Extended Data Fig. 12. Determination of immune responses and BCG in tissues 1 month after immunization.
NHPs (cohort 6, n = 2) were immunized with 5 × 107 BCG CFUs (IDhigh (a), IV (b), AE (c), or EB (d)); BCG CFUs and antigen-responsive T cells were measured in various tissues 1 month later. Before euthanasia, a fluorochrome-conjugated anti-CD45 antibody was injected intravenously (ivCD45) such that circulating (intravascular) leukocytes were uniformly stained (ivCD45+) while leukocytes in the tissue remained protected from staining (ivCD45−). To investigate whether antigen-responsive (IFNγ+) CD4 T cells were located in the ivCD45− lung tissue compartment, cells isolated from lung lobes were re-stimulated in vitro with Mtb WCL and analysed by intracellular cytokine staining. FACS plots show memory CD4 T cells in all tissues collected from one of two NHPs per BCG regimen, organized by type/location (systemic, peripheral LN, lung LN, BAL and lung lobes). The BAL and lung responses from the IV BCG NHPs, shown in the bottom row of b, is reproduced from Fig. 4b. Pre-infusion PBMC indicates PBMCs isolated from whole blood collected just before anti-CD45 injection. Bar graphs show the number of BCG CFUs in each respective tissue for each animal (colour-coded by vaccine), if detected.