Fig. 3: Properties of backbones at recruiting APC and inducing antigenic presentation by both the MHC-I and-II pathways.

a, c BM-DCs from BALB/c (H-2^d) or C57BL/6 (H-2^b) mice were transduced (MOI = 20) with LV::M40-H, -HA, -HAP, or -HAPE (a) or LV::S40-H, -HAPEHR, or -HAPEHR-20 (c) under the transcriptional control of the BCUAG promoter. Control cells were transduced with LV::EsxH alone. b, d BM-DCs from BALB/c or C57BL/6 mice were incubated with successive dilutions of supernatants of HEK-293T cells transduced (MOI = 20) for 48 h with each of the indicated LVs. On day 3 after addition of the LVs or day 1 after incubation with the HEK293T cell supernatants, the presentation of MHC-I- or -II-restricted epitopes of the EsxH, EsxA, PE19, or EspC mycobacterial antigens by DCs was assessed by their co-culture with T-cell hybridomas specific for EsxH:20-28 (YB8 cell line, restricted by K^d), EsxH:74-88 (1G1 cell line, restricted by I-A^d), EsxA:1-20 (NB11 cell line, restricted by I-A^b), PE:19:1-18 (IF6 cell line, restricted by I-A^b), or EspC:45:54 (IF1 cell line, restricted by I-A^b). Results are presented as the concentration of IL-2 produced by the T-cell hybridomas 24 h after the beginning of the co-cultures. The amount of IL-2 found in the co-culture supernatants is proportional to the efficacy of antigenic presentation by DCs and TCR triggering. This assay does not measure the physiological response induced in the T cells but only provides an indicator of the stimulation of the T-cell hybridoma TCR. e Impact of HAPEHR and HAPEHR-20 proteins on migration of BM-DCs in a transwell system. Supernatant of HEK293T cells transduced (MOI = 100) with LV::S40-HAPEHR or LV::S40-HAPEHR-20 (n = 6). Statistical significance was evaluated using the Mann–Whitney test (* = p < 0.05, ** = p < 0.01, *** = p < 0.001, ns non-significant).