Fig 1: PTPN2-Deficient KLRG1+ T Cells Can Undergo Robust Secondary Expansion(A) Experimental outline: CD45-congenic C57BL/6J host mice received 104 WT or PTPN2-deficient T cells and 24 h later 1,000 CFUs of Lm-N4. At 7 days post infection, either KLRG1+ effector or CD127+ memory precursor PTPN2-deficient or WT OT-I T cells were sorted and adoptively transferred into new hosts.(B–D) Hosts were sacrificed and OT-I numbers were analyzed 24 h (B, KLRG1+ grafts only) or 3 weeks after the transfer (C) (shown are data for KLRG1+ grafts). (D) In addition, 3 weeks after the transfer, the host mice were infected with 1,000 CFUs Lm-N4 and analyzed 5 days later. Shown are data for KLRG1+ and CD127+ grafts. The data are representative of three independent experiments with four to five mice each. One dot represents one mouse, and the horizontal line the mean in all plots. Statistical analysis: unpaired t test, **p = 0.001, *p = 0.01, nsp = 0.05.
Fig 2: PTPN2 Alters the Ratio of Terminal Effector versus Memory Precursor T CellsCD45-congenic C57BL/6J host mice were grafted with 104 WT or KO OT-I T cells and infected with 1,000 colony-forming units (CFUs) Lm-N4 24 h later.(A and B) Peripheral blood T cells were analyzed by flow cytometry at 7 and 28 days post infection (dpi) and splenic T cells at 28 days post infection. (A) The depicted flow cytometry plots are representative blood samples. (B) The dot plots show the frequencies of CD127+ (upper row) or KLRG1+ (lower row) cells within the OT-I T cell population.(C) CD45-congenic C57BL/6J host mice received 104 OT-I;Lck-Cre;Ptpn2fl/fl (KO) and OT-I;Ptpn2fl/fl (WT) cells and were infected 24 h later with 1,000 CFUs Lm-N4. The dot plots show the ratio of total PTPN2-deficient versus WT T cells at the day of infection and at 28 dpi.(D) Splenic OT-I T cells were analyzed by flow cytometry for CD25 expression 4 days after infection. Representative histogram overlays of PTPN2-deficient (solid, light blue) versus WT (dotted line) OT-I T cells, and geometric mean fluorescence intensity (MFI) data for all mice are shown.(E) Splenic WT and KO OT-I T cells were isolated 7 days post infection and co-incubated with DAPI-labeled peptide-pulsed splenocytes at titrated doses for 18 h. Shown is the fraction of target cells that were lysed by WT or PTPN2-deficient OT-I T cells. The data are representative of at least two independent experiments with three to five mice in each group, and the horizontal line represents the mean. Statistical analysis: unpaired t test, ****p = 0.00001, ***p = 0.0001, nsp = 0.05. ns, not significant.
Fig 3: Tet-regulated GFP reporter expression in hematopoietic stem cells and early progenitors of tet-transactivator transgenic mice.Flow cytometry profiles of GFP expression in hematopoietic stem and progenitor cells isolated from the bone marrow of various transgenic mouse strains. Profiles from a representative mouse (n = 2 mice analysed per genotype) carrying the indicated transactivator transgene along with the TRE-GFP-shLuc reporter transgene are shown in green, with wild type controls shown in black. Tet-on bitransgenic reporter mice (CAG-rtTA3, CMV-rtTA, ROSA26-M2rtTA, Vav-rtTA3) were given Dox food for 7 days before analysis, whereas tet-off bitransgenic reporter mice (Eµ-tTA and Vav-tTA) were untreated. The percentage of GFP+ cells in each population is indicated. HSC: Lin–Sca1+Kit+ (LSK) hematopoietic stem cell. CLP: Lin–KitIntSca1+CD127+ common lymphoid progenitor. CMP: Lin–Sca1–Kit+CD34+Fc?RII/III– common myeloid progenitor. GMP: Lin–Sca1–Kit+CD34+Fc?RII/III+ granulocyte/macrophage progenitor. MEP: Lin–Sca1–Kit+CD34–Fc?RII/III– megakaryocyte/erythroid progenitor. MkP: Lin–Sca1–Kit+CD41+CD150+ megakaryocyte progenitor. Gating strategies are shown in Figure S2.
Fig 4: GFP reporter expression in T cell subsets of CAG-rtTA3 mice.Flow cytometry profile of GFP expression in thymic and splenic T cell subsets from a representative CAG-rtTA3 bitransgenic reporter mouse (green) compared with a control mouse (black). Mice were given Dox food for 7 days before analysis. The percentage of GFP+ cells in each population is indicated. (A) Reporter expression during thymocyte differentiation through DN (CD4–CD8–) to DP (CD4+CD8+) to SP (CD4+CD8– and CD4–CD8+) stages. (B) Reporter expression in splenic T cell subsets. Naïve: CD62L+CD44–, Effector: CD62L–CD44+, Effector memory: CD44+CD127+CD62L–, Central memory: CD44+CD127+CD62L+. Gating strategies are shown in Figure S5.
Fig 5: Mds1CreERT2Rosa26LSL-YFP/E9.5 TAM labeling demonstrates increasing presence of HSC-dependent hematopoiesis through E16.5(A) Flow cytometric analysis of E14.5 liver cells. Levels of YFP positivity were normalized to the level found in LSK (Lin-Kit+Sca1+, average 23.3% YFP+) to control for excision rates. Averages ± SEM of 5 individual embryos are plotted for LK (Lin-Kit+Sca1-), erythroid (Ter119+FSChi), granulocyte (Ly6G+), monocyte (Ly6C+), and B (CD19+) cells. See Figures S3A and S3B for gating strategy.(B) Flow cytometric analysis of circulating blood cells and liver cells at E16.5. Averages ± SEM of 3 individuals are plotted. Gating is as in (A) with the addition of circulating definitive erythroid (EryD, Ter119+) cells gated as in Figure S3C. Levels of YFP positivity were normalized to the level found in LSK average 8.1% YFP+) to control for excision rates.(C) Flow cytometric analysis of E16.5 progenitors in the liver. LSK was further refined into LT-HSC, ST-HSC, MPP2, MPP3, and MPP4 based on Flt3, CD150, and CD48 positivity (see Figures S3A and S3D). LK were further refined into lymphoid (CD127+), myeloid (CD16/32+), and erythroid/megakaryocyte (CD16/32-) progenitors. Values normalized to YFP levels in LSK (B). Averages ± SEM, n = 3.(D) Analysis by flow cytometry of YFP+ F4/80+ macrophages from E14.5 and E16.5 brain and liver normalized to YFP positivity of LSK (panels A and B). Details of gating are found in Figure S3E. Average ± SEM, n = 3. Unpaired two tail Student’s t test was performed comparing YFP labeling in cell populations to LSK. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
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