Figure 2. BCG-containing cells found outside the granuloma in contact with P25 T-cells during acute infection.

CD11c-eYFP mice were IP infected with tdTomato BCG and 3 weeks (acute infection, (A–C)) or 10 weeks (chronic infection, (D)) later adoptively transferred (by IV injection) with 5 × 10⁵ DsRed P25 T-cells. Mice were harvested 7 days after cell transfer. (A) Representative low-magnification micrograph from 50 μm-thick sections of acutely-infected liver containing CD11c+ and P25 T-cells (bottom left image). Five representative granulomas (white dotted boxes, magnified in peripheral images) show the morphology and size (15–30 cells in diameter) of mature granulomas during acute infection. (B) Representative micrographs of BCG-containing, CD11c+ cells found outside mature granulomas and in contact with P25 T-cells. Clusters containing 1–4 cells (group 1, left panels), 5–10 cells (group 2, middle panels) and 11–20 cells (group 3, right panels) were identified. Images were taken from 50 μm-thick liver sections. (C) Comparison between of P25 T-cell to CD11c+ cell ratio in mature granulomas (15+ cells in diameter) and small cell clusters (groups 1,2,3), showing the unique cellular composition of each type of aggregate. 100 mature granulomas and 50 small cell clusters were randomly selected among n = 6 mice from 2 replicate experiments. The # of CD11c+ and P25 T-cells in each aggregate was counted using image J. Error bars are mean +/− s.e.m. student’s t-test used to determine statistical significance. ***P < 0.001. (D) Representative low-magnification micrographs from 50 μm-thick sections of chronically-infected liver containing CD11c+ and P25 T-cells (bottom left image, granulomas in white dotted circles). Higher magnification of 5 representative chronic granulomas shown in peripheral images.