Journal: Journal of autoimmunity

Article Title: Autoreactive memory Th17 cells are principally derived from T-bet + RORγt + Th17/1 effectors

doi: 10.1016/j.jaut.2022.102816

Figure Lengend Snippet: Naïve CD4 + T cells were isolated from wild-type (WT) or Tbx21 −/− mice and then i.v. injected into Rag1 −/− mice. After adoptive transfer, these Rag1 −/− recipients were immediately subjected to desiccating stress for 14 d and then relocated to normal non-desiccating environment where they were maintained until day 35. (A) Clinical disease severity was evaluated by corneal fluorescein staining (CFS) and representative images show baseline, acute (day 14) and chronic (day 35) stage of DED. (B) Kinetic DED scores in each group are summarized as mean ± SEM in linear graphs (n = 28 eyes per group at acute stage, 16 eyes per group at chronic stage, pooled from two independent experiments). (C) Representative flow cytometry plots show frequencies of eTh17/1 cells in draining lymph nodes (DLN) of Rag1 −/− recipients in acute DED (day 14). The frequencies of eTh17/1 cells (mean ± SEM) are summarized in the bar graphs (n = 6 per group). (D) Flow cytometry plots show frequencies of eTh17/1 cells in conjunctiva (CONJ) of Rag1 −/− recipients in acute DED (day 14). Ocular tissues were pooled from 6 animals in each group for the analysis, and data shown are one representative out of two performed. (E) Representative flow cytometry plots show frequencies of CD44 hi mTh17 cells in DLNs of Rag1 −/− recipients in chronic DED (day 35). (F) The proportion of CD44 hi mTh17 cells among Th17 population and the expression intensity of CD44 (MFI) by Th17 cells in DLNs of chronic DED from one representative experiment out of two independent experiments performed are summarized as mean ± SEM in the bar graphs (n = 3–6 per group). (G) Flow cytometry plots show frequencies of CD44 hi mTh17 in conjunctiva of Rag1 −/− recipients in chronic DED (day 35). Ocular tissues were pooled from 4 animals in each group for the analysis, and data shown are one representative out of two performed.*, p <0.05; **, p <0.01; ***, p <0.001.

Article Snippet: Tbx21 F/F mice (Jackson Laboratory) were crossbred in house to generate T-bet conditional knockout IL-17A cre Tbx21 F/F ( IL-17A ΔTbx21 ) mice.

Techniques: Adoptive Transfer Assay, Isolation, Injection, Staining, Flow Cytometry, Expressing

Journal: Journal of autoimmunity

Article Title: Autoreactive memory Th17 cells are principally derived from T-bet + RORγt + Th17/1 effectors

doi: 10.1016/j.jaut.2022.102816

Figure Lengend Snippet: (A) IL-17A ΔTbx21 and control (Tbx21 F/F ) mice were exposed to desiccating stress for 14 d and then transfer to normal non-desiccating vivarium. Clinical disease severity was evaluated by corneal fluorescein staining (CFS) scores and representative images at day14 (acute disease) and day 42 (chronic disease) are shown. (B) Kinetic disease scores in each group are summarized as mean ± SEM in linear graphs (n = 44 eyes in IL-17A ΔTbx21 , 36 eyes in Tbx21 F/F group at acute stage, 28 eyes per group at chronic stage, pooled from three independent experiments). (C) Representative flow cytometry plots show frequencies of CD44 hi mTh17 cells in DLNs at day 42 (chronic disease), and summarized as mean ± SEM in the bar graph (n = 14 per group, pooled from 3 independent experiments). *, p <0.05; ***, p <0.001; n.s., not significant.

Article Snippet: Tbx21 F/F mice (Jackson Laboratory) were crossbred in house to generate T-bet conditional knockout IL-17A cre Tbx21 F/F ( IL-17A ΔTbx21 ) mice.

Techniques: Control, Staining, Flow Cytometry

Journal: Journal of autoimmunity

Article Title: Autoreactive memory Th17 cells are principally derived from T-bet + RORγt + Th17/1 effectors

doi: 10.1016/j.jaut.2022.102816

Figure Lengend Snippet: (A) Chronic DED mice (desiccating stress from day 1 to 14, followed by normal non-desiccating environment until day 42) were re-challenged with another short-term desiccating stress (6 days in duration until day 48). Kinetic scores summarized the disease scores immediately before re-challenge and after 6-day re-challenge. (n=20 per group, pooled from three independent experiments). (B) Representative flow cytometry plots show frequencies of Th17 effector cells (CD4 + IL17 + ) in draining lymph nodes (DLN) after re-challenge. (C) The frequencies of effector cells (mean ± SEM) from one representative experiment out of two performed are summarized in the bar graphs (n = 4 per group). (D) Representative flow cytometry plots showing CD4 + T cells infiltration in conjunctiva (CONJ) upon re-challenge. Six eye tissues were pooled together. The frequencies of CD4 + T cells are summarized in the bar graphs (n = 3 per group). (E) CD4 + CD44 hi T cells were isolated from chronic DED mice (day 42) and in vitro stimulated with corneal tissue homogenates. Cells were then evaluated for CD154 expression by flow cytometry, and the culture supernatants were assayed for IL-17 and IFN-γ by ELISA (n = 3–4 per group). (F) mTh17 cells isolated from chronic DED mice were adoptively transferred to normal Rag1 −/− mice which were subsequently exposed to desiccating stress, and the kinetic disease scores are summarized (n = 6 per group). ( G ) Representative flow cytometric plots showing CD4 + T cells infiltration in DLN and the CONJ, and the frequencies of T cells are summarized in the bar graph (n = 3 per group). *, p < 0.05; **, p <0.01, ***, p <0.001.

Article Snippet: Tbx21 F/F mice (Jackson Laboratory) were crossbred in house to generate T-bet conditional knockout IL-17A cre Tbx21 F/F ( IL-17A ΔTbx21 ) mice.

Techniques: Functional Assay, Flow Cytometry, Isolation, In Vitro, Expressing, Enzyme-linked Immunosorbent Assay