MSC inhibit the up-regulation of CD86 and MHC class II (Ia^b) in M stimulated by LPS and impairs antigen presentation to T CD4+ cells.

<p>(A) M were cultured overnight in the absence or presence of MSC (M: MSC ratio = 10∶1). Cells were then washed and incubated for 18 h with or without LPS (30 ng/ml), and the expression of CD86, MHC class II (Ia^b), CD40 and MHC class I (H2D^b) in M was analyzed using FITC-labeled antibodies directed to CD11b and PE-labeled antibodies directed to CD86, Ia^b, CD40, and H2D^b, in the gate of CD11b-positive cells. Results are expressed as the mean fluorescence intensity (MFI) values and represent the arithmetic mean ± SEM of 4-5 experiments. *, p<0.05 for M+MSC+LPS vs M+LPS. (B) M (1x10⁵ cells/100 µl) were cultured overnight in 96 well-flat bottom plates with culture medium alone or MSC-conditioned medium (50% V/V). After washing, cells were treated with LPS (30 ng/ml) in the presence of OVA (500 µg/ml) for 18 hs. After this period, M were washed and fresh culture medium was added. Spleen T CD4+ cells from OT-II mice were purified and labeled with CFSE, as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009252#s2" target="_blank">Materials and Methods</a>. Macrophages and CFSE-labeled OT-II T CD4+ cells were cultured together (M∶T cell ratio 1∶5) for 72 h. Cells were stained with PE-labeled anti-CD4 mAb, and the proliferation of the CFSE-labeled T CD4+ cells was analyzed by flow cytometry. A representative experiment is shown (n = 5). No proliferation was observed when LPS-stimulated M were cultured without OVA.