Fig 2: TKO Tregs Display Marked Defects in Mobilization In Vitro and In Vivo(A) CD4+CD25- T cells from spleens of WT and TKO mice after 12 weeks of HFD were treated with anti-CD3 antibodies and TGF-ß1 for differentiation into in vitro differentiated Tregs (iTregs). Percentage of WT and TKO CD4+CD25+Foxp3+ Tregs were measured by flow cytometry at the indicated time points (n = 6 per group). (B and C) CD4+CD25- T cells from spleens of WT and TKO mice after 12 weeks of HFD were activated by anti-CD3 antibodies for 24 h and subjected to qRT-PCR analysis (B) or ELISA from supernatants (C) for the indicated cytokines, chemokines, and growth factors (n = 5–9 per group).(D and E) Transwell migration study of CD4+CD25+ Tregs isolated from WT and TKO mice after 12 weeks of HFD. Cells were assessed for migration in the presence or absence of CCL19 (D) or CCL20 (E) (n = 3 per group).(F and G) Flow cytometry for CCR7 (F) or CCR6 (G) expression in WT and TKO Tregs (n = 6 per group).(H) Schematic of PKH26-labeled HFD WT and TKO Tregs adoptively transferred to HFD C57BL/6 mice. Flow cytometry shows percentage of PKH26-expressed cells in liver, VAT, and SAT of recipient mice (n = 6 per group).(I and J) Schematic of glucose uptake study of differentiated 3T3-L1 cells co-cultured with HFD WT and TKO iTreg supernatant (supe) (I). (J) Fluorescence intensity of 2-Deoxy-D-glucose (2-DG) uptake by differentiated 3T3-L1 cells co-cultured with supernatants of WT and TKO CD4+ Tregs in the presence or absence of insulin stimulation (n = 4 per group).(K and L) Schematic of glucose production study of mouse primary hepatocytes co-cultured with HFD WT and TKO iTreg supernatants (K). (L) Glucose production by mouse primary hepatocytes co-cultured with supernatants of HFD WT and TKO CD4+ Tregs (n = 6 per group).Statistical differences are indicated as *p<0.05, **p<0.01, and ***p<0.001. Results are reported as mean ± SEM.Related to Figures S3 and S4.

Fig 3: GM-CSF-Producing ILCs in Inflamed Joints(A) Flow cytometry analysis of GM-CSF expression by CD4+ T cells and CD4- cells among CD45+ joint infiltrating cells in Rag2-/- mice transferred with CD4+ T cells from WT or Csf2-/- SKG mice.(B) Quantitative RT-PCR analysis of Csf2, Il1b, and Tnf in CD11b+Ly-6G- (CD11b+), CD11b+Ly-6G+ (Ly-6G+), CD4+ T cells, and ILCs sorted from arthritic joints of mannan-treated SKG mice (n = 3). mRNA expression is presented relative to the expression of Hprt1.(C) Flow cytometry of joint infiltrating cells in Rag2-/- mice transferred with CD4+ T cells from WT or Csf2-/- SKG mice. Cells were stained for CD45.2 and lineage markers (a cocktail of CD3, CD4, CD8, CD11b, CD11c, CD19, and DX-5).(D) Proportion of ILCs in Rag2-/- mice transferred with CD4+ T cells as shown in (C). Each symbol represents an individual mouse. Horizontal bars indicate the means.(E) Total cell number of ILCs from healthy or inflamed joints of SKG mice (n = 3).(F) Flow cytometry of synovial ILCs (CD45.2+ lineage markers-negative Thy1.2+ cells as shown in C) for Ki-67 expression.(G) Flow cytometry of synovial ILCs (CD45.2+ lineage markers-negative Thy1.2+ cells as shown in C) for cell surface expression of IL-7Ra, CD25, CCR6, c-kit, IL-33Ra, CD44, and MHC2.(H) Flow cytometry of synovial ILCs (as shown in C) for intranuclear expression of the transcription factor T-bet, Gata-3, Ror?t, and Foxp3.(I) Proportion of the transcription factor-expressing synovial ILCs (n = 3) as shown in (H).(J) Flow cytometry of synovial ILCs (as shown in C) for the expression of GM-CSF, Gata-3, and IL-13.(K) Total cell numbers of ILCs from healthy or inflamed joints of C57/BL6 (B6) mice with collagen antibody-induced arthritis (n = 3). Data are representative of two independent experiments.(L) Flow cytometry of synovial ILCs for the expression of GM-CSF and FP635 in arthritic Il17aCre R26RFP635 SKG mice.(M) Quantitative RT-PCR analysis of Csf2 and Bhlhe40 in splenic naive CD25-CD44loCD4+ T cells (naive CD4+ T) and synovial ILCs (n = 3) as shown in (C).(N) The effects of ILC depletion on arthritis development. CD4+ T cells (1 × 106) from Thy1.1+ SKG mice were adoptively transferred into Thy1.2+Rag2-/- mice, which were i.v. injected with 500 µg anti-Thy1.2 mAb or control Rat IgG every week (n = 19 each). The severity of arthritis was monitored every week.*p < 0.05, **p < 0.01. Data are representative of three independent experiments in (A)–(C), (E)–(J), (L), and (M) and pooled from more than two experiments in (D), (F), and (N). Vertical bars mean SD in (B), (E), (I), (K), (M), and (N).

