scholarly journals P148 Differential methylation of the TNF gene promoter, potential for the development of a diagnostic biomarker for RA

Rheumatology ◽  
2021 ◽  
Vol 60 (Supplement_1) ◽  
Author(s):  
Rujiraporn Pitaksalee ◽  
Paul Emery ◽  
Richard Hodgett ◽  
Frederique Ponchel
Keyword(s):  
T Cells ◽  
Cd4 T Cells ◽  
Tissue Bank ◽  
Gene Promoter ◽  
Classification Performance ◽  
Differential Methylation ◽  
Diagnostic Biomarker ◽  
Clinical Model ◽  
Early Ra ◽  
Tnf Gene

Abstract Background/Aims  Diagnosing rheumatoid arthritis (RA) early is difficult despite the use of anti-citrullinated protein antibodies in the new EULAR-2010 classification criteria, particularly for antibody-negative patients. Alterations in epigenetic patterns have been associated with RA CD4+T-cells at the TNF gene locus (1). The second aim of my PhD project was to select a CpG site to develop a quantitative methylation sensitive PCR (qMSP) assay as a diagnostic biomarker test for RA classification. Methods  Patients attending an early arthritis clinic were used (with full ethical approval). An 450K Illumina methylation genome-wide dataset analysing DNA from CD4+T-cells identified CpG on TNF promoter region as a differentially methylated CpG between controls and RA patients (1). A qMSP assay was designed using a TaqMan approach (primers and probe), using a reference gene (GAPHD) for normalisation. The assay was validated in DNA extracted from PBMC. The performance of the assay for RA classification was accessed using binary logistic regression. Results  We noted differences in levels of methylation in CD4+T-cells in RA, notably affecting the TNF gene promoter in about 15% of cells. We validated this using bisulphite sequencing showing about 30% of cells with differential methylation in early RA compared to controls. This allowed us to select cg11484872, cg21370522, and cg01569083 as candidates. We optimised a qMSP assay using fully methylated/un-methylated DNA. PBMC samples were retrieved from our tissue bank for 65 early RA, and 64 non-RA patients (including 11 reactive arthritis, 37 undifferentiated arthritis, and 16 psoriatic arthritis). The methylation levels (%) detected by the TNF-qMSP assay were significantly lower in RA (Δ-DM =-10.28%, p = 7x10-7). The loss of TNF promoter gene methylation was associated with RA classification (p < 0.0001, unadjusted OR = 0.900 (95% CI: 0.85-0.94)) with a good classification performance (AUROC = 0.76 (95% CI: 0.67, 0.84)). An adjusted regression model using the demographic and clinical parameters associated with RA classification in this group of patients (age, smoking, counts for tender and swollen joints) showed that adding TNF to the clinical model improved the model’s performance (increasing AUROC from 0.86 , to 0.91). Comparing classification accuracy, adding the TNF-qMSP resulted in an increased correct classification of 5.7 % compared to the clinical model. A similar result was obtained in the antibody negative patients, with TNF-qMSP assay improving the model’s classification from AUROC 0.91 to 0.96 and 3.3% more patients correctly classified. Conclusion  The loss of DNA methylation on the TNF gene promoter identified in early RA patients provided a valuable target for a biomarker assay development. The results will need to be confirmed in a larger cohort but the TNF-qMSP assay offers good opportunities for RA versus non-RA classification particular in antibody negative disease. Disclosure  R. Pitaksalee: Grants/research support; ROYAL THAI GOVERNMENT SCHOLARSHIP. P. Emery: None. R. Hodgett: None. F. Ponchel: None.

Abstract 033: Differential Vasopressin Receptor Expression on CD4+ T Cells from Mouse and Human Preeclamptic Pregnancies

Hypertension ◽  
2016 ◽  
Vol 68 (suppl_1) ◽  
Author(s):  
Sabrina M Scroggins ◽  
Donna A Santillan ◽  
Jenna M Peterson ◽  
Nicole A Pearson ◽  
Jeremy A Sandgren ◽  
...  
Keyword(s):  
T Cells ◽  
Cd4 T Cells ◽  
Immune Modulation ◽  
Inflammatory Cytokine ◽  
Mononuclear Cells ◽  
Cytokine Production ◽  
Cell Activity ◽  
Tissue Bank ◽  
Receptor Expression ◽  
Inflammatory Cytokine Production

