Stx18, VAMP4, and Rab6 silencing alter TGN structure and intracellular MR1 localization.

<p>a) BEAS-2B cells treated with missense, Stx18, Rab6, or VAMP4 siRNA were transfected with pCI:MR1-GFP for 48 hours, then fixed and imaged. Shown are representative images of the cells used to enumerate MR1-GFP⁺ EC. TGN46 staining is shown at 1.5x magnification in greyscale below each image to demonstrate dispersion or contraction of the TGN. b) MR1-GFP⁺ EC were identified and quantified as previously described. On the left, each dot represents the number of MR1-GFP⁺ EC in a single cell, and the data shown is representative of 4 independent experiments. On the right, each dot represents the mean intensity of GFP signal in one individual MR1-GFP⁺ EC. The data represent all endosomes from the cells plotted on the left, and are representative of 4 independent experiments. The Mann-Whitney test was used to determine statistical significance, *p<0.01.</p

Cell surface translocation of MR1 in the presence of ligand.

<p>a) BEAS-2B or primary NHBE cells were transfected with pCI:MR1-GFP and incubated for 30 hours. Transfected cells were then incubated with 6-FP for 18 hours. For imaging, cells were fixed and surface stained with α-MR1 (26.5), then imaged. In NHBE cells, MR1 surface staining (red) in MR1-GFP-expressing cells (green) is denoted by the arrows in the enlarged insets. Images shown are representative of at least three independent experiments. b) BEAS-2B or NHBE cells were treated as described in a). Following 6-FP treatment cells were harvested and surface stained on ice with α-MR1 (26.5). After staining, cells were fixed and analyzed by flow cytometry. Data shown are representative of at least three independent experiments. Where indicated, cells were treated with 100ng/ml brefeldin A (BFA) for 2 hours prior to 6-FP addition. Shown is a representative histogram demonstrating BFA blockade of 6-FP-dependent surface stabilization and the geometric mean and SEM of from three independent experiments. c) BEAS-2B or NHBE cells were transfected with pCI:MR1-GFP and treated with 6-FP as described above. Where indicated, cells were treated with10ug/ml cycloheximide (CHX) for 2 hours prior to 6-FP addition. Cells were fixed, and where indicated, stained with α-β2M, and imaged. Shown are results from one of at least three independent experiments. The Mann-Whitney test was used to determine the statistical significance of CHX treatment. For all other statistical comparisons, a Student’s t-test was used, *p<0.01. d) MR1-GFP⁺ β2M⁺ EC were identified and quantified as described in the Materials and Methods. Each dot represents the number of endosomes in one cell for any of the conditions. Shown are results from one of at least three independent experiments, *p<0.01.</p

Identification of trafficking molecules involved in presentation of Mtb ligands on MR1.

<p>a) A lentiviral shRNA library of trafficking molecules was screened by IFNγ ELISPOT assay with MR1, HLA-E, and HLA-B45 restricted T cell clones as described. Each bar represents separate shRNA wells and each well was analyzed individually in relation to the mean and characterized as a hit if it was at least 25% below the mean. The mean was normalized to 100% and is represented by red line. Results from shRNA knockdown of Rab2b, Rab7L1, or Stx18 from three independent experiments are shown. Stars indicate gene specific shRNA wells that met the candidate selection criteria. Candidates were selected if at least 2 of 5 independent shRNAs resulted in reduced T cell response. b) BEAS-2B cells were treated with missense or Stx18 siRNA, infected with Mtb (MOI:5), and used as APC in an IFNγ ELISPOT assay with 1x10⁴ MR1, HLA-E, or HLA-B45 restricted T cells clones per well. Shown are the results using 5 x 10³ APC that have been normalized to the response to the Missense siRNA treated cells (100%). **p<0.01. Error bars represent the mean and SEM from four independent experiments. qRT-PCR was performed on cDNA synthesized from mRNA isolated from missense or Stx18 siRNA silenced BEAS-2B cells that were uninfected or infected with Mtb (MOI:5). Error bars represent the mean and SEM for three independent experiments. Mtb colony forming units (CFU) from 1x10⁵ infected cells were determined by lysing and plating serial dilutions on 7H10 agar plates. Error bars represent the mean and SEM from 3 independent experiments. c) NHBE cells were treated with missense or Stx18 siRNA and infected with Mtb (MOI:10). IFNγ ELISPOT and qRT-PCR analyses were performed as described for BEAS-2B cells. Error bars represent the mean and SEM from two independent experiments. d) BEAS-2B cells were treated with missense, VAMP4, or Rab6 siRNA and infected with Mtb (MOI:5). IFNγ ELISPOT and qRT-PCR analyses were performed as described above. Error bars represent the mean and SEM from at least three independent experiments, *p<0.01 compared to missense. e) BEAS-2B cells were treated with missense, Sec22b, BNIP1, or Use1 siRNA and infected with Mtb (MOI:5). IFNγ ELISPOT and qRT-PCR analyses were performed as described above. Error bars represent the mean and SEM from at least two independent experiments, *p<0.01 compared to missense.</p

Mtb-derived ligands are loaded on MR1 using a pathway distinct from that of 6-FP.

<p>a) BEAS-2B cells treated with missense, Stx18, Rab6 or VAMP4 siRNA were transfected with pCI:MR1-GFP for 30 hours, then incubated or not with 6-FP for 18 hours. Cells were then harvested, surface stained with α-MR1 (26.5) and examined by flow cytometry. Cells were gated on expression of MR1-GFP based on an untransfected control. Shaded histograms represent the isotype control. Black solid histograms represent the cell surface expression of MR1 after treatment with missense control siRNA, while the dashed histograms represent the cell surface expression of MR1 in the after treatment with the indicated siRNA. Data is representative of three independent experiments. b) For Stx18, BEAS-2B cells were transfected with MR1-GFP and incubated for 24 hours, then transfected with Stx18-RFP and incubated for 16 hours. For VAMP4, BEAS-2B cells stably expressing MR1-GFP were transfected with VAMP4-RFP and incubated for 18 hours. Cells co-expressing MR1-GFP and Stx18 or VAMP4 were imaged live. Images are representative of at least three independent experiments. c) BEAS-2B cells were infected with Mtb (MOI:8) for 18 hours or incubated with supernatant from <i>M</i>. <i>smegmatis</i> cultures (Msm Sup). Where indicated, cells were pretreated with 6-FP for 2 hours prior to infection or addition of supernatant. Cells were then used as APC in an IFNγ ELISPOT assay with IFNγ production by an MR1-restricted T cell clone as a readout for antigen presentation. Data are representative of two independent experiments, *p<0.01, n.s. not significant.</p

MR1 candidates evaluated using siRNA gene knockdown.

<p>MR1 candidates evaluated using siRNA gene knockdown.</p