**1. Introduction**

Rheumatoid arthritis (RA) is a chronic autoimmune disease that results in the destruction of cartilage and bone in joints. The murine Collagen-Induced Arthritis (CIA) model has been used to investigate the pathogenesis of RA representatively, because it shares many features with RA (Courtenay, et al., 1980; Luross & Williams, 2001). Susceptibility to arthritis of both CIA and RA is associated with the specific major histocompatibility complex class II allele (Gregersen, et al., 1987; Wooley, et al., 1989). In addition, autoantibodies to type II collagen (CII) have been detected from the synovial fluid of RA patients and it has an aggravating effect in both CIA mice and RA (Clague & Moore, 1984; Cook, et al., 1996; Mullazehi, et al., 2007; Tarkowski, et al., 1989). Finally, pathogenic contributions of CD4+ helper T (Th) cells have been reported in both CIA mice and RA (Weyand & Goronzy, 1999; Weyand, et al., 1998). More recently, the SKG mouse which carries a point mutation of the gene encoding zeta-chain-associated protein kinase 70 (ZAP-70) has been reported as a new model of RA. However, there is little information available about the similarities and differences in the pathogenesis of arthritis among these murine models and RA.

Interleukin-17 (IL-17) is a cytokine secreted by T cells, natural killer (NK) cells, and neutrophils (Ferretti, et al., 2003), and it induces IL-6, IL-8, chemokine, and metalloproteinase production by target cells (Weaver, et al., 2007). Central pathogenic roles of IL-17 in CIA have also been reported recently. For example, systemic or local IL-17 gene transfer aggravated CIA, whereas administration of an IL-17-blocking antibody ameliorated CIA even after the onset of arthritis (Lubberts, et al., 2001; Lubberts, et al., 2004) and IL-17 deficient mice also showed reduced severity of CIA (Nakae, et al., 2003). Furthermore, IL-23 deficient mice, which show impaired Th17 response, do not exhibit CIA because IL-23 is an essential factor for the maintenance of Th17 cells (Murphy, et al., 2003). Consequently, although the pathological contribution of IL-17 to CIA is evident, it remains unclear with RA.

In the present study, we analyzed the phenotypes and cytokine profiles of T cells in the joints of CIA mice kinetically, for the first time, and then analyzed those of SKG mice and RA.

### **2. Most of the IL-17-producing T cells in the swollen joints of CIA were gamma/delta T cells**

To analyze the phenotype and cytokine profile of T cells in the joints of CIA mice, we choose foot pad injection for CII-CFA (complete Freund's adjuvant) instead of tail base injection to

Cytokine Profile of T Cells in the Joints of Rheumatoid Arthritis and Its Murine Models 5

then reached their highest counts with the peak of arthritis. The absolute numbers of IL-17 producing gamma/delta T cells were consistently larger than those of Th17 cells at most time points analyzed. In contrast to the swollen joints, IFN-gamma-producing T cells were detected in the immunized joints after immunization (Fig. 2A and B). For both swollen and immunized joints, the percentages of IL-17-producing gamma/delta T cells among IL-17 producing cells were larger than those in their DLNs (Fig. 2A). In the non-swollen joints, both IL-17-producing T cells and IFN-gamma-producing T cells were rarely observed. In addition, IFN-gamma-producing gamma/delta T cells were a minor population in the joints of CIA (Fig. 2A). These data suggest that CIA is an IL-17-driven arthritis that is produced mainly by gamma/delta T cells, while IFN-gamma-producing T cells are probably

Fig. 2. Distribution and kinetics of IL-17- and IFN-gamma-producing T cells in the CIA joints **A**, Cells were obtained from joints, their DLNs, and the spleens of mice with CIA at the peak of arthritis. IL-17-producing T cells and IFN-gamma-producing T cells were detected by intracellular cytokine staining (top row). IL-17-producing T cells (second row) or IFNgamma-producing T cells (bottom row) were gated and plotted by their expression of gamma/delta TCR and CD4. The absolute number and the percentage of T cells among

dispensable.

distinguish the inflammatory effect of adjuvant itself from real CIA. Each joint was thus designated as immunized joint (CII-CFA injected site), swollen joint (real CIA), or nonswollen joint (Figure 1A). First, the phenotypes and cytokine profiles of T cells in swollen joints of CIA mice were analyzed using intra-cellular cytokine staining by FACS at the peak of CIA (Figure 1B). Surprisingly, most of the IL-17-producing T cells were gamma/delta TCR+ T cells, and did not express CD4, CD8, and DX5. The remainder of the IL-17 producing cells were Th17 cells which express alpha/beta TCR+ (data not shown) and CD4, and neither CD8+ cells nor DX5+ NK cells produced IL-17.

