Journal: Frontiers in Immunology

Article Title: T cells in ARAP-deficient mice present defective T cell receptor signaling and reduced severity in an experimentally-induced autoimmune disease

doi: 10.3389/fimmu.2025.1556616

Figure Lengend Snippet: Reduced severity and incidence of EAE in ARAP-deficient mice. (A, B) WT (+/+) and ARAP-deficient (-/-) mice were immunized with MOG peptide 35–55 (200 μg) emulsified with complete Freund’s adjuvant, as described in the Materials and Methods. The severity of EAE (A) is presented as mean clinical scores. Data are pooled from four independent experiments and presented as the mean ± SEM (n = 16). Statistical significance was analyzed using an unpaired t -test. ** p < 0.01, *** p < 0.001. The incidence of EAE (B) is shown by the percentage of mice that developed the disease. (C, D) Anti-IgM, anti-IgG, and anti-MOG-specific Abs in sera were collected on day 15 after an ELISA-measured immunization. The data are the mean ± SEM of four independent experiments performed in triplicate. Unpaired t -tests analyzed statistical significance. * p < 0.05, ** p < 0.01, *** p < 0.001. (E) ELISA was used to measure the IFN-γ and IL-17 cytokine levels in sera collected on day 15 after immunization. The data are the mean ± SEM of four independent experiments performed in triplicate. Unpaired t -tests analyzed statistical significance. ** p < 0.01. (F–K) CD4 + T cells were isolated from the spleen (F–H) or draining lymph nodes (I–K) of WT (+/+) and ARAP-deficient (-/-) mice 15–18 days after immunization. Cells were stained for intracellular cytokines and surface proteins using various fluorochrome-conjugated Ab combinations and analyzed by flow cytometry, as shown in Figure 2 (F, I) . The percentages of IFN-γ<συπ>+ T cells (Q1), IL17 + T cells (Q4), and IFN-γ<συπ>+ IL-17 + double-positive T cells (Q2) are presented as the mean ± SEM from three independent experiments for spleen cells (G) and four independent experiments for draining lymph node cells (J) . Statistical significance was determined using unpaired t -tests ( ** p < 0.01, * p < 0.05). The absolute cell numbers in Q1, Q2, and Q4 were obtained from a total of 10,000 cells analyzed by flow cytometry. The absolute cell counts are presented as the mean ± SEM from three independent experiments for spleen cells (H) and four independent experiments for draining lymph node cells (K) . Statistical significance was determined using unpaired t -tests.

Article Snippet: Human fibronectin, GolgiStopTM (monensin), Cytofix/CytopermTM reagent from BD Biosciences (San Jose, CA, USA), ICAM-1-Fc chimera from R&D Systems Inc. (Minneapolis, MN, USA), magnetic particle-labeled streptavidin, pan- or CD4 + -T cell isolation kit from Miltenyi Biotech Inc. (Auburn, CA, USA), and mouse IL-2, IFN-γ, IL-17 Ready-SET-Go ELISA sets from eBioscience were also used.

Techniques: Adjuvant, Enzyme-linked Immunosorbent Assay, Isolation, Staining, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: T cells in ARAP-deficient mice present defective T cell receptor signaling and reduced severity in an experimentally-induced autoimmune disease

doi: 10.3389/fimmu.2025.1556616

Figure Lengend Snippet: Severity and incidence of EAE in Rag1-deficient mice adoptively transferred with lymphocytes. Rag1-deficient mice were reconstituted with T cells from WT (+/+) or ARAP-deficient (-/-) mice and B cells from WT mice. EAE was induced in these mice, as in Figure 5 . (A) The clinical scores are pooled from three independent experiments (mean ± SEM, n = 13). Unpaired t -tests were used to determine statistical significance. ** p < 0.01, *** p < 0.001. (B) The incidence of EAE is shown by the percentage of mice that developed the disease in a group. (C, D) ELISA was used to measure the anti-IgM, anti-IgG, anti-MOG-specific Ab levels in sera collected on day 15 after immunization. The data are presented as the mean ± SEM of three independent experiments performed in triplicate. Unpaired t -tests analyzed statistical significance. * p < 0.05. (E) ELISA was used to measure the IFN-γ and IL-17 cytokine levels in sera collected on day 15 after immunization. The data are the mean ± SEM of three independent experiments performed in triplicate. Unpaired t -tests analyzed statistical significance. * p < 0.05. (F–K) CD4 + T cells were isolated from the spleen (F–H) or draining lymph nodes (I–K) of Rag1-deficient mice reconstituted with T cells from WT (+/+) or ARAP-deficient (-/-) mice and B cells from WT mice 15–18 days after immunization. Cytokine-secreting cells were analyzed as shown in Figure 5 . The percentages and absolute counts of cytokine-secreting cells are presented as the mean ± SEM from three independent experiments. Statistical significance was determined using unpaired t -tests.

Article Snippet: Human fibronectin, GolgiStopTM (monensin), Cytofix/CytopermTM reagent from BD Biosciences (San Jose, CA, USA), ICAM-1-Fc chimera from R&D Systems Inc. (Minneapolis, MN, USA), magnetic particle-labeled streptavidin, pan- or CD4 + -T cell isolation kit from Miltenyi Biotech Inc. (Auburn, CA, USA), and mouse IL-2, IFN-γ, IL-17 Ready-SET-Go ELISA sets from eBioscience were also used.

Techniques: Enzyme-linked Immunosorbent Assay, Isolation