Fig. 3: Assessments of ΔΔ gene expression comparisons of blood CD4⁺ T cells in response to in vitro BCG stimulation identified multiple immune themes in LTBI individuals and recipients of ID, but not PO, BCG.

BCG-induced changes in global gene expression of cultured CD4⁺ T cells from LTBI, ID BCG, and PO BCG study groups (n = 15, 14, and 15, respectively) were compared to those of the pooled Mtb/BCG-naïve (pre-vaccination) group (n = 29) and displayed as models indicating the connectedness of red spheres that represent distinct immune themes. As detailed in the methods section, the size of each theme sphere is determined by the rank of that theme’s absolute enrichment score within the full map, and the thickness of the connecting edges reflects the number of genes shared between themes. BCG-induced ΔΔ gene expression profiles identified multiple immune themes in CD4⁺ T cell from both LTBI individuals (a) and recipients of ID BCG (b), whereas no themes demonstrated significant enrichment in recipients of PO BCG. Figure 3c displays a table (upper right) of the mean ΔΔ responses for genes associated with immune-related themes detected in LTBI, ID BCG and PO BCG groups. As illustrated, both LTBI individuals and recipients of ID BCG displayed enrichment for themes of T cell stimulation, IL23 signaling, NK cells, germinal centers, and histiocytes. LTBI individuals uniquely displayed significant enrichments for pathogen response, necroptosis and immune checkpoint themes, whereas themes of lymphocytes, opsonization, lymphoid/IL-9 and CD30 signaling themes were unique to recipients of ID BCG; again, no immune themes were significantly enriched in recipients of PO BCG. Visual comparisons of differential expression of individual genes within representative enriched immune themes are displayed in the associated heatmaps (c). These present the ΔΔ responses for each participant for the top ten genes within selected immune themes (or fewer when a theme was defined by less than ten genes) for which enrichment was shared by LTBI and ID BCG groups (T-cell stimulation), unique to LTBI individuals (necroptosis), and unique to recipients of ID BCG (lymphoid/IL9). Of note, both LTBI and ID BCG participants generally display upregulation of IL9 (pink arrow). Consistent with the other representations of these data, for all portions of the heatmap, expression of these genes is substantially less striking in recipients of PO BCG than in the other study groups.