Article Title: Autoimmune Th17 Cells Induced Synovial Stromal and Innate Lymphoid Cell Secretion of the Cytokine GM-CSF to Initiate and Augment Autoimmune Arthritis
Figure Lengend Snippet: 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, Rorγt, 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 Il17a Cre R26R FP635 SKG mice. (M) Quantitative RT-PCR analysis of Csf2 and Bhlhe40 in splenic naive CD25 − CD44 lo CD4 + 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 × 10 6 ) 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
Article Snippet: The following monoclonal antibodies were used for flow cytometry analysis (BD FACSCantoII) and cell sorting (BD FACSAria SORP): anti-mouse CCR6 (29-2L17, Biolegend), CD3e (145-2C11, BD Biosciences), CD4 (RM4-4, Biolegend), CD8 (53-6.7, Biolegend), CD11b (M1/70, Biolegend), CD11c (HL3, BD Biosciences), CD16/32 (2.4G2, BD Biosciences), CD19 (1D3, BD Biosciences), CD25 (PC61, BD Biosciences), CD44 (IM7, BD Biosciences), CD45.2 (104, Biolegend), c-Kit (2B8, Biolegend), Foxp3 (FJK-16S, eBioscience), Gata-3 (TWAI, eBioscience), GM-CSF (MP1-22E9, BD Biosciences), GM-CSFRa (698423, R & D systems), IFN-γ (XMG1.2, eBioscience), IL-13 (eBio13A, eBioscience), IL-17 (TC11-18H10.1, Biolegend), IL-7Ra (SB/199, BD Biosciences), Ki-67 (MKI67, BD Biosciences), IL-33Ra (D1H9, Biolegend), Ly-6C (AL-21, BD Biosciences), Ly-6G (1A8, Biologend), MHC2 (M5/114.15.2, Biolegend), Pan-NK (DX-5, eBioscience), Podoplanin (8.1.1, Biolegend), Rorγt (AFKJS-9, eBioscience), TCR-β (H57-597, Biolegend), T-bet (4B10, Biolegend), Thy1.1 (OX-7, BD Biosciences), Thy1.2 (53-2.1, Biolegend), anti-human CD3 (UCHT1, Biolegend), CD4 (OKT4, Biolegend), CD8 (SK1, Biolegend), CD11b (M1/70, eBioscience), CD11c (3.9, Biolegend), CD19 (HIB19, Biolegend), CD45 (HI30, Biolegend), CD56 (HCD56, Biolegend), GM-CSF (BVD2-21C11, Biolegend), IFN-γ (4S.B3, Biolegend), IL-13 (JES10-5A2, Biolegend), IL-17 (BL168, Biolegend), PECy7-Streptavidin (BD Biosciences).
Techniques: Flow Cytometry, Cytometry, Expressing, Mouse Assay, Quantitative RT-PCR, Staining, Injection