Rab7 accumulates in Msm-containing vacuoles.

<p>From 0–2 hours following synchronized infection, individual Rab7-GFP expressing BEAS-2B cells containing RFP-Msm were imaged sequentially. Each cell was imaged every 30 sec for 5 minutes. Shown is a representative 5 minute time-lapse sequence of Rab7 accumulation on an RFP-Msm compartment. In each experiment, we obtained time-lapse images from 8–10 cells. This experiment was repeated four times. The graph represents the GFP relative fluorescent units (RFU) calculated at the perpendicular transection of the bacterium in a single 0.2 uM z-plane at each time point, as shown in the first panel. Scale bar = 3 uM.</p

Mtb-infected human AEC are efficiently recognized by Mtb-specific CD8⁺ T cells.

<p>A–B) Mtb-infected DC, BEAS-2B cells, or LAEC (MOI:10) were used as APCs in an IFN-γ ELISPOT assay. A) IFN-γ release by the HLA-B45-restricted CD8⁺ T cell clone D466H4 was assessed by ELISPOT assay. 10,000 T cells were used with a titration of APCs from 10,000 - 1,250 cells per well. Results are representative of all experiments (N = 3). Error bars represent the mean and standard error from duplicate wells. B) IFN-γ release by the MR1-restricted (D426B1), HLA-E-restricted (D160 1-23), and HLA-B45-restricted (D466H4) CD8⁺ T cell clones was assessed for Mtb-infected DC, BEAS-2B, or primary LAEC (except D466H4). The fold-difference in IFN-γ release was normalized to the T cell response to Mtb-infected DC (5,000 DC/well) for each APC type and T cell clone. Results are representative of all experiments (N = 3 for DC and BEAS-2B cells and N = 1 for LAEC). C–F) Following fixation in 4% PFA, uninfected or infected BEAS-2B cells, DC, or LAEC were surface stained with either the pan-HLA-I antibody W6/32 (C-F) or the anti-MR1 antibody 26.5 (G) and analyzed by flow cytometry. C-E: shaded histogram, UI; black line, Mtb. F-G: shaded histogram, isotype staining for each cell type. Results are representative of all experiments (N = 5 for W6/32 and N = 4 for 26.5). H) DC or BEAS-2B cells were pulsed with CFP10_2-9 peptide at the indicated concentrations for 1 hour and assessed for their ability to stimulate IFN-γ production by the HLA-B45 restricted, CFP10_2-9 specific, D466H4 T-cell clone. APCs were added at 10,000 cells/well, while T cells were kept constant at 500 T cells per well. Results are representative of all experiments (N = 3). Error bars represent the mean and standard error from duplicate wells.</p

The Mtb vacuole in epithelial cells acquires Class I molecules.

<p>A–C) BEAS-2B cells were infected with dsRED-expressing Mtb or RFP-expressing M. smegmatis at MOI:5 for 4 or 18 hours. Infected cells were fixed and stained for Class I molecules (W6/32). At least 200 intracellular bacteria were counted from 3 independent experiments. Mtb-containing vacuoles were categorized as positive or negative, the mean percent positive and standard error were determined, and a Student's t-test was used to determine statistical significance between groups. A (Mtb); B (Msm). Scale bar = 10 uM. D) Primary LAEC were infected with dsRED-expressing Mtb at MOI:5 for 18 hours and analyzed as described above. Infected cells were fixed and stained with W6/32. Scale bar = 10 uM. Images are representative of Mtb or Msm associated with HLA-I. Error bars represent the mean and SEM for all events.</p

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

The Mtb vacuole in BEAS-2B cells acquires Lamp1, but does not acidify.

