Fig 2: Thymocytes with lower self-reactivity exhibit delayed in situ positive selection kinetics relative to those with high self-reactivity.(a) Expression of CD5 on CXCR4−CCR7 + CD4 CD8+TG6 (96 hr), F5 (72 hr and 96 hr), and OT-1 (72 hr and 96 hr) donor cells developed in thymic slices. Values for each experiment are normalized to the average CD5 expression of the CXCR4−CCR7 + CD4 CD8+polyclonal slice resident (SR) population at the same time points. (b–d) Preselection TG6 (white bars, triangle), F5 (gray bars, square), or OT-1 (dark grey bars, circle) thymocytes were overlaid onto selecting or nonselecting thymic slices, harvested at 2, 24, 48, 72, or 96 hr post-thymocyte overlay, and analyzed using flow cytometry. Left three graphs show individual values for each transgenic. Horizontal lines in (a) and (b) indicate the average value for nonselecting slices. Right line graph shows the average for all three transgenics overlaid. (b) Percent of CD69+ cells within the CD4+CD8+ and CD4−CD8+populations. (c) Percentage of CXCR4−CCR7 + cells within CD4+CD8+ and CD4−CD8+populations. (d) Percentage of CXCR4−CCR7 + CD4 CD8+ cells out of the donor population. Values for each experiment are normalized to the average at the time point with the maximum CD8SP development (72 hr or 96 hr). For (a), data are presented as average ± SD and analyzed using an ordinary one-way ANOVA with Tukey’s multiple comparisons (****p<0.000). For (b–d), data are presented as average ± SD and analyzed using an ordinary one-way ANOVA with Dunnett’s multiple comparisons (**p<0.01, ***p<0.001, and ****p<0.0001) comparing to 2 hr time point. All data are compiled from three or more experiments. Preselection thymocyte populations were obtained from TG6tg Rag2−/− H2b mice, irradiated β2M−/− mice reconstituted with F5tg Rag1−/− bone marrow, and OT-1tg Rag2−/− β2M−/− mice. See also Figure 3—figure supplements 1 and 2.

Fig 3: Mature thymocytes with lower self-reactivity appear later post-birth than those with higher self-reactivity.Thymuses were harvested from TG6 (triangle, white bar), F5 (square, gray bar), or OT-1 (circle, black bar) transgenic neonatal mice at 7, 14, and 21 days post-birth and from adult mice (6–9 weeks old), and analyzed using flow cytometry. (a) Percentage of CD4−CD8+ cells. (b) Total number of CD4−CD8+ thymocytes at the indicated time point post-birth, relative to adult, for each transgenic. (c) Percentage of CXCR4−CCR7+ cells within CD4+CD8+ and CD4−CD8+ populations. Data are presented as average ± SD and analyzed using an ordinary one-way ANOVA, Dunnett’s multiple comparisons (*p<0.05, **p<0.01, and ****p<0.0001) comparing to adult. All data are compiled from three or more experiments.

Fig 4: Lineage Potency of FS Cells and Responsiveness to Differentiation Cues(A) Neural differentiation assayed by quantification of Sox1::GFP-positive cells. Error bars represent SD from 4 independent experiments.(B) Immunostaining of FS cells and EpiSCs during neural differentiation; DAPI in white. Scale bars, 100 μm.(C) Lateral plate mesoderm differentiation and representative quantifications of the Flk1+Ecad− fractions by flow cytometry.(D) Average efficiency of Flk1-positive cell production from FS cells and EpiSCs. n, independent cell lines assayed. Error bars represent the SD. ∗∗p < 0.01.(E) Definitive endoderm differentiation protocol and representative quantifications of the Cxcr4+Ecad+ fraction.(F) Average proportion of Cxcr4+Ecad+ double-positive cells from differentiation of FS and EpiSC lines. Error bars represent SD; ∗p < 0.05.(G) T expression analyzed by qRT-PCR 6 h and 24 h after transfer into N2B27 medium with the indicated supplements; 2 μM XAV939, 20 ng/ml activin A, 10 ng/ml BMP2, 12.5 ng/ml Fgf2, and 3μM CH. Relative expression is normalized to GAPDH. Error bars are SD from two independent cell lines and two technical replicates.

Fig 5: Neutrophil tissue damaging properties are modulated by bone marrow B cells via Cxcr4.(A) Principle componen analysis (PCA) of the top 1000 most variable genes expressed by neutrophils isolated from blood or bone marrow, of mice pretreated with lipopolysaccharide (LPS) or NaCl 2 weeks earlier. (B–C) Gene ontology (GO) enrichment analysis of blood and bone marrow neutrophil differentially expressed genes (DEGs). (D) Cxcr4 mRNA expression in sorted bone marrow neutrophils from WT and JHT mice, pretreated with NaCl or LPS 2 weeks earlier. (E) Schematic depiction of the treatment procedure for the therapeutic application of a Cxcr4 agonist (ATI2341) and a Cxcr4 antagonist (ADM3100) at indicated doses. (F) Aspartate aminotransferase (ASAT) plasma levels of mice 18 hr p.i. with E. coli, which were treated with depicted doses of Cxcr4 ligands (agonist ATI2341 or antagonist ADM3100, respectively) 6 hr p.i. (G) Representative liver histology (H&E stain) of mice 18 hr p.i. with E. coli, treated with PBS or the indicated Cxcr4 ligands (agonist ATI2341, antagonist ADM3100, at 2000 ng/kg) 6 hr p.i. (H) Liver and blood CFUs of mice 18 hr p.i. with E. coli of mice, which were i.v. treated with depicted doses of Cxcr4 ligands (agonist ATI2341 and antagonist ADM3100, respectively) at 6 hr p.i. Data in (A–C) are from a single experiment (n=4–5/group). Data in (D) are from an independent experiment (n=3–4), versus data shown in (A–C). Data in (F–H) are representative of two independent experiments (n=3–8/experimental group). All data are and presented as mean +/-SEM. * p≤0.05 and ** p≤0.01.