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OX40 regulates double-negative regulatory T cells dependently of its effects on conversion and function

ABSTRACT: Methods: To better understand the role of OX40 in the regulation of double-negative regulatory T cells (DNT), OX40 deficiency and wild-type DNT converted from naïve CD4 T cells were compared in a transcriptome sequencing study. Total RNA was isolated from FACS-separated DNT. Transcriptome sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and sequenced on an Illumina Hiseq platform (Illumina, San Diego, CA). Sequences were aligned to the reference genome with TopHat and processed with Cufflinks, which quantifies each transcript in each sample using reference annotations produced by the University of California Santa Cruz UCSC. Differentially expressed genes with a fold change of >=2.0 and padj < 0.05 between OX40 deficiency and control DNT were submitted to GO and KEGG enrichment analysis, which uses unbiased methods to assess pathway enrichment. Results: Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that OX40 knockout are involved in regulatory function of DNT; including suppressive function, apoptosis, proliferation, and so on. Compared to control DNT, OX40 knockout DNT exhibited up-regulation of 52 genes and down-regulated of 46 genes. The most enriched GO term were Signal Transduction, Protein Binding, and Banding.

ORGANISM(S): Mus musculus

PROVIDER: GSE107908 | GEO | 2020/12/08

REPOSITORIES: GEO

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Project description:Methods: To better understand the role of OX40 in the regulation of monocyte activation and polarization, OX40/Fc- or control IgG-treated liver monocytes were compared in a transcriptome sequencing study. Total RNA was isolated from FACS-separated hepatic monocytes. Transcriptome sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and sequenced on an Illumina Hiseq platform (Illumina, San Diego, CA). Sequences were aligned to the reference genome with TopHat and processed with Cufflinks, which quantifies each transcript in each sample using reference annotations produced bythe University of California Santa Cruz UCSC. Differentially expressed genes with a fold change of >=2.0 and padj < 0.05 between OX40 treated and control monocytes were submitted to GO and KEGG enrichment analysis, which uses unbiased methods to assess pathway enrichment. Results: Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that upregulated genes from OX40-stimulated monocytes are involved in inflammatory response; cytokine production regulation; lipid, cytokine and bacteria responses; innate immune response; enhanced cellular response to cytokines and the cytokine-cytokine receptor interaction; and the phagosome and chemokine signaling pathway, among others. Compared to control monocytes, OX40/Fc-treated monocytes exhibited upregulated levels of cytokines (Tnf, Il1a, Il1b, Il6 and Il12a), cytokine receptors (Il1rl1, Il2r, Il18r and Il23r), chemokines (Ccl3, Ccl4, Ccl22, Cxcl1, Cxcl2, Cxcl10 and Cx3cl1), chemokine receptors ( Ccrl2, Cxcr5 and Cxcr6) and antigen processing and presentation molecules (Cd40, Cd80, Cd86, Tlr1, Tlr2, and Ox40l). The coordinated action of various inflammatory modulators, signaling molecules, and transcription factors, including Nlrp3, Tank, Jak2, Stat5a, Hif1a, Nos2, Socs3, Mir155hg and Pparg, was determined to be involved in the OX40 regulation of monocyte M1 polarization. MyD88, Irf3, Irf5, Stat1, Nod1 and Nod2 may not be involved in OX40-triggered monocyte M1 polarization.

Project description:Primary biliary cholangitis (PBC) is a liver-specific autoimmune disease. Treatment of PBC with ursodeoxycholic acid (UDCA) is not sufficient to prevent disease progression, new and creative approaches to treating patients with PBC are urgently required. Double-negative T (DNT) cells, unique regulatory T cells, play crucial roles in maintaining immune system homeostasis. However, both the absolute number and proportion of liver DNT cells were reduced in PBC patients. Hence, it is worth pursuing that whether replenishment of DNT cells can improve the hepatic function and pathology of PBC. In this study, adoptive transfer of DNT cells significantly decreases plasma levels of ALT, AST, AMA-M2 and IgM in both dnTGFβ RII and 2OA-BSA-immunized PBC mouse models. In addition, DNT cells exhibit a strong suppression on liver T cells. While combination therapy with DNT cells and UDCA predominantly ameliorate liver inflammation and extensively inhibit hepatic T and B cells. In vitro study further reveals that UDCA promotes the proliferation of DNT cells, increased functional molecule perforin and reduced inhibitory molecule NKG2A and EPCR expression, resulting in augmenting the suppressive function of DNT cells via FXR/JNK signaling pathway in both mice and human DNT cells. In conclusion, UDCA augments the suppressive function of DNT cells via FXR/JNK. A single transfer of DNT cells in combination with UDCA strongly ameliorates PBC in mice. This study provides a potential application of DNT cell-based therapy for PBC. Methods: The DNT cells incubation with anti-CD3/CD28 antibodies were stimulated with UDCA (60uM) for 48 hours, then transcriptome sequencing studies were performed on DNT cells RNA.Transcriptome sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and sequenced on an Illumina Hiseq platform (Illumina, San Diego, CA). Sequences were aligned to the reference genome with TopHat and processed with Cufflinks, which quantifies each transcript in each sample using reference annotations produced by the University of California Santa Cruz UCSC. Differentially expressed genes with a fold change of >=1.41 and pvalue <= 0.05 between UDCA treated and control DNT cells were submitted to GO and KEGG enrichment analysis, which uses unbiased methods to assess pathway enrichment.

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OX40 regulates double-negative regulatory T cells dependently of its effects on conversion and function

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