Fig 2: PD-1 is expressed in human TB granulomas.(A) Analysis of PD-1 expression by T cells in the lung and peripheral circulation of thirty-five TB patients undergoing medically indicated lung resection. PD-1 shows a trend towards higher expression by lung CD4+ (i) and is significantly higher on lung CD8+ (ii) T cells. Significance analysed by one-tailed unpaired Mann-Whitney test. (B) Flow cytometric analysis of lung parenchyma CD4+ (i) and CD8+ (ii) T cells from TB patients based on the expression of PD-1, CD69 and CD103 demonstrates increased PD-1 expression in the resident T cells in the lung parenchymal cells. Significance analysed by Kruskal-Wallis test with Dunn’s multiple comparison test. (C) Immunohistochemical staining for PD-1+, CD4+ and CD8+ expression in human lung TB granulomas. PD-1 is expressed around the central macrophage core in the same region as CD4+ (ii) and CD8+ (iii) T cells. Co-localization of PD-1 (blue), CD4+ (red) and CD8+ (yellow) using false colour of the immunostaining shows co-localisation of PD-1 with both CD4+ and CD8+ cells (purple and green respectively) (iv). Scale bar 100 µm. (D) PD-1 is not expressed in caseating granulomas where immunopathology is present in human lung biopsies (i). Six biopsies taken as part of routine clinical care were studied. CD4+ (ii) and CD8+ (iii) expressing cells are present in the same area, and so absence of PD-1 immunoreactivity is not due to lack of viable cells. Scale bar 200 µm.

Fig 3: Flow-cytometry plots for in vitro syngeneic T cell expansion assay (with PFA fixed DC), related to Figure 6Representative flow cytometry plots of in vitro syngeneic T cell expansion of fetal mLN T cells (treated with bacteria primed DC fixed with 1% PFA) showing the:(A) frequency of CD4 and CD8 T cells ratio,(B) CD45RO+ memory T cell plotted against CTV dye to show the dye dilution post 6 days, and(C) CD69+ activated memory T cell plotted against CTV dye to show the dye dilution post 6 days.The conditions shown are T cells + fetal staphylococci primed DC (blue), T cells + fetal staphylococci primed PFA fixed DC (orange), T cells + unprimed DC alone (gray), and T cells + CD3/28 dynabeads (magenta).Fetal T cell expansion and memory activation assay (with PTI treated DC)(D) Schematic representation of the syngeneic T cell expansion assay. DC isolated from fetal mLN were primed with heat-killed fetal bacteria (for 8 hours) followed by Protein Transport Inhibitor (PTI) treatment (a combination of Monensin and Brefeldin A). PTI treated DC were then presented to CTV labeled T cells from the same organ (mLN). At day 6, T cell expansion is assayed by dye dilution using flow cytometry.(E) Representative flow-cytometry histograms for T cell expansion (17 weeks EGA fetal mLN). CellTrace Violet (CTV) dye labeled T cells were incubated with bacteria (fetal staphylococci) primed DC for 6 days. For controls, T cells were incubated with unprimed DC (no bacteria) or CD3/28 dynabeads as a negative and positive control, respectively. T cell expansion is assayed by measuring dye dilution using flow cytometry. The conditions shown are T cells + DC alone (gray), T cells + fetal staphylococci primed DC (blue), T cells + fetal staphylococci primed PTI treated DC (orange) and T cells + CD3/28 dynabeads (magenta).(F) Total T cell number (absolute count) as counted by CountBright beads, post 6 day incubation, plotted for all the conditions (n = 4). Statistical significance between experimental groups was determined by paired t test (****p < 0.0001, ***p < 0.001, **p < 0.01)(G) Frequency of CTVneg expanded memory T cells (CD45RO+, CTVneg) plotted for all conditions.(H) Frequency of expanded CD69+ active memory T cells (CTVneg) plotted for all conditions. Statistical significance between experimental groups was determined by two-tailed, unpaired Student’s t test (****p < 0.0001, ***p < 0.001, **p < 0.01)

