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

High-density cartilage proteoglycan (aggrecan) was purified from human cartilage, by CsCl gradient centrifugation, and depleted of glycosaminoglycan side chains as described previously [21]. Female BALB/c mice (National Cancer Institute, Friedrich, MD, USA, or The Jackson Laboratory, Bar Harbor, ME, USA) and CD28-deficient mice (The Jackson Laboratory) were immunized intraperitoneally with cartilage proteoglycan (100 μg protein) in complete Freund's adjuvant on day 0, and boosted with proteoglycan in incomplete Freund's adjuvant on days 21 and 42. Female SCID mice on a BALB/c background (NCI/NCrC.B-17-scid/scid), aged 8–12 weeks or young retired breeders, were purchased from the National Cancer Institute and maintained under germ-free conditions.

A standard scoring system, based on swelling and redness of each paw, was used for the assessment of the severity of disease. The first clinical symptom of swelling was recorded as the time of onset of arthritis. Joint swelling was scored (ranging from 0 to 4 in each paw) and expressed as acute cumulative arthritis score, resulting in a possible maximum severity score of 16. Typically, in the primary form of PGIA, BALB/c mice developed swelling and redness in one or more limbs 7–14 days after the third injection with proteoglycan and adjuvant. In the transfer system, recipient SCID mice developed a more uniform disease from day 10 after transfer, with involvement of nearly all peripheral joints.

The CD4⁺CD25⁺-expressing T cells were identified by staining spleen cells with fluorescein isothiocyanate (FITC)-labeled anti-CD4 and biotin-labeled anti-CD25 followed by CyChrome-labeled streptavidin (BD PharMingen, San Diego, CA, USA), and analyzed on a flow cytometer (Beckton Dickinson, San Jose, CA, USA). For analysis of intracellular CTLA-4, spleen cells were first stained with the above fluorescence-labeled antibodies, fixed and rendered permeable, and stained with R-phycoerythrin (PE)-labeled anti-CTLA-4 using a Cytofix/Cytoperm kit (BD PharMingen).

The CD4⁺CD25⁺ T cells were purified using a protocol described previously [6]. Briefly, CD4⁺ T cells were isolated from spleens of 10- to 12-week-old, naïve, BALB/c mice by negative selection using CD4-enrichment columns (R&D System, Minneapolis, MN, USA). Purified CD4⁺ T cells were incubated with PE-labeled anti-CD25, and the stained cells were incubated with anti-PE microbeads (Miltenyi Biotech Inc., Auburn, CA, USA). The CD25⁺ cells were selected on an LS⁺ column (Miltenyi Biotech Inc.). The purity of the CD4⁺CD25⁺ T cells was approximately 90%. The CD4⁺CD25^- T cells, which did not bind to magnetic beads, were collected from the flow through the washing steps (purity >98%). A total of 5 × 10⁵ CD4⁺CD25⁺ or CD4⁺CD25^- T cells were injected intraperitoneally, along with 1 × 10⁷ spleen cells from arthritic animals that were depleted of CD25⁺ cells and 100 μg proteoglycan, into SCID mice.

Spleen cells from arthritic animals were depleted of CD25⁺ cells using negative selection as described above (0.5 μg PE-labeled anti-CD25 per 1 × 10⁶ cells). The depletion of CD4⁺CD25⁺ cells was detected by flow cytometry. Typically, less than 0.3% of the depleted cells expressed CD25. a total of 1 × 10⁷ CD25⁺ and CD25^- spleen cells, together with 100 μg proteoglycan, were injected intraperitoneally into SCID mice.

CD4⁺CD25⁺ T cells (1 × 10⁴) isolated from naïve BALB/c mice were mixed with CD4⁺CD25^- T cells (5 × 10⁴) from arthritic mice in the presence of irradiated syngenic splenocytes as antigen-presenting cells (7.5 × 10⁴) with 1 μg/ml soluble anti-CD3 for 3 days or 50 μg/ml proteoglycan for 5 days, and T-cell proliferation was determined by [³H]thymidine incorporation.

Antigen-specific T-cell responses were measured in quadriplicate samples of spleen cells (3 × 10⁵ cells/well) cultured in the presence of 50 μg proteoglycan protein/ml. T-cell proliferation was assessed on day 5 by [³H]thymidine incorporation [14, 22]. Antigen (proteoglycan)-specific interferon-γ, IL-10, and IL-4 production by T cells was determined in media collected on day 4 using capture enzyme-linked immunosorbent assays (ELISAs) from BioSource International, Inc. (Camarillo, CA, USA). For induction of TGF-β₁, spleen cells were cultured in serum-free medium X-Vivo-20 (BioWhittaker, Inc., Walkersville, MD, USA) in the presence of proteoglycan. Total TGF-β₁ was measured after acidification to activate latent TGF-β, followed by neutralization using an ELISA kit from Promega (Madison, WI, USA).

Maxisorp immunoplates (Nunc, Rochester, NY, USA) were coated with human or mouse cartilage proteoglycans (0.1 μg protein/100 μl per well) for ELISA, and free binding capacity of the wells was blocked by 1% fat-free milk in phosphate-buffered saline [23‐25]. Sera were applied at increasing dilutions and isotypes of proteoglycan-specific antibodies were determined using peroxidase-conjugated rat antimouse IgG₁ or IgG_2a secondary antibodies (Zymed, South San Francisco, CA, USA) as previously described [24, 26]. Serum antibody levels were calculated according to mouse IgG₁ and IgG_2a standards. Mouse IgG₁ and IgG_2a standards were purified by sepharose-coupled protein G from irrelevant (non-proteoglycan-specific) monoclonal antibody-containing ascites fluids and coated directly onto the microplate's surface.

