Intracellular distribution of CD1b versus CD1c in immature DCs. (A) Confocal microscopy of immature DCs that were fixed, permeabilized, and double labeled with CY3-conjugated anti-CD1c mAb (F10/21A3) and CY2-conjugated anti-CD1b mAb (BCD1b3). Single channel analysis is shown for CD1c (left) and for CD1b (middle). The overlay (right) revealed vesicles containing both CD1b and CD1c (yellow), and vesicles containing only CD1c (red) or CD1b (green) (arrowheads). Bars, 2 μm. (B) Electron micrographs of cryosections of immature DCs labeled with CD1b-specific mAb BCD1b3 (top) or CD1c-specific mAb L161 (bottom). CD1b labeling was found at the PM, in endosomes (arrows) and MIICs, but not in mitochondria (m) or nuclei (n). Expression of CD1c was restricted to PM and endosomes (arrows) and was not found in MIICs (arrowhead). Bar, 0.5 μm. (C) Quantitation of the CD1b or CD1c staining in immunoelectron micrographs. The mean PM expression (the number of gold particles [Q] over a unit length of membrane [p]; left) for CD1b and CD1c was not significantly different (P = 0.48; for CD1b, n = 11, and for CD1c, n = 10). Quantitation of endosomal (End) distribution (including lysosomes and MIICs) of the CD1 proteins in the same samples revealed fourfold more CD1b labeling compared with CD1c (P = 0.0001; middle). In addition, for each individual section the ratio of endosomal to PM staining (End/PM) was determined (right), and again the mean of these ratios was three- to fourfold higher for CD1b compared with CD1c (P = 0.0002). The plasma membrane gold particle count was 404 for CD1b and 310 for CD1c labeling on surface areas of 615 and 544 U, respectively. Similarly, endosomal gold particles counted were 231 for CD1b and 27 for CD1c on surface areas of 535 and 262 U, respectively.

Accumulation of CD1b but not CD1c in lysosomal compartments and MIICs. Immature DCs were labeled for 22 h with radioactive [³⁵S]methionine/cysteine, homogenized, and subsequently fractionated by Percoll gradient centrifugation. (A) The relative distribution of the enzymes alkaline phosphodiesterase (APDE, ▵) and β-hexosaminidase (B-HEX, •) was determined to monitor the presence of plasma membrane or lysosomes in the Percoll gradient fractions, respectively. The distribution of total proteins (▪) is also shown. Membranes of each fraction were isolated and deglycosylated with PNGase F before immunoblotting with antibodies specific for TfR, Lamp-1, MHC class I, and CD1b and detection by chemiluminescence and autoradiography. (B) The distribution of MHC class II, MHC class I, CD1b, and CD1c molecules in Percoll fractions was compared by immunoprecipitation. Proteins from the postnuclear supernatant (PNS) were immunoprecipitated with either the specific antibody (Sp) or an isotype control antibody (Co). Only the CD1b and CD1c molecules were deglycosylated with PNGase F before SDS-PAGE.

Cytoplasmic tail dependence of the endosomal distribution of CD1c. (A) Transfected HeLa cells stably expressing CD1c.WT (top) and CD1c.TD (bottom) were fixed, permeabilized, and labeled with the specific anti-CD1c mAb F10/21A3. Bars, 10 μm. (B) HeLa cells were transiently transfected with equimolar quantities of plasmid encoding CD1c.WT, CD1c.TD, or with vector alone. After 48 h, intact cells were analyzed by flow cytometry for surface expression of CD1c. Results shown are mean values of the mean fluorescence intensity (MFI) for three independent samples of both transfections.

Characterization of CD1c-mediated lipid antigen presentation. (A) C1R B lymphoblastoid cells stably transfected with CD1b (circles) or CD1c (squares) were labeled with ⁵¹Cr and coincubated overnight at a constant antigen concentration (CD1b: 60 μg/ml mycolic acid; CD1c: 13 μg/ml of M. tuberculosis total lipid extract) and various concentrations of either chloroquine (0.02–62.5 μM; filled symbols) or concanamycin B (0.02–62.5 nM; open symbols). These target cells were then analyzed by a standard 4-h chromium release cytolysis assay as described in Materials and Methods using the CD1c-specific T cell line CD8.1 and the CD1b-specific T cell line DN1. (B) HeLa cells transfected with vector alone (mock; ▴) or with CD1c.WT (▪) or CD1c.TD (•) plasmid for stable expression were labeled with ⁵¹Cr. These cells were incubated overnight with increasing doses of antigen (M. tuberculosis total lipid extract), then harvested, washed once with PBS, and combined with the T cell line CD8.1 at an E/T ratio of 15:1 for 4 h. (C) Immature DCs were incubated with antigens presented by CD1c (10 μg/ml of total lipid extract from M. tuberculosis containing the Hex-1–PIP antigen) or MHC class II (0.1 mg/ml tetanus toxoid protein [TTprot] or 75 ng/ml of an antigenic 15-mer peptide from tetanus toxoid [TTpep]) for various times. The presentation of antigens was measured by the increase in [Ca²⁺]_i in the T cells specific for the CD1c-presented lipid antigen Hex-1–PIP (▪) or MHC class II–presented TTpep (▴) or TTprot (•), and was expressed as a percentage of maximal Indo-1 index after the subtraction of background values (T cells plus APCs without antigen). Specific T cell lines used were CD8.1 for CD1c and SP-14 for MHC class II. Results shown are representative of three independent experiments.