Fig 2: Migration efficiency of IV transferred monocytes and cMDM to acutely inflamed brain tissue is linked to monocyte-to-macrophage differentiation.(A) MDM cultured from day 0 to day 28 were introduced IV (5×106 cells) into recipient mice bearing LPS-induced acute neuroinflammaiton, and cMDM in vivo brain homing efficiency deceased as the in vitro cultivation time went up. Female gDNA = female mouse brain tissue genomic DNA (negative control). No cell IV = LPS injected control animal that received no cell IV transplant. Final data presented here represents mean values ± SD. Data was analyzed by one-way ANOVA, with resulting p-value <0.05. (B–D) Flow cytometry analysis on cMDM cell populations at different time points post in vitro cultivation. (B) Example of phenotype identification of cMDM using D5 cMDM. The green cluster reflects macrophage phenotype (CD11b+ F4/80hi Ly6Clow to neg), blue cluster reflects Ly6clow monocytes (CD11b+ F4/80low to med Ly6Clow to neg), and yellow cluster reflects Ly6Chi monocytes (CD11b+ F4/80low to med Ly6Chigh). (C) The ratio of macrophages (CD11b+ F4/80high Ly6Cneg) and total monocytes (CD11b+ F4/80low to med Ly6Clow to high), and (C) the ratio of Ly6Chi monocytes (CD11b+ F4/80med Ly6Chigh CCR2+) and Ly6Clo monocytes (CD11b+ F4/80low Ly6Clow CCR2low to neg) in culture were evaluated. (E) Phenotyping of freshly isolated EnMO by flow cytometry analysis. Expression level of CD115, CD11b, F4/80, and Ly6c were measured to determine the purity of the enriched cells. (F) Freshly isolated EnMO showed superior brain homing efficiency over cMDM. 5x106 of EnMO (D0 EnMO), or MDM cultured for 2-, 5-, or 12- days (D2 cMDM, D5 cMDM, D12 cMDM, respectively) were infused IV to animals with acute neuroinlfammation, and the number of donor cells present in the LPS-injected brain hemisphere was quantified at 48 hour following cell IVI. Final data presented here represents mean values ± SD. Data was analyzed by one-way ANOVA, with resulting p-value all < 0.05.

Fig 3: ILC3s are enriched in lung-draining mediastinal lymph nodes (MedLNs) and have high expression of MHC class II(A and B) Representative plots and quantitative graphs of frequencies (A) or total numbers of ILC2s and ILC3s (B) in mouse lung parenchyma and MedLNs (pooled from 2 independent assays, n = 8–10). Cells are gated on live CD45+ Lin− CD127+ (lineage markers: CD3e, CD5, CD8a, NK1.1, CD11c, CD11b, B220, and F4/80) as in Figure S1A.(C) Representative histograms of specific markers on gated ILC2s and ILC3s in mouse MedLNs (representative of 6 mice).(D) Representative plots and quantitative graphs of ratios of ILC2s and ILC3s in human lung and MedLN tissues. Cells were gated on live CD45+ Lin− (CD3, CD4, CD5, CD11b, CD11c, CD14, CD19, FcεRI) CD127+ populations from matched lung and MedLN tissues pooled from 5 donors.(E) Representative histograms of specific markers on ILC2s and ILC3s from human MedLN tissues (representative from 5 donors). Data are represented as mean ± SEM. Statistics are calculated by Student’s t test. See also Figure S1.

Fig 4: JPYZXZ reversed expansion and differentiation of MDSCs caused by GC cell exosomal PD-L1. (A) Transmission electron microscopy image of exosomes. (B) NTA analysis confirmed the exosomes. (C) Western blot of the exosome-related markers in GC cell-derived exosomes. (D,E) Western blot analysis of exosomal PD-L1 in GC cells. (F) Correlation of PD-L1 expression with infiltrating levels of MDSCs and immune cells in STAD. (G,H) Fluorescence images of PD-L1 after lentivirus transfection of MKN-74. (I,J) Western blot analysis of the PD-L1 content in GC cells. (K,L) Western blot analysis of the exosomal PD-L1 content in culture supernatant of GC cells. (M) Immunofluorescence staining for DAPI (nucleus), Dil (exosomes), and PD-L1 in MDSCs. Scale bar = 20 μm. (N,O) Percentages of CD11b+Gr-1+ MDSCs detected using flow cytometry. Data are expressed as mean ± SEM, n = 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 5: MHC class II+ ILC3s limit airway Th17 cell responses to microbes upon HDM exposure(A) Representative plots and quantitative graphs of the frequency and number of Th17 cells in lung parenchyma and MedLN from H2-Ab1fl/fl control mice and MHC class IIΔILC3 mice after exposure to HDM or PBS. Cells were gated on live CD45+ CD19− CD3/CD5+ CD4+ (data pooled from 4 independent assays, n = 6).(B) Quantitative graph of the number of neutrophils in lung parenchyma from mice in (A). Neutrophils were identified as live CD45+ CD19− TCRβ− CD11b+ Ly6G+ (data pooled from 4 independent assays, n = 7).(C) ELISA analysis for IL-5 and IL-17A levels from supernatants of microbiota-extract-restimulated MedLN cells from HDM-exposed MHC class IIΔILC3 mice (data pooled from 2 independent assays).(D) Quantitative graphs of the number of Th17 cells in lung parenchyma and MedLNs from H2-Ab1fl/fl control mice and MHC class IIΔILC3 mice after exposure to sterile papain (data pooled from 2 independent assays, n = 4).(E) Bar graphs of major bacterial composition in HDM from four different vendors. Data are represented as mean ± SEM. Statistics are calculated by two-way ANOVA. See also Figure S4.