Figure 5
Suppression of CD4+ T cells by H. polygyrus bakeri-induced CD11c−CD11b+Gr1+ cells is nitric oxide (NO) dependent. (a) CD11c−CD11b+Gr1+ cells purified from mesenteric lymph node (MLN) and spleens of infected mice on day 7 after primary infection were co-cultured with carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled splenic CD4+ T cells purified from OT-II mice in the presence of mitomycin-C-treated splenic CD11c+ dendritic cell (DC) purified from naive mice. The co-cultures were stimulated with 1 nM OVA323–339 peptide, and the cells from triplicate wells were harvested 72 h later and pooled. The cells were stained with APC-conjugated anti-CD4 monoclonal antibody (mAb), and the CFSE dilution was analyzed by flow cytometry in gated CD4+ cells. (b) Supernatants were collected from the triplicate wells for each culture condition in a, and NO levels were determined using Griess reagent. Data are presented as mean±s.e.m. ***P<0.001. Culture conditions indicated as numbers are as follows: (1) CD4+ T cells+DC with medium; (2) CD4+ T cells+myeloid-derived suppressor cells (MDSC) with medium; (3) CD4+ T cells+DC+MDSC with medium; (4) CD4+ T cells+DC with N(omega)-hydroxy-nor-L-arginine (nor-NOHA) (300 μM); (5) CD4+ T cells+DC+MDSC with nor-NOHA (300 μM); (6) CD4+ T cells+DC with 1400W (100 μM); (7) CD4+ T cells+DC+MDSC with 1400W (100 μM); (8) CD4+ T cells+DC with both inhibitors; and (9) CD4+ T cells+DC+MDSC with both inhibitors. Data from one of two independent experiments are presented.
