Mycobacterial infection promotes ATP-dependent shedding of MHC-II. C57BL/6 macrophages (2 × 10⁶ cells/well in six-well plates) were activated with IFN-γ (2 ng/ml) for 24 h. Cells were then primed for 4 h with LPS or infected overnight with mycobacteria (MOI of 3). Cells were transferred into BSS and stimulated with or without 5 mM ATP for 20 min. Extracellular medium was collected, and released proteins were precipitated with TCA. TCA-precipitated proteins and cell lysate were analyzed for MHC-II by Western blotting. (A) Analysis of MHC-II in TCA-precipitated proteins and cell lysates from macrophages activated with or without IFN-γ, primed with or without LPS (1 μg/ml), and stimulated with or without ATP. (B) Analysis of MHC-II in TCA-precipitated proteins and cell lysates from macrophages activated with IFN-γ, primed with various concentrations of LPS, and stimulated with ATP. (C) Analysis of MHC-II in TCA-precipitated proteins and cell lysates from macrophages activated with IFN-γ, primed with or without LPS (10 ng/ml) or M. tuberculosis H37Ra (Mtb Ra), and stimulated with or without ATP. (D) Analysis of MHC-II in TCA-precipitated proteins from macrophages activated with IFN-γ; primed with or without LPS (10 ng/ml), M. tuberculosis H37Ra, M. bovis BCG, and M. tuberculosis H37Rv (Mtb Rv); and stimulated with or without ATP. Results are representative of data from four independent experiments.

Microscopy reveals ATP-dependent shedding of membrane vesicles. Macrophages from C57BL/6 MHC-II-EGFP mice (green is MHC-II-EGFP) were plated into eight-chamber slides and activated with IFN-γ (2 ng/ml) for 24 h. Cells were incubated with 10 ng/ml LPS for 4 h (A and B) (magnification, ×189) or infected for 4 h with Bact-Light Red-labeled M. tuberculosis H37Ra at an MOI of 3 (C and D) (magnification, ×126). Cells were then transferred into BSS and monitored every 30 s for 10 min before and after the addition of 5 mM ATP using a high-speed Leica SP5 broadband confocal microscope. Images were acquired by using Leica Application Suite software and analyzed by using the Imaris software suite. Results are representative of data from independent experiments. Movies S1A to S1D in the supplemental material depict time-lapse microscopy under these conditions. Since Bact-Light Red-labeled M. tuberculosis H37Ra was highly susceptible to bleaching, the red laser was activated initially to assess M. tuberculosis infection but was not activated when analyzing the release of vesicles over the 10-min period. For each LPS experiment, five separate chambers of cells were analyzed with or without ATP. Within each chamber, a field containing approximately 10 cells was analyzed. Since two independent experiments were performed, the analysis included a total of approximately 100 cells, including those shown in C to E. For each M. tuberculosis experiment, three separate chambers containing cells were analyzed with or without ATP. Within each chamber, a field containing approximately 20 cells was analyzed. Since two independent experiments were performed, the analysis included a total of approximately 120 cells, including those shown in C to E.

Shed MHC-II is included in two distinct released membrane types, microvesicles and exosomes. C57BL/6 macrophages were activated with IFN-γ (2 ng/ml) for 24 h. Cells were then primed with LPS (10 ng/ml) for 4 h (A, left, B, and C) or 12 h (A, right) or infected with M. tuberculosis H37Ra (MOI of 3) for 4 h (A, left) or 12 h (A, right, and C). Cells were transferred into BSS and stimulated with 5 mM ATP for 20 min. The extracellular medium was collected and sequentially centrifuged at 300 × g, 2,000 × g, 10,000 ×g, and 100,000 × g. The 10,000 × g pellet (microvesicles) and the 100,000 × g pellet (exosomes) were analyzed by Western blotting (A and C) or electron microscopy (B). M, microvesicles; E, exosomes. Results are representative of data from two or more independent experiments.

Exosomes and microvesicles can present peptide-MHC-II complexes to T cells. C57BL/6 macrophages were activated with IFN-γ (2 ng/ml) for 24 h. Cells were then primed with LPS (10 ng/ml) for 4 h or infected for 12 h with M. tuberculosis H37Ra (MOI of 1 to 3). Cells were transferred into BSS and stimulated with 5 mM ATP for 20 min. Microvesicles and exosomes were isolated as described in the legend of Fig. , resuspended in standard medium, and incubated as follows. (A) LPS-induced microvesicles plus BB7 T hybridoma cells (10⁵) with or without 10 μM peptide [M. tuberculosis Ag 85B(241-256)]. (B) LPS-induced exosomes plus BB7 T hybridoma cells (10⁵) with or without 10 μM peptide [M. tuberculosis Ag 85B(241-256)]. (C) BB7 T hybridoma cells (10⁵) alone or with M. tuberculosis-induced microvesicles. (D) BB7 T hybridoma cells (10⁵) alone, with M. tuberculosis-induced exosomes, or with M. tuberculosis-induced exosomes plus 10 μM peptide [M. tuberculosis Ag 85B(241-256)]. (E) Fixed CBA/J macrophages (10⁵) and BB7 T hybridoma cells (10⁵) with or without M. tuberculosis-induced exosomes. M, microvesicles; E, exosomes. Results are displayed as means ± standard deviations (SD) from T-cell assays that were performed with duplicate samples of exosomes or microvesicles. Statistical analysis was performed by including data from three independent experiments. For each experiment, the mean value for each condition was normalized to the maximum CTLL-2 assay response, and normalized values from three independent experiments were assessed for statistical significance by an unpaired Student's t test. Differences were statistically significant for LPS microvesicles without peptide versus with peptide (as in A; P < 0.001), LPS exosomes without peptide versus with peptide (as in B; P < 0.005), M. tuberculosis microvesicles without peptide versus with peptide (as in C; P < 0.005), M. tuberculosis exosomes without peptide versus with peptide (as in D; P < 0.001), and fixed macrophages with or without M. tuberculosis exosomes (as in E; P < 0.001).

Naïve T cells are stimulated by endogenously processed M. tuberculosis Ag 85B presented by microvesicles but not exosomes from M. tuberculosis-infected macrophages. Naïve CFSE-labeled P25 CD4⁺ T cells were activated with M. tuberculosis-induced microvesicles (10.8 μg, 5.4 μg, or 2.7 μg per well) or exosomes (20.8 μg or 10.4 μg per well) for 6 days, and the percentage of cells undergoing CFSE dilution was assessed by flow cytometry. (A) Proliferation of naïve CFSE-labeled P25 T cells incubated with or without 2.7 μg microvesicles per well. Limiting amounts of material (and decreased primary T-cell survival in the absence of a stimulus) limited the number of events that could be analyzed to 5,116 for T cells stimulated with exosomes and 1,877 for control T cells (CFSE labeled, no stimulus). To compare the extents of proliferation of the two T-cell populations, event counts are normalized to the maximum event count under the condition. Since the experiment was designed to allow ample time for T cells to divide to enhance sensitivity, peaks for early stages of CFSE dilution are not present, and proliferating T cells have divided six or more times to dilute the CFSE label to the background fluorescence level. (B) Proliferation of naïve CFSE-labeled P25 T cells incubated with or without 20.8 μg exosomes per well. Results were analyzed as described above for A, with 6,654 events for the microvesicle conditions and 1,454 events for the control conditions (CFSE labeled, no stimulus). Results of both panels represent a single flow cytometry sample from one experiment that is representative of data from three independent experiments.