To elucidate the role of these regulatory T cells in autoimmune arthritis, we first followed up the CD4⁺CD25⁺-expressing regulatory T cells in mice with PGIA during the entire course of immunization. To exclude the possibility that different CD4⁺CD25⁺-expressing T cells may be a strain- and/or antigen-dependent phenomenon, we immunized BALB/c mice (PGIA-susceptible strain) and C57BL/6 (arthritis-resistant strain) with human cartilage proteoglycan or ovalbumin. As shown in Fig. 1, BALB/c mice immunized with human cartilage proteoglycan started to develop arthritis after the third injection and the incidence of arthritis reached 100% at day 72 following the third immunization, whereas BALB/c mice immunized with ovalbumin or C57BL/6 mice immunized with proteoglycan were completely resistant to PGIA. The CD4⁺CD25⁺-expressing T cells detected 14 days after each immunization from each experimental group were similar during the entire course of immunization (Fig. 2a). This observation suggests that the CD4⁺CD25⁺ regulatory T cells might not be involved in the development of PGIA. Although it has been shown that the CD4⁺CD25⁺ regulatory T cells can suppress the development of autoimmunity, a recent report [27] demonstrated that depletion of the CD4⁺CD25⁺ T cells is necessary but not sufficient for induction of autoimmune gastritis.

It has been shown that the CD4⁺CD25⁺ regulatory T cells express CTLA-4 [6‐8], which is a negative regulator of T-cell activation. The suppressive activity of the CD4⁺CD25⁺ regulatory T cells may in part be ascribed to signaling through CTLA-4. CTLA-4 may transduce an activating signal to CD4⁺CD25⁺ regulatory T cells [2]. Although we did not detect any significant decrease in numbers of the CD4⁺CD25⁺ regulatory T cells between proteoglycan-immunized BALB/c and C57BL/6 mice, this might be due to reduced expression of CTLA-4 in the CD4⁺CD25⁺ regulatory T cells from proteoglycan-immunized BALB/c mice. To test this possibility, we then detected CTLA-4 expression on the CD4⁺CD25⁺ regulatory T cells using intracellular staining. There was no significant difference between levels of CTLA-4 expression in the CD4⁺CD25⁺ regulatory T cells from each group (Fig. 2b). This finding excludes the possibility that reduced expression of CTLA-4 in the CD4⁺CD25⁺ regulatory T cells from BALB/c mice with PGIA may mediate the induction of the disease.

To further investigate whether the CD4⁺CD25⁺ T cells play a role in PGIA, we performed adoptive transfer experiments using SCID mice as recipients. Purified CD4⁺CD25⁺ regulatory T cells (3–20 × 10⁵) from naïve BALB/c mice, together with 10⁷ spleen cells from arthritic mice that were depleted of CD25⁺ T cells and 100 μg proteoglycan, were injected intraperitoneally into SCID mice [18], and the incidence and severity of disease were monitored. Note that we used different ratios of the CD4⁺CD25⁺ regulatory T cells to the effector cells, and we did not observe any suppression of arthritis development. As a control, the same number of CD4⁺CD25^- T cells, together with arthritogenic spleen cells that were depleted of CD25⁺ cells, and proteoglycan, were also adoptively transferred into SCID mice. The transfers of CD4⁺CD25⁺ T cells did not have any impact on incidence and severity of the disease in SCID mice as compared with transfer of CD4⁺CD25^- T cells (Fig. 3a), suggesting that the CD4⁺CD25⁺ regulatory T cells did not protect SCID mice from arthritis. To further verify these results, a depletion experiment was conducted. Spleen cells from arthritic animals, either depleted of CD25⁺ cells or without depletion, were injected into SCID mice together with proteoglycan. The incidence and severity of arthritis in SCID mice receiving spleen cells containing CD25⁺ cells or spleen cells depleted of CD25⁺ cells were identical (Fig. 3b). Proteoglycan-specific T-cell proliferation and production of IL-4, interferon-γ, IL-10, TGF-β₁ and antiproteoglycan autoantibodies were comparable in both transfer groups (Fig. 3c). The inability of the CD4⁺CD25⁺ regulatory T cells to suppress PGIA was not due to loss of their suppression activity, because in vitro the CD4⁺CD25⁺ T cells could effectively inhibit the proliferation of the CD4⁺CD25^- T cells (Fig. 3d, left panel). Interestingly, proteoglycan-induced proliferation of CD4⁺CD25^- T cells could not be inhibited by the CD4⁺CD25⁺ T cells (Fig. 3d, right panel), which is consistent with a recent report that the effective function of the CD4⁺CD25⁺ regulatory T cells is T-cell receptor (TCR) specific [28]. Collectively, our data suggest that the CD4⁺CD25⁺ regulatory T cells may not be involved in the development of PGIA.

It has been shown that CD28–B7 interaction is essential for the homeostasis of the CD4⁺CD25⁺ regulatory T cells that control autoimmune diabetes [6]. Consistent with that report, the expression of CD4⁺CD25⁺ regulatory T cells was found to be significantly reduced as compared with that in wild-type BALB/c mice (Fig. 4a). To investigate whether a deficiency in the CD4⁺CD25⁺ regulatory T cells may lead to an increase in the development of PGIA, we immunized wild-type and CD28-deficient BALB/c mice with proteoglycan, and disease incidence was monitored. In contrast to the report in autoimmune diabetes [6], CD28-deficient BALB/c mice were highly resistant to induction of arthritis, albeit at a low percentage of the CD4⁺CD25⁺ regulatory T cells observed in these mice (Fig. 4b). Note that T cells from proteoglycan-immunized CD28-deficient mice proliferate in response to proteoglycan stimulation, albeit at a rate lower than that in wild-type T cells (Fig. 4c), suggesting that CD28-deficient T cells can be effectively primed.