Fig 1: Protein-level confirmation of key cytokine responses significantly upregulated in the blood and BAL gene expression data.To confirm key gene expression findings, we assessed BCG/Mtb-induced production of T cell-associated cytokines within supernatants of blood and BAL cell cultures. All graphs present cytokine concentrations in pg/mL. Responses from blood CD4+ T cells are shown in the left column. Results for individual participants are shown for both uninfected cultures (open symbols) and BCG-infected samples (shaded symbols). Statistical assessment compared infection-induced changes in cytokine levels of LTBI individuals (triangles, n = 15) and recipients of ID and PO BCG vaccination (diamonds, n = 14 and squares, n = 15, respectively) as compared with samples from pre-vaccination Mtb/BCG-naïve controls (circles, n = 29). Blood CD4+ T cells from LTBI individuals displayed significant BCG-induced production of IFN-γ, TNF, IL-2 and IL-9. In contrast, BCG stimulation of blood CD4+ T cells from recipients of ID BCG resulted in significant production of TNF, IL-2 and IL-9, but not IFN-γ; none of these cytokines were produced to significant levels in blood CD4+ T cells from PO BCG recipients. BAL cell cytokine responses are presented in the right column. Again, results for individual participants for uninfected cultures (open symbols) and infected samples (shaded symbols) are displayed, here for Mtb/BCG naïve controls (circles, n = 6), LTBI individuals (triangles, n = 10) and recipients of ID BCG (diamonds, n = 8). For the PO ± ID BCG group (n = 8), results for two participants who received PO and ID BCG are displayed as pink stars in contrast to the squares representing results of recipients of PO BCG alone. Following Mtb infection, significant production of IFN-γ, TNF and IL-2 was observed in cells from LTBI individuals. In contrast, BAL from the ID BCG group displayed minimal concentrations of these cytokines at baseline or in response to Mtb. However, BAL cells from the PO ± ID BCG group demonstrated significant Mtb-induced production of TNF and IL-9. Notably, Mtb-induced production of IL-2 was not significant in this group. Overall, the BAL cytokine protein-level data support the transcriptomic findings in demonstrating a strong local immune response in BAL cells from LTBI individuals and recipients of PO ± ID BCG, but not of ID BCG alone. *p < 0.05 and **p < 0.01 by 2-tailed Mann-Whitney U tests; error bars indicate median and 95% CI values.
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