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RNA-seq analysis of GCBC and NBC that had been stimulated for either 2h or 4h with IL-21, CD40 ligation, BCR ligation, or combinations of these.

ABSTRACT: RNA-seq analysis of GCBC and NBC that had been stimulated for either 2h or 4h with IL-21, CD40 ligation, BCR ligation, or combinations of these.

PROVIDER: PRJNA811522 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR18183501.fastq.gz	Fastqsanger.gz
	SRR18183501_1.fastq.gz	Fastqsanger.gz
	SRR18183501_2.fastq.gz	Fastqsanger.gz
	SRR18183502.fastq.gz	Fastqsanger.gz
	SRR18183502_1.fastq.gz	Fastqsanger.gz

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RNA-seq analysis of GCBC and NBC that had been stimulated for either 2h or 4h with IL-21, CD40 ligation, BCR ligation, or combinations of these.

Project description:Purpose: To gain insight into both similarities and differences in the gene expression programs induced by IL-21R, BCR and CD40 signals, we performed RNA-seq on both GCBC and NBC that had been stimulated for either 2h or 4h with IL-21, CD40 ligation, BCR ligation, or combinations of these. Methods : Purified GCBC from d14 NP-CGG immunized MEG mice and NBC from naïve MEG mice were warmed at 37C for 30 minutes and then stimulated with 10ng/mL IL-21, 20μg/ml ⍺-IgM or 2.5μg/mL ⍺-CD40 (prepared as tetramer) alone or combined IL21/⍺-CD40 or ⍺-IgM/⍺-CD40 for 2 and 4 hours. After stimulation, cells were processed for RNA isolation using QIAshredder columns (Qiagen) and the RNeasy Plus Mini Kit (Qiagen) following the manufacturer’s instructions. Samples were sequenced using Illumina NextSeq 500 with 75 bp paired-end reads and aligned to the mm10 genome using the STAR aligner (Dobin et al., 2013). Gene-level counts were determined using featureCounts (Liao et al., 2014), and raw counts were quantile normalized to each other for differential expression using the voom method (Law et al., 2014) in the Limma R package (Ritchie et al., 2015). For normalization of the datasets, the Quantile method was used. Results: Like BCR and CD40 signals, IL-21R plus CD40 signals also synergize to induce c-Myc in GCBC. However, IL-21R plus CD40 stimulation differentially affects GCBC fate compared to BCR plus CD40 ligation—engaging unique molecular mechanisms

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Project description:The interaction between the transmembrane glycoprotein surface receptor CD40 expressed by skin keratinocytes (KCs) and its T cell-expressed ligand CD154 was suggested to exacerbate inflammatory skin diseases. However, the full spectrum of CD40-mediated effects by KCs underlying this observation is unknown. Therefore, changes in gene expression after CD40 ligation of KCs were studied by microarrays. CD40-mediated activation for 2 hours stimulated the expression of a coordinated network of immune-involved genes strongly interconnected by IL8 and TNF, while after 24 hours anti-proliferative and anti-apoptotic genes were upregulated. CD40 ligation stimulated the production of chemokines that attracted lymphocytes and myeloid cells from peripheral blood mononuclear cells (PBMCs). Thus, CD40-mediated activation of KCs resulted in a highly coordinated response of genes required for the local development and sustainment of adaptive immune responses. The importance of this process was confirmed by a study on the effects of human papilloma virus (HPV) infection to the KCâs response to CD40 ligation. HPV infection clearly attenuated the magnitude of the response to CD40 ligation and consequently the KCâs capacity to attract PBMCs. The fact that HPV attenuates CD40 signalling in KCs indicates the importance of the CD40-CD154 immune pathway in boosting cellular immunity within epithelia. Total RNA from eight 72 hours 50 IU/ml IFNgamma pre-stimulated primary undifferentiated keratinocyte cultures, four uninfected and four HPV-positive, that were either left unstimulated, or stimulated for 2 hours with IFNgamma and Control- or CD40L-expressing L-cells (mouse fibroblasts), or stimulated for 24 hours with IFNgamma and Control- or CD40L-expressing L-cells. The stimulated samples were generated in duplo. 72 samples were generated in total.

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Single cell RNA-seq (scRNA-seq) of 48h GCBC cultures with either of these two stimuli or with the combination of IL-21R/CD40 and BCR stimulation.

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