Fig 4: Mincle and Syk in DCs Are Needed for Intestinal IL-17 and IL-22 Steady-State Production(A–E) Representative plots and summary graph of IL-17 and IL-22 (A and B), IFN-? (C and D), and IL-17, IFN-?, and IL-10 (E) production by intracellular staining after phorbol 12-myristate 13-acetate (PMA) and ionomycin stimulation in CD4+ T cells from PPs in the indicated genotypes.(F and G) Frequency of CCR6+ ILCs from PPs producing IL-22 and IL-17 in the steady state by intracellular staining in the indicated genotypes.At least two independent experiments were pooled. Each symbol represents an individual mouse. The arithmetic mean for each group is indicated. **p < 0.01, ***p < 0.001 (unpaired two-tailed Student’s t test). See also Figure S4.

Fig 5: Pattern of systemic inflammation in DIAMOND mice as they age on a regular chow diet(A) Sera were collected from DIAMOND mice at weeks 19, 24, or 40 of being on a CD or WD three to five mice/group) and analyzed for 44 cytokines and chemokines in association with time using multivariate linear regression. The overall time trend, summarized across subjects, is presented for all cytokines and chemokines, which have a significant time trend during a CD. Data represent five to nine mice/group.(B) FVS- viable splenocytes were gated for CD3+ T cells to show the proportion of CD8+ T cells and CD4+ T cells in males or females (three to five mice/group) being on a CD for 24 weeks (24MCD or 24FCD) and 40 weeks (40MCD or 40FCD) and were analyzed for M1 (F4/80+CD68+CD206-) and M2 macrophages (F4/80+CD68+/-CD206+), NK cells (CD3-CD4-CD8-CD49b+), NKT cells CD3+CD4-CD8-CD49b+), and CD11b+Gr1+ MDSCs.(C) FVS- viable splenocytes gated for CD3+CD4+ T cells were analyzed for the percentage of Th1 (CXCR3+CCR5-), Th2 (CCR8+IL-3Ra-), or Th17 (CCR6+CD161-) cells: percentage of Th1 cells that do not express TIM-3 was determined.(D) FVS- viable splenic CD8+ or CD4+ T cells were analyzed for percentage of T cell phenotypes, including Te (CD44+CD62L-), Tem (blue line, CD44+CD62Llow), Tcm (red line, CD44+CD62Lhigh), and Tn (CD44-CD62L+) cells.Error bars are SEM.