Fig. 1. Phenotypes of IL-17 producing T cells in the swollen joints of CIA

**A**, Schema of analyzed joints and their DLNs of mice with CIA. **B**, Infiltrated cells of swollen joint of CIA mice were obtained by collagenase digestion, and stained with antibodies against CD3, CD4, CD8, DX5, and gamma/delta TCR. IL-17-producing cells were detected by intracellular cytokine staining. The percentage of cells in each region or quadrant is noted.

#### **3. Distribution and kinetics of IL-17- and IFN-gamma-producing T cells in the joints of CIA**

We next analyzed the kinetics of phenotypes and cytokine profiles of infiltrated T cells in the joints of CIA mice at six distinct phases: before immunization (naïve mice), one day after immunization (day 1), before onset (day 10), onset (day 32), peak (day 42), and ankylosing phase (day 70) of arthritis. At each phase, cells were collected from the swollen joint, immunized joint and non-swollen joint, as well as the DLN and spleen (Fig. 1A).

In the swollen joints, absolute numbers of IL-17-producing gamma/delta T cells were larger than those of Th17 cells (CD4+, alpha/beta TCR+ T cells) with the maximal counts at the peak of arthritis (Fig. 2A and B). Surprisingly, neither IFN-gamma-producing CD4+ (Th1) cells nor IFN-gamma-producing gamma/delta T cells were detected at any phase analyzed in the swollen joints. In contrast, IFN-gamma-producing T cells were detected in their DLNs (Fig. 2A). In the immunized joints, IL-17-producing gamma/delta T cells and Th17 cells were already observed on Day 1, reached the first peak on day 10 after immunization, and

distinguish the inflammatory effect of adjuvant itself from real CIA. Each joint was thus designated as immunized joint (CII-CFA injected site), swollen joint (real CIA), or nonswollen joint (Figure 1A). First, the phenotypes and cytokine profiles of T cells in swollen joints of CIA mice were analyzed using intra-cellular cytokine staining by FACS at the peak of CIA (Figure 1B). Surprisingly, most of the IL-17-producing T cells were gamma/delta TCR+ T cells, and did not express CD4, CD8, and DX5. The remainder of the IL-17 producing cells were Th17 cells which express alpha/beta TCR+ (data not shown) and CD4,

and neither CD8+ cells nor DX5+ NK cells produced IL-17.

Fig. 1. Phenotypes of IL-17 producing T cells in the swollen joints of CIA

noted.

**joints of CIA** 

**A**, Schema of analyzed joints and their DLNs of mice with CIA. **B**, Infiltrated cells of swollen joint of CIA mice were obtained by collagenase digestion, and stained with antibodies against CD3, CD4, CD8, DX5, and gamma/delta TCR. IL-17-producing cells were detected by intracellular cytokine staining. The percentage of cells in each region or quadrant is

**3. Distribution and kinetics of IL-17- and IFN-gamma-producing T cells in the** 

We next analyzed the kinetics of phenotypes and cytokine profiles of infiltrated T cells in the joints of CIA mice at six distinct phases: before immunization (naïve mice), one day after immunization (day 1), before onset (day 10), onset (day 32), peak (day 42), and ankylosing phase (day 70) of arthritis. At each phase, cells were collected from the swollen joint,

