The extent of T cell activation in each condition is shown as the division index relative to that of LPS-treated DCs in the absence of IL-10 at a DC:T of 1 1:10. studies, values were calculated using a two-tailed paired Student’s test. values 0.05 were considered significant. values are indicated by * ( 0.05), and non-significant differences are indicated by ns. Results IL-10 Reduces MHC-II and CD86 Expression on Activated M To begin to examine the mechanism(s) by which IL-10 suppressed the ability of M to activate CD4 T cells, bone marrow-derived M were activated by treatment with IFN- for 24 h and then cultured alone or with IL-10 for an additional 18 h. Unstimulated M did not express MHC-II and experienced very low expression of CD86 and CD40 (Fig. 1and 0.05; and and and and and 0.05; and and 0.05; 0.05; histogram. histogram). The extent of T cell activation in each condition is usually shown as the division index relative to that of LPS-treated DCs in the absence of IL-10 at a DC:T of 1 1:10. The data shown are the mean S.D. (of DC maturation and whether March-I played any role in the effect of IL-10 on DC maturation and/or function. BMDCs obtained from wild-type mice or March-I-deficient mice were cultured alone, with LPS only, or with LPS and IL-10 together for 18 h, and expression of various DC activation markers was quantitated by FACS analysis. IL-10 inhibited (but did not completely prevent) the LPS-induced up-regulation of surface MHC II, CD86, and CD40 expression on wild-type BMDCs (Fig. 6histogram. The geometric mean fluorescence intensity of FLT3 the indicated protein was expressed relative to that in cells treated with LPS alone. The data shown are the mean S.D. (represents the baseline expression of each marker in untreated cells. *, 0.05. 0.05. 0.05. We also examined the effects of IL-10 around the maturation of mouse spleen DCs 0.05; 0.05; and histogram). Data shown are the CD4 T cell division index relative to that using DCs cultured with LPS alone at a DC:T of 1 1:20. 17 alpha-propionate The mean S.D. ( 0.05. The ability of IL-10 to suppress activation-induced reduction in March-I mRNA was also resolved using a functional assay. MHC-II is usually oligo-ubiquitinated in immature DCs, and activation of immature DCs with LPS for as little as 4 h resulted in a dramatic reduction in MHC-II ubiquitination (Fig. 7and that IL-10 stimulates March-I expression in LPS-treated DCs, in reality, IL-10 just suppresses the well documented process of DC activation that leads to March-I down-regulation. Our finding that down-regulation of MHC-II ubiquitination by LPS was partially reversed by IL-10 provides functional biochemical evidence for the lack of total DC activation by IL-10. In conclusion, our study discloses fundamental differences in the molecular mechanisms by which IL-10 mediates immunosuppression in DCs and M. Whereas suppression of antigen presentation by M is usually March-I-dependent, suppression of antigen presentation by DCs is not. Our study also suggests that March-I may be an important target for modulating the immunosuppressive effects of IL-10 in M but not DCs, and therefore for the development of maximally effective DC-based vaccines, other (non-March-I) targets must be recognized. Author Contributions S. K. M. performed experiments, analyzed data, and published the manuscript. K.-J. C. performed experiments and analyzed data. S. I. provided experimental mice and published the manuscript. P. A. R. analyzed data and published the manuscript. Acknowledgments We thank Dr. Michael Kruhlak of the Experimental Immunology Branch Microscopy and Digital Imaging Facility for assistance with confocal microscopy studies. We thank Drs. Dragana Jankovic, Alfred Singer, and Richard Hodes for scientific guidance and helpful feedback during the course of these studies. *This work was supported by the Intramural Research Program of the National Institutes of Health (to P. A. R.), the Ministry of Education, Culture, Sports, Science and 17 alpha-propionate Technology of Japan (to 17 alpha-propionate S. I.), and the Japan Society for the Promotion of Science (to S. I.). The authors declare that they have no conflicts of interest with the contents of this article. 2The abbreviations used are: APCantigen-presenting cellDCdendritic cellMmacrophage(s)TLRToll-like receptorpMHC-IIpeptideMHC-II complex(es)BMDCbone marrow-derived dendritic cellCFSEcarboxyfluorescein succinimidyl esterOVAovalbumin..