Figure 4

The suppressive effect of GMPs is NO-dependent.

(A) Minimal effect of separating cell-cell contact on the GMPs-mediated suppression. 1 × 10⁶ CFSE-labeled B6 splenocytes were co-cultured with 2.5 × 10⁵ BM cells in a transwell system with splenocytes and BM cells in the lower and upper compartment, respectively. After 3-day incubation in the presence of anti-CD3/anti-CD28 antibodies, cells were recovered and analyzed by FACS. (B) Relative expression of iNOS and Arg.1 by GMPs. FACS-sorted cells were extracted for total RNA and mRNA levels were determined by RT-PCR using a commercial kit. Representative data of three independent experiments are shown. (C) Production of NO by GMPs. Samples were collected from non-specific T cell proliferation assay cultures as described in Fig. 1A and the levels of NO in supernatants were determined using Greiss reagents per the manufacturer’s protocol. Values are mean ± SD of triplicate samples and representative of three experiments. (D,E) Impact of iNOS- and arginase-specific inhibitors on the suppression of GMPs. Non-specific T cell proliferation assays were performed and cells were cultured in the absence or presence of various concentration of L-NMMA (iNOS inhibitor, (D)) or nor-NOHA (arginase inhibitor, (E)). Data shown are mean ± SD of triplicate samples and representative of two reproducible experiments. ^***p < 0.001; ^**p < 0.01; ^*p < 0.05.