Gene Expression
Nebulas
Gene Expression NebulasSummary: Dysregulated IL-23/IL-17 responses have been linked to psoriatic arthritis and other forms of spondyloarthritides (SpA). RORγt, the key Thelper17 (Th17) cell transcriptional regulator, is also expressed by subsets of innate-like T cells, including invariant natural killer T (iNKT) and γδ-T cells, but how they contribute to disorders such as SpA is still unclear. Here we describe the presence of particular RORγt+T-betloPLZF- iNKT and γδ-hi T cell subsets in healthy peripheral blood. RORγt+ iNKT and γδ-hi T cells showed profound IL-23 mediated Th17-like immune responses and were clearly enriched within inflamed joints of SpA patients where they act as major IL-17 secretors. SpA derived iNKT and γδ-T cells showed a unique Th17 skewed phenotype and gene expression profile. Strikingly, RORγt inhibition blocked γδ17 and iNKT17 cell function while selectively sparing IL-22+ subsets. Overall, these findings highlight a unique diversity of human RORγt+ T cells and underscore the potential of RORγt antagonism to modulate aberrant type 17 responses.
Overall Design: iNKT (CD3+TCRVb11+6B11+), γδ-T (CD3+TCRγδ+) cells and Tconv (CD3+CD161-; negative for iNKT and γδ-T markers) cells were sorted from peripheral blood samples of SpA patients (n=7) and RA patients (n=5). Sequence-libraries of each sample were sequenced on a NextSeq500 system (Illumina).
| Strategy: |
|
| Species: |
|
| Tissue: |
|
| Healthy Condition: |
|
| Cell Type: |
|
| Growth Protocol: | For iNKT assays, PBMC were cultured in 24-well plates (1.5 × 106 cell/well) for 14days in the presence of αGalCer (100 ng/mL, in house made) and IL-23 (20 ng/mL), IL-1β (10 ng/mL), TGFβ1 (5 ng/mL; all eBioscience), and IL-2 (5 U/mL, Roche) or IL-2 alone. Similar experiments were performed with sorted iNKT and γδ-T cell in U bottom 96 well plates (50,000 cell/well) where we added 50,000 irradiated (40 Gy) T cell depleted PBMC cell (using CD2 dynabeads, ThermoFisher) as feeder/antigen presenting cell. Cytokine titers in supernatants were then determined by ELISA (ebioscience) or multiplex protein assays (MSD).; - |
| Treatment Protocol: | Patients were treatment naïve or under treatment with a NSAID and/or DMARD (Methotrexate, Sulfasalazin) |
| Extract Protocol: | RNA was isolated by means of the RNeasy Micro kit following manufacturer’s instructions (Qiagen). |
| Library Construction Protocol: | From the sheared material, sequencing libraries were prepared with the NEBNext Ultra DNA Library Prep Kit for Illumina (version 6.0–2/18), according to the manufacturer’s protocol including a size selection to 250bp insert size. |
| Molecule Type: | poly(A)+ RNA |
| Library Source: | |
| Library Layout: | SINGLE |
| Library Strand: | - |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina NextSeq 500 |
| Strand-Specific: | Unspecific |