Journal: eLife

Article Title: Synaptotagmin 1 and Synaptotagmin 7 promote MR1-mediated presentation of Mycobacterium tuberculosis antigens

doi: 10.7554/eLife.108318

Figure Lengend Snippet: ( a ) Relative gene expression levels of SYT1 , SYT7 , and GAPDH in BEAS-2B ( n =3) and PMA-differentiated THP-1 cells ( n =3-4). ( b ) Genome editing efficiency and percent distribution of individual indels of Syt1 and Syt7 KO BEAS-2B cells as determined by Sanger sequencing and Interference of CRISPR Edits (ICE) ( n =1). ( c ) IFN-γ release by T cell clones (MR1- and HLA-B45-restricted) co-cultured with H37Rv Mtb-infected (MOI=8) WT, Syt1 KO, or Syt7 KO BEAS-2B cells, represented as spot forming units (SFU). ( d ) IFN-γ release by T cell clones (MR1- and HLA-B45-restricted) co-cultured with WT, Syt1 KO, or Syt7 KO BEAS-2B cells in the presence of Msmeg supernatant, CFP10 2–9 peptide, or ( e ) 5-A-RU prodrug, represented as SFU. All data are plotted as mean ± SEM and pooled from n =3 independent experiments. For ( c–e ), the means of technical duplicates were pooled, and non-linear regression analysis of pairwise comparison to WT on best-fit values of top and EC 50 was used to calculate p-values by extra sum-of-squares F test. A p value of <0.05 was considered statistically significant. Figure 1—source data 1. Source data corresponding to .

Article Snippet: Taqman FAM-MGB probes for SYT11 (Hs00383056_m1), ESYT1 (Hs00248693_m1), ESYT2 (Hs00294020_m1), SYT1 (Hs00194572_m1), SYT7 (Hs01590513_m1) , and MR1 (Hs00155420_m1) were obtained from Thermo Fisher Scientific.

Techniques: Gene Expression, Sequencing, CRISPR, Clone Assay, Cell Culture, Infection, Comparison

Journal: eLife

Article Title: Synaptotagmin 1 and Synaptotagmin 7 promote MR1-mediated presentation of Mycobacterium tuberculosis antigens

doi: 10.7554/eLife.108318

Figure Lengend Snippet: ( a ) Gating strategy of BEAS-2B cells infected overnight with auxotrophic strain mEmeraldRFP-AuxMtb (MOI=8) by gating on cells, excluding doublets using forward scatter properties, and selecting Live/Dead Near-IR stain negative cells. ( b ) Representative gate on GFP + population to indicate live BEAS-2B cells infected with mEmeraldRFP-AuxMtb. ( c ) Percent Mtb uptake measured as proportion of live WT, Syt1 KO, or Syt7 KO BEAS-2B cells that are GFP + . ( d ) Colony-forming units (CFU) of H37Rv Mtb in WT, Syt1 KO, or Syt7 KO BEAS-2B cells after overnight infection (MOI=8). ( e ) Relative gene expression levels of MR1 and GAPDH in WT, Syt1 KO, or Syt7 KO BEAS-2B cells. All data are plotted as mean ± SEM. ( c, d ) are pooled from n =3 independent experiments and ( e ) is pooled from n =2 independent experiments. For ( c, d ), ordinary one-way ANOVA with Dunnett’s multiple comparisons test was used to analyze significant differences. ns=not significant (p > 0.05). Figure 2—source data 1. Source data corresponding to .

Article Snippet: Taqman FAM-MGB probes for SYT11 (Hs00383056_m1), ESYT1 (Hs00248693_m1), ESYT2 (Hs00294020_m1), SYT1 (Hs00194572_m1), SYT7 (Hs01590513_m1) , and MR1 (Hs00155420_m1) were obtained from Thermo Fisher Scientific.

