Journal: Frontiers in Immunology

Article Title: Transcriptome Analysis of CCR9+ T Helper Cells From Primary Sjögren’s Syndrome Patients Identifies CCL5 as a Novel Effector Molecule

doi: 10.3389/fimmu.2021.702733

Figure Lengend Snippet: Validation of selected target genes from the blue module in replication cohorts confirms differences between Th subsets, but not between HC and pSS patients. According to the criteria shown in Figure 3 and based on pathway enrichment analysis ( Figure 2 ) and literature, genes were selected from the blue module and were replicated by qPCR and flow cytometry. RNA sequencing data is shown in (A, C) , qPCR and flow cytometry data are shown in (B, D) , respectively. HOPX, SOX4, ITGA1, C3AR1 and ABCB1 were evaluated by qPCR in an additional replication cohort (B) . Genes encoding for surface proteins for which antibodies were available were replicated by flow cytometry: ITGAE (CD103), CD73 (NT5E), NCR3 (NKp30), CCR5 and ABCB1 (D) . NRC, normalized read counts; FC, fold change, DN HC mean is set at 1. *, **, *** indicates statistical significance of p < 0.05, 0.01, 0.001, respectively.

Article Snippet: To validate RNA sequencing results, TaqMan assays were performed for selected target genes ( ITGA1 Hs00235006_m1, C3AR1 Hs00269693_s1, ABCB1 Hs00184500_m1, CCL5 Hs00982282_m1, SOX4 Hs04987498_s1 and HOPX Hs04188695_m1, and B2M Hs00187842_m1, all from ThermoFisher, LifeTechnologies) using RNA extracted from FACS sorted CCR9+, CXCR5+ and CCR9-CXCR5- Th cell subsets from HC and pSS patients as described above.

Techniques: Biomarker Discovery, Flow Cytometry, RNA Sequencing