The pathogenesis of preeclampsia (PreE) involves the failure of the maternal immune system to normally tolerate the pregnancy. Inflammatory cytokines are elevated in PreE-affected women with a concurrent decrease in anti-inflammatory cytokine production. Consistent with what other groups have observed in mouse models of hypertension during pregnancy and in human PreE-affected pregnancies, we observed increased inflammatory cytokine production and CD4+ T helper populations in our chronic infusion of vasopressin (AVP) mouse model of PreE. The mechanisms of immune modulation by AVP have not been elucidated. As increased T cell activity is involved in the development of PreE, the objective of this study was to investigate if CD4+ T cells express AVP receptors. Splenic CD4+ T cells were negatively purified from C57BL/6J saline and AVP-infused (24 ng/hour) dams. Expression of AVP receptors (AVPR) 1a, 1b, 2, and the aminopeptidase LNPEP (catalyzes AVP degradation) was determined via qPCR. Raw cycle threshold (Ct) values were normalized (ΔCt) against the 18S rRNA endogenous control. Mouse CD4+ T cells express all AVP receptors and LNPEP. By ANOVA, AVPR2 is the highest expressed receptor in CD4+ T cells from saline (N=7, p=0.002) and AVP-infused (N=10, p<0.0001) dams. Human maternal mononuclear cells, obtained from the University of Iowa Maternal-Fetal Tissue Bank (IRB #200910784) from control and PreE-affected women, were similarly analyzed. As in mouse CD4+ T cells, human control (N=27, p<0.0001) and PreE-affected (N=26, p<0.0001) CD4+ T cells most highly expressed AVPR2. AVPR1a was also highly expressed while AVPR1b was the least expressed. CD4+ T cells isolated from human PreE-affected women expressed significantly lower AVPR1a (10.0±0.3 N=27 vs. 11.1±0.2 N=0.23, p=0.009) and increased LNPEP (17.2±0.5 N=27 vs. 15.1±0.3 N=26, p=0.001) than controls. Here, we demonstrate CD4+ T cells, both mouse and human, express AVP receptors and that 1a and 2 are highest expressed. Although the actions of AVP on the vasculature are primarily mediated through AVPR1a, these data suggest AVP may differentially act through AVPR1a to mediate immune responses during PreE.

Epigenetic Modulation of STAT3 by Histone Deacetylase 6 (HDAC6) Regulates IL-10 Gene Expression and Immune Tolerance Mediated by Antigen-Presenting Cells (APCs)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 519-519 ◽  
Author(s):  
Fengdong Cheng ◽  
Zi Wang ◽  
Hongwei Wang ◽  
Karrune V. Woan ◽  
Eva Sahakian ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Cd4 T Cells ◽  
Transcriptional Activity ◽  
Gene Promoter ◽  
B Cell Lymphoma ◽  
Gene Promoters ◽  
Enhanced Production ◽  
Stat3 Phosphorylation

Abstract Abstract 519 We have previously shown that the pan-HDAC inhibitors LAQ824 and LBH589 inhibit IL-10 production in APCs, rendering these cells more inflammatory and capable of effectively priming naïve antigen-specific CD4+ T-cells and restoring the responsiveness of tolerant T-cells1. These findings led us to explore which HDAC(s) might be involved in the regulation of IL-10 gene transcription and be the putative target(s) of HDI-mediated IL-10 inhibition. To answer these questions we subjected the macrophage cell line RAW264.7 to shRNA screening using specific shRNAs to knockdown each known HDAC. We found that among all the HDACs, knocking down HDAC6 (HDAC6KD) was associated with a significant decrease in IL-10 mRNA and protein in response to LPS stimulation. Furthermore, HDAC6KD clones display an enhanced expression of the co-stimulatory molecule B7.2. Functionally, HDAC6KD cells were better activators of anti-HA (hemagglutinin-influenza) transgenic CD4+ T cells, leading to significantly enhanced production of IL-2 and IFN-g in response to cognate antigen. More importantly, anti-HA CD4+ anergic T cells isolated from animals bearing HA-expressing A20 B-cell lymphoma regained their ability to produce IL-2 and IFN-g when cultured in vitro with HDAC6KD cells. These results have been confirmed in APCs isolated from HDAC6 knock-out mice and in wild type APCs treated in vitro with isotype-selective HDAC6 inhibitors. Given that HDACs do not bind to DNA and they need to interact with transcription factors to regulate gene expression, we investigate next which transcription factor(s) HDAC6 might be associated with, to regulate IL-10 transcriptional activity. One likely candidate was Stat3, a well-known transcriptional activator of IL-10 gene expression that we have previously shown to play a central role in tolerance induction by APCs2. By co-immunoprecipitation studies we found that HDAC6 indeed interacts physically with Stat3. Of note, knocking down HDAC6 in APCs resulted in absence of Stat3 phosphorylation and decreased recruitment of Stat3 to the IL-10 gene promoter which might explain the inability of HDAC6KD cells to produce IL-10. The additional findings that IL-10 production by HDAC6KD cells was restored when these cells were transfected with a constitutively active mutant version of Stat3 (Stat3c) provides additional support for the important role of HDAC6 upon Stat3 activation. Further confirmation for a concerted regulatory mechanism involving HDAC6 and Stat3 in IL-10 gene regulation was provided by studies using CPA-7, a specific Stat3 inhibitor that disrupts Stat3 recruitment and binding to gene promoters. As expected, a complete abrogation of Stat3 recruitment to the IL-10 gene promoter was observed in CPA-7 treated APCs. Interestingly, such an effect was accompanied by a parallel decrease in HDAC6 recruitment to the IL-10 promoter and inhibition of IL-10 gene transcriptional activity. Taken together, we have shown for the first time that HDAC6 interacts physically with Stat3 and is required for its phosphorylation. Since Stat3 phosphorylation is absolutely necessary for activation of Stat3 target genes, HDAC6 inhibition is an enticing molecular approach to disrupt the Stat3/IL-10 axis and overcome tolerogenic mechanisms in APCs. Disclosures: No relevant conflicts of interest to declare.