Techniques: Infection, Staining, Gene Expression

Journal: eLife

Article Title: Synaptotagmin 1 and Synaptotagmin 7 promote MR1-mediated presentation of Mycobacterium tuberculosis antigens

doi: 10.7554/eLife.108318

Figure Lengend Snippet: ( a ) Genome editing efficiency and percent distribution of individual indels of Syt1 and Syt7 KO THP-1 cells as determined by Sanger sequencing and ICE analysis ( n =1). ( b ) IFN-γ release by MAIT cell clones co-cultured with H37Rv Mtb-infected (MOI=1) WT, Syt1 KO, or Syt7 KO THP-1 cells following PMA differentiation, represented as SFU. ( c ) IFN-γ release by MAIT cell clones co-cultured with WT, Syt1 KO, or Syt7 KO THP-1 cells following PMA differentiation in the presence of Msmeg supernatant, represented as SFU. For ( b, c ), the means of technical duplicates were pooled, data were plotted as mean ± SEM and pooled from n =3 independent experiments, and non-linear regression analysis of pairwise comparison to WT on best-fit values of top and EC 50 was used to calculate p-values by extra sum-of-squares F test. A p value of <0.05 was considered statistically significant. Figure 3—source data 1. Source data corresponding to .

Article Snippet: Taqman FAM-MGB probes for SYT11 (Hs00383056_m1), ESYT1 (Hs00248693_m1), ESYT2 (Hs00294020_m1), SYT1 (Hs00194572_m1), SYT7 (Hs01590513_m1) , and MR1 (Hs00155420_m1) were obtained from Thermo Fisher Scientific.

Techniques: Sequencing, Clone Assay, Cell Culture, Infection, Comparison

Journal: eLife

Article Title: Synaptotagmin 1 and Synaptotagmin 7 promote MR1-mediated presentation of Mycobacterium tuberculosis antigens

doi: 10.7554/eLife.108318

Figure Lengend Snippet: ( a ) BEAS-2B cells transfected with Syt1- or Syt7-RFP (magenta) plasmids and incubated with CellLight BacMam 2.0 reagents for Rab5a, Rab7a, and LAMP1 (yellow) overnight. Images are representative of n =2 independent experiments ( b ) Percent co-localization of Rab5a ( n =11), Rab7a ( n =12), and LAMP1 ( n =12) with Syt1 or Syt7. Data are pooled from n =2 independent experiments and plotted as mean ± SEM. Each dot represents one cell. ( c ) Polyclonal BEAS-2B:TET-MR1GFP (green) cells transfected overnight with Syt1- or Syt7-RFP (magenta) plasmids. Images are representative of n =3 independent experiments. ( d ) Percent co-localization of Syt1 or Syt7 with MR1 ( n =17). Data are pooled from n =3 independent experiments and plotted as mean ± SEM. Each dot represents one cell. Two-way ANOVA with Sidak’s multiple comparisons test ( b ) and two-tailed unpaired Student’s t -test ( d ) were used to calculate p-values. A p value of <0.05 was considered statistically significant. All scale bars represent 10 µm. Figure 5—source data 1. Source data corresponding to .

Article Snippet: Taqman FAM-MGB probes for SYT11 (Hs00383056_m1), ESYT1 (Hs00248693_m1), ESYT2 (Hs00294020_m1), SYT1 (Hs00194572_m1), SYT7 (Hs01590513_m1) , and MR1 (Hs00155420_m1) were obtained from Thermo Fisher Scientific.

Techniques: Transfection, Incubation, Two Tailed Test

Journal: eLife

Article Title: Synaptotagmin 1 and Synaptotagmin 7 promote MR1-mediated presentation of Mycobacterium tuberculosis antigens

