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. Author manuscript; available in PMC: 2017 Oct 15.
Published in final edited form as: J Immunol. 2016 Sep 21;197(8):3076–3085. doi: 10.4049/jimmunol.1600197

Fig. 2. Intracellular RAGE binding augments pro-inflammatory cytokine production in activated T1D T cells.

Fig. 2

(A) Serum HMGB1 levels were similar in at-risk progressors (open circles), at-risk non-progressors (filled circles), established T1D (triangles) patients and HC (squares) individuals (mean±SEM). (B) Enriched T1D T cells were analyzed for RAGE-HMGB1 interaction using a proximity ligation assay (PLA) in CD8+ T cells from a patient with T1D. After 4 hours, in culture with his-tagged HMGB1 or HSA proximity of the his-tagged protein and RAGE is detected by PLA expression (red, arrows), nuclear staining by DAPI (blue) in CD8+ T cells (green). Data from a single experiment representative of 3 is shown. (C) Cytokine secretion from activated T1D (black bars, n = 3) and HC (white bars, n = 4) T cells were compared as fold induction in the presence of HMGB1 (100 ng/mL) after 72h stimulation. There was greater release of cytokines from cells from patients with T1D vs HC (p<0.0001). Statistical significance was determined using a two-way ANOVA. (**** < 0.0001, *<0.05, mean±SEM). (D-E) Intracellular IFNγ (D, red=CD4, blue=CD8) and IL-17a (E, CD8 only) in activated patient cells with or without HMGB1 stimulation (**p=0.0078 Wilcoxon signed-rank test). (F) HMGB1 induced cytokine secretion was also measured in enriched T cells from T1D patients transfected with RAGE (black) or Ctl (white) siRNA as above (n=6). There was significant reduction in cytokine release in cells that had received RAGE vs Ctl siRNA (p<0.0001)(*p<0.05 ***p<0.001, mean±SEM).