Methylation differences at the HLA-DRB1 locus in CD4+ T-Cells are associated with multiple sclerosis

2013 ◽  
Vol 20 (8) ◽  
pp. 1033-1041 ◽  
Author(s):  
MC Graves ◽  
M Benton ◽  
RA Lea ◽  
M Boyle ◽  
L Tajouri ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
Dna Methylation ◽  
T Cells ◽  
Cd4 T Cells ◽  
Cpg Island ◽  
Major Effect ◽  
Differential Methylation ◽  
Genome Wide ◽  
A Genome ◽  
Drb1 Locus

Background: Multiple sclerosis (MS) is thought to be caused by T-cell mediated autoimmune dysfunction. Risk of developing MS is influenced by environmental and genetic factors. Modifiable differences in DNA methylation are recognized as epigenetic contributors to MS risk and may provide a valuable link between environmental exposure and inherited genetic systems. Objectives and methods: To identify methylation changes associated with MS, we performed a genome-wide DNA methylation analysis of CD4+ T cells from 30 patients with relapsing–remitting MS and 28 healthy controls using Illumina 450K methylation arrays. Results: A striking differential methylation signal was observed at chr. 6p21, with a peak signal at HLA-DRB1. After prioritisation, we identified a panel of 74 CpGs associated with MS in this cohort. Most notably we found evidence of a major effect CpG island in DRB1 in MS cases ( pFDR < 3 × 10−3). In addition, we found 55 non-HLA CpGs that exhibited differential methylation, many of which localise to genes previously linked to MS. Conclusions: Our findings provide the first evidence for association of DNA methylation at HLA-DRB1 in relation to MS risk. Further studies are now warranted to validate and understand how these findings are involved in MS pathology.

scholarly journals Differential methylation at MHC in CD4+ T cells is associated with multiple sclerosis independently of HLA-DRB1

2017 ◽  
Vol 9 (1) ◽  
Author(s):  
Vicki E. Maltby ◽  
Rodney A. Lea ◽  
Katherine A. Sanders ◽  
Nicole White ◽  
Miles C. Benton ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
T Cells ◽  
Cd4 T Cells ◽  
Differential Methylation

scholarly journals THU0033 ALTERATIONS IN THE PHENOTYPIC LANDSCAPE AND SPECIFICITY OF CD4+ T CELLS IN CCP+ AT RISK SUBJECTS BEFORE THE ONSET OF RHEUMATOID ARTHRITIS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 230.1-230
Author(s):  
C. Rims ◽  
V. Muir ◽  
K. Deane ◽  
S. Nagpal ◽  
N. Rao ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
At Risk ◽  
Flow Cytometry ◽  
T Cells ◽  
Immune Responses ◽  
Cd4 T Cells ◽  
Research Support ◽  
Phenotypic Analysis ◽  
Tetramer Staining ◽  
Early Ra

