Figure 2.

Lymphocyte populations after incubation with BCG.

PBMC from healthy donors were incubated with the sub-strains of BCG iBCG or Oncotice at a 6:1 ratio (total bacteria to PBMC). After 7 days in culture, cells in suspension were recovered and analyzed. a-c. Flow cytometry. The percentage and standard deviation of the different lymphocyte subpopulations, analyzed by flow cytometry, before and after BCG incubation are plotted. Total lymphocytes (a), NK cells with high or dim CD56 expression (b) and T-cell subpopulations, including γδ T-cells (c) are plotted. This figure includes data from 16 independent donors. d-f Transcriptomic. For scRNA-seqanalysis, three donors were used. Individual data are shown in Supplementary Figure 4D. After quality control (see Materials and Methods, Supplementary Figure 4), raw data from 6383 iBCG-treated PBMC (51% of the pool) and 6097 of Oncotice-treated PBMC (49% of the pool) were combined. Twelve single clusters of lymphocyte subpopulations were identified. d. Lymphocyte clusters. UMAP plots represent the 12 clusters identified across the PBMC from 3 donors, stimulated either with iBCG or Oncotice, as indicated (left). The same clusters are expressed as percentage in bar graphs (right), showing the average of the three donors. Individual values are shown in Supplementary Figure 4D. Differences in the proportion of γδ T-cell cluster between iBCG and Oncotice were significant (paired sample t-test, *p < .05). e. Cluster annotation. The graph represents the mean standardized expression values per variable (blue color intensity, as indicated) and the fraction of cells (diameter of the circle) expressing the transcript of the indicated genes (x-axis) within each cluster (y-axis). The cluster labels were added manually. f. Heat-map. Heat-map represents the scaled mean expression values of differentially expressed genes across the samples per treatment, showing each donor (sample #1, 2, 3) and treatment (iBCG and oncotice) in different colors.