Figure 4

Knockdown of endosomal trafficking proteins can distinguish MR1 trafficking pathways between Mtb and exogenously added antigens. (A) qPCR of BEAS-2B after 48 hours of knockdown with missense or Syntaxin 4 (STX4) siRNA. The data are pooled from 3 independent experiments. (B) IFN-γ ELISPOT assay results of Syntaxin 4 knockdown in BEAS-2B using Msmeg supernatant as the antigen. Data are pooled from 2 independent experiments. The difference in Log EC₅₀ was statistically significant (P = 0.0055). (C) IFN-γ ELISPOT assay results of Syntaxin 4 knockdown in BEAS-2B using Mtb infection as the antigen. Data are pooled from 2 independent experiments. The difference in Log EC₅₀ was not statistically significant (P = 0.5888). (D) IFN-γ ELISPOT assay results of Syntaxin 4 knockdown in BEAS-2B using Mtb infection as the antigen and an HLA-B45-restricted T cell clone. Data are pooled from 2 independent experiments. The difference in Log EC₅₀ was not statistically significant (P = 0.7840). (E) IFN-γ ELISPOT assay results of VAMP2 and Syntaxin 16 (STX16) knockdown in BEAS-2B. Mean and SEM from technical replicates are plotted. Data are representative of 2 independent experiments. Left) Mtb infection. An unpaired two-tailed t-test was used to calculate statistical significance (Mis to VAMP2 P = 0.0011; Mis to Syntaxin 16 P = 0.0003). Right) Msmeg supernatant. An unpaired two-tailed t-test was used to calculate statistical significance (Mis to VAMP2 P = 0.0096; Mis to Syntaxin 16 P = 0.0033).