Skip to main content
. 2021 May 5;8(14):2004973. doi: 10.1002/advs.202004973

Figure 2.

Figure 2

The lysine motif of CTLA‐4 is required for Foxp3 induction and Treg‐mediated EAE suppression. a–c) Experimental autoimmune encephalomyelitis (EAE) was induced by MOG35–55 in CFA emulsion with PTX. After immunization, C57BL/6 mice were treated with different doses of CTLA‐4 signaling peptide (50, 100, 200 µg, i.p.) from days 7 to 12 (5 mice per group, representative data from two independent experiments). a) Experimental scheme used to induce EAE. b) Clinical score of EAE. c) Lymphocytes that had infiltrated the spinal cord were analyzed by flow cytometry on day 13. d–f) EAE was induced in mice depleted of Tregs using α‐CD25 antibody (250 µg, two times i.p. injections). After immunization, C57BL/6 mice were treated with CTLA‐4 signaling peptide (100 µg, i.p.) every day from days 7 to 17 (5 mice per group, representative data from two independent experiments). d) Experimental scheme of Treg depletion in the EAE mouse model. e) Clinical score of Treg‐depleted EAE mice. f) Percent of EAE incidence in the Treg‐depleted EAE model. g–k) EAE was induced by MOG35–55 in the CFA emulsion with PTX. After immunization, mice were treated with CTLA‐4 WT, KA, or YF peptide (100 µg, i.p.) every day from days 7 to 13 (4–10 mice per group, two independent experiments). g) Schematic images of the WT synthetic CTLA‐4 signaling peptide (WT), the lysine mutant (KA), and the tyrosine mutant (YF). h) EAE clinical score. i) Number of infiltrated lymphocytes (left) and Treg/Teff ratio (right) in the spinal cord were analyzed at day 14. j) Histological analysis of spinal cord tissue stained with Luxol fast blue at day 14. The area of demyelination was marked with a dashed black line. k) Quantification of infiltrated lymphocytes in a section of spinal cord using ImageJ software. Data are presented as mean ± S.E.M. in b,e,h) and mean ± S.D. in c,i,k). Statistical significance was determined by Friedman test with post‐hoc Dunn's test in b,e,h) or Kruskal–Wallis test in c,i,k). n.s. = nonsignificant, *p < 0.05, **p < 0.01, ***p < 0.001.