fragments representing 10,820 unique proteins. Based on these results of ours and other previous studies of autoantibodies related to psychosis, we selected 226 fragments with an average length of 80 amino acids, representing 127 unique proteins. Tissue-based analysis of IgG reactivities using antigen suspension bead arrays was performed in a multiplex and parallel fashion for all 125 subjects. Among the detected autoantigens, higher IgG reactivity in subjects with schizophrenia, as compared to psychiatrically healthy subjects, was found against the glutamate ionotropic receptor NMDA type subunit 2D (anti-GluN2D). In a separate cohort with serum samples from 395 young adults with a wider spectrum of psychiatric disorders, higher levels of serum autoantibodies targeting GluN2D were found when compared to 102 control individuals. By further validating GluN2D and additional potential autoantigens, we will seek insights into how these are associated with severe mental illnesses.

443 An immunotherapy trio in advanced HNSCC for coordinated B and T cell antigen response

BackgroundOutcomes for recurrent or metastatic (R/M) head and neck squamous cell carcinoma (HNSCC) are dismal and responses to anti-PD-1 appear best in tumors with PD-1+ T cells in proximity to PD-L1+ cells, arguing that improved outcome is associated with a pre-existing anti-cancer immune response. Based on this, we hypothesize that vaccines which prime and/or expand T cells to a spectrum of antigens overexpressed by HNSCC combined with T cell agonists, like anti-GITR, that provide costimulatory signals will improve the anti-PD-1 response rates. We have developed a cancer vaccine, DPV-001, that contains more than 300 proteins for genes overexpressed by HNSCC, encapsulated in a CLEC9A-targeted microvesicle and containing TLR/NOD agonists and DAMPs. Recently, we reported that combining anti-GITR + vaccine + anti-PD-1 augmented therapeutic efficacy in a preclinical model and now plan a phase 1b trial of this combination in patients with advanced HNSCC.MethodsSera from patients receiving DPV-001 as adjuvant therapy for definitively treated NSCLC, were analyzed for IgG responses to human proteins by MAP bead arrays and results compared to TCGA gene expression data sets for HNSCC. HNSCC cell lines were evaluated by RNASeq and peptides were eluted from HLA, analyzed by mass spectroscopy and correlated against MAP bead arrays and TCGA data sets. Tumor-reactive T cells from a vaccinated patient were enriched and expanded, and used in cytokine release assay (CRA) against autologous NSCLC and partially HLA matched allogeneic HNSCC cell lines.ResultsPatients receiving DPV-001 (N=13) made 147 IgG responses to at least 70 proteins for genes overexpressed by HNSCC. Preliminary evaluation of the HNSCC peptidome against the results of MAP bead array identify antigens that are target of a humoral immune response. Additionally, tumor-reactive T cells from DPV-001 vaccinated patient recognize two partially HLA-matched HNSCC targets, but not a mis-matched target.ConclusionsRecent observations from our lab and others have correlated IgG Ab responses with T cell responses to epitopes of the same protein. Based on the data summarized above, we hypothesize that we have induced T cell responses against a broad spectrum of shared cancer antigens that are common among adenocarcinomas and squamous cell cancers. Our planned clinical trial will vaccinate and boost the induced responses by costimulation with anti-GITR and then sequence in delayed anti-PD-1 to relieve checkpoint inhibition. MAP bead arrays and the peptidome library generated above will be used to assess anti-cancer B and T cell responses.Trial RegistrationNCT04470024Ethics ApprovalThe original clinical trial was approved by the Providence Portland Medical Center IRB, approval # 13-046. The proposed clinical trial has not yet been reviewed by the IRB.

T helper cells in synovial fluid of patients with rheumatoid arthritis primarily have a Th1 and a CXCR3+Th2 phenotype

Background The majority of CD4+ T helper (Th) cells found in the synovial fluid (SF) of patients with rheumatoid arthritis (RA) express CXCR3, a receptor associated with Th1 cells. In blood, subsets of Th2 and Th17 cells also express CXCR3, but it is unknown if these cells are present in RA SF or how cytokines from these subsets affect cytokine/chemokine secretion by fibroblast-like synoviocytes (FLS) from patients with RA. Methods We examined the proportions of Th1, Th2, CXCR3+Th2, Th17, CXCR3+Th17, Th1Th17, peripheral T helper (TPh) and T follicular helper (TFh) cells in paired SF and blood, as well as the phenotype of TPh and TFh cells in RA SF (n = 8), by the use of flow cytometry. We also examined the cytokine/chemokine profile in paired SF and plasma (n = 8) and in culture supernatants of FLS from patients with chronic RA (n = 7) stimulated with Th-associated cytokines, by the use of cytometric bead arrays and ELISA. Cytokine receptor expression in FLS (n = 3) were assessed by the use of RNA sequencing and qPCR. Results The proportions of Th1 and CXCR3+Th2 cells were higher in SF than in blood (P < 0.05). TPh and PD-1highTFh in RA SF were primarily of a Th1 and a CXCR3+Th2 phenotype. Moreover, the levels of CXCL9, CXCL10, CCL20, CCL2, CXCL8, IL-6 and IL-10 were higher in SF than in plasma (P < 0.05). Lastly, IL-4, IL-13 and IL-17A induced RA FLS to secrete proinflammatory IL-6, CCL2, CXCL1 and CXCL8, while IFNγ mainly induced CXCL10. Conclusion These findings indicate that not only Th1 but also CXCR3+Th2 cells may have a pathogenic role in RA synovial inflammation.