Background:The “Targeting Immune Responses for Prevention of RA” (TIP-RA) collaboration studies individuals at high risk for developing rheumatoid arthritis (RA) because of serum anti-citrullinated protein antibody (ACPA) positivity in absence of arthritis at baseline, and is focused on defining how they transition from at-risk to classifiable disease. One potential mechanism is the expansion of antigen specific T cells that recognize self-antigens and acquisition of disease associated T cell phenotypes. ACPA emerge years prior to clinically apparent disease and subsequently increase in their titer and breadth of specificity. However, few studies have characterized T cells during this transition.Objectives:To identify features associated with progression to RA by examining the specificity and surface phenotype of CD4+ T cells in individuals from the TIP-RA cohort by HLA class II tetramer staining and multi-parameter flow cytometry.Methods:Tetramer staining and flow cytometry were performed on peripheral blood samples from a baseline visit from CCP3- controls (n=34), CCP3+ at-risk (n=26), CCP3+ positive individuals who transitioned in the near-term to RA (called “RA converters”, n=4), and seropositive early-RA (n=21). Our staining panel allowed us to measure the frequencies of T cells specific for citrullinated alpha-enolase, aggrecan, cartilage intermediate layer protein (CILP), fibrinogen and vimentin. We then applied both supervised phenotyping and a cluster-based computational approach to compare the phenotypic landscape and specificity of antigen specific and total CD4+ T cells in each cohort.Results:We observed higher overall frequencies of T cells that recognize citrullinated epitopes in CCP3+ at-risk subjects than CCP- controls (p< 0.05). Among the individual specificities, elevated frequencies prior to disease onset were most prominent for CILP specific T cells. Supervised phenotypic analysis revealed an increase in CCR4+ CD4+ T cells in CCP3+ at risk subjects (p< 0.001) and a corresponding decrease in CXCR3+ CD4+ T cells that was most pronounced in RA converters and seropositive early-RA (p< 0.05). Cluster-based phenotypic analysis defined ten distinct phenotypic states present within all subjects. Each of these ten immunotypes contained T cells that recognize citrullinated epitopes. However, the predominant immunotype varied for different antigens. During progression, the frequencies of Ag specific T cells diminished when onset was imminent, but rebounded shortly after diagnosis. Concomitantly, Ag specific T cells with memory phenotypes were diminished, but subsequently reverted to TSCM, Th1, and Th1-17 like phenotypes.Conclusion:Our data show that disease associated changes in the antigen specificity of CD4+ T cells are present in CCP3+ at-risk subjects. Furthermore, the number of antigen specific T cells and their phenotype are perturbed before the onset of symptoms and development of classified RA. These findings support a continuum of immunologic changes that underlie risk and drive disease, motivating new approaches for early intervention.Acknowledgments:We gratefully acknowledge the Targeting Immune Responses for Prevention of Rheumatoid Arthritis (TIP-RA) for designing and executing this collaborative studyDisclosure of Interests:Cliff Rims: None declared, Virginia Muir: None declared, Kevin Deane Grant/research support from: Janssen, Consultant of: Inova, ThermoFisher, Janseen, BMS and Microdrop, Sunil Nagpal Shareholder of: Janssen Pharmaceuticals, Employee of: Janssen Pharmaceuticals, Navin Rao Shareholder of: Janssen Pharmaceuticals, Employee of: Janssen Pharmaceuticals, Frederic Baribaud Shareholder of: Janssen Research & Development, LLC, Employee of: Janssen Research & Development, LLC, George Vratsanos Shareholder of: Janssen Pharmaceuticals, Employee of: Janssen Pharmaceuticals, V. Michael Holers Grant/research support from: Janssen, Celgene, and BMS, Peter Linsley Consultant of: BMS, Eddie A. James Grant/research support from: Janssen, Pfizer, Sanofi, Novartis, Jane Buckner Grant/research support from: Bristol-Myers Squibb, Janssen

scholarly journals PM2.5 exposure and cold stress exacerbates asthma in mice by increasing histone acetylation in IL-4 gene promoter in CD4+ T cells

