A.He. performed experiments and analyzed data. L.B. generated and provided critical reagents. T.S. and A.Ts assisted with mRNAseq and ATAC-seq data analysis, interpretation and T.S. generated figures. J.Z. and A.Te. performed and analyzed the targeted proteomics experiments. V.K., P.G. and M.B. contributed in data analysis and interpretation. T.C. and D.B. interpreted data. P.V. designed and supervised the study, performed data analysis, and wrote the manuscript.
Persistent viral infections are characterized by the simultaneous presence of chronic inflammation and T cell dysfunction. In prototypic models of chronicity--infection with human immunodeficiency virus (HIV) or lymphocytic choriomeningitis virus (LCMV)--we used transcriptome-based modeling to reveal that CD4(+) T cells were co-exposed not only to multiple inhibitory signals but also to tumor-necrosis factor (TNF). Blockade of TNF during chronic infection with LCMV abrogated the inhibitory gene-expression signature in CD4(+) T cells, including reduced expression of the inhibitory receptor PD-1, and reconstituted virus-specific immunity, which led to control of infection. Preventing signaling via the TNF receptor selectively in T cells sufficed to induce these effects. Targeted immunological interventions to disrupt the TNF-mediated link between chronic inflammation and T cell dysfunction might therefore lead to therapies to overcome persistent viral infection.
Regulatory T cells (Treg cells) are important for preventing autoimmunity and maintaining tissue homeostasis, but whether Treg cells can adopt tissue-or immune-context-specific suppressive mechanisms is unclear. Here, we found that the enzyme hydroxyprostaglandin dehydrogenase (HPGD), which catabolizes prostaglandin E 2 (PGE 2 ) into the metabolite 15-keto PGE 2 , was highly expressed in Treg cells, particularly those in visceral adipose tissue (VAT). Nuclear receptor peroxisome proliferator-activated receptor-g (PPARg)-induced HPGD expression in VAT Treg cells, and consequential Treg-cell-mediated generation of 15-keto PGE 2 suppressed conventional T cell activation and proliferation. Conditional deletion of Hpgd in mouse Treg cells resulted in the accumulation of functionally impaired Treg cells specifically in VAT, causing local inflammation and systemic insulin resistance. Consistent with this mechanism, humans with type 2 diabetes showed decreased HPGD expression in Treg cells. These data indicate that HPGD-mediated suppression is a tissue-and context-dependent suppressive mechanism used by Treg cells to maintain adipose tissue homeostasis. (C) Time course of relative HPGD mRNA expression in human Treg and Tconv cells in the presence of IL-2. (D) Immunoblotting for HPGD (top) and b-actin (bottom) in human Treg and Tconv cells after isolation (0 h) or cultivated for 48 or 72 h without stimulation (unstim) or stimulated with IL-2 (left) and densitometric analysis (right). (E and F) Relative HPGD mRNA expression in unstimulated or IL-2-stimulated human Treg cells cultured for 24 h in the presence of DMSO (control) or increasing doses of a STAT5 inhibitor (E) or JAK3 inhibitor (F). (G) ChIP qPCR analysis of human IL-2-stimulated Treg and Tconv cells with a STAT5-specific antibody. Relative enrichment of STAT5 ChIP over input normalized to immunoglobulin G (IgG) is shown. (H and I) IL-2-and STAT5-dependent activation of luciferase reporter constructs. (H) IL-2-induced HPGD promoter activity. (I) STAT5-dependent HPGD induction. (J) ChIP qPCR analysis of human expanded cord blood Treg cells with a FOXP3-specific antibody. Relative enrichment of FOXP3 ChIP over input normalized to IgG was calculated. A region within intron 4 was used as a negative control. (K) Luciferase assay of FOXP3 binding to the respective BRs at the HPGD locus. Numbers indicate Foxp3-binding motifs within each region. (L) Relative HPGD mRNA expression in human Treg cells after silencing of FOXP3. Treg cells were transfected and cultivated for 48 h without stimulation. (A, B, G, J, and L) *p < 0.05 (paired Student's t test); (C) *p < 0.05 (two-way ANOVA with false-discovery rate [FDR]); (D-F) *p < 0.05 (one-way ANOVA with FDR); (H) *p < 0.05 (Mann-Whitney U test); (I and K) *p < 0.05 (unpaired Student's t test). Data are representative of fourteen experiments (A; mean and SEM), six experiments (B; mean and SEM), two to five experiments (L; mean and SEM), four experiments (C-F; mean and SEM), three experiments (G and J; mean and SEM), each with ...
