Fig 2: TMEM, TINT, and TEFF cells reside in distinct areas of the spleen(A–C) TG6-GFP chimeric mice infected with T. gondii and their spleens were analyzed by confocal microscopy after 6 weeks. Sections were stained with antibodies against TCF1 (A), Ki67 (B), and KLRG1 (C) to identify the location of TMEM, T, and TINT/TEFF TG6-GFP cells, respectively. B220 staining was used to identify B cell zones (B) and T cell zones (T) to map the white pulp (WP) and red pulp (RP) regions. In some images, bridging channels (BCs) are marked with dotted lines. Dashed rectangles indicate the area in the magnified regions on the right. Examples of cells staining positively with the indicated marker are highlighted with yellow arrows and negative examples with white arrows.(D) Data summarizing the location of TG6-GFP cells that stained positively for each protein.(E) Data showing the proportion of each GRA6-specific population labeled after i.v. anti-CD8 antibody injection.(F) Summary data showing the location of TMEM, TINT, and TEFF cells in the spleen after adoptive transfer.Images in (A)–(C) and data in (D) are representative of images taken from three or more separate mice. Data in (E) are taken from two separate infections and are representative of data from three separate experiments. Data in (F) are summarized from multiple recipient mice across three infections (TMEM cells, n = 3; TINT cells, n = 3; TEFF cells, n = 4).

Fig 3: CXCR3-deficient CD8+ T cells retain a memory phenotype in the RP(A–C) CXCR3 KO TG6-GFP neonatal chimeras were infected with T. gondii and analyzed by confocal microscopy after 6 weeks. Sections were stained with antibodies against TCF1 (A), Ki67 (B), and KLRG1 (C). B220 staining was used to identify B cell zones (B) to map the T cell zone (T) and RP regions. Yellow arrows highlight examples of cells staining positively for the specified marker; white arrows highlight examples of negative staining.(D) Summary data showing the proportion of GFP cells found in the WP or RP that are identified as positive or negative for expression of the indicated marker. Bar graphs display the proportion of WT or CXCR3 KO GFP cells expressing the protein of interest (TCF1, Ki67, or KLRG1) within the WP, BC, or RP.Data for WT cells are combined from at least three mice across two different experiments, and data for KO cells are combined from four mice taken across two experiments.

Fig 4: TNF-a, via TNFR2 Signaling, Enhances ILC2 Survival and Activation In Vivo(A) Rag2-/- mice received i.v. injections of 100 µg aTNFR2 or an isotype control and 0.5 µg rmIL-33 or PBS i.n. on days 1–3. On day 4, lung ILC2 numbers, proliferation, and apoptosis were measured.(B) Representative flow cytometry plots of lung ILC2s in isotype control- or aTNFR2-treated mice. ILC2s were gated as lineage- CD45+ ST2+ CD127+.(C) Total number of lung ILC2s, presented as mean numbers ± SEM.(D and E) Representative flow cytometry plots of AnnexinV/DAPI (D) and intranuclear Ki67 expression (E) with corresponding quantitation, presented as frequency of Ki67+ ILC2s ± SEM (D) and AnV+DAPI+ ILC2s ± SEM (E).(F) Representative flow cytometry plots of intracellular IL-5 and IL-13 expression by lung ILC2s cultured for 4 h with phorbol 12-myristate 13-acetate (PMA), ionomycin, and GolgiPlug and corresponding quantitation, presented as mean frequency ± SEM.(G) Representative flow cytometry plots of intranuclear GATA-3 in lung ILC2s (CD45+Lin-ST2+CD127+) and corresponding quantitation, presented as MFI ± SEM.(H) Representative flow cytometry plots of ST2 expression in GATA-3+ ILC2s and corresponding quantitation, presented as mean MFI ± SEM.(I) C57BL/6 and TNFR2-/- mice were challenged i.n. on days 1–3 with 0.5 µg rmIL-33. On day 4, aILC2s were FACS sorted as lineage- CD45+ ST2+ CD127+ and cultured in vitro for 3 days with rmIL-2 (10 ng/mL), rmIL-7 (10 ng/mL), and rmIL-33 (50 µg/mL) prior to transfer of 50 × 103 aILC2s in 2 separate cohorts of Rag-/- Il2rg-/- mice, followed by i.n. administration of 50 ng rmTNF-a on days 1–3. On day 4, lung function, BAL, and lung ILC2s were analyzed.(J) Lung resistance in response to increasing doses of methacholine.(K) Total number of eosinophils in the BAL fluid, presented as mean numbers ± SEM.(L) Total number of ILC2s in the lungs, presented as mean numbers ± SEM.Data are representative of 3 individual experiments (n = 4–5). *p < 0.05, **p < 0.01. See also Figures S4–S6.

Fig 5: Sirt1 overexpression in MPCs partially rescued defects of osteogenesis and hematopoiesis in Bmi1 KO mice. (A) ALP staining on paraffin sections of proximal tibiae. Images at higher magnification (the bottom row) showed the area framed in images at lower magnification (the top row). (B) Histomorphometric analysis of ALP+ area in total tissue area (ALP+ A/T.A). n=6 mice/group. (C) Representative images of H&E-stained paraffin sections of tibiae and (D) total bone marrow cell numbers in two femora were counted. n=6 mice/group. (E) FACS gates of LSK cells in BM of mouse femora. (F-G) Flow analysis of LSK cells in BM single-cell suspension from femora (F) and spleen (G) of various mice. n=6 mice/group. (H) FACS gates of Ki67+GFP+LSK cells in BM of various recipient mice. (I-K) Statistic analysis of frequencies of GFP+LSK cells (I) and Ki67+ cells in GFP+LSK cells (J) and Ki67+GFP+LSKcells (K) in BM of various recipient mice. n=4 samples. *p<0.05, **p<0.01, ***p<0.001. Data are mean ± SD. One-way ANOVA with Tukey's post-hoc test.