Fig 2: Absence of RANKL expression leads to mouse uMf dysfunction and fetal loss. (a) RANK expression on uMf from CBA/J? × DBA/2? matings (the abortion-prone model) and CBA/J? × BALB/c? matings (normal pregnancy model) at days 5 and 9 of gestation (n=6 mice per group). Moreover, the expression of CD80, CD86, CD206 and MHCII on F4/80+uMf from CBA/J? × DBA/2? matings (the abortion-prone model) and CBA/J? × BALB/c? matings (normal pregnancy model) at days 5 and 9 of gestation (n=6 mice per group); (adjusted t-test). (b) FCM analysis of CD206, CD209, IL-10, CD80 and CD86 in uMf of wild-type and RANKL knockout pregnant mice at day 10 (n=6 mice per group); (Student’s t-test). (c) FCM analysis of GATA-3, T-bet, IL-4, IL-10, IFN-? and TNF-a in uCD4+T cells of WT and RANKL-/- pregnant mice at day 10 (n=5 mice per group); (Student’s t-test). (d) FCM analysis of the phosphorylation level of Akt and STAT6 in uMf cells of WT and RANKL-/- pregnant mice at day 10 (n=6 mice per group); (Student’s t-test). (e) uMf were isolated from mouse uterus (n=20 mice per group) from WT and RANKL-/- mice at day 10 of gestation by MACS, and then used to analyze the transcription of Jmid3 and IRF4 in uMf. (Student’s t-test). (f) FCM analysis of IRF4 levels in uMf cells of WT and RANKL-/- pregnant mice at day 10 (n=6 mice per group); (Student’s t-test). (g and h) The embryo absorption rate in WT and RANKL-/- pregnant mice (n=6 mice per group) was determined on day 14 of gestation. Fetal loss sites could be identified as hemorrhagic spots and necrosis (red arrows, left); (adjusted t-test). uMf: Mf from mouse uterus; uCD4+T cells: CD4+T cells from mouse uterus; Normal: normal pregnant mouse model; Abortion: abortion mouse model. D5: day 5 of gestation; D9: day 9 of gestation. WT: wild-type pregnant mice; RANKL-/-: RANKL knockout pregnant mice. Data are expressed as the mean±S.E.M. *P<0.05, **P<0.01 and ***P<0.001

Fig 3: Presence and frequency of ILC subtypes within the meninges of severe TBI patients.(A) Dura was collected from consecutive, severe TBI patients undergoing decompressive craniectomy to alleviate elevated intracranial pressure. ILCs were sorted using forward scatter (FSC)/side scatter (SSC) and identified as CD45+, lineage-negative (Lin-), CD127+ lymphoid cells. ILCs subtypes were further defined as ILC1:, CD45+Lin-CD127+CD161+NKp44+; ILC2, CD45+Lin-CD127+GATA3+CRTH2+; and ILC3, CD45+Lin-CD127+ROR?t+AhR+, as shown in representative flow cytometry scatterplots. Gray shaded areas indicate isotype controls. To demonstrate functionality, ILCs were further stimulated with cytokine cocktails, and production of signature cytokines was assessed (ILC1, IFN-?; ILC2, IL-5/IL-13; ILC3, IL-17). (B and C) Frequency of ILC subtypes from individual patients, expressed as total cell number (B) and % leukocytes (C) (n = 5). Scatterplots depict mean ± SD. (D) Computed tomography scan of a TBI patient before (Pre-) and after (Post-) decompressive craniectomy surgery. The dura was collected during surgery at the time of bone flap removal.