Fig 4: Gating strategy for in vitro syngeneic T cell expansion assay, related to Figure 5(A) Reference input gating strategy for the Figure 5C, showing the live CD45+ CD3+ T cells gate obtained from fetal mesenteric lymph nodes T cells, post 6 days of incubation with the T cells + DC alone, T cells + DC primed with fetal Staphylococcus, T cells + DC primed with fetal Lactobacillus and T cells + CD3/28 dynabeads respectively (color coded in the figure).(B) As shown, the CD4/CD8 population frequencies post culturing, and CTV staining, gated on total CD3+ cells from the above panel a.(C) Another representative flow-cytometry plots for T cell expansion (15 weeks EGA fetal mLN). CellTrace violet (CTV) dye labeled T cells were incubated with bacteria (fetal Staphylococci and Lactobacilli) primed DC for 6 days. For controls, T cells were incubated with unprimed DC (no bacteria) or CD3/28 dynabeads as a negative and positive control, respectively. T cell expansion is assayed by measuring dye dilution using flow cytometry. The conditions shown are T cells + DC alone (gray), T cells + DC primed with fetal staphylococci (blue), T cells + DC primed with fetal lactobacilli (green) and T cells + CD3/28 dynabeads (magenta). For comparisons, T cell profile at day zero is shown at the left.(D) CD45RO+ memory T cell expansion post 6 day incubation measured by CD45RO expression and dye dilution (CTVneg) for all the conditions in comparison to T cell + DC alone (gray). For comparisons, T cell at day zero is shown at the left.(E) CD69+ activated memory T cell expansion post 6 day incubation measured by CD69 expression and dye dilution (CTVneg) for all the conditions, in comparison to T cell + DC alone (gray). For comparisons, T cell at day zero is shown at the left.(F) Levels of TNF released in the culture supernatant of T cells expanded post 6 day exposure to fetal bacteria as determined by drop array-based Luminex assays (n = 4)(G) Levels of IFN? released in the culture supernatant of T cells expanded post 6 day exposure to fetal bacteria as determined by drop array-based Luminex assays (n = 4)

Fig 5: Fetal T cell expansion and memory activation with PFA-fixed DCs(A) Schematic representation of the syngeneic T cell expansion assay. DCs isolated from fetal mLN were primed with heat-killed fetal bacteria (for 8 h) followed by 1% PFA-induced fixation. Fixed DCs were then presented to CTV-labeled T cells from the same organ (mLN). At day 6, T cell expansion is assayed by dye dilution using flow cytometry.(B) Representative flow cytometry histograms for T cell expansion (17-weeks EGA fetal mLN). CTV-labeled T cells were incubated with bacteria (fetal Staphylococci) primed DCs for 6 days. For controls, T cells were incubated with unprimed DCs (no bacteria) or CD3/28 Dynabeads as a negative and positive control, respectively. T cell expansion is assayed by measuring dye dilution using flow cytometry. The conditions shown are T cells + DCs alone (gray), T cells + fetal Staphylococci primed DCs (blue), T cells + fetal Staphylococci primed PFA-fixed DCs (orange), and T cells + CD3/28 Dynabeads (magenta).(C) Total T cell number (absolute count) as counted by CountBright beads, post 6 day incubation, plotted for all the conditions (n = 4). Statistical significance between experimental groups was determined by paired t test (****p < 0.0001, ***p < 0.001, **p < 0.01).(D) Frequency of CTVneg-expanded memory T cells (CD45RO+, CTVneg) plotted for all conditions.(E) Frequency of expanded CD69+ active memory T cells (CTVneg) plotted for all conditions. Statistical significance between experimental groups was determined by two-tailed, unpaired Student’s t test (****p < 0.0001, ***p < 0.001, **p < 0.01).See also Figure S6.