Phagosomal TAP transports peptides into the lumen.

<p>A) Isolated GFP-Mtb-phagosomes were incubated with TMR-conjugated CFP10_2–12 peptide (blue), then fixed and stained with anti-TAP1 (red) and analyzed by fluorescence microscopy. B) TMR-conjugated CFP10_2–12 peptide (500 nM) and UL49.5-NP (10 uM) or UL49.5-SCR (10 uM) were added simultaneously to isolated Mtb phagosomes and incubated at 37° C for 30 min. Washed and fixed phagosomes were analyzed by FACS. FACS plot is representative of three independent experiments. C) Prior to infection, Mtb was coupled to UL49.5-NP or UL49.5-SCR to target the inhibitor specifically to the Mtb phagosome. Isolated phagosomes were then incubated with TMR-conjugated CFP10_2–12 peptide as indicated above. FACS plot is representative of two independent experiments.</p

UL49.5 peptide inhibits TAP-dependent presentation of antigen to CD8⁺ T cell clones.

<p>A-B) Murine DCs were incubated with 0.5 mg/ml OVA and the indicated concentrations of the UL49.5-NP. DCs were then co-cultured with OT-I T (A) or OT-II (B) cells and supernatants were analyzed for IL-2 by ELISA. Shown is the mean and standard error of 2 independent experiments. * p = 0.07; ** p = 0.01; *** p = 0.007 (Student's two-tailed t test). C) Human monocyte derived DC were incubated for 1 hr with UL49.5-NP (5 uM) or UL49.5-SCR (5 uM) peptides and then infected with H37Rv-eGFP (MOI = 10) overnight. IFNγ production by the TAP-dependent HLA-E-restricted T cell clone D160 1-23 or the TAP-independent Class II clone D454 E12 was assessed by ELISPOT. IFNγ response by each T cell clone following UL49.5-NP treatment was normalized to the response in the presence of UL49.5-SCR. Shown is the mean response and standard error from at least 4 independent experiments. *** denotes significantly reduced IFNγ production by D160 1–23 in the presence of UL49.5-NP compared to D454 E12 (Student's two-tailed t test, p<0.001).</p

Mtb antigens are processed by the cytosolic pathway.

<p>(A,B) Human monocyte-derived DC were treated with epoxomicin, BFA, or bafilomycin for one hour before infection with Mtb H37Rv-eGFP (A) or addition of CFP10 and CFP10_3–11 (B). After 15–16 hours in the presence of the inhibitor, DC were harvested, fixed, washed extensively, and used as APC in an IFN-γ ELISPOT assay where T cell clones are effectors. DC were added to an excess of T cells so that antigen was the limiting factor (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000374#s4" target="_blank">Materials and Methods</a>). The mean number of spots produced by each clone was: D160 1-23 (228.4±39.3 to Mtb–infected DC, 19.6±6 to uninfected DC), D480 F6 (623.5±73.7 to Mtb–infected DC, 6.7±2.5 to uninfected DC), D454 E12 (453±52.4 to Mtb–infected DC, 13±4.4 to uninfected DC). Data have been normalized to the untreated control, and each bar reflects the mean±SEM of at least three experiments per clone (*p<0.05, **p<0.01 using two-tailed Student's t test compared to untreated controls, except where indicated). (C,D) DC were transduced with either empty vector or adenoviral ICP47 using Lipofectamine 2000. After 6–26 hours, DC were washed and infected with H37Rv-eGFP (C) or pulsed with antigen (D). Following overnight incubation, T cell clones were added and IFN-γ production was assessed by intracellular cytokine staining. The mean percentage of IFN-γ⁺ clones was: D160 1-23 (8.2±1.1 to Mtb–infected DC, 1.1±0.2 to uninfected DC), D480 F6 (46.4±3.9 to Mtb–infected DC, 1.5±0.4 to uninfected DC), D454 E12 (64±6 to Mtb–infected DC, 0.7±0.3 to uninfected DC). Each bar represents the mean±SEM of seven independent experiments.</p

Mtb phagosomes contain minimal contamination.

