Fig 2: ESAT-6-specific effectors in Mtb memory mice exhibit increased differentiation within the lung parenchyma early during infection compared to H56 memory mice.A) KLRG1 expression among lung localized I-Ab:ESAT-6-specific CD4 T cells. Representative FACS plots showing KLRG1 expression on ESAT-64-17 tet + cells from Mtb (upper plot) and H56 (lower plot) memory mice infected for ten weeks with Mycobacterium tuberculosis relative to their localization in the lung vasculature (CD45.2 IV + ve) or lung parenchyma (CD45.2 IV –ve). Numbers in parentheses represent the percentage of cells expressing KLRG1 in the IV + ve (Red) and the IV - ve population (Blue). Graphs show the proportion of KLRG1 + of I-Ab:ESAT-64-17 tetramer + CD4 T cells in the lung vasculature (IV + ve; upper graph) and in the lung parenchyma (IV –ve, lower graph) over the course of infection (unperfused lungs). Mean ± s.d. of 3–4 mice per group at any given time-point. Two-way ANOVA with Sidak's multiple comparison test for simple row effects. Differences between Mtb and H56 memory. ****P < 0.0001, ***P < 0.001, **P < 0.01.B) PD-1 expression among lung localized I-Ab:ESAT-6-specific CD4 T cells. Representative FACS plots showing PD-1 expression on ESAT-64–17 tet + cells from Mtb (upper plot) and H56 (lower plot) memory mice infected for ten weeks with Mycobacterium tuberculosis relative to their localization in the lung vasculature (CD45.2 IV + ve) or lung parenchyma (CD45.2 IV –ve). Numbers in parentheses represent the percentage of cells expressing PD-1 in the IV + ve (Red) and the IV - ve population (Blue). Graphs show the proportion of PD-1 + of I-Ab:ESAT-64-17 tetramer + CD4 T cells in the lung vasculature (IV + ve; upper graph) and in the lung parenchyma (IV –ve, lower graph) over the course of infection (unperfused lungs). Mean ± s.d. of 3–4 mice per group at any given time-point. Two-way ANOVA with Sidak's multiple comparison test for simple row effects (ns).C) ICOS expression among lung localized I-Ab:ESAT-6-specific CD4 T cells. Representative FACS plots showing ICOS expression on ESAT-64-17 tet + cells from Mtb (upper plot) and H56 (lower plot) memory mice infected for ten weeks with Mycobacterium tuberculosis relative to their localization in the lung vasculature (CD45.2 IV + ve) or lung parenchyma (CD45.2 IV –ve). Numbers in parentheses represent the percentage of cells expressing ICOS in the IV + ve (Red) and the IV –ve population (Blue). Graphs show the proportion of ICOS + of I-Ab:ESAT-64–17 tetramer + CD4 T cells in the lung vasculature (IV + ve; upper graph) and in the lung parenchyma (IV –ve, lower graph) over the course of infection (unperfused lungs). Mean ± s.d. of 3–4 mice per group at any given time-point. Two-way ANOVA with Sidak's multiple comparison test for simple row effects. Differences between Mtb and H56 memory. **P < 0.01.Gating as depicted in Supplementary Fig. 4.One of two comparable experiments shown.

Fig 3: PD-1 and LAG-3 Restrict Re-expansion of Fetal-Specific CD8+ T Cells Selectively during Secondary Pregnancy(A) Schematic showing the administration of aPD~L1 and/or aLAG-3 antibodies mid-gestation (E10.5) during primary or secondary pregnancies sired by Act-OVA male mice on the BALB/c background.(B) Percentage of fetal wastage 3 days after the administration of aPD-L1 and/or aLAG-3 antibodies for mice described in (A).(C) Number of H-2Kb:OVA257–264 CD8+ T cells in the spleen and pooled peripheral lymph nodes for each group of mice described in (A).(D) Fetal wastage 3 days after the administration of aPD-L1/LAG-3 antibodies with or without co-administration of aCD8- and/or aCD4-depleting antibody during secondary pregnancy sired by Act-OVA male mice on the BALB/c background.(E) Schematic showing the administration of aPD-L1 and/or LAG-3 antibodies mid-gestation (E10.5) during allogeneic compared with syngeneic secondary pregnancies.(F) Percentage of fetal wastage 3 days following aPD-L1/LAG-3 antibody treatment mid-gestation for groups of mice described in (E).Data are from at least 3 independent experiments, each with similar results, with each point representing data from an individual mouse.Bar, mean ± SEM. **p < 0.01 and ****p < 0.0001.

