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. 2008 Nov 26;105(49):19396–19401. doi: 10.1073/pnas.0806855105

Fig. 4.

Fig. 4.

CTLA-4-mediated regulation of TCR-associated signaling is impaired in RA Treg. (A) FACS-sorted Tresp (CD4+CD25−CD127+) from healthy controls and RA patients were sorted and stimulated with anti-CD3 (1 μg/106 cells)/CD28 (1 μg/106 cells) antibodies for 10 min at 37 °C. Results represent the mean ± SE from 6 healthy individuals and 6 patients with RA. (B) FACS-sorted Treg (CD4+CD25+CD127−) were stimulated with anti-CD3 (1 μg/106 cells)/CD28 (1 μg/106 cells) ± anti-CTLA-4 (5 μg/106 cells) antibodies for 10 min. Phosphorylation of signaling molecules was evaluated by flow cytometry after intracellular staining with phosphospecific antibodies. Shown are percentage increases in protein tyrosine-phosphorylation (p) levels of TCR-associated signaling proteins in stimulated Treg. Results represent the mean ± SE from 6 healthy individuals and 6 patients with RA. (C) PBMC were stimulated as in B and the induction of phosphorylation was evaluated in the CD4+Foxp3hi subset. Results represent the mean ± SE from 6 healthy individuals, 6 patients with RA, and 6 patients with PsA. (D) Analysis of total protein-tyrosine phosphorylation in Treg from patients with RA and healthy controls. FACS-sorted Treg were stimulated for 10 min at 37 °C with anti-CD3 (1 μg/106 cells)/CD28 (1 μg/106 cells) ± anti-CTLA-4 (5 μg/106 cells). Samples from 3 different healthy individuals or 3 different RA patients (matched for disease activity score) were pooled before loading onto gels for protein phosphorylation analysis by Western blot. The lower gels show incubation of membranes with anti-S 473-Akt and anti-β-actin antibodies. Also indicated are the phospho Akt/β-actin ratios obtained from densitometry analysis. (E) Induction of phosphorylation of Akt in CD4+Foxp3hi T cells, PBMC stimulated as in B. Results represent the mean ± SE from 6 healthy individuals, 6 patients with RA, and 6 patients with PsA. (F) Purified Treg were mixed with beads coated with anti-CD3/CD28 antibodies. Activation was stopped at 10 min with 2% paraformaldehyde, and cells were stained for Foxp3 and CTLA-4. Recruitment of CTLA-4 (blue) to the immunological synapse (beads:Foxp3+ cell depicted) is indicated (arrow). Confocal microscopy images are representative of at least 25 conjugates from 4 healthy controls and 4 patients with RA. (Magnification: 63×.) *, P < 0.05.