Skip to main content
Fig. 4 | Journal for ImmunoTherapy of Cancer

Fig. 4

From: Tumor-released autophagosomes induces CD4+ T cell-mediated immunosuppression via a TLR2–IL-6 cascade

Fig. 4

Hsp90α on intact TRAPs is essential for IL-6 induction in CD4+ T cells. a, b ELISA of IL-6 secretion by CD4+ T cells treated with TRAPs (3 μg/ml), proteinase K-digested TRAPs (a) or sonicated TRAPs (b) for 72 h in the presence of anti-CD3/CD28. c Flow cytometric determination of the Hsp60, Hsp70, Hsp90α, or HMGB1 expression levels on the surface of TRAPs from B16F10 tumor cells. d ELISA of IL-6 secretion by CD4+ T cells treated with TRAPs (3 μg/ml) or blocking antibody-pretreated TRAPs (anti-HMGB1, anti-Hsp60, anti-Hsp70, anti-Hsp90α antibodies) for 72 h in the presence of anti-CD3/CD28. e-g CFSE-labeled TRAPs were pretreated with the indicated dose of functional an anti-Hsp90α antibody or an isotype-matched control antibody overnight at 4 °C and then co-cultured with purified CD4+ T cells in the presence of anti-CD3/CD28. Twenty-four hour later, the percentage of CFSE+ CD4+ T cells was assessed by flow cytometry (e, f). Seventy-two hour later, IL-6 levels in supernatants were determined by ELISA (g). h The Hsp90α level in tumor cell lysates, an equal amount of TRAPs, sonicated TRAPs, and proteinase K-pretreated TRAPs was determined by western blot. Purified CD4+ T cells were co-cultured with the above stimulators for 72 H. IL-6 levels in the supernatants were determined by ELISA. i ELISA of IL-6 secretion by human CD4+ T cells treated with 3 μg/ml human TRAPs (hTRAPs) from 3 human tumor cell lines (A375, MDA-MB-231 and HepG2 cells) or 8 tumor patient effusions and ascites, respectively for 72 h in the presence of anti-CD3/CD28. j ELISA of IL-6 secretion by human CD4+ T cells treated with hTRAPs (3 μg/ml) or anti-Hsp90α-pretreated hTRAPs for 72 h in the presence of anti-CD3/CD28. Data (mean ± SEM) represent 3 independent experiments. *, P < 0.05; **, P < 0.01; ns, not significant by one-way ANOVA with the Tukey-Kramer multiple test

Back to article page