<p>(A) HLA-A2⁻ or HLA-A2⁺ DC were infected with H37Rv-eGFP for 20 minutes, washed, and incubated for an additional 40 minutes. HLA-A2⁻ DC were mixed with uninfected HLA-A2⁺ DC, homogenized, and homogenate separated using 27% percoll as described or pelleted without percoll separation. Phagosomes were stained with an antibody to HLA-A2. Shaded histograms represent isotype staining. Data are representative of three experiments. (B) HLA-A2⁻ or HLA-A2⁺ LCL were fixed, permeabilized, and stained with an antibody to HLA-A2. (C,D) Mtb phagosomes (C) or intact DC (D) were analyzed for the presence of cis- and trans-golgi markers GM130 and golgin-97, respectively. Data in C are representative of three experiments each after a 40 minute or overnight chase. Data in D are representative of two experiments.</p

Mtb proteins require retrotranslocation for presentation.

<p>(A,B) DC were treated with exoA or cycloheximide for one hour prior to infection with H37Rv-eGFP (A) or addition of CFP10 and CFP10_3–11 (B). DC were harvested, fixed, and assessed for their ability to stimulate T cell clones by IFN-γ ELISPOT as described. Each bar reflects the mean±SEM of at least three experiments. ND, not done. (C) DC were treated with exoA, exoA/PJ34, or BSA/PJ34 for one hour prior to infection with vaccinia virus expressing eGFP. After 16–18 hours, DC were harvested and GFP expression analyzed by flow cytometry. Data are representative of three experiments. (D,E) DC treated with exoA, BSA, exoA/PJ34, or BSA/PJ34 for one hour were subsequently infected with H37Rv-eGFP (D) or pulsed with antigen (E) overnight, harvested, fixed, and assessed for their ability to stimulate T cell clones by IFN-γ ELISPOT. Data are representative of two experiments. (F) DC were treated with exoA, exoA/PJ34, or BSA/PJ34 for one hour prior to infection with vaccinia virus expressing HIV p24. After 16–18 hours, DC were harvested, fixed and used to stimulate the HIV p24_306–316-specific CD8⁺ clone 16A7 in an IFN-y ELISPOT assay. Data are representative of two experiments.</p

HLA-I, loading machinery, and HLA-I:peptide complexes are present in highly pure Mtb phagosomes.

<p>(A) Phagosomes were isolated by percoll gradient or magnetic purification and prepared for electron microscopy as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000374#s4" target="_blank">Materials and Methods</a>. (B) Magnetic bead-isolated phagosomes were analyzed by flow cytometry to assess HLA-II-PE (plasma membrane) and H37Rv-eGFP fluorescence as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000374#s4" target="_blank">Materials and Methods</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000374#ppat-1000374-g006" target="_blank">Figure 6</a>. The events shown represent a small proportion of the population of phagosomes isolated and the experiment is representative of three experiments. (C) DC were pulsed with magnetically-labeled H37Rv-eGFP for 20 minutes, washed, and incubated for 18 hr. After magnetic separation of Mtb phagosomes, flow organellometry was performed as described previously. Data are representative of three experiments. (D) Magnetically-isolated Mtb phagosomes were freeze-thawed and tested for their ability to stimulate D160 1-23 CD8⁺ T cell clones in the absence of additional APC. IFN-γ production was measured using ELISPOT. Data are representative of two experiments.</p

The Mtb phagosome contains HLA-I loading accessory molecules.

<p>(A) DC were pulsed with H37Rv-eGFP for 20 minutes, washed, and incubated for 40 minutes. Phagosomal fractions were prepared as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000374#ppat-1000374-g003" target="_blank">Figure 3</a> and stained with the indicated antibodies (top panel). Intact DC were fixed, permeabilized, and stained with the indicated antibodies (bottom panel). Data are representative of three experiments. (B) Quantitative analysis of Mtb phagosomes over time. The percent positive number represents Overton cumulative histogram subtraction of the isotype control from the indicated stain. Each bar represents the mean±SEM from three experiments per timepoint.</p

The Mtb phagosome retains characteristics of an early endosome.

