Figure 4.

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.