2019 ◽  
Vol 316 ◽  
pp. 147-153 ◽  
Author(s):  
Ji Zhou ◽  
Fuhai Geng ◽  
Jianming Xu ◽  
Li Peng ◽  
Xiaofang Ye ◽  
...  
Keyword(s):  
T Cells ◽  
Cold Stress ◽  
Histone Acetylation ◽  
Cd4 T Cells ◽  
Gene Promoter ◽  
Pm2.5 Exposure

scholarly journals SAT0013 PIM-1 KINASE IS A MEASURABLE MEDIATOR OF CD4+ T CELL DYSREGULATION AND THERAPEUTIC TARGET IN EARLY RHEUMATOID ARTHRITIS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 936.1-937
Author(s):  
N. Maney ◽  
H. De Paula-Lemos ◽  
B. Barron-Millar ◽  
A. Mellor ◽  
J. D. Isaacs ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
T Cells ◽  
T Cell ◽  
Therapeutic Target ◽  
Cd4 T Cells ◽  
Kinase Inhibitors ◽  
Early Arthritis ◽  
Early Ra ◽  
Sat 1

Background:As well as being an established oncoprotein and a therapeutic target in cancer,Proviral Integration site for murine Moloney leukemia virus-1(pim-1) has been implicated in human autoimmunity. We previously confirmed this serine-threonine protein kinase to be strikingly upregulated in circulating CD4+ T cells of untreated rheumatoid arthritis (RA) patients as a consequence of IL-6 signalling1-2. Evidence for the relevance of pim-1 signalling in the disruption of RA synovial fibroblast (RASF) homeostasis3further supports its candidacy as a therapeutic target.Objectives:To investigate PIM1 and its family members (PIM2 and PIM3) as potential candidates for drug repurposing in RA.Methods:A flow cytometric assay for PIM1 transcript measurement in circulating CD4+ T cells of early arthritis patients was validated against real-time PCR in paired cells isolated by bead selection. Synovial protein expression in tissue from the same cohort of untreated RA patients and disease controls was determined by quantitative multiplex immunofluorescence. The functional consequences of manipulating pim kinase family expression in freshly purified T cell receptor (TCR)-stimulated CD4+ T cells from early RA patients was explored. The impact of pim-1 specific and pim-1-3 (pan-pim) kinase inhibition on progression of the IL-6 dependent collagen-induced arthritis (CIA) model was assessed.Results:The percentage of circulating CD4+ T cells positive forPIM1transcript by flow cytometry proved a faithful surrogate for gene expression in early arthritis (Figure 1A), distinguishing RA from other pathologies (Figure 1B). Pim-1 protein expression was increased in the synovium of untreated RA compared with disease controls, including amongst infiltrating CD4+ T cells (Figure 1C-D).In vitro, exposure of TCR-stimulated early RA CD4+ T cells to pim kinase inhibitors restrained their activation and proliferative capacity; diminished pro-inflammatory cytokine production (IFN-g and IL-17) and an expanded CD25hiFoxP3+ regulatory T cell (Treg) fraction were also observed in treatedversusun-treated cells. Finally, administration of pim inhibitors robustly attenuated clinical scores of arthritis in the CIA model, with reduced cartilage loss observed in animals treated with a pan-PIM inhibitor compared with vehicle control (Figure 2).Conclusion:Our data highlight pim kinases as plausible therapeutic targets for a subgroup of early RA patients that may be identifiable using tractable in vitro assays. Pim kinase inhibitors could simultaneously target immune inflammation and RASF dysregulation; consideration should now be given to their repurposing for this condition.References:[1] Anderson AE et al Annals of the Rheumatic Diseases 2016; 75:466-73.[2] Anderson AE et al Rheumatology 2019; 58:1250-1258[3] Ha YJ et al Rheumatology 2019; 58:154-64Disclosure of Interests:Nicola Maney Consultant of: Current employee of Eli Lilly, Henrique De Paula-Lemos: None declared, Ben Barron-Millar: None declared, Andrew Mellor Shareholder of: NewLink Genetics PLC, and has received patent licensing income from this source., John D Isaacs Consultant of: AbbVie, Bristol-Myers Squibb, Eli Lilly, Gilead, Janssen, Merck, Pfizer, Roche, Amy Anderson: None declared, Arthur Pratt Grant/research support from: Pfizer, GlaxoSmithKlein

Activated CD4+ T cells enriched from spleen or colon induce apoptosis in intestinal epithellal cells via fas/fas ligand interactions

2001 ◽  
Vol 120 (5) ◽  
pp. A192-A192
Author(s):  
H TAKAISHI ◽  
T DENNING ◽  
K ITO ◽  
R MIFFLIN ◽  
P ERNST
Keyword(s):  
T Cells ◽  
Cd4 T Cells ◽  
Fas Ligand ◽  
Ligand Interactions
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