In the swollen joints, absolute numbers of IL-17-producing gamma/delta T cells were larger than those of Th17 cells (CD4+, alpha/beta TCR+ T cells) with the maximal counts at the peak of arthritis (Fig. 2A and B). Surprisingly, neither IFN-gamma-producing CD4+ (Th1) cells nor IFN-gamma-producing gamma/delta T cells were detected at any phase analyzed in the swollen joints. In contrast, IFN-gamma-producing T cells were detected in their DLNs (Fig. 2A). In the immunized joints, IL-17-producing gamma/delta T cells and Th17 cells were already observed on Day 1, reached the first peak on day 10 after immunization, and

immunized joint and non-swollen joint, as well as the DLN and spleen (Fig. 1A).

then reached their highest counts with the peak of arthritis. The absolute numbers of IL-17 producing gamma/delta T cells were consistently larger than those of Th17 cells at most time points analyzed. In contrast to the swollen joints, IFN-gamma-producing T cells were detected in the immunized joints after immunization (Fig. 2A and B). For both swollen and immunized joints, the percentages of IL-17-producing gamma/delta T cells among IL-17 producing cells were larger than those in their DLNs (Fig. 2A). In the non-swollen joints, both IL-17-producing T cells and IFN-gamma-producing T cells were rarely observed. In addition, IFN-gamma-producing gamma/delta T cells were a minor population in the joints of CIA (Fig. 2A). These data suggest that CIA is an IL-17-driven arthritis that is produced mainly by gamma/delta T cells, while IFN-gamma-producing T cells are probably dispensable.

Fig. 2. Distribution and kinetics of IL-17- and IFN-gamma-producing T cells in the CIA joints

**A**, Cells were obtained from joints, their DLNs, and the spleens of mice with CIA at the peak of arthritis. IL-17-producing T cells and IFN-gamma-producing T cells were detected by intracellular cytokine staining (top row). IL-17-producing T cells (second row) or IFNgamma-producing T cells (bottom row) were gated and plotted by their expression of gamma/delta TCR and CD4. The absolute number and the percentage of T cells among

Cytokine Profile of T Cells in the Joints of Rheumatoid Arthritis and Its Murine Models 7

Fig. 3. IL-23 and IL-1 beta efficiently stimulate IL-17-producing gamma/delta t cells without

**5. IL-17-producing gamma/delta T cells were maintained by IL-23 but not by** 

Since IL-23 plays important roles in the maintenance of Th17 cells (Bettelli, et al., 2006; Harrington, et al., 2005; Infante-Duarte, et al., 2000; Mangan, et al., 2006; Park, et al., 2005; Veldhoen, et al., 2006), we next analyzed the maintaining effect of IL-23 or CII on IL-17 producing gamma/delta T cells. To test this, cells from DLNs of swollen joints of CIA mice were cultured with IL-23, CII, or medium alone (Fig. 4A). Both IL-17-producing gamma/delta T cells and Th17 cells were maintained by the presence of IL-23. However, IL-17-producing gamma/delta T cells were not maintained by the presence of CII, and Th17 cells showed CII-dependency. To further investigate the factors that enhance the accumulation of IL-17-producing gamma/delta T cells in the inflamed joints, the numbers of IL-17-producing gamma/delta T cells were counted in the joints of differently-immunized mice on day 10. Mice were immunized with PBS, IFA+solution (0.05 mM of acetic acid for CII solvent), IFA+CII, or CFA+CII (Fig. 4B). The numbers of IL-17-producing gamma/delta T cells were not significantly different between mice immunized with IFA+solution, IFA+CII, or CFA+CII. In contrast, the numbers of IL-17-producing gamma/delta T cells were significantly smaller in mice immunized with PBS than in the other three conditions, while the numbers of Th17 cells were significantly larger in mice immunized with IFA+CII than those in mice treated with IFA+solution. These data suggested that IL-17-producing

TCR stimulation

**CII in vitro and induced by IFA in vivo** 

them are indicated in the joint panels. In panels of DLNs and spleens, the percentages of cells in each quadrant are noted. **B**, Cells were recovered from swollen joints, immunized joints, and non-swollen joints of CIA mice at the six distinct phases of arthritis. IL-17 producing T cells and IFN-gamma-producing T cells were detected by intracellular cytokine staining, and their absolute numbers were counted (open circle; gamma/delta T cell, cross; CD4+ T cells).
