1930 Richard Apps et al. Eur. J. Immunol. 2007. 37: 1924–1937 trophoblast and untransfected cells as controls. MDDC maturation was stimulated by TLR4 ligand (LPS), inflammatory cytokines (TNF-a, IL-6 and IL-1b) or soluble CD40 ligand. During MDDC differentiation, HLA-G was presented either concomitantly with the maturation stimulus or preceding it. The results were essentially similar. MDDC expressed the LILRB1 and B2 receptors, and these were down-regulated in co-culture with cells expressing HLA-G but not with control cells (Fig. 6A). This is consistent with an interaction occurring between the LILR and HLA-G. HLA-DR, costimulatory molecules (CD40, CD80, CD86) and the activation marker CD83 were up-regulated on MDDC in response to all three maturation stimuli (Fig. 6A). Up- regulation of these markers was not affected by HLA-G and is consistent with maturation to an antigen- presenting phenotype. However, analysis of the cyto- kines produced by these activated MDDC did show changes when HLA-G was present. MDDC activated in the presence of HLA-G demonstrated a significant increase in IL-6 and IL-10 production (p = 0.04 and p = 0.01, respectively) (Fig. 6B). In contrast, there was no effect on TNF-a (Fig. 6B) or IL-8 secretion (not shown). These experiments indicate that when DC are activated, although HLA-G does not affect the acquisi- tion of maturation markers, it does alter their cytokine secretion by increasing IL-6 and IL-10 production. HLA-G stimulating LILRB on HLA-DR+ cells results in inhibited allogeneic lymphocyte proliferation We next investigated how DC exposed to HLA-G would function in the presentation of allogeneic stimuli to T cells. Previously, the presence of HLA-G in an MLR has Figure 6. MDDC activated in the presence of HLA-G show normal up-regulation of maturation markers but altered cytokine production. (A) MDDC, co-cultured with HLA-I 721.221 cells, were analysed for expression of the indicated LILR receptors, differentiation and activation markers in the absence of stimulation (filled histogram) or after exposure to LPS (green trace). The same markers were analysed after exposure to LPS in the presence of HLA-G transfectants instead of HLA cells (red trace). Isotype control staining for each marker is shown in orange. (B) Levels of the indicated cytokines in these co-culture supernatants were measured by ELISA on two separate occasions. A representative experiment is shown here, SD of triplicate measurements. Culture supernatants from at least four independent experiments were tested and analysed together for statistical significance. f 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu