Fig. 1

Mycobacterium tuberculosis (Mtb)-derived CFP-10 and ESAT-6 proteins show a differential activation of antigen-presenting cells in vitro. a, b Amino acid sequences of the whole proteins and overlapping peptides synthesized from CFP-10 and ESAT-6 are shown. CFP-10-derived first-stage synthesis peptides were subjected to an ELISA validation for their ability to stimulate antigen presentation (lower box), and “resynthesized” as peptides C4-C7 (upper box) and ELISA validation of the peptide designated as C5 in Fig. 2f. c C57Bl/6 mouse bone marrow-derived macrophages (MΦs) and dendritic cells (DCs) (APCs) were treated with recombinant CFP-10 and ESAT-6 whole proteins (BEI Resources Repository, NIH) at doses indicated, followed by infection with Mycobacterium bovis BCG (Pasteur; MOI = 1), and overlaid with antigen-85B-specific CD4 BB7 hybridoma T cells, for antigen presentation. The supernatants were tested for IL-2 levels using sandwich ELISA (one of three similar experiments shown; p-values, one-way ANOVA with Dunnett’s multiple comparisons post test). d A constant amount of CFP-10 (C; 0.5 µM; eq.ng/mL indicated) was mixed with ESAT-6 (E) in varying amounts (0.5–2.0 µM) in PBS at 37 °C for 15 min and added to MΦs, followed by infection with BCG and antigen presentation. CFP-10 and ESAT-6 were added alone as internal controls. Addition of ESAT-6 reduces the ability of CFP-10 to enhance in vitro antigen presentation (two experiments, *p-values, one-way ANOVA with Dunnett’s post test). e Three truncated proteins of ESAT-6 protein were prepared and added to CFP-10 protein with BCG vaccine, and antigen presentation determined using MΦs. The inhibitory motif of ESAT-6 lies near N-terminus (E1) and extends to middle (E2), but not at C-terminus (E3). Potential binding to CFP-10 is shown, which allows CFP-10 C-terminus to extend. Arrows above the ESAT-6 indicate the truncated regions