Fig 2: ZEB2 Controls Tissue Identity of KCs and AMs(A) Schematic of experimental set up.(B) Expression of CD45.1 (donor) and Tim4 in total Clec4F+ KCs in Clec4f-crexZeb2fl/fl chimeras. Percentage of total chimerism of KCs in Zeb2fl/fl and Clec4f-crexZeb2fl/fl mice. Data are pooled from two experiments with n = 6–10 per group.***p < 0.001 Student’s t-test.(C) Expression of CD45.1 (donor) and CD45.2 (host) in total lung AMs in Itgax-crexZeb2fl/fl mice. Percentage of total chimerism of AMs in Zeb2fl/fl and Itgax-crexZeb2fl/fl mice. Data are pooled from two experiments with n = 5–8 per group. NS; non-significant. Student’s t-test. Percentage total chimerism calculated as ratio over the chimerism in blood Ly6Chi monocytes in the same mouse.(D and E) Heatmap of expression of cell death-associated genes per group of KCs (D) or AMs (E) from SC-RNA-seq data.(F) Representative western blots (n = 2) for RIPK3, pMLKL, and Tubulin expression by total BAL cells isolated from CD45.1 mice that were irradiated (8 Gy) and reconstituted with Rosa26-creert2xZeb2fl/fl BM. 33 weeks post reconstitution, mice were fed 5 mg tamoxifen or corn oil as a control for 5 days. 27 days after the last dose, mice were sacrificed and BAL fluid isolated. BAL fluid from 3 or 4 mice was pooled per replicate and 150,000 cells were used per lane. Ratio of band intensity was calculated using ImageJ. Data are pooled from two experiments with n = 6–8 per group. Student’s t-test was used to calculate indicated p values.(G) Venn diagram showing DE genes specific to Zeb2-/- KCs (group 3), Zeb2-/- AMs (group 3), or shared between both mac populations.(H and I) Heatmap showing expression of top core genes across KC (H) or AM (I) groups from SC-RNA-seq data. Genes in red are significantly differentially expressed. See also Figure S3.

Fig 3: CCRF-CEM and Treg migrate towards Raji in a CCR4-dependent manner.(A) Chemotaxis assays were performed on CCRF-CEM cells towards recombinant CCL22 or Raji cell supernatant with or without the CCR4 antagonist CCR4-351. Relative luciferase units (RLU) are shown as a proxy for migrated cell number. (B) Migration assays were performed with human iTreg. (C) Raji and Daudi xenograft tumors grown in NOD/SCID mice (n = 5) were assayed for chemokine levels by ELISA. (D) Human iTreg were transferred into Daudi or Raji tumor-bearing mice (n = 8) and allowed to migrate for 5 days, followed by harvesting of the tumors. Mice were treated with vehicle or CCR4-351. Fractions of human CD45+ cells which were iTreg (identified as CD45+ CD4+ CD19-) were quantitated by FACS.

Fig 4: T cell senescence in various adipose tissues of old mice. (A,B) Flow cytometric analysis of CD45+CD3+ T cells in AT of young and old mice and their quantitation. (C,D) Representative flow cytometric analysis of CD44 subsets in T cells isolated from adipose tissue and their quantification. (E,F) Representative flow cytometry of CD28+ T cells in adipose tissue from young or old mice and their quantification. *P < 0.05; **P < 0.01; ***P < 0.001 vs. young mice, n = 3~6 per group.

Fig 5: Desiccation stress recruits monocytes into the conjunctiva in a CCR2 dependent manner. The gating strategy used for these experiments is provided in Supplementary Figure 1A . (A) Single cell suspensions prepared from conjunctivae of C57BL/6 (B6) and Ccr2-/- strains were stained with anti-CD45, CD11b, MHCII, CD64, Ly6c and MerTK. Squares within the representative plots show frequency of CD45+CD11b+MHCII-CD64+Ly6Chigh (classical monocytes) and CD45+CD11b+MerTK+ (tissue resident macrophages) cells (n = 6/group); (B) Experimental design, bone marrow cells from Pepc/BoyJ (2x10^6) were adoptively transfer via intra-orbital injection at 2 day time intervals and the recipient mice either remained in normal environmental conditions (NS) or were exposed to desiccating stress for 5 days (DS5) (n = 6/group); (C) relative fold change in CCL2 mRNA after desiccating stress for 1 day (DS1) (n = 6/group); (D) Adoptively transferred cells were tracked by gating on CD45.1+, squares within the representative plots showing frequency of classical monocytes (top) and CD45+CD11b+MHCII+ cells (bottom). The bar graphs on the right side indicate the percentages of these cells with each dot representing one animal (n = 6/group); (E) Ccr2-/-/Ccr2+/+ bone marrow chimera. Bone marrow ablation in Pepc/BoyJ mice was accomplished with 137Cs irradiation with 1300 cGy, followed by intraorbitally injection of 2x10^6 bone marrow cells from Ccr2-/-/CD45.2 (2x10^6) or wild type B6 (Ccr2+/+/CD45.2) donors. Flow cytometry scatter plot shows the number of endogenous (CD45.1) or transplanted (CD45.2) immune cells in the conjunctiva two weeks post transfer; (F) Donor CD45.2+CD11b+ myeloid cells were measured in the conjunctiva of Pepc/BoyJ recipient mice after exposure to desiccating stress for 5 days (n = 6/group). (G) Donor CD45.2+CD11b+MHCII+ cells were measured in the conjunctiva of Pepc/BoyJ recipient mice after exposure to desiccating stress for 5 days (n = 6/group). The error bars indicate the standard error of mean (SEM), Student T-test was used for statistical comparison. ***p < 0.0001.