Figure 5: TNFi-exposed IL-17+ CD4+ T cells express increased levels of the transcription factor IKZF3 associated with IL10 expression.

CD4+ T cells and monocytes were co-cultured with anti-CD3 mAb in the absence or presence of adalimumab, and IL-17+ CD4+ T cells were sorted on day 3. (a) The RMA-normalized expression intensity for IKZF3 is shown for n=9 paired samples (mean with s.e.m.). (b) IKZF3 expression (mean with s.e.m.) was determined in three independent samples by real-time PCR, normalized to GAPDH. (c) Confocal microscopy of sorted control and TNFi-exposed IL-17+ CD4+ T cells stained for Aiolos (green). Nuclei were stained with DAPI (blue). Inserts (right) show single cells, demonstrating co-localization of Aiolos with DAPI in both conditions. Scale bars, 40 μm; 10 μm in inserts. (d) Quantification (mean grey value with 95% CI, corrected for background) of Aiolos staining per nucleus from confocal microscopy images. The experiment was repeated in three independent donors with similar results. (e,f) Western blot analysis of lysates from sorted control and TNFi-exposed IL-17+ CD4+ T cells probed with mAbs against Aiolos or β-actin. A single blot (e) and cumulative data (mean with s.e.m.) quantified by densitometry (n=4. Normalized to actin by densitometry) (f) are shown. Full gels are shown in Supplementary Fig. 8. (g) Correlation between IKZF3 and IL10 expression in array samples (see Fig. 4 for description), analysed by Pearson test. (h) IKZF3 expression (normalized to PPIA, mean with s.e.m., n=3) was measured by real-time PCR in sorted control or TNFi-exposed IL-17-IL-10- (Negs), IL-17+IL-10− (IL-17+), IL-17−IL-10+ (IL-10+) and IL-17+IL-10+ (dp+) CD4+ T cells. (i) Correlation between IKZF3 and IL10 expression in primary CD4+ T cells isolated from healthy donors as measured by real-time PCR (normalised to SDHA) and analysed by Pearson test.