<p>A–C) BEAS-2B cells were infected with dsRED-expressing Mtb or RFP-expressing Msm at MOI:5 for 4 or 18 hours. Infected cells were fixed and stained for the lysosomal associated protein, Lamp1. Over 200 intracellular bacteria were counted from 4 independent experiments. Mtb-containing vacuoles were categorized as positive or negative, the mean percent positive and standard error were determined, and a Student's t-test was used to determine statistical significance between groups. Error bars represent the mean and SEM for all events (**p<0.01) D) Primary LAEC were infected with dsRED-expressing Mtb at MOI:5 for 18 hours and analyzed as described above. Scale bar = 10 uM. Images are representative of Lamp1-positive (A,D) or Lamp1-negative (B) Mtb containing compartments. E-F) DC or BEAS-2B cells were infected with GFP-expressing Mtb or GFP-expressing Msm linked to the pH sensitive dye pHrodo Red. Cells were fixed 18 hours after infection and images were acquired on a DeltaVision Core DV wide-field microscope. E) pHrodo signal from perpendicular transection on a single 0.2 µm z-stack was used to generate the relative fluorescence unit (RFU) data point for each individual bacterium. The image on the left is an example of a GFP+ bacterium (green) associated with low pHrodo Red (red) signal, while the image on the right is an example of a bacterium with low GFP signal, but high pHrodo Red signal. Lines on the image indicate the perpendicular transection of the bacterium used to generate the RFU for GFP and pHrodo Red indicated in the panels below the images. F) The pHrodo Red RFU for at least 200 individual bacteria from 4 independent experiments for each condition was plotted on a log scale. Error bars represent the mean RFU and SEM for all events. (*p<0.001)</p

Prevalence and degree of Mtb infection in DC and BEAS-2B cells.

<p>Prevalence and degree of Mtb infection in DC and BEAS-2B cells.</p

Intracellular Mtb are required for T cell activation.

<p>A) BEAS-2B cells were seeded in a 12-well tissue culture plate and a 0.4 uM pore size transwell insert. Cells in the transwell were infected with Mtb (MOI:10) for 18 hrs. Cells from both chambers were then used as APCs (10,000 cells/well) in an IFN-γ Elispot assay. Results are representative of three independent experiments. B-C) Mtb were labeled with streptavidin coated magnetic microbeads prior to infection (MOI:30). A fraction enriched for infected BEAS-2B cells was then obtained by magnetic sorting (adherent). Mtb CFUs were enumerated from each fraction (B) and cells were used as APCs (5,000 cells/well) in an IFN-γ Elispot assay (C). (N = Non-adherent, A = Adherent). Results are representative of three independent experiments. D) The T cell response to the enriched fraction of Mtb-infected BEAS-2B cells (MOI:30, 2,000 cells/well) was compared to an equal number of Mtb-infected DC (MOI:30) in an IFN-γ Elispot assay. Results are representative of two independent experiments. For all assays, error bars represent the mean and standard error from duplicate wells.</p

Transferrin receptor (TfR) does not accumulate at high levels in the Mtb vacuole.

<p>A–C) BEAS-2B cells were infected with dsRED-expressing Mtb or RFP-expressing Msm at MOI:5 for 4 or 18 hours. Infected cells were fixed and stained for TfR. At least 200 intracellular bacteria were counted from 3 independent experiments. Mtb-containing vacuoles were categorized as positive or negative, the mean percent positive and standard error were determined, and a Student's t-test was used to determine statistical significance between groups. A (Mtb); B (Msm). Images are representative of TfR-positive compartments for Mtb and Msm. Error bars represent the mean and SEM for all events. Scale bar = 10 uM. D) Primary LAEC were infected with dsRED-expressing Mtb at MOI:5 for 18 hours. Infected cells were fixed and stained for TfR and analyzed as described above. Image is representative of a TfR-negative compartment. Scale bar = 10 uM. E-F) BEAS-2B cells were infected with dsRED-Mtb or RFP-Msm (MOI:10). Infection was synchronized by centrifugation. After a 2 hour infection, cells were washed three times with PBS, and wells replenished with media. Cells were harvested and counted, and lysates were plated in triplicate after the 2 hour infection, and at 24, 48, and 72 hours following infection. Error bars represent the mean and SEM from three experiments (Mtb) or the mean and standard error from triplicate wells (Msm).</p

The human lung epithelial cell line BEAS-2B is less efficiently infected with Mtb than human DC.

<p>A–B) Primary human DC or the human bronchial epithelial cell line, BEAS-2B, were infected with dsRED Mtb at an MOI of 30, 10, and 3. Fixed cells were assessed for dsRED-H37Rv infection by flow cytometry after 18 hours. FlowJo (TreeStar) was used to calculate the change in geometric mean in BEAS-2B cells or DC for MOI:10. Results are representative of all experiments (N = 3). C–E) DC, BEAS-2B cells, or primary LAEC were infected with Mtb (MOI:7). Infected cells were imaged in an unbiased manner, and the number of bacteria per cell was enumerated. Results are representative of all experiments (N = 3 for DC and BEAS-2B cells, N = 2 for LAEC).</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