Fig 4: Maternal CD8+ T Cells with Fetal Specificity Expand during Pregnancy and Persist after Parturition(A) Female mice on the C57BL/6 (H-2b) background were mated with Act-OVA male mice on the BALB/c (H-2d) background. For analysis, females were sacrificed either before mating (virgin), at embryonic day 10.5 (E10.5) (pregnant), or 21 days following delivery (postpartum).(B) Number of H-2Kb:OVA257–264-specific CD8+ T cells in the spleen and pooled peripheral lymph nodes of female C57BL/6 virgin or mid-gestation (E10.5) mice compared with postpartum week 1 (PP7), week 3 (PP21), and week 8 (PP56) after allogeneic pregnancy sired by Act-OVA male on the BALB/c background.(C) H-2Kb:OVA257–264 tetramer geometric mean fluorescence intensity (gMFI) and ratio of tetramer gMFI to CD3 gMFI for CD8+ T cells from female C57BL/6 virgin, mid-gestation (E10.5) pregnant, and PP21 after allogeneic pregnancy sired by Act-OVA male mice on the BALB/c background.(D) Percentage of CD44hi, CD62Llo, CCR7, PD-1, LAG-3, CTLA-4, and TIM-3 expression levels by H-2Kb:OVA257–264 CD8+ T cells for each group of mice described in (C).(E) Representative flow cytometry plots showing the identification of H-2Kb:OVA257–264 CD8+ T cells by tetramer staining, and their production of IFN-?, TNF-a, or IL-2 following PMA-ionomycin stimulation compared with no stimulation controls for cells isolated from the spleen and pooled peripheral lymph nodes for each group of mice described in (C).(F) Frequency of IFN-?, TNF-a, or IL-2 producing H-2Kb:OVA257–264 CD8+ T cells from female C57BL/6 mice at the indicated time points following allogeneic pregnancies sired by Act-OVA male mice on the BALB/c background.Data are from at least 3 independent experiments, each with similar results, with each point representing data from an individual mouse.Bar, mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.001.

Fig 5: Resting memory cells after a cleared Mycobacterium tuberculosis infection show a higher degree of differentiation than memory cells primed by H56/CAF01 vaccination.A) Schematic overview of the experimental outline. C57BL/6 mice, aerosol infected for 6 weeks, were subjected to a 12 week Isoniacid/Rifabutin (INH/RIF) treatment. ESAT-6 specific memory cells in the spleen were phenotypically characterized at week 12/End of Treatment (EoT) by ESAT-64-17:I-Ab pulldown using magnetic bead-based tetramer enrichment and compared to E6 memory responses 12 week post three s.c. immunizations with H56/CAF01, each spaced by two weeks. ESAT-6 specific memory cells in the spleen were similarly characterized and compared between the two groups of resting memory mice at week 40 after immunization/start of treatment. In order to study protective efficacy, H56 and Mtb memory mice were allowed to rest for half a year and then aerosol infected with Mtb Erdman. Protective efficacy was evaluated at week 2, 10 and 20 after challenge. B) The number of ESAT-64-17-specific CD4 T cells in the spleen was determined by Tetramer pulldown in Mtb (upper panel) and H56 (lower panel) memory mice at week 6, 12 and 40. Symbols, mean ± s.d. of 3–4 mice per time point. For Mtb memory mice, the experiment was repeated twice at week 6 and once at week 12 and 40. For H56 mice, the experiment was repeated twice at week 6 and 12 and once for week 40. C) Representative flow cytometry plots depicting KLRG1, PD-1, ICOS and CXCR3 (wk 40) expression by ESAT-6 tetramer-binding CD4 T cells in the spleen of Mtb (upper panels) and H56 (lower panels) memory mice at week 12 and 40.