Figure 3

The incidence and severity of arthritis in severe combined immunodeficient (SCID) mice. (a) SCID mice (six to eight mice/group) were intraperitoneally injected with 5 × 10⁵ purified CD4⁺CD25⁺ or CD4⁺CD25^- T cells from naïve BALB/c mice, together with 1 × 10⁷ CD25-depleted spleen cells from arthritic animals and 100 μg proteoglycan (PG). The incidence and severity of arthritis was determined. One representative experiment of two is shown. (b) A total of 1 × 10⁷ spleen cells or spleen cells depleted of CD25-expressing cells from arthritic animals, together with 100 μg PG, were transferred intraperitoneally into SCID mice. Disease incidence and severity were determined. (c) Spleen cells from SCID mice that received cell transfer of the CD4⁺CD25⁺ or CD4⁺CD25^- T cells and CD25-depleted spleen cells from arthritic mice were cultured in 96-well plate for 5 days in the presence of 25 μg/ml PG, and PG-specific T-cell proliferation was determined using [³H]thymidine incorporation, and expressed as stimulation index (SI; a ratio of incorporated [³H]thymidine [counts per minute] in antigen-stimulated cultures relative to counts per minute in nonstimulated cultures). PG-specific interferon-γ, IL-10 and IL-4 production by T cells was determined in media collected at day 4 using capture enzyme llinked immunosorbent assays (ELISAs). For induction of transforming growth factor (TGF)-β₁, spleen cells were cultured in serum-free medium X-Vivo-20 (BioWhittaker, Inc.) in the presence of PG. Total TGF-β₁ was measured after acidification to activate latent TGF-β, followed by neutralization using an ELISA kit from Promega. Serum antibody levels were determined by ELISA using mouse IgG₁ and IgG_2a as standards. (d) Purified splenic CD4⁺CD25⁺ T cells from naïve BALB/c mice were cocultured with CD4⁺CD25^- T cells from arthritic mice in the presence of irradiated splenocytes and anti-CD3 for 72 hours or PG for 5 days at 37°C, and T-cell proliferation was determined by [³H]thymidine incorporation. **P < 0.005.