CD4+CD25+ regulatory T cells in autoimmune arthritis

Abstract

CD4(+)CD25(+) regulatory T (Treg) cells can play a critical role in the prevention of autoimmunity, as evidenced by the cataclysmic autoimmune disease that develops in mice and humans lacking the key transcription factor forkhead box protein 3 (Foxp3). At present, however, how and whether Treg cells participate in the development of rheumatoid arthritis (RA), which has both systemic manifestations and a joint-targeted pathology that characterizes the disease, remains unclear. In this review, we describe work that has been carried out aimed at determining the role of Treg cells in disease development in RA patients and in mouse models of inflammatory arthritis. We also describe studies in a new model of spontaneous autoimmune arthritis (TS1 x HACII mice), in which disease is caused by CD4(+) T cells recognizing a neo-self-antigen expressed by systemically distributed antigen-presenting cells. We show that TS1 x HACII mice develop arthritis despite the presence of CD4(+)CD25(+)Foxp3(+) Treg cells that recognize this target autoantigen, and we outline steps in the development of arthritis at which Treg cells might potentially act, or fail to act, in the development of inflammatory arthritis.

Fig. 1. HA is synthesized and presented by APCs in TS1×HACII mice

(A) Cell-surface expression of PR8 HA by B220⁺ and CD11c⁺ cells from BALB/c, HA104, and HACII mice analyzed directly ex vivo or following in vitro activation (anti-IgM for B cells, 20 h on plastic for DCs). (B) CFSE-labeled CD4⁺ T cells from TS1 mice were transferred into BALB/c, HA104, or HACII mice, and after 48 h, LN cells were isolated and analyzed by flow cytometry. Left panels show staining with 6.5 versus CFSE on CD4⁺ T cells, right panels show expression of MHC class II or CD86 on B220⁺ cells.

Fig. 2. TS1×HACII mice develop spontaneous autoimmune inflammatory arthritis

(A) Pictures show 20-week-old TS1 and TS1×HACII littermates, with panels showing front and rear paws. (B) Graph shows ankle widths of TS1 and TS1×HACII mice at different ages, in weeks. **P≤0.01. (C) Hematoxylin and eosin (H&E) stained knee sections from 23-week-old TS1 and TS1×HACII littermates; b, bone; c, cartilage; s, synovium; p, pannus; arrowhead, bone erosion.

Fig. 3. B cells are not required for arthritis development in TS1×HACII mice

(A) Histograms show CD86 and MHC class II expression on B220⁺ splenocytes from TS1 (grey lines), TS1×HA104 (dashed lines), and TS1×HACII (black lines) mice. (B) Bar graphs show the numbers of B220⁺ splenocytes and serum IgG with circles representing individual mice. Bars indicate the averages for TS1 (white bars), TS1×HA104 (light grey bars), and TS1xHACII (dark grey bars) mice. (C) Graph indicates the largest ankle widths from individual TS1.JH^−/− (open bars) and TS1×HACII.JH^−/− (filled bars) mice. The mean ankle width of TS1×HACII.JH^−/− mice is significantly greater than that of TS1.JH^−/− mice (P<0.01, Student's t-test). Mice used in this analysis ranged in ages from 6 to 23 weeks. Picture shows H&E stained knee section from an arthritic TS1×HACII.JH^−/− mouse. p, pannus; arrowhead, bone erosion.

Fig. 4. Recognition of HA by 6.5⁺CD4⁺ T cells drives arthritis in TS1×HACII mice

(A) Graph indicates the largest ankle widths from individual 9 to 13 week-old TS1.RAG^−/− (open bars) and TS1×HACII.RAG^−/− (filled bars) mice. The mean ankle width of TS1×HACII.RAG^−/− mice is significantly greater than that of TS1.RAG^−/− mice (P<0.01). (B) Picture shows H&E stained wrist section from an arthritic TS1xHACII.RAG^−/− mouse. p, pannus.

