MTP is present in APCs. (A) cDNA was synthesized from 1 μg RNA from the indicated tissues. MTP transcripts were amplified by RT-PCR, and the volume of sample loaded was normalized to β-actin transcript levels. MTPΔ DCs were obtained from the liver of a pIpC-injected MTPmx1 mouse. (B) Protein was harvested from LMNCs isolated from MTPMx1 or C57BL/6 mice either injected or not injected with pIpC and B cell lines C1R and 721 transfected with human and mouse CD1d, respectively. 25 μg of cell lysate was loaded per lane, separated by reducing SDS-PAGE, and immunoblotted with polyclonal antisera to MTP:PDI. (C) Triglyceride transfer activity in 100 μg splenocyte lysate (▵), 50 μg splenocyte lysate (○), 100 μg splenocyte lysate with BMS197636 (▴), and 50 μg splenocyte lysate with BMS197636 (•) is shown. Assays were in triplicate with SDs less than 10% of the percent transfer at 3 h. Each well contained 100 picomoles of NBD-labeled triglyceride.

Chemical inhibition of mouse MTP causes a selective defect in CD1d antigen presentation. (A) MODE-K cells cultured in the presence of BMS212122 (MTPi) or vehicle were incubated with α-galcer for 3 h, washed, and co-cultured with DN32 cells. (B) Splenocytes were isolated from wild-type mice, incubated for 24 h with BMS212122, and washed. The exogenous antigens ovalbumin or α-galcer were added as indicated. Splenocytes were then co-cultured with autoreactive 24.8 NKT cells (left), DN32 NKT cells (center), or CD4⁺ cells from an OT-II transgenic mouse (right). *, P < 0.05. Results are representative of two independent experiments. (C) CD11c⁺ splenocytes were incubated for 24 h with BMS212122, DMSO, or 9-fluorenyl carboxylic acid, washed, and co-cultured with 50,000 24.8 NKT cells per well. *, P < 0.005; **, P < 0.05 compared with DMSO values. Results are representative of five independent experiments. (D) CD11c⁺ splenocytes were incubated for 4 d with BMS212122 or DMSO, pulsed with the indicated concentrations of α-galcer, washed, and co-cultured with DN32 NKT cells (E/T = 100,000:30,000). *, P < 0.05. (E) 30,000 CD11c⁺ splenocytes incubated for 24 h with BMS212122 or DMSO were washed and co-cultured with 100 μg/ml ovalbumin and 100,000 CD4⁺ cells from an OT-II transgenic mouse. Values are ±SD.

MTP in human cells is critical for CD1d antigen presentation. (A) U937 cells were treated with irrelevant or MTP-specific siRNA oligomers. RNA was isolated 48 h after silencing, and transcript levels of mtp and β-actin were determined by RT-PCR. (B) Silenced and mock-silenced U937 cells were incubated with α-galcer for 3 h, washed, and co-cultured with DN32 cells (E/T = 1:1). *, P < 0.001. Results are representative of two independent experiments. (C) U937 cells cultured with BMS212122 (MTPi) or vehicle for 3 d were incubated with α-galcer for 3 h, washed, and co-cultured with DN32 cells (E/T = 1:1). *, P < 0.001. (D) C1Rd cells cultured with BMS212122 (MTPi) or vehicle for 4 d were incubated with α-galcer for 4 h, fixed with 0.05% glutaraldehyde for 30 s, washed, and co-cultured with DN32 cells (E/T = 2:1). *, P < 0.02. Results are representative of three independent experiments. (E) C1Rd cells cultured with BMS197636 or vehicle were incubated with NKT cell lines derived from human peripheral blood in the presence of 1 ng/ml PMA. NKT cells co-cultured with C1R mock transfected cells in the same assay yielded 33.6 ± 9.8 pg/ml IFN-γ. (F) Human monocyte-derived DCs were stained with 42.1 (CD1d), anti–HLA-A,B,C (MHC class I), or isotype control antibodies (dashed lines) after 6 d of differentiation in the presence of BMS212122 (gray lines, MTPi) or vehicle control (black lines). Results are representative of two independent experiments. (G) Day 6 human moDCs differentiated in the presence of BMS212122 (○, MTPi) or vehicle (•) were incubated with α-galcer for 3 h, washed, and co-cultured with 50,000 DN32 cells. *, P < 0.05. Results are representative of two independent experiments. Values are ±SD.

Purified MTP transfers a lipid to recombinant CD1d in vitro. (A) 2 μg BSA or recombinant CD1d was coated onto microtiter wells and incubated with either lipid vesicles alone or lipid vesicles plus 0.5 μg purified MTP. Lipid vesicles contained either PE:NBD or TG:NBD. Donor PE vesicles were composed of 450 nmol unlabeled PE and 14 nmol PE:NBD per milliliter. Results are representative of six independent experiments. *, P < 0.01. (B) 2 μg of recombinant CD1d was coated onto microtiter wells and incubated with PE:NBD vesicles and increasing concentrations of purified MTP. *, P < 0.005; **, P < 0.001 compared with 0 μg MTP. Values are ±SD.

Chemical inhibition of MTP in mouse BMDCs causes a selective defect in CD1d antigen presentation. (A) BMDCs cultured for 6 d with BMS212122 were washed and co-cultured with 50,000 24.8 NKT cells per well. *, P < 0.01. Results are representative of three experiments. (B) BMDCs cultured for 4 d with BMS212122 were pulsed with α-galcer, washed, and co-cultured with 100,000 DN32 NKT cells. *, P < 0.05. Results are representative of three experiments. (C) BMDCs cultured for 4 d with BMS212122 or 9-fluorenyl carboxylic acid were stained with the FITC-conjugated mAb 1B1 and analyzed by flow cytometry. The dashed line represents the isotype control. Results are representative of three independent experiments. (D) 50,000 BMDCs incubated for 3 d with BMS212122 or DMSO were washed and co-cultured with 100 μg/ml ovalbumin and 100,000 CD4⁺ cells from an OT-II transgenic mouse. Values are ±SD.