doi: 10.7554/eLife.108318

Figure Lengend Snippet: Syt1 KO and Syt7 KO were generated in the background of BEAS-2B MR1KO:tetMR1-GFP clone D4 cells. ( a ) Syt1 and Syt7 KO clones were verified by Sanger sequencing and analyzed using the ICE tool ( n =1). ( b ) IFN-γ release by T cell clones (MR1- and HLA-B45-restricted) co-cultured with H37Rv Mtb-infected cells (MOI=8) is represented as SFU. The means of technical duplicates were pooled from n =3-4 independent experiments, and non-linear regression analysis comparing best-fit values of top and EC 50 was used to calculate p-values by extra sum-of-squares F test. ( c, d ) WT, Syt1 KO, and Syt7 KO BEAS-2B MR1KO:tetMR1-GFP cells were incubated overnight with doxycycline and Ac-6-FP or NaOH (solvent control). Images ( c ) and histograms (d, left) representative of n =3 independent experiments with pooled geometric mean fluorescence intensity (GeoMFI) (d, right) of surface MR1 and HLA-Ia expression. ( e ) Representative images of WT, Syt1 KO, and Syt7 KO EAS-2B MR1KO:tetMR1-GFP cells incubated overnight with doxycycline and ( f ) measurement of area of MR1 vesicles, classified into small (1 vesicle) or large (>1 vesicle) vesicles ( n =15). Each dot represents one cell. Data are pooled from n =3 independent experiments. ( g ) Representative images of WT, Syt1 KO, and Syt7 KO BEAS-2B MR1KO:tetMR1-GFP cells incubated overnight with doxycycline and CellLight BacMam 2.0 reagents for Rab5a and LAMP1 (yellow). ( h ) Percent co-localization of Rab5a ( n =16) and LAMP1 ( n =16) with MR1 vesicles. Each dot represents one cell. Data are pooled from n =4 independent experiments. For ( d, f, h ), p-values were analyzed by two-way ANOVA with Dunnett’s multiple comparisons test. All data are plotted as mean ± SEM. A p value of <0.05 was considered statistically significant. All scale bars represent 10 µm. Figure 6—source data 1. Source data corresponding to .

Article Snippet: Taqman FAM-MGB probes for SYT11 (Hs00383056_m1), ESYT1 (Hs00248693_m1), ESYT2 (Hs00294020_m1), SYT1 (Hs00194572_m1), SYT7 (Hs01590513_m1) , and MR1 (Hs00155420_m1) were obtained from Thermo Fisher Scientific.

Techniques: Generated, Clone Assay, Sequencing, Cell Culture, Infection, Incubation, Solvent, Control, Fluorescence, Expressing

Journal: eLife

Article Title: Synaptotagmin 1 and Synaptotagmin 7 promote MR1-mediated presentation of Mycobacterium tuberculosis antigens

doi: 10.7554/eLife.108318

Figure Lengend Snippet: ( a ) WT, Syt1 KO, and Syt7 KO BEAS-2B MR1KO:tetMR1-GFP (green) cells were infected with AuxMtb (MOI=5) labeled with Alexa Fluor 555 Succinimidyl Ester (AuxMtb-Alexa Fluor555; magenta). Images representative of n =3 independent experiments are shown. Scale bars represent 10 µm. ( b ) Number of MR1 vesicles within 1 µm of the center of the AuxMtb surface ( n =51–53). ( c ) Total number (left, n =51–53) and average speed (µm/s) (right, n =14–16) of MR1 vesicles. ( d ) Overlapped area ratio of MR1 to Mtb surfaces ( n =51–53). For ( b–d ), data are plotted as mean ± SEM and pooled from n =8 independent experiments. Each dot represents one cell. p-values were analyzed by a one-way ANOVA with Dunnett’s multiple comparisons test. ( e ) WT, Syt1 KO, and Syt7 KO BEAS-2B MR1KO:tetMR1-GFP cells were infected overnight with AuxMtb (MOI=10) labeled with Alexa Fluor 647 Succinimidyl Ester. Mtb-containing vacuoles for each fraction from flow organellometry assay were identified by gating on vesicles, excluding doublets using forward scatter properties, and selecting the AuxMtb + LAMP1 + population (left). Total Lamp1 + and MR1 + populations were gated following the same strategy (right). Representative histograms comparing fractions 40 and 46 are shown. ( f ) Graphs representative of n =3 independent experiments showing the frequency of total LAMP1 + , total MR1 + , and AuxMtb + LAMP1 + vesicles of all vesicles from fractions 36–50. ( g ) Representative histogram and geometric mean fluorescence intensity (GeoMFI) of MR1 in the subcellular fraction with the highest percentage of AuxMtb + LAMP1 + vesicles. Data are pooled from n =3 independent experiments and plotted as mean ± SEM. p-values were analyzed by a one-way ANOVA with Dunnett’s multiple comparisons test. A p value of <0.05 was considered statistically significant. Figure 7—source data 1. Source data corresponding to .

Article Snippet: Taqman FAM-MGB probes for SYT11 (Hs00383056_m1), ESYT1 (Hs00248693_m1), ESYT2 (Hs00294020_m1), SYT1 (Hs00194572_m1), SYT7 (Hs01590513_m1) , and MR1 (Hs00155420_m1) were obtained from Thermo Fisher Scientific.

Techniques: Infection, Labeling, Fluorescence