Fig. 4. Mtb infection induces differential expression of RAB GTPases and cathepsin proteases in human macrophages affecting autophagy-dependent ex vivo antigen presentation to CD4 T cells.

a Differential expression of RAB GTPases and transcripts are shown as FPKMs (fragments per kilobase per million reads) for duplicate samples; *p < 0.01; t test. b MΦs were infected with rfpMtb, followed by staining with an isotype or specific antibodies to RAB7 or LAMP1 proteins and counterstained using fluorescein isothiocyanate anti-IgG conjugates. Confocal microscopy analysis of phagosomes colocalizing with RAB7 is illustrated (full panels shown in Supplementary Fig. 3c); bar graph indicates quantitation of RAB7 colocalization (*p < 0.009; t test; one of 2 similar experiments shown). c MΦs indicated were treated with siRNA for RAB7 or its scrambled control followed by Mtb infection and overlay with F9A6-CD4 T cell line for antigen presentation and IL-2 assay (**p < 0.009; t test; one of 2 similar experiments shown). d, e Differential gene expression of cathepsins (CTS) expressed as FPKMs shown for duplicate samples (*p < 0.01; t test). f MΦs indicated were treated with non-cytotoxic doses of either CTS specific inhibitors or pan-specific inhibitors followed by Mtb infection and antigen presentation using F9A6-CD4 T cells (**p < 0.009, t test; one of 2 similar experiments shown). g MΦs treated with CTS inhibitors were infected with Mtb followed by CFU assay on day 5, maintaining >90% viability of MΦs (**p < 0.009, one-way ANOVA with Tukey’s post-hoc test; one of 2 similar experiments shown). h Replicates of MΦs used in panel (g) were infected using Mtb or BCG and supernatants collected at 18 h were tested for IL-2 and antigen presentation (**p < 0.009; t test). i PBMC-derived macrophages from healthy donors, household contacts, and TB patients (TB) were treated or not treated with Rapamycin (10 µM) followed by infection with Mtb and CFU counts on day 3 in vitro. j Washed Mtb-infected MΦs (as in panel i) were overlaid with F9A6-CD4 T cells for antigen presentation with or without Rapamycin and IL-2 assay. Horizontal bars indicate median (interquartile range) for IL-2 (non-normal distribution) or mean (SD) for CFUs (*p < 0.05; **p < 0.001. Wilcoxon paired ranked signed test, and Kruskal-Wallis test). k Healthy adult donor MΦs from the TB endemic area differentiated into M1-, M2-, or M0-MΦs were infected with Mtb followed by CFU counts on day 3 (**p < 0.01; Kruskal–Wallis test). l Five donor MΦs from each group of panel k were treated with siRNA vs. beclin1 or its scrambled control, followed by infection with Mtb and CFU counts on day 3 (**p < 0.009, Kruskal-Wallis test; triplicate wells per donor; one of 2 similar experiments shown). All Mtb CFU experiments used MOI of 1.