The epigenome and transcriptome constitute a critical element of a tightly regulated, cell-type specific gene expression program, and subtle perturbations in the regulation of this program can result in pathology. Epigenetic features such as DNA accessibility dictate transcriptional regulation in a cell type- and cell state- specific manner, and mapping this in health vs. disease in clinically relevant material is opening the door to new mechanistic insights and new targets for therapy. Assay for Transposase Accessible Chromatin Sequencing (ATAC-seq) allows profiling of chromatin accessibility with low cell input, making it amenable to the clinical setting, such as peripheral blood from clinical trials, and this makes it applicable to rare cell populations, such as regulatory T (Treg) cells. However, there is little known about the compatibility of the assay on materials recovered from cryopreserved rare cell populations. In the context of tolerance or autoimmunity, regulatory T cells play a critical role in maintaining immune homeostasis, and loss of numbers or function is linked to many diseases, making them a clinically relevant population to analyse using genomic platforms. Here we demonstrate the robustness and reproducibility of an ATAC-seq protocol comparing fresh or cryopreserved primary Treg cells, and comparing their profile in the steady state and in response to stimulation. We extend this method to explore the feasibility of conducting simultaneous quantitation of chromatin accessibility and transcriptome from a single aliquot of 50,000 Treg cells from cryopreserved PBMCs. Profiling of chromatin accessibility and gene expression in parallel within the same pool of cells controls for cellular heterogeneity and will be particularly beneficial for experiments constrained by limited input material, such as biobanked PBMC from clinical trials. This approach will be complementary to single-cell experiments as libraries used to profile chromatin accessibility and transcriptome are derived from the same population of cells, controlling for stochastic gene fluctuation in different cells in a population at any given time. Overall, we observed a high correlation of accessibility patterns and transcription factor (TF) dynamics between fresh Treg cells and cells recovered from cryopreservation samples. The distribution of fragment size, enrichment of transcription start sites (TSS) and genomic features of thawed Treg cells recapitulate that of the fresh cells. Furthermore, highly consistent global chromatin and transcriptional changes in response to stimulation were observed in both fresh and frozen samples. Lastly, highly similar transcriptomic profiles were obtained from whole cells and from the supernatants recovered from ATAC-seq reactions. This report highlights the feasibility of applying these techniques to profile the epigenomic landscape of cells recovered from cryopreservation biorepositories. Implementation of this approach is suitable in biorepositories and will contribute to advances in the field of translational research and personalized medicine.
Epigenetic features such as DNA accessibility dictate transcriptional regulation in a cell type- and cell state- specific manner, and mapping this in health vs. disease in clinically relevant material is opening the door to new mechanistic insights and new targets for therapy. Assay for Transposase Accessible Chromatin Sequencing (ATAC-seq) allows chromatin accessibility profiling from low cell input, making it tractable on rare cell populations, such as regulatory T (Treg) cells. However, little is known about the compatibility of the assay with cryopreserved rare cell populations. Here we demonstrate the robustness of an ATAC-seq protocol comparing primary Treg cells recovered from fresh or cryopreserved PBMC samples, in the steady state and in response to stimulation. We extend this method to explore the feasibility of conducting simultaneous quantitation of chromatin accessibility and transcriptome from a single aliquot of 50,000 cryopreserved Treg cells. Profiling of chromatin accessibility and gene expression in parallel within the same pool of cells controls for cellular heterogeneity and is particularly beneficial when constrained by limited input material. Overall, we observed a high correlation of accessibility patterns and transcription factor dynamics between fresh and cryopreserved samples. Furthermore, highly similar transcriptomic profiles were obtained from whole cells and from the supernatants recovered from ATAC-seq reactions. We highlight the feasibility of applying these techniques to profile the epigenomic landscape of cells recovered from cryopreservation biorepositories.
CD4+ T cells play a central role in the adaptive immune response through their capacity to activate, support and control other immune cells. Although these cells have become the focus of intense research, a comprehensive understanding of the underlying regulatory networks that orchestrate CD4+ T cell function and activation is still incomplete. Here, we analyzed a large transcriptomic dataset consisting of 48 different human CD4+ T cell conditions. By performing reverse network engineering, we identified six common denominators of CD4+ T cell functionality (CREB1, E2F3, AHR, STAT1, NFAT5 and NFATC3). Moreover, we also analyzed condition-specific genes which led us to the identification of the transcription factor MEOX1 in Treg cells. Expression of MEOX1 was comparable to FOXP3 in Treg cells and can be upregulated by IL-2. Epigenetic analyses revealed a permissive epigenetic landscape for MEOX1 solely in Treg cells. Knockdown of MEOX1 in Treg cells revealed a profound impact on downstream gene expression programs and Treg cell suppressive capacity. These findings in the context of CD4+ T cells contribute to a better understanding of the transcriptional networks and biological mechanisms controlling CD4+ T cell functionality, which opens new avenues for future therapeutic strategies.
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