Fig. 1: Outline of the study: study groups, design and analytical assessment*.

In studies of both peripheral blood CD4⁺ T-cell responses and of unsorted BAL cells, gene expression of LTBI individuals and of recipients of ID or PO BCG vaccination were compared to those of Mtb/BCG-naïve participants. In peripheral blood studies (Fig. 1a, top), pre-vaccination samples from participants who later received ID or PO vaccination were pooled to form the Mtb/BCG-naïve comparator group. Subsequent PBMC samples for all BCG recipients were obtained 56 days following their last vaccine dose. CD4⁺ T-cells were selected from thawed samples as were blood monocytes (MN) that were cultured in vitro with IL-4 and GM-CSF to develop autologous CD14^-/CD11c⁺ monocyte-derived dendritic cells (MDDC). MDDC were then co-cultured with CD4⁺ T cells in the presence of BCG or in medium alone, as indicated, and samples frozen for subsequent RNA extraction. In contrast, for the BAL cell substudy (1a, lower panel), a separate group of Mtb/BCG-naïve individuals was recruited specifically as control participants; additional study groups included LTBI individuals and recipients of ID and/or PO BCG in prior vaccine studies. Because two of the eight individuals recruited following PO BCG vaccine also received ID BCG on the same date, we designate this group as recipients of PO ± ID BCG. Unsorted BAL cells were then incubated overnight with virulent Mtb strain H37Rv or medium alone and frozen for eventual RNA extraction. For both substudies, RNASeq was performed and data evaluated for comparisons between the study groups with regard to both baseline gene expression and differential responses to in vitro infection. *Bacterial cell adapted from “E. coli, without flagella and pili”, Blood vial adapted from “Icon Pack - Haematology”, 96well plate adapted from “Icon Pack - Engineering”, Sequencer adapted from “Icon Pack - Nucleic Acid Sequencing”, and Lungs: Adapted from “ Lung with Labels (Layout)”, by BioRender.com (2023). All retrieved from https://app.biorender.com/biorender-templates. The terminology used to describe gene expression comparisons in both PBMC and BAL studies is detailed in the formulas presented in b. As indicated, delta (Δ) responses indicate the quotient of gene expression (in counts per million reads, or CPM) between infected and uninfected cells of a single study group in blood or BAL studies. Delta-delta (ΔΔ) responses indicate the quotient of the in vitro infection vs. uninfected baseline (Δ) in the treatment groups (LTBI or BCG-vaccine recipients) with that of the Mtb/BCG-naïve controls (Δ). Protein coding genes with a median expression ≥1.0 CPM were evaluated. For (Δ) and (ΔΔ) calculations, expression data were floored to a minimum value of 0.5 CPM. Absolute fold changes ≥1.3 with p-value < 0.05 (Mann-Whitney two-tailed) were required for inclusion in subsequent analysis.