Fig 2: Transplantation of HSCs cultured with various bile acids(A) The gating strategy for isolating CD150+CD48-KSL cells from murine bone marrow.(B) Experimental design of the competitive reconstitution assay. Fifty (50) CD150+CD48-KSL cells were isolated from BM of Ly5.1 mice (donor, two mice per experiment were used) and cultured in Stemspan SFEM medium supplemented with 100 ng/mL mSCF and 100 ng/mL hTPO with or without 100 µM of different types of bile acids (BA) for 7 or 14 days. Cells were then transplanted into lethally irradiated Ly5.2 mice (recipient, 5–18 mice) along with 2 × 105 total BM cells derived from Ly5.1/5.2 (F1) mice (competitor). Donor contribution (chimerism) was monitored by analyzing peripheral blood (PB) every month.(C) Peripheral blood analysis of the transplanted mice. Chimerism in PB at 16 weeks of transplanted mice are shown. Mean ± SD from three independent experiments (n = 5–18) are displayed. Significance was calculated using one-way ANOVA within the same culture days. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.(D–F) Cell surface expression of CD48 on KSL fraction of HSCs cultured with or without BA. Representative FACS plots (C) and summary of the flow cytometry analyses (D and E) are shown. Mean ± SD (n = 3) are displayed.

Fig 3: The designs show in vivo activity in zebrafish and mouse models.A, B Interval plots of the fluorescent neutrophils in zebrafish (Tg:mpxGFP) that were either not injected, or injected with the inactive protein Moevan_control, rhG-CSF, Boskar4_t2, or Boskar4_st2, after (A) 24 h or (B) 72 h of treatment. Data show mean ± standard deviation, and the number of treated embryos under each condition is show as N (under the x-axis), *p < 0.05, or ****p < 0.0001 vs. the Moevan_control group (p-values are listed in Supplementary Table 4). C Representative images of 1- and 3-day embryos that were not injected, or injected with inactive protein (Moevan_control), Boskar4_t2, or Boskar4_st2. Fluorescently labelled neutrophils can be observed and quantified across the tail region (scale bar: µm). D Ly5.1 mice were treated with PBS, the inactive protein Moevan_control, rhG-CSF, Boskar4, Boskar4_t2, or Boskar4_st2 (n = 7 per group for each condition). Fold change of mouse neutrophils (DAPI-CD45+Ly6G+Ly6C+) in the bone marrow of treated mice compared to PBS group is shown. Statistical significance was calculated by Fisher’s two-sided t-test (*p < 0.05, or **p < 0.01 vs. the PBS group). The exact p-values are provided in the source data files.

Fig 4: TET2 promotes inflammatory cytokine-mediated demethylation of murine Ifng during effector CD8 T cell differentiation(A) Experimental setup showing adoptive transfer of CD45.1 OT-1 CD8 T cells into WT C57BL/6 CD45.2 mice. One day later, the mice were either infected with LM-OVA or co-infected with LM-OVA and LCMV Armstrong. At the effector (8 days) and memory (>2 months) stages, CD45.1 OT1 CD8 T cells were FACS purified for phenotypic and DNA methylation analysis.(B) Representative and summary graphs of IFNg CNS bisulfite sequencing methylation profiles of naive CD8 T cells and LM-specific and LCMV-specific effector CD8 T cells at day 8 postinfection. The bottom bar graphs show the percentage of CpG methylation (mean ± SEM) at each individual CpG site of the IFNg locus. n = 3 for LM and n = 2 for LM+LCMV summary graphs.(C) Experimental setup showing infection of WT C57BL/6 or TET2cKO mice with LCMV Armstrong. At the effector (8 days) stage, LCMV-specific effector CD8 T cells were FACS purified for DNA methylation analysis.(D) Representative and summary bisulfite sequencing methylation profiles of the Ifng 3' CNS in LCMV-specific effector CD8 T cells from WT or TET2cKO mice. Error bars are defined based on the means ± SEM.(E) Experimental setup showing adoptive transfer of Thy1.1+ P14 CD8 T cells into Thy1.2+ WT C57BL/6 or TET2cKO mice. One day later, the mice were infected with LCMV Armstrong. At the effector (8 day) stages, endogenous and adoptively transferred GP33-specific CD8 T cells were phenotypically characterized and FACS purified for DNA methylation analysis.(F) Representative FACS analysis and summary graph characterizing the terminal effector (KLRG1+ CD127-) and memory precursor (KLRG1- CD127+) phenotype of either endogenous or adoptively transferred LCMV-specific CD8 T cells isolated from WT C57BL/6 or Tet2cKO mice. n = 4 for Tet2cKO and n = 4 for WT C57BL/6 summary graphs. ****p < 0.0001.(G) Targeted methylation profiling of the IFNg 3' CNS in naive, endogenous, and WT P14s CD8 Tcells isolated from WT and TET2cKO mice at the effector time point.

Fig 5: Characterization of cutaneous Langerhans cell (LC) and dendritic cell (DC) subpopulations.(A) Representative flow cytometry dot plots for LC characterization in the epidermis. Cells from epidermis were first gated for FSC, SSC, and CD45 (not shown). Then, CD45+ cells were analysed for CD11b and F4/80 expression. The CD11bhiF4/80hi cell fraction was further analysed for CD207 and CD326 expression to identify classical bona fide LCs. (B) Representative flow cytometry dot plots for dermal LC and DC subpopulations. Isolated dermis cells were first gated for FSC, SSC, and CD45 (not shown). CD45+ cells were then analysed for CD11b and F4/80 expression. The CD11bhi F4/80hi fraction contained classical CD207+CD326+ LCs. The remaining cells were gated for CD11c+MHC II+ DCs and separated into three subsets based on CD103 and CD11b expression: CD103+CD11b- cells (labelled cDC1), CD103-CD11bhi DCs (labelled CD11bhi), and CD11blow/neg. CD207 and CD326 expression was detectable on cDC1 but not CD11bhi DCs, whereas CD11blow cells were further separated into CD207-CD326- (labelled triple negative [TN]) and CD207+CD326+ (labelled LClike). (C) Representative flow cytometry dot plots for cutaneous DC subpopulations in auricular skin-draining LNs. LN cells were first gated for FSC and SSC to exclude small lymphocytes before F4/80 and CD11b staining. The cell fraction excluding F4/80hi/CD11bhi cells was separated by CD11c and MHCII. CD11chiMHCIIhi migratory DCs were gated and analysed for CD103, CD11b, CD207, and CD326 expression. Four subsets were detected: CD103+CD11b-CD207+CD326+ (cDC1), CD103-CD11blowCD207-CD326- (TN), CD103-CD11blowCD207+CD326+ (LClike), and CD103-CD11bhiCD207-CD326-/+(CD11bhi). (D) Frequency of each DC subpopulation (LC, cDC1, LClike, TN, and CD11bhi) present in epidermis, dermis, and cutaneous lymph node (LN), respectively.