Fig 4: RANKL induces dMf differentiation by activating the Akt/STAT6-Jmjd3/IRF4 signaling pathway. (a-c) dMf were co-cultured with RANKL+D+J or Ctrl-D+J at a 1 : 1 : 1 ratio, and treated with or without 10 µM Akt signaling inhibitor (Akti) Ly294002 for 24 h. The intracellular phosphorylation level of Akt and or STAT6 in dMf (n=5) was then analyzed by FCM. (d) The secretion level of IL-10, IL-12p40 and IL-23 in the co-culture of dMf and RANKL+-D+J or Ctrl-D+J (at a 1 : 1 : 1 ratio), which was treated with or without Akti (10 µM) or the STAT6 signaling inhibitor (STAT6i) AS1517499 (21 nM) for 48 h. (One-way ANOVA). (e) After intraperitoneal injection of STAT6i (200 µl at the concentration of 2 mg/kg) in pregnant C57BL/6 mice (n=5 mice per group) at day 4, the levels of CD206, CD209, IL-10, CD80 and CD86 in uMf, and the ratios of GATA-3 to T-bet and IL-4 to IFN-? in uCD4+T cells at day 10 were detected by FCM. (Student’s t-test). (f) The transcription levels of IRF4, Jmjd3 and IRF5 in dMf treated as described in Figure 3d. (One-way ANOVA). (g) After intraperitoneal injection of STAT6i or the Jmjd3 selective inhibitor (JMJD3i, 200 µl at a concentration of 4.48 mg/kg) GSK J4 HCl in pregnant C57BL/6 mice (n=5 mice per group) at day 4, the IRF4 level in uMf cells at day 10 was detected. (h and i) After intraperitoneal injection of JMJD3i in pregnant C57BL/6 mice (n=5 mice per group) at day 4, the levels of CD206, CD209, IL-10, CD80 and CD86 in uMf, and the ratios of GATA-3 to T-bet and IL-4 to IFN-? in uCD4+T cells at day 10 were detected by FCM. (Student’s t-test). dMf: human dMf; uMf: mouse uterus macrophages. Data are expressed as the mean±S.E.M. *P<0.05, **P<0.01 and ***P<0.001

Fig 5: Frequencies of CD4+ T-cells expressing Gata3 and Th2 cytokines in asthma and helminth infection are reduced in Bcl11bfl/fl dLck-iCre mice by an intrinsic defect. a Flow cytometry analysis of IL-4 (left column), IL-5 (middle column), and IL-13 (right column) against GATA3 in the CD4+ T-cell population of the lung parenchyma of HDM asthma-induced mice. b Frequencies of IL-4+ (n = 4), IL-5+ (n = 4), and IL-13+ (n = 8) (top panel) and GATA3+ (n = 10) (bottom panel) CD4+ T-cells from the lungs of the indicated groups. Data are derived from two (IL-4, IL-5, and IL-13 data) or five (GATA3 data) independent experiments. c Flow cytometry analysis of IL-4 (left column), IL-5 (middle column), and IL-13 (right column) against GATA3 in the CD4+ T-cell population from the mesenteric (m)LNs of H. polygyrus infected mice. d Frequencies of IL-4+ (n = 4), IL-5+ (n = 4), and IL-13+ (n = 4) (top panel) and GATA3+ (n = 8) (bottom panel) CD4+ T-cells from mLNs of the indicated groups. Data are derived from two (IL-4, IL-5, and IL-13) or four (GATA3) independent experiments. e Flow cytometry analysis of GATA3 in CD4+ T-cells (left column) and IL-4 (middle and right columns) in the GATA3hi (middle column) and GATA3lo (right column) CD4+ T-cells from the mLNs of H. polygyrus bakeri infected mice. f The ratio of GATA3hi/GATA3lo CD4+ T-cells (top) and frequencies of IL-4 secreting cells within the GATA3hi and GATA3lo CD4+ T-cells (bottom) from the mLNs of H. polygyrus bakeri infected WT (n = 4) and Bcl11bfl/fl dLck-iCre (n = 3) mice. Data shown are from two independent experiments. g Flow cytometry analysis of IL-4 and GATA3 in CD4+ T-cells from the mLNs of mixed bone-marrow chimeric mice infected with H. polygyrus bakeri. Cells from WT donors are CD45.1/CD45.2, while cells from Bcl11bfl/fl dLck-iCre donors are CD45.2. h Frequencies of IL-4+GATA3+ cells from the mLNs of mixed bone-marrow chimeric mice (n = 6) infected with H. polygyrus bakeri. Data shown are from two independent experiments. All statistical values in this figure were calculated using Student’s t-test. All error bars shown in this figure are of standard deviation