<p>(A) Representative figure showing organelle distribution after percoll separation of homogenate from Mtb–infected DC. The plasma membrane was labeled with a PE-conjugated antibody to HLA-II prior to homogenization and fluorescence was detected by fluorometry. For detection of ER, fractions were assessed for the presence of TAP1 and PDI by western blot. An enzymatic assay for β-hexosaminidase was used for detection of lysosomes. Finally, fractions were examined for the presence of H37Rv-eGFP by flow cytometry and quantified using a reference latex bead population. For flow cytometric analysis of Mtb phagosomes, the final 2 ml (fractions 23–28) of the gradient were pelleted, fixed, permeabilized, and stained with antibodies of interest. (B) Magnetic bead and Mtb phagosomes were gated based on FSC/SSC (not shown) and then on LAMP-1/HLA-I (beads) or LAMP-1/GFP (Mtb). Arrows indicate the gated population. Analysis of phagosome maturation on one hour LAMP-1^lo/−/HLA-I⁺ magnetic bead phagosomes (top panel), one hour LAMP-1⁺/HLA-I^lo/− magnetic bead phagosomes (second panel), overnight LAMP-1⁺ magnetic bead phagosomes (third panel), and overnight Mtb phagosomes (bottom panel). Plots include isotype staining (shaded histograms) as well as staining with the indicated antibody (red lines). The amplified HLA-I signal on the HLA-I-FITC gated events is due to the use of primary and secondary antibody combination, with which we routinely see up to a log shift in signal over conjugated primary. (C,D) Quantitative analysis of phagosomes over time. The percent positive number represents Overton cumulative histogram subtraction of the isotype control from the indicated stain. Each bar represents the mean±SEM of three experiments per timepoint.</p

DC from adults and neonates are equivalent in their ability to cross present cell-associated antigen.

<div><p><b>4A</b>. To confirm inactivation of vaccinia virus, HLA-A2⁺LCL (30,000 cells/well) were co-incubated for 24 hrs with vvpp65-infected HLA-A2⁻ LCL (60,000 cells/well) prior to (left well) or after (right well) heat-inactivation (30 minutes at 56C) and UV-treatment (200mJ²). Direct presentation was then detected by IFN-γ ELISPOT after an overnight incubation with CD8⁺ T cell clones D2 1-D2 (10,000/well), specific for the HLA-A2-restricted antigen HCMV pp65.</p><p><b>4B.</b> Representative ELISPOT wells of the cross presentation assay are shown. DC (30,000/well) from five individual adult (top row) or neonatal donors (bottom row) were incubated overnight with cell-associated antigen, namely, vvpp65-infected LCL (60,000/well) that were heat (30 minutes at 56C) and UV-treated (200 mJ²). CD8⁺ T cell clones D2 1-D2, specific for the pp65 antigen, were added (10,000 cells/well) and IFN-γ was detected the following day by ELISPOT. Only HLA-A2⁺ DC cross presented the pp65 antigen (right 4 columns) and HLA-A2⁻ DC (left column) never cross presented the antigen. HLA-A2⁺ DC incubated with uninfected HLA-A2⁻ LCL never elicited a response by CD8⁺ T cell clone D2-1-D2 (not shown). In addition, vvpp65-infected HLA-A2⁻ LCL alone in the absence of HLA-A2+ DC never elicited a response by CD8⁺ T cell clone D2-1-D2 (not shown).</p><p><b>4C</b>. Combined data from 3 separate cross presentation experiments using adult (<i>n = </i>18) and neonatal (<i>n = </i>14) HLA-A2⁺ DC. No significant differences were observed (p = 0.8587).</p></div

DC from adults and neonates are comparable in their ability to present the <i>M. tuberculosis</i> antigen pronase-digested cell wall to HLA-E-restricted clone D160-1-23.

<p>DC from adult (<i>n = </i>5) and neonatal (<i>n = </i>6) blood were incubated overnight with or without the pronase-treated cell wall fraction from <i>M.tuberculosis</i>. The DC were diluted over a range of concentrations (90,000 to 1100 cells/well), and incubated with the cognate CD8⁺ HLA-E-restricted clone D160-1-23 (10,000 cells/well) overnight. IFN-γ was detected by ELISPOT. There was no statistically significant difference between adult and neonatal DC in presentation of pronase-digested cell wall to HLA-E-restricted CD8⁺ T cells (p = 0.7943).</p