Syt1 and Syt7 specifically mediate MR1 presentation of intracellular Mtb.

(a) Relative gene expression levels of Syt1 and Syt7 compared to Gapdh in BEAS-2B and PMA-differentiated THP-1 cells.

(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)50. (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, CFP102-9 peptide, or (e) 5-A-RU prodrug, represented as SFU. All data are plotted as mean±SEM and pooled from three 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 EC50 were used to calculate p-values by extra sum-of-squares F test.

Syt1 and Syt7 do not affect Mtb uptake and growth.

(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 compared to Gapdh in WT, Syt1 KO, or Syt7 KO BEAS-2B cells. All data are plotted as mean±SEM. (c-d) are pooled from three independent experiments and (e) is pooled from two independent experiments. For (c-e), ordinary one-way ANOVA with Dunnett’s multiple comparisons test were used to analyze significant differences. ns=not significant (p>0.05).

Syt1 and Syt7 also mediate MR1 presentation of Mtb in THP-1 cells.

(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 analysis50. (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. All data are plotted as mean±SEM and pooled from three independent experiments. For (b-c), the means of technical duplicates were pooled, and non-linear regression analysis of pairwise comparison to WT on best-fit values of top and EC50 were used to calculate p-values by extra sum-of-squares F test.

Syt11 and ER-associated Esyt1 and Esyt2 do not solely affect MR1 presentation of Mtb.

(a) Relative gene expression levels of Syt11, Esyt1, and Esyt2 compared to Gapdh in BEAS-2B cells. (b) Knockdown efficiency of Syt11, Esyt1, and Esyt2 after 48 hours of knockdown with missense (Mis) or gene-specific (KD) siRNA. (c-e) IFN-γ release by T cell clones (MR1- and HLA-B45-restricted) co-cultured with BEAS-2B cells following siRNA knockdown of Syt11 (c), Esyt1 (d), or Esyt2 (e). Cells were either infected overnight with H37Rv Mtb (MOI=8) or incubated with exogenously added antigens (Msmeg supernatant and CFP102-9 peptide). IFN-γ release is represented as SFU. All data are plotted as mean±SEM and pooled from three independent experiments. For (c-e), the means of technical duplicates were pooled, and non-linear regression analysis comparing best-fit values of top and EC50 were used to calculate p-values by extra sum-of-squares F test.

Syt1 and Syt7 localize to late endo-lysosomes and MR1 vesicles.

(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 two independent experiments (b) Percent co-localization of Rab5a (n=9), Rab7a (n=12), and LAMP1 (n=10) with Syt1 or Syt7. Data are pooled from two 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 three independent experiments. (d) Percent co-localization of Syt1 or Syt7 with MR1 (n=17). Data are pooled from three 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. All scale bars represent 10 µm.

Absence of Syt1 and Syt7 alters MR1 cellular distribution in lysosomal compartments.

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 tool50. (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 three independent experiments, and non-linear regression analysis comparing best-fit values of top and EC50 were 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 three 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, 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 three independent experiments. (g) Representative images of WT, Syt1 KO, 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 four 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. All scale bars represent 10 µm.

Syt1 and Syt7 mediate trafficking of MR1 vesicles from the Mtb-containing vacuole.

(a) WT, Syt1 KO, 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 three 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=16). (c) Total number (left, n=15) and average speed (µm/s) (right, n=14) of MR1 vesicles. (d) Overlapped area ratio of MR1 to Mtb surfaces (n=23). For (b-d), data are plotted as mean±SEM and pooled from four to five 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, 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 three independent experiments showing the frequency of total LAMP1+, total MR1+, and AuxMtb+LAMP1+ vesicles of all vesicles from fractions 36 to 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 three independent experiments and plotted as mean±SEM. P-values were analyzed by a one-way ANOVA with Dunnett’s multiple comparisons test.