Formation and Kinetics of MHC Class I-Ovalbumin Peptide Complexes on Immature and Mature Murine Dendritic Cells  Nicole A. Kukutsch, Susanne Roßner, Jonathan M. Austyn, Gerold Schuler, Manfred B. Lutz  Journal of Investigative Dermatology  Volume 115, Issue 3, Pages 449-453 (September 2000) DOI: 10.1046/j.1523-1747.2000.00084.x Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Dose-dependent binding of OVA peptide to MHC class I molecules. Duplicate samples of BM-DC were incubated for 8 h without or with the indicated concentrations of OVA peptide. Thereafter cells were double-stained for MHC class II and peptide/Kbcomplexes. (A) Gates were set according to MHC class II expression to distinguish between the precursors/granulocytes (MHC IIneg), immature DC (MHC IIlow), and mature DC (MHC IIhigh). Staining with 25D1.16 antibodies without peptide (backround) and with 10 μM and 100 μM peptide is shown. (B) Cells were pulsed with peptide in serum-free (HL1) or serum-containing (R10) medium. Mean fluorescence values are shown for each condition and plotted in a graph. Peptide/Kb complex expression was clearly seen at a concentration of 1 μM peptide on cells within the mature DC gate. A 10-fold higher concentration of peptide was neccessary to receive a clear staining on cells within the immature DC gate. No difference was found between pulse in serum-free or serum-containing medium. The data shown are representative for five independent experiments. Journal of Investigative Dermatology 2000 115, 449-453DOI: (10.1046/j.1523-1747.2000.00084.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Time kinetics of peptide pulse for the expression of peptide/Kb complexes and total Kb molecules in media with and without fetal calf serum. Duplicate samples of BM-DC were pulsed with 50 μM OVA peptide SIINFEKL in serum-free (HL1) and serum-containing (R10) medium for different time durations. (A) Mature DC gave a significant higher staining of peptide/MHC I complexes that increased during the pulse time as compared with immature DC. (B) Total MHC class I expression was stable during the pulse for all subsets. No differences were observed between the pulse in serum-free or serum-containing medium. The data shown are representative for five independent experiments. Journal of Investigative Dermatology 2000 115, 449-453DOI: (10.1046/j.1523-1747.2000.00084.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 LPS and Poly I:C induce maximal upregulation of MHC class II and MHC class I molecules on DC. On day 6 BM-DC were treated with either TNF-α, LPS, Poly I:C, or PBS for 16 h, harvested, and double-stained for MHC class II and MHC class I expression. (A) MHC I was low on the MHC IIneg gate but expression was increased during further DC development via MHC IIlow and MHC IIhigh stages. (B) The percentage of the MHC class IIhigh subpopulation increased after pretreatment with TNF-α, LPS, or Poly I:C. (C) The MHC class I upregulation expressed by MFI was strikingly higher on the MHC IIhigh subpopulation after LPS or Poly I:C but not after TNF-α pretreatment. Journal of Investigative Dermatology 2000 115, 449-453DOI: (10.1046/j.1523-1747.2000.00084.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 The surface expression of peptide/Kb complexes on mature DC can be prolonged by pretreatment with LPS or Poly I:C. BM-DC were pretreated with TNF-α, LPS, Poly I:C, or PBS for 16 h, washed, and pulsed with or without 50 μM OVA peptide for 6 h, washed again, and chased for the indicated times. (A) Peptide/Kb complexes were increased after pretreatment with LPS and Poly I:C on all DC subsets (MFI at time 0). Complex expression was higher on mature DC than on immature DC and precursors. After 72 h still a significant staining for peptide/Kb complexes could be observed on mature DC after pretreatment with LPS or Poly I:C (**). This MFI value was about equal to the value after 24 h chase for the TNF-α and PBS pretreated cells (*). (B) After 0, 24, 48 h of chase total Kb expression was slightly increased on mature DC after pretreatment with LPS and Poly I:C compared with untreated and TNF-α-treated cells. It was also increased on immature DC after 0 and 24 h chase after LPS and Poly I:C pretreatment. LPS and Poly I:C pretreatment increased the level of MHC class I surface expression to a similar extent and were higher compared with TNF-α pretreated cells. Journal of Investigative Dermatology 2000 115, 449-453DOI: (10.1046/j.1523-1747.2000.00084.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions