Figure 5A . Mice were co - colonized with E1 and either N1 WT ( n = 4 ) or N1 ΔtssC ( n = 3 ) . Five days post - inoculation , fecal RNA was extracted and tested for BFT expression via qRT - PCR . B . Four weeks after co - colonization with E1 and either N1 WT or N1 ΔtssC ( n = 4 mice per group ) , the sera were collected , tested via ELISA for anti - BFT IgG and end point titer calculated . C - F . Mice pre - treated with DSS were inoculated with no organisms ( sham ) , E1 only , or E1 competed with N1 WT or N1 ΔtssC . Five days post - inoculation , the ceca were weighed ( C ) and fixed for histopathological examination after sham ( D ) , E1 only ( E ) and E1 - N1 WT ( F ) colonizations . Scale bars denote 100μm ( main image ) and 200μm ( inset ) .

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Figure 5A. Mice were co-colonized with E1 and either N1 WT (n=4) or N1 ΔtssC (n=3). Five days post-inoculation, fecal RNA was extracted and tested for BFT expression via qRT-PCR.B. Four weeks after co-colonization with E1 and either N1 WT or N1 ΔtssC (n= 4 mice per group), the sera were collected, tested via ELISA for anti-BFT IgG and end point titer calculated.C-F. Mice pre-treated with DSS were inoculated with no organisms (sham), E1 only, or E1 competed with N1 WT or N1 ΔtssC. Five days post-inoculation, the ceca were weighed (C) and fixed for histopathological examination after sham (D), E1 only (E) and E1-N1 WT (F) colonizations. Scale bars denote 100μm (main image) and 200μm (inset).


Figure 1T cells were collected from age - and gender - matched WT , EGR1KO and EGR4KO mice . ( A ) CD4 + T cells were isolated from the spleen by negative selection before incubating with anti - CD3 / CD28 antibodies for the indicated time periods . Levels of expression of EGR1 , EGR2 , EGR3 and EGR4 were measured by qPCR . T cells were collected from age - and gender - matched WT , EGR1KO and EGR4KO mice . ( B ) WBC were collected from WT , EGR1KO and EGR4KO mice before staining and FACS analysis to determine composition ( gating strategies in Appendix Fig S1 ) . Subclasses of CD8 + , CD4 + and Treg populations were determined as shown .

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Figure 1T cells were collected from age- and gender-matched WT, EGR1KO and EGR4KO mice. (A) CD4+ T cells were isolated from the spleen by negative selection before incubating with anti-CD3/CD28 antibodies for the indicated time periods. Levels of expression of EGR1, EGR2, EGR3 and EGR4 were measured by qPCR.T cells were collected from age- and gender-matched WT, EGR1KO and EGR4KO mice. (B) WBC were collected from WT, EGR1KO and EGR4KO mice before staining and FACS analysis to determine composition (gating strategies in Appendix Fig S1). Subclasses of CD8+, CD4+ and Treg populations were determined as shown.


Figure 2WT and EGR4 - / - CD4 + and CD8 + T cells were isolated from the spleen by cell sorting and stained with CFSE . A Cells were incubated under unstimulated conditions or stimulated with either anti - TCRβ or anti - CD3 / CD28 antibodies for 0 to 5 days ( mean ± SEM ; a minimum of 3 biological replicates were examined ; each biological replicate includes 2 technical replicates ) . WT and EGR4 - / - CD4 + and CD8 + T cells were isolated from the spleen by cell sorting and stained with CFSE . B Dose dependence of EGR4 - dependent differences in proliferation were also determined in both CD4 + and CD8 + cells aft er treatment with anti - TCRβ ( E , F ) or anti - CD3 / CD28 ( G , H ) at the indicated concentrations before FACS analysis ( Appendix Fig S2 ) ( mean ± SEM ; a minimum of 3 biological replicates were examined ; each biological replicate includes 2 technical replicates ) . WT and EGR4 - / - CD4 + and CD8 + T cells were isolated from the spleen by cell sorting and stained with CFSE . C , D Generation analysis was then assessed in WT , EGR1 - / - and EGR4 - / - CD4 + ( C ) or CD8 + ( D ) T cells after a 4 day incubation , under unstimulated conditions , or stimulated with either anti - TCRβ or anti - CD3 / CD28 antibodies . Generation analysis was determined using FlowJo as depicted in Appendix figure S3 ( mean ± SEM ; a minimum of 3 biological replicates were examined ; each biological replicate includes 2 technical replicates ) . . EGR4 deletion significantly shifted the number of generations as determined by two - way ANOVA ; loss of either EGR1 or EGR4 significantly changed the number of generations under resting or stimulated conditions ( p < 0 . 0001 ) .

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Figure 2WT and EGR4-/- CD4+ and CD8+ T cells were isolated from the spleen by cell sorting and stained with CFSE. A Cells were incubated under unstimulated conditions or stimulated with either anti-TCRβ or anti-CD3/CD28 antibodies for 0 to 5 days (mean±SEM; a minimum of 3 biological replicates were examined; each biological replicate includes 2 technical replicates).WT and EGR4-/- CD4+ and CD8+ T cells were isolated from the spleen by cell sorting and stained with CFSE. B Dose dependence of EGR4-dependent differences in proliferation were also determined in both CD4+ and CD8+ cells aft er treatment with anti-TCRβ (E,F) or anti-CD3/CD28 (G,H) at the indicated concentrations before FACS analysis (Appendix Fig S2) (mean±SEM; a minimum of 3 biological replicates were examined; each biological replicate includes 2 technical replicates).WT and EGR4-/- CD4+ and CD8+ T cells were isolated from the spleen by cell sorting and stained with CFSE. C,D Generation analysis was then assessed in WT, EGR1-/- and EGR4-/- CD4+ (C) or CD8+ (D) T cells after a 4 day incubation, under unstimulated conditions, or stimulated with either anti-TCRβ or anti-CD3/CD28 antibodies. Generation analysis was determined using FlowJo as depicted in Appendix figure S3 (mean±SEM; a minimum of 3 biological replicates were examined; each biological replicate includes 2 technical replicates).. EGR4 deletion significantly shifted the number of generations as determined by two-way ANOVA; loss of either EGR1 or EGR4 significantly changed the number of generations under resting or stimulated conditions (p < 0.0001).


Figure 3CD4 + and CD8 + T cells were isolated from the spleens of WT , EGR1KO and EGR4KO mice by cell sorting . ( A - F ) Cytokine secretion was measured by ELISA after incubating CD4 + ( A , C , E ) and CD8 + ( B , D , F ) T cells for 48 hrs in resting conditions ( A , B ) or stimulated with either anti - TCRβ ( C , D ) or anti - CD3 / CD28 antibodies ( E , F ) . CD4 + and CD8 + T cells were isolated from the spleens of WT , EGR4KO mice by cell sorting . ( G ) Representative FACS plots depicting IFNγ levels after intracellular staining in WT and EGR4KO CD4 + and CD8 + T cells . CD4 + and CD8 + T cells were isolated from the spleens of WT , and EGR4KO mice by cell sorting . ( H ) Comparison of IFNγ levels in WT vs . EGR4 - / - T cells . CD4 + T cells were isolated from the spleens of WT , EGR1KO and EGR4KO mice by cell sorting . ( I ) Representative FACS plots of CD4 + T cells incubated for 24 hrs under Th1 polarizing conditions before intracellular staining with IFNγ and IL - 17 antibodies . CD4 + T cells were isolated from the spleens of WT , EGR1KO and EGR4KO mice by cell sorting . ( J ) Comparison of the number of Th1 polarized cells between WT , EGR1 - / - and EGR4 - / - CD4 + T cells . CD4 + T cells were isolated from the spleens of WT , EGR1KO and EGR4KO mice by cell sorting . ( K ) CD4 + T cells were incubated for 5 days under polarizing conditions before analysis of cytokine production by ELISA .

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Figure 3CD4+ and CD8+ T cells were isolated from the spleens of WT, EGR1KO and EGR4KO mice by cell sorting. (A-F) Cytokine secretion was measured by ELISA after incubating CD4+ (A,C,E) and CD8+ (B,D,F) T cells for 48 hrs in resting conditions (A,B) or stimulated with either anti-TCRβ (C,D) or anti-CD3/CD28 antibodies (E,F).CD4+ and CD8+ T cells were isolated from the spleens of WT, EGR4KO mice by cell sorting. (G) Representative FACS plots depicting IFNγ levels after intracellular staining in WT and EGR4KO CD4+ and CD8+ T cells.CD4+ and CD8+ T cells were isolated from the spleens of WT, and EGR4KO mice by cell sorting. (H) Comparison of IFNγ levels in WT vs. EGR4-/- T cells.CD4+ T cells were isolated from the spleens of WT, EGR1KO and EGR4KO mice by cell sorting. (I) Representative FACS plots of CD4+ T cells incubated for 24 hrs under Th1 polarizing conditions before intracellular staining with IFNγ and IL-17 antibodies.CD4+ T cells were isolated from the spleens of WT, EGR1KO and EGR4KO mice by cell sorting. (J) Comparison of the number of Th1 polarized cells between WT, EGR1-/- and EGR4-/- CD4+ T cells.CD4+ T cells were isolated from the spleens of WT, EGR1KO and EGR4KO mice by cell sorting. (K) CD4+ T cells were incubated for 5 days under polarizing conditions before analysis of cytokine production by ELISA.


Figure 4 ( A ) NFAT nuclear localization was measured was measured by immunocytochemistry in CD4 + and CD8 + T cells isolated by negative selection from WT , EGR1KO and EGR4KO mice . Cells were stimulated with anti - CD3 / CD28 antibodies for the indicated time periods before fixation and staining . Nuclear localization of NFATc1 in CD4 + and CD8 + T cells was determined based on colocalization of NFATc1 and DAPI measured by Pearson analysis ( mean ± SEM ; data are based upon 13 to 20 cells / data point from one experiment ; experiment was repeated 3 times ) . EGR4 - mediated differences in NFATc1 localization were determined by two - way ANOVA . NFAT localization was dependent upon EGR4 expression in both CD4 + ( p = 0 . 025 ) and CD8 + ( p = 0 . 039 ) T cells . Post - hoc analysis revealed EGR4 - dependent differences at specific timepoints * p < 0 . 05 ; * * * p < 0 . 001 . ( B ) WT , EGR1KO and EGR4KO CD4 + T cells were isolated from the spleen by negative selection before plating on poly - lysine ( control ) or CD3 / CD28 and loading with fura2 . Ca2 + levels shown are from representative single cells measured from multiple experiments . ( C ) Scatter plots showing area under the curve ( AUC ) and variance for each cell measured under all conditions .

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Figure 4(A) NFAT nuclear localization was measured was measured by immunocytochemistry in CD4+ and CD8+ T cells isolated by negative selection from WT, EGR1KO and EGR4KO mice. Cells were stimulated with anti-CD3/CD28 antibodies for the indicated time periods before fixation and staining. Nuclear localization of NFATc1 in CD4+ and CD8+ T cells was determined based on colocalization of NFATc1 and DAPI measured by Pearson analysis (mean±SEM; data are based upon 13 to 20 cells/data point from one experiment; experiment was repeated 3 times). EGR4-mediated differences in NFATc1 localization were determined by two-way ANOVA. NFAT localization was dependent upon EGR4 expression in both CD4+ (p=0.025) and CD8+ (p=0.039) T cells. Post-hoc analysis revealed EGR4-dependent differences at specific timepoints * p < 0.05; *** p < 0.001.(B) WT, EGR1KO and EGR4KO CD4+ T cells were isolated from the spleen by negative selection before plating on poly-lysine (control) or CD3/CD28 and loading with fura2. Ca2+ levels shown are from representative single cells measured from multiple experiments. (C) Scatter plots showing area under the curve (AUC) and variance for each cell measured under all conditions.


Figure 5CD4 + and CD8 + T cells were isolated by negative selection from WT or EGR4 - / - mice . ( A ) CD4 + and CD8 + T cells were loaded with Fura - 2AM and plated on poly - D - lysine ( 2 hrs ) before treating with biotinylated anti - CD3ε , followed by streptavidin where indicated . The average response of the majority of CD4 + ( WT 74 . 2 ± 3 . 2 % ; EGR4 - / - 77 . 8 ± 2 . 9 % ) and CD8 + ( WT 79 . 4 ± 3 . 5 % ; EGR4 - / - 78 . 2 ± 2 . 5 % ) cells that responded to streptavidin crosslinking of CD3 ( mean ± SEM ; 40 - 60 cells / coverslip ; 7 to 8 separate experiments ) . CD4 + and CD8 + T cells were isolated by negative selection from WT or EGR4 - / - mice . ( B ) Cells were plated on anti - CD3 / CD28 antibodies for 2 hrs ; BTP2 ( 10 μM ) was added where marked by the arrow . CD4 + T cells were isolated by negative selection from WT or EGR4 - / - mice . ( C ) CD4 + T cells were incubated with anti - CD3 / CD28 antibodies for 6 hrs before measuring expression of IFNγ , IL - 2 , IL - 17 and IL - 4 by FACS analysis . Data are mean ± SEM ; a minimum of 3 biological replicates were examined ; each biological replicate includes 2 technical replicates .

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Figure 5CD4+ and CD8+ T cells were isolated by negative selection from WT or EGR4-/-mice. (A) CD4+ and CD8+ T cells were loaded with Fura-2AM and plated on poly-D-lysine (2 hrs) before treating with biotinylated anti-CD3ε, followed by streptavidin where indicated. The average response of the majority of CD4+ (WT 74.2±3.2%; EGR4-/- 77.8±2.9%) and CD8+ (WT 79.4±3.5%; EGR4-/- 78.2±2.5%) cells that responded to streptavidin crosslinking of CD3 (mean±SEM; 40-60 cells/coverslip; 7 to 8 separate experiments).CD4+ and CD8+ T cells were isolated by negative selection from WT or EGR4-/-mice. (B) Cells were plated on anti-CD3/CD28 antibodies for 2 hrs; BTP2 (10 μM) was added where marked by the arrow.CD4+ T cells were isolated by negative selection from WT or EGR4-/-mice. (C) CD4+ T cells were incubated with anti-CD3/CD28 antibodies for 6 hrs before measuring expression of IFNγ, IL-2, IL-17 and IL-4 by FACS analysis. Data are mean±SEM; a minimum of 3 biological replicates were examined; each biological replicate includes 2 technical replicates.


Figure 6CD4 + T cells were isolated by negative selection from WT , EGR4 - / - mice . ( A ) WT or EGR4 - / - cells were loaded with Fura - 2AM and plated on anti - CD3 / CD28 antibodies for 2 hrs . Senicapoc ( 4 μM ) or Tram - 34 ( 5 μM ) were added where marked by the arrow . CD4 + T cells were isolated by negative selection from WT , EGR4 - / - mice . ( B ) CD4 + T cells were isolated by negative selection from WT or EGR4 - / - mice and incubated with anti - CD3 / CD28 antibodies for 6 hrs in the presence or absence of either senicapoc , TRAM - 34 or Shk - Dap22 before measuring expression of IFNγ levels by FACS analysis . Data are mean ± SEM ; 3 biological replicates were examined ; each biological replicate includes 2 technical replicates . * * * p < 0 . 001 ; * * * * p < 0 . 0001 . CD4 + T cells were isolated by negative selection from WT , EGR4 - / - mice . ( C ) To measure the effect of KCa3 . 1 inhibition of cell proliferation , WT and EGR4 - / - CD4 + T cells were isolated from the spleen by cell sorting and stained with CFSE . Cells were incubated with anti - TCRβ antibodies in the presence of vehicle , Senicapoc ( 5 µM ) or TRAM - 34 ( 5 µM ) for 4 days and then collected for FACS analysis . ( D ) Generation analysis was completed for all data using FlowJo software . Data are mean ± SEM ; 3 biological replicates were examined ; each biological replicate includes 2 technical replicates .

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Figure 6CD4+ T cells were isolated by negative selection from WT, EGR4-/- mice. (A) WT or EGR4-/- cells were loaded with Fura-2AM and plated on anti-CD3/CD28 antibodies for 2 hrs. Senicapoc (4 μM) or Tram-34 (5 μM) were added where marked by the arrow.CD4+ T cells were isolated by negative selection from WT, EGR4-/- mice. (B) CD4+ T cells were isolated by negative selection from WT or EGR4-/- mice and incubated with anti-CD3/CD28 antibodies for 6 hrs in the presence or absence of either senicapoc, TRAM-34 or Shk-Dap22 before measuring expression of IFNγ levels by FACS analysis. Data are mean±SEM; 3 biological replicates were examined; each biological replicate includes 2 technical replicates. *** p < 0.001; **** p < 0.0001.CD4+ T cells were isolated by negative selection from WT, EGR4-/- mice. (C) To measure the effect of KCa3.1 inhibition of cell proliferation, WT and EGR4-/- CD4+ T cells were isolated from the spleen by cell sorting and stained with CFSE. Cells were incubated with anti-TCRβ antibodies in the presence of vehicle, Senicapoc (5 µM) or TRAM-34 (5 µM) for 4 days and then collected for FACS analysis. (D) Generation analysis was completed for all data using FlowJo software. Data are mean±SEM; 3 biological replicates were examined; each biological replicate includes 2 technical replicates.


Figure 7B16N melanoma cells stably expressing GFP - luciferase were injected into syngeneic WT and EGR4 - / - mice by tail vein injection . ( A ) Luciferase expression was monitored by IVIS imaging at the indicated timepoints after luciferin injections . B16N melanoma cells stably expressing GFP - luciferase were injected into syngeneic WT and EGR4 - / - mice by tail vein injection . ( B - D ) Mice were sacrificed on day 20 and tumors were counted . Data is presented as total / mouse ( B ) , N = 9 for each gender and genotype . Differences in luciferase activity measured by IVIS in Panel A were determined by two - way ANOVA . Changes in luciferase activity were significantly altered in EGR4 knockout mice ( P < 0 . 0112 ) . B16N melanoma cells stably expressing GFP - luciferase were injected into syngeneic WT and EGR4 - / - mice by tail vein injection . Mice were sacrificed on day 20 and tumors were counted . ( B ) , lung tumors ( C ) and metastatic tumors ( all tumors found outside of the lungs ; D ) . N = 9 for each gender and genotype . Differences in luciferase activity measured by IVIS in Panel A were determined by two - way ANOVA . Changes in luciferase activity were significantly altered in EGR4 knockout mice ( P < 0 . 0112 ) . B16N melanoma cells stably expressing GFP - luciferase were injected into syngeneic WT and EGR4 - / - mice by tail vein injection . All lung tumors were isolated from both WT and EGR4KO mice along with spleens and analysed for CD45 + cells . WT lung tumors were 5 . 407 ± 1 . 843 % CD45 + , while EGR4KO mice were 12 . 445 ± 2 . 239 % CD45 + . WT spleens were 83 . 92 ± 3 . 344 % CD45 + , while EGR4KO mice were 95 . 13 ± 1 . 856 % CD45 + . Relative distributions of T and myeloid cells within the CD45 + populations were determined by flow cytometric analysis . ( E , F ) Distributions of T and myeloid cells found within lung tumors ( E ) vs . the spleen ( F ) . B16N melanoma cells stably expressing GFP - luciferase were injected into syngeneic WT and EGR4 - / - mice by tail vein injection . All lung tumors were isolated from both WT and EGR4KO mice along with spleens and analysed for CD45 + cells . ( G ) Representative FACS plots depicting gating strategies for defining Treg cell populations in Panels E and

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7B16N melanoma cells stably expressing GFP-luciferase were injected into syngeneic WT and EGR4-/- mice by tail vein injection. (A) Luciferase expression was monitored by IVIS imaging at the indicated timepoints after luciferin injections. B16N melanoma cells stably expressing GFP-luciferase were injected into syngeneic WT and EGR4-/- mice by tail vein injection. (B-D) Mice were sacrificed on day 20 and tumors were counted. Data is presented as total/mouse (B), N=9 for each gender and genotype. Differences in luciferase activity measured by IVIS in Panel A were determined by two-way ANOVA. Changes in luciferase activity were significantly altered in EGR4 knockout mice (P < 0.0112).B16N melanoma cells stably expressing GFP-luciferase were injected into syngeneic WT and EGR4-/- mice by tail vein injection. Mice were sacrificed on day 20 and tumors were counted. (B), lung tumors (C) and metastatic tumors (all tumors found outside of the lungs; D). N=9 for each gender and genotype. Differences in luciferase activity measured by IVIS in Panel A were determined by two-way ANOVA. Changes in luciferase activity were significantly altered in EGR4 knockout mice (P < 0.0112).B16N melanoma cells stably expressing GFP-luciferase were injected into syngeneic WT and EGR4-/- mice by tail vein injection. All lung tumors were isolated from both WT and EGR4KO mice along with spleens and analysed for CD45+ cells. WT lung tumors were 5.407 ± 1.843% CD45+, while EGR4KO mice were 12.445 ± 2.239% CD45+. WT spleens were 83.92 ± 3.344% CD45+, while EGR4KO mice were 95.13 ± 1.856% CD45+. Relative distributions of T and myeloid cells within the CD45+ populations were determined by flow cytometric analysis. (E,F) Distributions of T and myeloid cells found within lung tumors (E) vs. the spleen (F).B16N melanoma cells stably expressing GFP-luciferase were injected into syngeneic WT and EGR4-/- mice by tail vein injection. All lung tumors were isolated from both WT and EGR4KO mice along with spleens and analysed for CD45+ cells. (G) Representative FACS plots depicting gating strategies for defining Treg cell populations in Panels E and F


Figure 8Tumors collected and analysed for exhaustion / anergy . Cells were collected and enriched for the immune cell population by Ficoll - Hypaque . WT cells were 36 . 9 ± 4 . 68 % Thy1 . 2 + , EGR4KO cells were 48 . 0 ± 0 . 58 % Thy1 . 2 + . ( A ) CD4 + and CD8 + T cells were stimulated in vitro with ionomycin / PMA before intracellular staining for IFNγ , IL - 17 and IL - 9 and FACS analysis . Tumors collected and analysed for exhaustion / anergy . Cells were collected and enriched for the immune cell population by Ficoll - Hypaque . WT cells were 36 . 9 ± 4 . 68 % Thy1 . 2 + , EGR4KO cells were 48 . 0 ± 0 . 58 % Thy1 . 2 + . ( B , C ) Representative FACS plots depicting gating strategies for defining cytokine expression in panels A to DTumors collected and analysed for exhaustion / anergy . Cells were collected and enriched for the immune cell population by Ficoll - Hypaque . WT cells were 36 . 9 ± 4 . 68 % Thy1 . 2 + , EGR4KO cells were 48 . 0 ± 0 . 58 % Thy1 . 2 + . CD4 + and CD8 + T cells were stained with CD44 , CD73 , FR4 and PD1 to monitor the formation of anergic / exhausted T cells . ( D ) Percentages of each population . Tumors collected and analysed for exhaustion / anergy . Cells were collected and enriched for the immune cell population by Ficoll - Hypaque . WT cells were 36 . 9 ± 4 . 68 % Thy1 . 2 + , EGR4KO cells were 48 . 0 ± 0 . 58 % Thy1 . 2 + . CD4 + and CD8 + T cells were stained with CD44 , CD73 , FR4 and PD1 to monitor the formation of anergic / exhausted T cells . ( E ) Gating strategies . Tumors collected and analysed for exhaustion / anergy . Cells were collected and enriched for the immune cell population by Ficoll - Hypaque . WT cells were 36 . 9 ± 4 . 68 % Thy1 . 2 + , EGR4KO cells were 48 . 0 ± 0 . 58 % Thy1 . 2 + . ( F ) FR4 and PD1 levels in WT vs . EGR4 - / - T cells .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8Tumors collected and analysed for exhaustion/anergy. Cells were collected and enriched for the immune cell population by Ficoll-Hypaque. WT cells were 36.9 ± 4.68% Thy1.2+, EGR4KO cells were 48.0 ± 0.58% Thy1.2+. (A) CD4+ and CD8+ T cells were stimulated in vitro with ionomycin/PMA before intracellular staining for IFNγ, IL-17 and IL-9 and FACS analysis.Tumors collected and analysed for exhaustion/anergy. Cells were collected and enriched for the immune cell population by Ficoll-Hypaque. WT cells were 36.9 ± 4.68% Thy1.2+, EGR4KO cells were 48.0 ± 0.58% Thy1.2+. (B,C) Representative FACS plots depicting gating strategies for defining cytokine expression in panels A to DTumors collected and analysed for exhaustion/anergy. Cells were collected and enriched for the immune cell population by Ficoll-Hypaque. WT cells were 36.9 ± 4.68% Thy1.2+, EGR4KO cells were 48.0 ± 0.58% Thy1.2+. CD4+ and CD8+ T cells were stained with CD44, CD73, FR4 and PD1 to monitor the formation of anergic/exhausted T cells. (D) Percentages of each population.Tumors collected and analysed for exhaustion/anergy. Cells were collected and enriched for the immune cell population by Ficoll-Hypaque. WT cells were 36.9 ± 4.68% Thy1.2+, EGR4KO cells were 48.0 ± 0.58% Thy1.2+. CD4+ and CD8+ T cells were stained with CD44, CD73, FR4 and PD1 to monitor the formation of anergic/exhausted T cells. (E) Gating strategies.Tumors collected and analysed for exhaustion/anergy. Cells were collected and enriched for the immune cell population by Ficoll-Hypaque. WT cells were 36.9 ± 4.68% Thy1.2+, EGR4KO cells were 48.0 ± 0.58% Thy1.2+. (F) FR4 and PD1 levels in WT vs. EGR4-/- T cells.


Figure 1 ( A ) Quantitative RT - PCR analysis of M . tb RNA in EVs from uninfected ( EVs _ Control ) or M . tb - infected ( EVs _ M . tb ) BMMs . ND , not detected . Scale bar , 20 μM . Data shown in ( A ) are the mean ± SD ( n = 3 wells per group ) and all data shown are representative of three independent experiments ( biological replicates ) . Quantitative RT - PCR analysis for IFN - β mRNA ( B ) levels in wild type BMMs 5 and 24 hr post - treatment with EVs respectively ( 1 μg / ml equals approximately 3 x 108 vesicles / ml ) . Data shown are the mean ± SD ( n = 3 wells per group ) and all data shown are representative of three independent experiments ( biological replicates ) . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) . IFN - β protein ( C ) levels in wild type BMMs 5 and 24 hr post - treatment with EVs respectively ( 1 μg / ml equals approximately 3 x 108 vesicles / ml ) . Data shown are the mean ± SD ( n = 3 wells per group ) and all data shown are representative of three independent experiments ( biological replicates ) . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) . ( D ) IFN - β mRNA level was quantified in wild type BMMs treated with EVs at a concentration of 10μg / ml for the times indicated . Data shown are the mean ± SD ( n = 3 wells per group ) and all data shown are representative of three independent experiments ( biological replicates ) . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) . Quantitative RT - PCR analysis for IFN - β mRNA ( E ) levels in either WT , MAVS - and / or RIG - I - knockdown BMMs treated for 4 hr ( E ) with 10µg / ml EVs from uninfected or M . tb - infected macrophages . ( Control ) negative control siRNA . Data shown are the mean ± SD ( n = 3 wells per group ) and all data shown are representative of three independent experiments ( biological replicates ) . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) . IFN - β protein ( F ) levels in either WT , MAVS - and / or RIG - I - knockdown BMMs treated for 24 hr ( F ) with 10µg / ml EVs from uninfected or M . tb - infected macrophages . ( Control ) negative control siRNA . Data shown are the mean ± SD ( n = 3 wells per group ) and all data shown are representative of three independent experiments ( biological replicates ) . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) . ( G ) Similar to above except using WT and Mavs - / - BMMs . Data shown are the mean ± SD ( n = 3 wells per group ) and all data shown are representative of three independent experiments ( biological replicates ) . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) . ( H ) Similar to above except using WT and Mavs - / - BMMs . Scale bar , 20 μM . Data shown in ( H ) are the mean ± SD ( n = 3 wells per group ) and all data shown are representative of three independent experiments ( biological replicates ) . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) . ( J ) Top chamber : WT BMMs ; Bottom chamber : WT or Mavs - / - BMMs . Data shown in ( J ) are the mean ± SD ( n = 3 wells per group ) and all data shown are representative of three independent experiments ( biological replicates ) . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) . ( K ) Western blot analysis for phospho - TBK1 ( Ser172 ) in WT and MAVS - knockdown BMMs treated for 4 hr with EVs from uninfected or M . tb - infected macrophages . β - actin served as a loading control . Densitometry of the western blots for ( K ) are shown . ( L ) Western blot analysis of IRF3 under the conditions described above for TBK1 . Histone H3 ( H3 ) and β - actin were used as loading controls for nuclear fraction and whole - cell lysate ( WCL ) respectively . Densitometry of the western blots are shown . ( M ) Fluorescence microscopy analysis for nuclear translocation of IRF3 in wild type BMMs untreated ( Mock ) or treated with EVs from uninfected or M . tb - infected macrophages . Scale bar , 20 μM . ( N ) IRF3 nuclear translocation was analyzed in RIG - I - or MAVS - knockdown BMMs . Scale bar , 20 μM .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Quantitative RT-PCR analysis of M.tb RNA in EVs from uninfected (EVs_Control) or M.tb-infected (EVs_M.tb) BMMs. ND, not detected. Scale bar, 20 μM. Data shown in (A) are the mean ± SD (n = 3 wells per group) and all data shown are representative of three independent experiments (biological replicates).Quantitative RT-PCR analysis for IFN-β mRNA (B) levels in wild type BMMs 5 and 24 hr post-treatment with EVs respectively (1 μg/ml equals approximately 3 x 108 vesicles/ml). Data shown are the mean ± SD (n = 3 wells per group) and all data shown are representative of three independent experiments (biological replicates). * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).IFN-β protein (C) levels in wild type BMMs 5 and 24 hr post-treatment with EVs respectively (1 μg/ml equals approximately 3 x 108 vesicles/ml). Data shown are the mean ± SD (n = 3 wells per group) and all data shown are representative of three independent experiments (biological replicates). * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).(D) IFN-β mRNA level was quantified in wild type BMMs treated with EVs at a concentration of 10μg/ml for the times indicated. Data shown are the mean ± SD (n = 3 wells per group) and all data shown are representative of three independent experiments (biological replicates). * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).Quantitative RT-PCR analysis for IFN-β mRNA (E) levels in either WT, MAVS- and/or RIG-I-knockdown BMMs treated for 4 hr (E) with 10µg/ml EVs from uninfected or M.tb-infected macrophages. (Control) negative control siRNA. Data shown are the mean ± SD (n = 3 wells per group) and all data shown are representative of three independent experiments (biological replicates). * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).IFN-β protein (F) levels in either WT, MAVS- and/or RIG-I-knockdown BMMs treated for 24 hr (F) with 10µg/ml EVs from uninfected or M.tb-infected macrophages. (Control) negative control siRNA. Data shown are the mean ± SD (n = 3 wells per group) and all data shown are representative of three independent experiments (biological replicates). * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).(G) Similar to above except using WT and Mavs -/- BMMs. Data shown are the mean ± SD (n = 3 wells per group) and all data shown are representative of three independent experiments (biological replicates). * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).(H) Similar to above except using WT and Mavs -/- BMMs. Scale bar, 20 μM. Data shown in (H) are the mean ± SD (n = 3 wells per group) and all data shown are representative of three independent experiments (biological replicates). * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).(J) Top chamber: WT BMMs; Bottom chamber: WT or Mavs -/- BMMs. Data shown in (J) are the mean ± SD (n = 3 wells per group) and all data shown are representative of three independent experiments (biological replicates). * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).(K) Western blot analysis for phospho-TBK1 (Ser172) in WT and MAVS-knockdown BMMs treated for 4 hr with EVs from uninfected or M.tb-infected macrophages. β-actin served as a loading control. Densitometry of the western blots for (K) are shown.(L) Western blot analysis of IRF3 under the conditions described above for TBK1. Histone H3 (H3) and β-actin were used as loading controls for nuclear fraction and whole-cell lysate (WCL) respectively. Densitometry of the western blots are shown.(M) Fluorescence microscopy analysis for nuclear translocation of IRF3 in wild type BMMs untreated (Mock) or treated with EVs from uninfected or M.tb-infected macrophages. Scale bar, 20 μM.(N) IRF3 nuclear translocation was analyzed in RIG-I- or MAVS-knockdown BMMs. Scale bar, 20 μM.


Figure 2 ( C ) Yield of purified EVs from BMMs infected with various M . tb CDC 1551 strains based on Nanosight analysis . Data shown are the mean ± SD ( n = 3 per group ) and all data shown are representative of three independent experiments . ( B ) EVs from cells infected with WT , ∆ esxA or esxA - complemented ( ∆ esxA - C ) M . tb Erdman strains . ( C ) Yield of purified EVs from BMMs infected with various M . tb CDC 1551 strains based on Nanosight analysis . Data shown are the mean ± SD ( n = 3 per group ) and all data shown are representative of three independent experiments . ( D ) EVs from cells infected with WT , ∆ esxA or esxA - complemented ( ∆ esxA - C ) M . tb Erdman strains . Data shown are the mean ± SD ( n = 3 per group ) and all data shown are representative of three independent experiments . qRT - PCR analysis for M . tb RNA in EVs from BMMs infected with various M . tb CDC 1551 strains ( E ) ND , not detected . Data shown are the mean ± SD ( n = 3 per group ) and all data shown are representative of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) . qRT - PCR analysis for M . tb RNA in EVs from BMMs infected with various M . tb Erdman strains ( F ) . ND , not detected . Data shown are the mean ± SD ( n = 3 per group ) and all data shown are representative of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) . qRT - PCR analysis for IFN - β mRNA ( G ) in WT BMMs treated with EVs for 4 hr and 24 hr respectively . The EVs were isolated from BMMs that were infected with the different M . tb CDC 1551 strains . Data shown are the mean ± SD ( n = 3 per group ) and all data shown are representative of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) . IFN - β protein ( H ) levels in WT BMMs treated with EVs for 4 hr and 24 hr respectively . The EVs were isolated from BMMs that were infected with the different M . tb CDC 1551 strains . Data shown are the mean ± SD ( n = 3 per group ) and all data shown are representative of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) . ( I ) ELISA analysis for IFN - β secreted by BMMs treated with EVs plus liposomes containing M . tb RNA . Data shown are the mean ± SD ( n = 3 per group ) and all data shown are representative of three independent experiments . qRT - PCR analysis for IFN - β mRNA ( J ) in WT BMMs treated with EVs for 4 and 24 hr respectively . The EVs were isolated from BMMs that were infected with the different M . tb Erdman strains . Data shown are the mean ± SD ( n = 3 per group ) and all data shown are representative of three independent experiments . IFN - β protein ( K ) levels in WT BMMs treated with EVs for 4 and 24 hr respectively . The EVs were isolated from BMMs that were infected with the different M . tb Erdman strains . Data shown in K ) are the mean ± SD ( n = 3 per group ) and all data shown are representative of three independent experiments .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(C) Yield of purified EVs from BMMs infected with various M.tb CDC 1551 strains based on Nanosight analysis. Data shown are the mean ± SD (n = 3 per group) and all data shown are representative of three independent experiments.(B) EVs from cells infected with WT, ∆esxA or esxA-complemented (∆esxA-C) M.tb Erdman strains.(C) Yield of purified EVs from BMMs infected with various M.tb CDC 1551 strains based on Nanosight analysis. Data shown are the mean ± SD (n = 3 per group) and all data shown are representative of three independent experiments.(D) EVs from cells infected with WT, ∆esxA or esxA-complemented (∆esxA-C) M.tb Erdman strains. Data shown are the mean ± SD (n = 3 per group) and all data shown are representative of three independent experiments.qRT-PCR analysis for M.tb RNA in EVs from BMMs infected with various M.tb CDC 1551 strains (E) ND, not detected. Data shown are the mean ± SD (n = 3 per group) and all data shown are representative of three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).qRT-PCR analysis for M.tb RNA in EVs from BMMs infected with various M.tb Erdman strains (F). ND, not detected. Data shown are the mean ± SD (n = 3 per group) and all data shown are representative of three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).qRT-PCR analysis for IFN-β mRNA (G) in WT BMMs treated with EVs for 4 hr and 24 hr respectively. The EVs were isolated from BMMs that were infected with the different M.tb CDC 1551 strains. Data shown are the mean ± SD (n = 3 per group) and all data shown are representative of three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).IFN-β protein (H) levels in WT BMMs treated with EVs for 4 hr and 24 hr respectively. The EVs were isolated from BMMs that were infected with the different M.tb CDC 1551 strains. Data shown are the mean ± SD (n = 3 per group) and all data shown are representative of three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).(I) ELISA analysis for IFN-β secreted by BMMs treated with EVs plus liposomes containing M.tb RNA. Data shown are the mean ± SD (n = 3 per group) and all data shown are representative of three independent experiments.qRT-PCR analysis for IFN-β mRNA (J) in WT BMMs treated with EVs for 4 and 24 hr respectively. The EVs were isolated from BMMs that were infected with the different M.tb Erdman strains. Data shown are the mean ± SD (n = 3 per group) and all data shown are representative of three independent experiments.IFN-β protein (K) levels in WT BMMs treated with EVs for 4 and 24 hr respectively. The EVs were isolated from BMMs that were infected with the different M.tb Erdman strains. Data shown in K) are the mean ± SD (n = 3 per group) and all data shown are representative of three independent experiments.


Figure 3M . tb CFU in WT mouse BMMs pre - treated with EVs minus co - treatment with IFN - ɣ . BMMs were treated with EVs from uninfected ( EVs _ Control ) or M . tb - infected ( EVs _ M . tb ) cells for 0 , 24 , 48 and 72 hr following a 1 hr M . tb infection . Mock , no EV treatment . Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by two - tailed Student ' s t - test . M . tb CFU in WT mouse BMMs pre - treated with EVs plus ( B ) co - treatment with IFN - ɣ . BMMs were treated with EVs from uninfected ( EVs _ Control ) or M . tb - infected ( EVs _ M . tb ) cells for 0 , 24 , 48 and 72 hr following a 1 hr M . tb infection . Mock , no EV treatment . Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by two - tailed Student ' s t - test . Immunofluorescence microscopy analysis for colocalization of M . tb ( GFP ) with lysosome marker Lamp - 1 in WT mouse BMMs . Cells were pre - treated for 5 hr with EVs from uninfected or M . tb - infected macrophages plus IFN - γ , and then infected with GFP - expressing M . tb for 24 hr prior to immunostaining . quantitative analysis of M . tb colocalization with Lamp - 1 in WT mouse BMMs Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . Scale bar , 5 μM n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by two - tailed Student ' s t - test . Immunofluorescence microscopy analysis for colocalization of M . tb ( GFP ) with lysosome marker Lamp - 1 in Mavs - / - ( E and F ) mouse BMMs . Cells were pre - treated for 5 hr with EVs from uninfected or M . tb - infected macrophages plus IFN - γ , and then infected with GFP - expressing M . tb for 24 hr prior to immunostaining . quantitative analysis of M . tb colocalization with Lamp - 1 in Mavs - / - mouse BMMs Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . Scale bar , 5 μM n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by two - tailed Student ' s t - test . Immunofluorescence microscopy as described above except using control siRNA - treated ( G and H ) BMMs . quantitative analysis of M . tb colocalization with Lamp - 1 in control siRNA - treated mouse BMMs Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . Scale bar , 5 μM n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by two - tailed Student ' s t - test . Immunofluorescence microscopy as described above except using RIG - I siRNA - treated ( I and J ) BMMs . quantitative analysis of M . tb colocalization with Lamp - 1 in RIG - I siRNA - treated mouse BMMs Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . Scale bar , 5 μM n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by two - tailed Student ' s t - test . M . tb CFU in infected Mavs - / - ( K ) mouse BMMs pre - treated with IFN - ɣ plus EVs from uninfected ( EVs _ Control ) or M . tb - infected ( EVs _ M . tb ) BMMs . CFU was determined immediately after the 1 hr infection or 24 and 72 hr post - infection . Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by two - tailed Student ' s t - test . M . tb CFU in control siRNA - treated ( L ) or RIG - I siRNA - treated ( M ) mouse BMMs pre - treated with IFN - ɣ plus EVs from uninfected ( EVs _ Control ) or M . tb - infected ( EVs _ M . tb ) BMMs . CFU was determined immediately after the 1 hr infection or 24 and 72 hr post - infection . Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by two - tailed Student ' s t - test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3M.tb CFU in WT mouse BMMs pre-treated with EVs minus co-treatment with IFN-ɣ. BMMs were treated with EVs from uninfected (EVs_Control) or M.tb-infected (EVs_M.tb) cells for 0, 24, 48 and 72 hr following a 1 hr M.tb infection. Mock, no EV treatment. Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. ; * p < 0.05, ** p < 0.01 and *** p < 0.001 by two-tailed Student's t-test.M.tb CFU in WT mouse BMMs pre-treated with EVs plus (B) co-treatment with IFN-ɣ. BMMs were treated with EVs from uninfected (EVs_Control) or M.tb-infected (EVs_M.tb) cells for 0, 24, 48 and 72 hr following a 1 hr M.tb infection. Mock, no EV treatment. Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. ; * p < 0.05, ** p < 0.01 and *** p < 0.001 by two-tailed Student's t-test.Immunofluorescence microscopy analysis for colocalization of M.tb (GFP) with lysosome marker Lamp-1 in WT mouse BMMs. Cells were pre-treated for 5 hr with EVs from uninfected or M.tb-infected macrophages plus IFN-γ, and then infected with GFP-expressing M.tb for 24 hr prior to immunostaining. quantitative analysis of M.tb colocalization with Lamp-1 in WT mouse BMMs Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. Scale bar, 5 μM n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by two-tailed Student's t-test.Immunofluorescence microscopy analysis for colocalization of M.tb (GFP) with lysosome marker Lamp-1 in Mavs-/- (E and F) mouse BMMs. Cells were pre-treated for 5 hr with EVs from uninfected or M.tb-infected macrophages plus IFN-γ, and then infected with GFP-expressing M.tb for 24 hr prior to immunostaining. quantitative analysis of M.tb colocalization with Lamp-1 in Mavs-/- mouse BMMs Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. Scale bar, 5 μM n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by two-tailed Student's t-test.Immunofluorescence microscopy as described above except using control siRNA-treated (G and H) BMMs. quantitative analysis of M.tb colocalization with Lamp-1 in control siRNA-treated mouse BMMs Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. Scale bar, 5 μM n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by two-tailed Student's t-test.Immunofluorescence microscopy as described above except using RIG-I siRNA-treated (I and J) BMMs. quantitative analysis of M.tb colocalization with Lamp-1 in RIG-I siRNA-treated mouse BMMs Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. Scale bar, 5 μM n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by two-tailed Student's t-test.M.tb CFU in infected Mavs-/- (K) mouse BMMs pre-treated with IFN-ɣ plus EVs from uninfected (EVs_Control) or M.tb-infected (EVs_M.tb) BMMs. CFU was determined immediately after the 1 hr infection or 24 and 72 hr post-infection. Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. ; * p < 0.05, ** p < 0.01 and *** p < 0.001 by two-tailed Student's t-test.M.tb CFU in control siRNA-treated (L) or RIG-I siRNA-treated (M) mouse BMMs pre-treated with IFN-ɣ plus EVs from uninfected (EVs_Control) or M.tb-infected (EVs_M.tb) BMMs. CFU was determined immediately after the 1 hr infection or 24 and 72 hr post-infection. Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 by two-tailed Student's t-test.


Figure 4 ( A ) Immunofluorescence microscopy analysis for colocalization of M . tb with Lamp - 1 in WT BMMs pre - treated with EVs from macrophages infected with WT , ∆ secA2 or secA2 - complemented ( ∆ secA2 - C ) M . tb CDC 1551 strains . The cells were pre - treated with EVs supplemented with recombinant mouse IFN - γ for 5 hr and subsequently infected for 24 hr with GFP - expressing M . tb . ( B ) quantitative analysis for the colocalization of M . tb with Lamp - 1 . Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . Scale bar , 5 μM ; * p < 0 . 05 and * * p < 0 . 01 by two - tailed Student ' s t - test . ( C ) M . tb CFU in WT BMMs pre - pretreated with recombinant mouse IFN - γ and EVs from macrophages infected with WT , ∆ secA2 or secA2 - complemented ( ∆ secA2 - C ) M . tb CDC 1551 strains . Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . n . s . , not significant ; * p < 0 . 05 and * * p < 0 . 01 by two - tailed Student ' s t - test . ( D ) and ( E ) liposomes containing RNA isolated from EVs released from BMMs infected with WT M . tb were also added to cells 5 hr prior to infection . Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . Scale bar , 5 μM n . s . , not significant ( F ) liposome - encapsulated RNA isolated from EVs released from BMMs infected with WT M . tb was included during EV and IFN - γ pre - treatment . Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . n . s . , not significant ; * p < 0 . 05 and * * p < 0 . 01 by two - tailed Student ' s t - test .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Immunofluorescence microscopy analysis for colocalization of M.tb with Lamp-1 in WT BMMs pre-treated with EVs from macrophages infected with WT, ∆secA2 or secA2-complemented (∆secA2-C) M.tb CDC 1551 strains. The cells were pre-treated with EVs supplemented with recombinant mouse IFN-γ for 5 hr and subsequently infected for 24 hr with GFP-expressing M.tb. (B) quantitative analysis for the colocalization of M.tb with Lamp-1. Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. Scale bar, 5 μM ; * p < 0.05 and ** p < 0.01 by two-tailed Student's t-test.(C) M.tb CFU in WT BMMs pre-pretreated with recombinant mouse IFN-γ and EVs from macrophages infected with WT, ∆secA2 or secA2-complemented (∆secA2-C) M.tb CDC 1551 strains. Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. n.s., not significant; * p < 0.05 and ** p < 0.01 by two-tailed Student's t-test.(D) and (E) liposomes containing RNA isolated from EVs released from BMMs infected with WT M.tb were also added to cells 5 hr prior to infection. Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. Scale bar, 5 μM n.s., not significant(F) liposome-encapsulated RNA isolated from EVs released from BMMs infected with WT M.tb was included during EV and IFN-γ pre-treatment. Data shown are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. n.s., not significant; * p < 0.05 and ** p < 0.01 by two-tailed Student's t-test.


Figure 5Immunofluorescence microscopy and quantitative analysis for colocalization of M . tb with autophagosome marker LC3 in control siRNA - treated BMMs that were untreated or pre - treated for 5 hr with recombinant mouse IFN - γ and EVs from uninfected ( EVs _ Control ) or M . tb - infected ( EVs _ M . tb ) macrophages followed by a 24 hr infection with GFP - expressing M . tb . Mock , untreated . Quantitative data are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . Scale bar , 5 μM . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by two - tailed Student ' s t - test . Immunofluorescence microscopy and quantitative analysis for colocalization of M . tb with autophagosome marker LC3 ( C and D ) in RIG - I siRNA - treated BMMs . Quantitative data are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . Scale bar , 5 μM . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by two - tailed Student ' s t - test . ( E and F ) Immunofluorescence microscopy and quantitative analysis for colocalization of M . tb with markers LC3 in WT BMMs that were left untreated or pre - treated for 5 hr with recombinant mouse IFN - γ and EVs from uninfected ( EVs _ Control ) or M . tb - infected ( EVs _ M . tb ) macrophages , followed by a 24 hr infection with GFP - expressing M . tb . Mock , untreated . Quantitative data are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . Scale bar , 5 μM . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by two - tailed Student ' s t - test . Immunofluorescence microscopy and quantitative analysis for colocalization of M . tb with markers LC3 in Mavs - / - BMMs . Quantitative data are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . Scale bar , 5 μM . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by two - tailed Student ' s t - test . Immunofluorescence microscopy quantitative analysis for colocalization of M . tb with NOX2 ( I and J ) as described above in WT BMMs . Quantitative data are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . Scale bar , 5 μM . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by two - tailed Student ' s t - test . Immunofluorescence microscopy quantitative analysis for colocalization of M . tb with p47phox ( K and L ) as described above in WT BMMs . Quantitative data are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . Scale bar , 5 μM . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by two - tailed Student ' s t - test . Immunofluorescence microscopy quantitative analysis for colocalization of M . tb similar to ( I - L ) , but using Mavs - / - BMMs . NOX2 ( M and N ) Quantitative data are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . Scale bar , 5 μM . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by two - tailed Student ' s t - test . Immunofluorescence microscopy quantitative analysis for colocalization of M . tb similar to ( I - L ) , but using Mavs - / - BMMs . p47phox ( O and P ) Quantitative data are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments . Scale bar , 5 μM . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by two - tailed Student ' s t - test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Immunofluorescence microscopy and quantitative analysis for colocalization of M.tb with autophagosome marker LC3 in control siRNA -treated BMMs that were untreated or pre-treated for 5 hr with recombinant mouse IFN-γ and EVs from uninfected (EVs_Control) or M.tb-infected (EVs_M.tb) macrophages followed by a 24 hr infection with GFP-expressing M.tb. Mock, untreated. Quantitative data are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. Scale bar, 5 μM. n.s., not significant; * p < 0.05 , ** p < 0.01 and *** p < 0.001 by two-tailed Student's t-test.Immunofluorescence microscopy and quantitative analysis for colocalization of M.tb with autophagosome marker LC3 (C and D) in RIG-I siRNA-treated BMMs. Quantitative data are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. Scale bar, 5 μM. n.s., not significant; * p < 0.05 , ** p < 0.01 and *** p < 0.001 by two-tailed Student's t-test.(E and F) Immunofluorescence microscopy and quantitative analysis for colocalization of M.tb with markers LC3 in WT BMMs that were left untreated or pre-treated for 5 hr with recombinant mouse IFN-γ and EVs from uninfected (EVs_Control) or M.tb-infected (EVs_M.tb) macrophages, followed by a 24 hr infection with GFP-expressing M.tb. Mock, untreated. Quantitative data are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. Scale bar, 5 μM. n.s., not significant; * p < 0.05 , ** p < 0.01 and *** p < 0.001 by two-tailed Student's t-test.Immunofluorescence microscopy and quantitative analysis for colocalization of M.tb with markers LC3 in Mavs -/- BMMs. Quantitative data are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. Scale bar, 5 μM. n.s., not significant; * p < 0.05 , ** p < 0.01 and *** p < 0.001 by two-tailed Student's t-test.Immunofluorescence microscopy quantitative analysis for colocalization of M.tb with NOX2 (I and J) as described above in WT BMMs. Quantitative data are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. Scale bar, 5 μM. n.s., not significant; * p < 0.05 , ** p < 0.01 and *** p < 0.001 by two-tailed Student's t-test.Immunofluorescence microscopy quantitative analysis for colocalization of M.tb with p47phox (K and L) as described above in WT BMMs. Quantitative data are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. Scale bar, 5 μM. n.s., not significant; * p < 0.05 , ** p < 0.01 and *** p < 0.001 by two-tailed Student's t-test.Immunofluorescence microscopy quantitative analysis for colocalization of M.tb similar to ( I -L), but using Mavs -/- BMMs. NOX2 (M and N) Quantitative data are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. Scale bar, 5 μM. n.s., not significant; * p < 0.05 , ** p < 0.01 and *** p < 0.001 by two-tailed Student's t-test.Immunofluorescence microscopy quantitative analysis for colocalization of M.tb similar to ( I -L), but using Mavs -/- BMMs. p47phox (O and P) Quantitative data are the mean ± SD of three independent infections and all data shown are representative of at least three independent experiments. Scale bar, 5 μM. n.s., not significant; * p < 0.05 , ** p < 0.01 and *** p < 0.001 by two-tailed Student's t-test.


Figure 6 ( A and B ) Quantitative analysis of immunofluorescence microscopy images for colocalization of M . tb with Lamp - 1 in WT ( A ) and Mavs - / - ( B ) BMMs infected with M . tb for 24 hr and then treated for an additional 24 hr with moxifloxacin ( MFX ) and / or EVs from M . tb - infected BMMs ( EVs + MFX ) in the presence of recombinant mouse IFN - ɣ . Mock , no EVs or moxifloxacin treatment ; EVs _ Control , EVs from uninfected BMMs . Data shown are representative of three independent infections . The results are the mean ± SD of three independent experiments . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) . LC3 ( C ) , colocalization with M . tb were analyzed . Data shown are representative of three independent infections . The results are the mean ± SD of three independent experiments . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) . NOX2 ( D ) colocalization with M . tb were analyzed . Data shown are representative of three independent infections . The results are the mean ± SD of three independent experiments . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) . p47phox ( E ) colocalization with M . tb were analyzed . Data shown are representative of three independent infections . The results are the mean ± SD of three independent experiments . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) . Mavs - / - BMMs were used and colocalization of M . tb with LC3 ( F ) were quantified . Data shown are representative of three independent infections . The results are the mean ± SD of three independent experiments . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) . Mavs - / - BMMs were used and colocalization of M . tb with NOX2 ( G ) were quantified . Data shown are representative of three independent infections . The results are the mean ± SD of three independent experiments . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) . Mavs - / - BMMs were used and colocalization of M . tb with p47phox ( H ) were quantified . Data shown are representative of three independent infections . The results are the mean ± SD of three independent experiments . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) . ( I and J ) M . tb CFU analysis in WT ( I ) and Mavs - / - ( J ) BMMs infected with M . tb for 24 hr followed by treatment with EVs , moxifloxacin or combination in the presence of IFN - ɣ for 24 and 72 hr . Data shown are representative of three independent infections . The results are the mean ± SD of three independent experiments . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A and B) Quantitative analysis of immunofluorescence microscopy images for colocalization of M.tb with Lamp-1 in WT (A) and Mavs -/- (B) BMMs infected with M.tb for 24 hr and then treated for an additional 24 hr with moxifloxacin (MFX) and/or EVs from M.tb-infected BMMs (EVs+MFX) in the presence of recombinant mouse IFN-ɣ. Mock, no EVs or moxifloxacin treatment; EVs_Control, EVs from uninfected BMMs. Data shown are representative of three independent infections. The results are the mean ± SD of three independent experiments. n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).LC3 (C), colocalization with M.tb were analyzed. Data shown are representative of three independent infections. The results are the mean ± SD of three independent experiments. n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).NOX2 (D) colocalization with M.tb were analyzed. Data shown are representative of three independent infections. The results are the mean ± SD of three independent experiments. n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).p47phox (E) colocalization with M.tb were analyzed. Data shown are representative of three independent infections. The results are the mean ± SD of three independent experiments. n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).Mavs -/- BMMs were used and colocalization of M.tb with LC3 (F) were quantified. Data shown are representative of three independent infections. The results are the mean ± SD of three independent experiments. n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).Mavs -/- BMMs were used and colocalization of M.tb with NOX2 (G) were quantified. Data shown are representative of three independent infections. The results are the mean ± SD of three independent experiments. n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).Mavs -/- BMMs were used and colocalization of M.tb with p47phox (H) were quantified. Data shown are representative of three independent infections. The results are the mean ± SD of three independent experiments. n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).(I and J) M.tb CFU analysis in WT (I) and Mavs -/- (J) BMMs infected with M.tb for 24 hr followed by treatment with EVs, moxifloxacin or combination in the presence of IFN-ɣ for 24 and 72 hr. Data shown are representative of three independent infections. The results are the mean ± SD of three independent experiments. n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).


Figure 7Representative histopathological analysis for lung sections of WT ( B ) mice that were infected with M . tb and subsequent left untreated ( Mock ) or treated with EVs from uninfected BMMs ( EVs _ Control ) , EVs from M . tb - infected BMMs ( EVs ) , moxifloxacin ( MXF ) , or combination of EVs and MXF ( EVs + MXF ) . Data shown is representative of two independent experiments . The results are the mean ± SD ( n = 4 mice per group ) . Scale bar , 100 μMM . tb CFU in the lung and spleen of WT ( C ) mice treated with EVs , MXF or combination of both . Data shown is representative of two independent experiments . The results are the mean ± SD ( n = 4 mice per group ) . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) . Representative histopathological analysis for lung sections of Mavs - / - ( D ) mice that were infected with M . tb and subsequent left untreated ( Mock ) or treated with EVs from uninfected BMMs ( EVs _ Control ) , EVs from M . tb - infected BMMs ( EVs ) , moxifloxacin ( MXF ) , or combination of EVs and MXF ( EVs + MXF ) . Data shown is representative of two independent experiments . The results are the mean ± SD ( n = 4 mice per group ) . Scale bar , 100 μMM . tb CFU in the lung and spleen of Mavs - / - ( E ) mice treated with EVs , MXF or combination of both . Data shown is representative of two independent experiments . The results are the mean ± SD ( n = 4 mice per group ) . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) . ELISA analysis for , IFN - β , TNF - α , IL - 1β and IFN - γ protein level in serum isolate from M . tb - infected WT ( F ) or Mavs - / - ( G ) mice treated with EVs , MXF or a combination of both . Data shown is representative of two independent experiments . The results are the mean ± SD ( n = 4 mice per group ) . n . s . , not significant ; * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 by Student ' s t - test ( two tailed ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Representative histopathological analysis for lung sections of WT (B) mice that were infected with M.tb and subsequent left untreated (Mock) or treated with EVs from uninfected BMMs (EVs_Control), EVs from M.tb-infected BMMs (EVs), moxifloxacin (MXF), or combination of EVs and MXF (EVs+MXF). Data shown is representative of two independent experiments. The results are the mean ± SD (n = 4 mice per group). Scale bar, 100 μMM.tb CFU in the lung and spleen of WT (C) mice treated with EVs, MXF or combination of both. Data shown is representative of two independent experiments. The results are the mean ± SD (n = 4 mice per group). n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).Representative histopathological analysis for lung sections of Mavs -/- (D) mice that were infected with M.tb and subsequent left untreated (Mock) or treated with EVs from uninfected BMMs (EVs_Control), EVs from M.tb-infected BMMs (EVs), moxifloxacin (MXF), or combination of EVs and MXF (EVs+MXF). Data shown is representative of two independent experiments. The results are the mean ± SD (n = 4 mice per group). Scale bar, 100 μMM.tb CFU in the lung and spleen of Mavs -/- (E) mice treated with EVs, MXF or combination of both. Data shown is representative of two independent experiments. The results are the mean ± SD (n = 4 mice per group). n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).ELISA analysis for, IFN-β, TNF-α, IL-1β and IFN-γ protein level in serum isolate from M.tb-infected WT (F) or Mavs -/- (G) mice treated with EVs, MXF or a combination of both. Data shown is representative of two independent experiments. The results are the mean ± SD (n = 4 mice per group). n.s., not significant; * p < 0.05, ** p < 0.01 and *** p < 0.001 by Student's t-test (two tailed).


figf1 ( a ) FLAG - AMBRA1 was transfected into HEK293 cells together with ULK1WT or ULK1K46I Myc - tagged proteins . Protein extracts were immunoprecipitated ( IP ) using anti - FLAG antibody . ( b ) HEK293 cells were grown either in normal or in EBS medium for 1 h ; protein extracts were immunoprecipitated using anti - ULK1 antibody or rabbit immunoglobulin as a negative control ( IgG ) . Autophagy induction is shown by p62 degradation . ( c ) HEK293 cells were treated as in b , in the presence of leupeptin and stained by anti - AMBRA1 ( red ) and anti - ULK1 ( green ) antibodies . The merge of the two signals and DAPI ( blue ) for nuclear staining is shown in the right panels . Scale bar , 6 μm . The outlined areas are shown at a higher magnification in the lower panels . White arrows point to the co - localization puncta . Measurements by Metamorph indicate a co - localization ratio of 46 % between ULK1 and AMBRA1 on autophagy induction . ( d , e ) ULK1 and AMBRA1 constructs are illustrated . Asterisks mark nonspecific bands corresponding to immunoglobulins . ( d ) HEK293 cells were co - transfected with vectors encoding AMBRA1 together with ULK1 - Myc FL ( full length ) or its deletion mutants encoding the kinase and the proline - rich domains ( Δ829 - 1 , 051 ) , and the ULK1 carboxy - terminal domain ( Δ1 - 828 ) . Protein extracts were immunoprecipitated using anti - Myc antibody . ( d , e ) ULK1 and AMBRA1 constructs are illustrated . Asterisks mark nonspecific bands corresponding to immunoglobulins . ( e ) HEK293 cells were co - transfected with vectors encoding ULK1 - HA together with β - galactosidase - Myc ( βgal ) , AMBRA1 full - length ( FL ) , and F1 , F2 or F3 Myc - tagged proteins . Protein extracts were immunoprecipitated using anti - HA antibody . The relative amounts of co - immunoprecipitatedULK1 and F1 or F3 , respectively , are the same ( see upper and lower left panel ) . ( f ) HEK293 cells were co - transfected with vectors encoding AMBRA1 , ULK1 - Myc and ATG13 - FLAG . Protein extracts were immunoprecipitated using anti - FLAG antibody . ( g ) HEK293 cells were transfected with scramble siRNA or ATG13 siRNA . Protein extracts were immunoprecipitated using ULK1 antibody or IgG . The band density ratio of immunoprecipitated AMBRA1 relative to immunoprecipitated ULK1 is analysed , with the control ratio arbitrarily defined as 1 . 00 . n = 3 extracts prepared from independent experiments ; data are presented as means ± s . d . and significance is P = 0 . 04 . Uncropped images of blots / gels are shown in Supplementary Fig . S6 . Source data of statistical analysis are shown in Supplementary Table S1 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf1(a) FLAG-AMBRA1 was transfected into HEK293 cells together with ULK1WT or ULK1K46I Myc-tagged proteins. Protein extracts were immunoprecipitated (IP) using anti-FLAG antibody.(b) HEK293 cells were grown either in normal or in EBS medium for 1 h; protein extracts were immunoprecipitated using anti-ULK1 antibody or rabbit immunoglobulin as a negative control (IgG). Autophagy induction is shown by p62 degradation.(c) HEK293 cells were treated as in b, in the presence of leupeptin and stained by anti-AMBRA1 (red) and anti-ULK1 (green) antibodies. The merge of the two signals and DAPI (blue) for nuclear staining is shown in the right panels. Scale bar, 6 μm. The outlined areas are shown at a higher magnification in the lower panels. White arrows point to the co-localization puncta. Measurements by Metamorph indicate a co-localization ratio of 46% between ULK1 and AMBRA1 on autophagy induction.(d,e) ULK1 and AMBRA1 constructs are illustrated. Asterisks mark nonspecific bands corresponding to immunoglobulins. (d) HEK293 cells were co-transfected with vectors encoding AMBRA1 together with ULK1-Myc FL (full length) or its deletion mutants encoding the kinase and the proline-rich domains (Δ829-1,051), and the ULK1 carboxy-terminal domain (Δ1-828). Protein extracts were immunoprecipitated using anti-Myc antibody.(d,e) ULK1 and AMBRA1 constructs are illustrated. Asterisks mark nonspecific bands corresponding to immunoglobulins. (e) HEK293 cells were co-transfected with vectors encoding ULK1-HA together with β-galactosidase-Myc (βgal), AMBRA1 full-length (FL), and F1, F2 or F3 Myc-tagged proteins. Protein extracts were immunoprecipitated using anti-HA antibody. The relative amounts of co-immunoprecipitatedULK1 and F1 or F3, respectively, are the same (see upper and lower left panel).(f) HEK293 cells were co-transfected with vectors encoding AMBRA1, ULK1-Myc and ATG13-FLAG. Protein extracts were immunoprecipitated using anti-FLAG antibody.(g) HEK293 cells were transfected with scramble siRNA or ATG13 siRNA. Protein extracts were immunoprecipitated using ULK1 antibody or IgG. The band density ratio of immunoprecipitated AMBRA1 relative to immunoprecipitated ULK1 is analysed, with the control ratio arbitrarily defined as 1.00. n = 3 extracts prepared from independent experiments; data are presented as means±s.d. and significance is P = 0.04. Uncropped images of blots/gels are shown in Supplementary Fig. S6. Source data of statistical analysis are shown in Supplementary Table S1.


figf2 ( a ) HEK293 cells were transfected with AMBRA1 or the empty vector , as a control . ULK1 , AMBRA1 and actin were analysed by western blotting . ( b ) Protein extracts from wild - type ( Ambra1 + / + ) and homozygous ( Ambra1gt / gt ) embryos for the Ambra1 gene - trap mutation at developmental day 14 . 5 were analysed by western blotting using anti - Ulk1 or anti - Hsp90 ( loading control ) antibodies . Quantification is shown on the right . n = 6 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 037 . ( c ) AMBRA1 , ULK1 and actin ( as a protein loading control ) were analysed by western blotting in HeLa cells transiently transfected with scramble siRNA or AMBRA1 siRNA . Quantification is shown on the right . n = 5 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 007 . ( d ) HeLa cells stably interfered for AMBRA1 were transiently transfected with empty vector or AMBRA1 vector ; AMBRA1 , ULK1 , ATG13 , p - ATG13 and actin levels were analysed by western blotting . Quantification of ULK1 and pATG13 bands is shown on the right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 006 for ULK1 and P = 0 . 044 and P = 0 . 011 for pATG13 . ( e ) HeLa cells were transfected or not with vector encoding AMBRA1 and treated with 35 μM cycloheximide ( CHX ) for 6 and 8 h before extraction . ULK1 , actin and AMBRA1 were analysed by western blotting . Quantification is shown below . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . and significance is P = 0 . 049 and P = 0 . 02 . ( f ) HEK293 cells were transfected as in a ; ATG13 , pATG13 , ULK1 , AMBRA1 and actin were analysed by western blotting . ( g ) Endogenous ULK1 is immunoprecipitated from HeLa cells transfected with β - galactosidase or AMBRA1 plasmid respectively using an anti - ULK1 antibody . As a control , rabbit IgG was used . The resulting precipitates were subjected to ULK1kinase assay using MBP as the substrate . Purified complexes were analysed by autoradiography and by western blotting using an anti - ULK1 antibody . Total extracts were analysed using an anti - AMBRA1 antibody .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf2(a) HEK293 cells were transfected with AMBRA1 or the empty vector, as a control. ULK1, AMBRA1 and actin were analysed by western blotting.(b) Protein extracts from wild-type (Ambra1+/+) and homozygous (Ambra1gt/gt) embryos for the Ambra1 gene-trap mutation at developmental day 14.5 were analysed by western blotting using anti-Ulk1 or anti-Hsp90 (loading control) antibodies. Quantification is shown on the right. n = 6 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.037.(c) AMBRA1, ULK1 and actin (as a protein loading control) were analysed by western blotting in HeLa cells transiently transfected with scramble siRNA or AMBRA1 siRNA. Quantification is shown on the right. n = 5 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.007.(d) HeLa cells stably interfered for AMBRA1 were transiently transfected with empty vector or AMBRA1 vector; AMBRA1, ULK1, ATG13, p-ATG13 and actin levels were analysed by western blotting. Quantification of ULK1 and pATG13 bands is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.006 for ULK1 and P = 0.044 and P = 0.011 for pATG13.(e) HeLa cells were transfected or not with vector encoding AMBRA1 and treated with 35 μM cycloheximide (CHX) for 6 and 8 h before extraction. ULK1, actin and AMBRA1 were analysed by western blotting. Quantification is shown below. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.049 and P = 0.02.(f) HEK293 cells were transfected as in a; ATG13, pATG13, ULK1, AMBRA1 and actin were analysed by western blotting.(g) Endogenous ULK1 is immunoprecipitated from HeLa cells transfected with β-galactosidase or AMBRA1 plasmid respectively using an anti-ULK1 antibody. As a control, rabbit IgG was used. The resulting precipitates were subjected to ULK1kinase assay using MBP as the substrate. Purified complexes were analysed by autoradiography and by western blotting using an anti-ULK1 antibody. Total extracts were analysed using an anti-AMBRA1 antibody.