(A) Expression of anergy-associated genes (qPCR) in Th1 cells activated
in the presence or absence of L/N or 3MA.
(B) Expression of anergy-associated genes (qPCR) in cells transfected
with non-targeting (Ctrl) or Atg7-specific siRNAs and activated
for 24 hr.
(C) Foxp3 gene expression (qPCR) in Th1 cells activated
in the presence or absence of 3MA. mRNA obtained from purified Tregs is included
as control.
(D)Th1 cells were anergized with anti-CD3 (Anerg), activated in the
absence (Preact) or presence of L/N (Preact-L/N) or 3MA (Preact-3MA) for 24 hr.
Cells were washed to remove inhibitors, cultured for 5 days with 50 ng/mL IL-2,
and re-stimulated. IL-2 production and cell proliferation were measured.
(E) IL-2 and cell proliferation were measured as in (D) in cells
transfected with Ctrl or Atg7 siRNAs 24 hr prior to
pre-activation.
(F) Wild-type or NFAT1-deficient Th1 cells were stimulated for 24 hr in
the presence or absence of 3MA. Cells were washed and re-stimulated after 5 days
to measure IL-2 production and cell proliferation.
Data represent mean + SEM from 3 (A–C), 4 (D and E), or 5 (F)
different experiments. *p < 0.05; **p < 0.01; ***p < 0.001,
ANOVA with Tukey post-test for (A), (C), (D), and (F); two-tailed t test for (B)
and (E). See also Figures
S2–S4.