CD4+CD25+ immunoregulatory T cells may not be involved in controlling autoimmune arthritis

Abstract

Accumulating evidence suggests that regulatory T cells play a crucial role in preventing autoimmunity. Recently, a naturally occurring CD4+CD25+ T-cell subset that is anergic and also suppressive has been shown to suppress autoimmunity in several animal models. We used proteoglycan-induced arthritis (PGIA) as a study model to investigate the role of the CD4+CD25+ regulatory T cells in autoimmune arthritis. There was no significant change in the percentage of CD4+CD25+ T cells during the immunization period when proteoglycan- or ovalbumin-immunized BALB/c and C57BL/6 mice were compared. An adoptive transfer study showed that the CD4+CD25+ T cells did not protect severe combined immunodeficient mice from arthritis when they were cotransferred with splenocytes from arthritic animals. Similarly, depletion of the CD4+CD25+ T cells did not enhance the onset of the disease or disease severity in severe combined immunodeficient mice. Moreover, CD28-deficient mice, which have very few CD4+CD25+ T cells, were highly resistant to PGIA. These findings indicate that the CD4+CD25+ regulatory T cells may not play a critical role in controlling PGIA.

Figure 1

Incidence and severity of arthritis in BALB/c mice. BALB/c and C57BL/6 mice (15 mice/group) were immunized with proteoglycan or ovalbumin in complete Freund's adjuvant (day 0), and boosted with proteoglycan or ovalbumin in incomplete Freund's adjuvant at days 21 and 42. The incidence and severity of arthritis was determined.

Figure 2

The number of CD4⁺CD25⁺-expressing regulatory T cells from BALB/c and C57BL/6 mice during immunization. (a) BALB/c and C57BL/6 mice, immunized with proteoglycan (PG) or ovalbumin (OVA), were killed at 14 days after each immunization, and levels of expression of the CD4⁺CD25⁺ T cells were determined by flow cytometry. *The CD4⁺CD25⁺ T cells from naïve BALB/c and C57BL/6 mice were also determined. (b) Cytotoxic T-lymphocyte antigen (CTLA)-4 expression on the CD4⁺CD25⁺ T cells from PG- or OVA-immunized animals (after the 3rd injection) was determined.

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.

Figure 4

Resistance to proteoglycan-induced arthritis (PGIA) in CD28-deficient mice. (a) The expression of CD4⁺CD25⁺ T cells in naïve wild-type (WT) and CD28-deficient BALB/c mice was determined. (b) Wild-type and CD28-deficient BALB/c mice were immunized with proteoglycan (PG) in complete Freund's adjuvant, and boosted in incomplete Freund's adjuvant at days 21 and day 42. The mice developed arthritis after the third immunization. The incidence of arthritis in WT and CD28-deficient BALB/c mice was monitored. (c) CD4⁺ T cells (1 × 10⁶/ml) from PG-immunized WT and CD28-deficient mice were cultured for 5 days with antigen-presenting cells (2500 rad-irradiated syngenic splenocytes, 2 × 10⁶/ml) in the presence of proteoglycan (10, 25 and 50 μg/ml). The cell proliferation was determined by [³H]thymidine incorporation.