Figure 7: LPS-induced iNOS and HIF-1α activity negatively affects GM-DC-induced T-cell responses.
From: Glucose represses dendritic cell-induced T cell responses
Hif1aflox/flox (WT) or Hif1aflox/flox VavCre (Hif1a−/−) GM-DCs (a,b,e,i,j) or WT and Nos2−/− GM-DCs (c,d,f–h,k,l) were pulsed with SIINFEKL peptide+/−LPS (100 ng ml−1) and cultured for a 1, 2 or 3 days. Nos2−/− GM-DCs were treated with S-nitroso-N-acetylpenicillamine (500 μM) every 5 h (c,d) or for the last 4 h of stimulation (f), cocultured with LPS+IFNγ-activated BMDMs (in a transwell cassette) for the last 4 h of stimulation (g) or treated with DMOG (200 μM) for the last 4 h of stimulation (h). (a–d) CD80 and CD86 expression was analysed by flow cytometry and (e–h) IL12a, IL10 and TNF mRNA by qPCR. (i–l) On the indicated day post LPS stimulation, GM-DCs were washed before the addition of purified carboxyfluorescein succinimidyl ester (CFSE)-labelled OT-I T cells. After a co-culture period of 48 h, the OT-I T cells were analysed by flow cytometry for proliferation as measured by CFSE dilution. Data are mean±s.e.m. or representative of three (e), four (i,j), five (a,b,g), six (f) or seven (c,d,k,l) separate experiments. qPCR analysis was performed in triplicate. Data were analysed using a one-way analysis of variance with Tukey’s post test (*P<0.05, **P<0.01, ***P<0.001).