Fig. 5. Development of 6.5⁺CD4⁺ T cells in TS1×HACII mice

(A) 6.5 expression and frequencies of thymocytes and 6.5⁺CD4SP thymocytes from 4 and >12 wk-old TS1 (light lines, open bars), TS1×HA104 (dashed lines, grey bars) and TS1×HACII (dark lines, closed bars) mice. (B) 6.5 expression and frequencies of splenocytes and 6.5⁺CD4⁺ splenocytes from 4 and >12 wk-old TS1 mice. TS1 (light lines, open bars), TS1×HA104 (dashed lines, grey bars), and TS1×HACII (dark lines, closed bars). (C) Histograms show 6.5 expression on CD4⁺ splenocytes from TS1 (grey line) and TS1.RAG^−/− (black line) mice (upper panel), and from TS1×HACII (grey line), TS1×HACII.RAG^−/− (black line), and TS1.RAG^−/− (dashed line) mice (lower panel).

Fig. 6. Autoreactive CD4⁺ T cells in TS1×HACII mice are activated and produce cytokines

(A) Expression of 6.5 versus CFSE on purified CD4⁺ T cells from TS1, TS1×HA104, and TS1×HACII mice following 72 h of culture with or without S1 peptide and/or IL-2. (B) Amounts of indicated cytokines in culture supernatants obtained following incubation of unfractionated LN cells from individual TS1 (open bars), TS1×HA104 (grey bars), and TS1×HACII (filled bars) mice for 3 days in the absence of exogenous peptide or cytokines. Bars indicate means. (C) Dot plots show intracellular cytokine staining of CD4⁺ (upper panels) and 6.5⁺CD4⁺ (lower panels) lymph node cells from TS1×HACII mice following stimulation immediately ex vivo either with PMA and ionomycin for 4 h (for IL-17, IFN-γ, IL-10 and IL-4) or with 3 μM S1 peptide for 4 (for IL-6 and TNF-α). Percentages of cells in indicated gates are shown.

Fig. 7. TS1×HACII mice contain CD4⁺CD25⁺Foxp3⁺ Treg cells, including a population that recognizes the target autoantigen

(A) Lymph node cells from TS1×HACII and TS1 mice were stained with CD4, CD25, and Foxp3. (B) Lymph node cells from a TS1×HACII mouse were stained with 6.5 to identify HA-specific cells, CD4, CD25, and Foxp3. (C) Lymph node cells from five, eight, and 12-week-old TS1×HACII mice were stained with CD4, CD25, and Foxp3. (D) Lymph node cells from TS1×HACII and BALB/c mice were stained with CD4, CD25, Foxp3, GITR, and CD103. A 6.5 stain was also included for TS1×HACII mice.

Fig. 8. CD4⁺CD25⁺ Treg cells from TS1×HACII mice suppress responder CD4⁺ T cells in vitro

(A) CFSE-labeled MACS-purified CD4⁺ T cells from TS1 mice were cultured with 0.1 μg/ml anti-CD3 and irradiated BALB/c splenocytes in the presence or absence of FACS-purified CD4⁺CD25⁺ cells from TS1×HACII mice. Cells were cultured at a Treg:responder cell ratio of 1:2. Responder CD4⁺ T-cell proliferation was analyzed via flow cytometry at day three of culture. (B) 6.5⁺CD4⁺CD25⁺ T cells were FACS-purified from TS1 (black circles) or TS1×HACII (gray circles) mice. Graded numbers of Treg cells were cultured with lymph nodes cells from TS1 mice and S1 peptide. Proliferation was assessed at day three of culture by [³H]-thymidine incorporation. (C) MACS-purified CD4⁺ T cells from TS1 mice were cultured with 0.1 μg/ml anti-CD3 and irradiated BALB/c splenocytes in the presence or absence of FACS-purified CD4⁺CD25⁺ cells from TS1×HACII mice. At day four of culture, IFN-γ production by the responder CD4⁺ T cells was determined by intracellular cytokine staining.

Fig. 9. Steps where regulatory T-cell activity may shape arthritis development in TS1×HACII mice

Step 1, Deletion/Treg cell formation among autoreactive 6.5⁺CD4⁺ thymocytes. Step 2, Lymphopenia-induced proliferation of 6.5⁺CD4⁺ T cells secondary to severe thymocyte deletion. Step 3, reciprocal activation of HA-specific CD4⁺ T cells by HA-expressing antigen presenting cells. Step 4, activation of B cells by HA-specific CD4⁺ T cells. Step 5, spontaneous differentiation and cytokine production by autoreactive HA-specific CD4⁺ T cells. Step 6, development of a regional immune response in the lymph nodes that drain the major joints.