**4. IL-23 and IL-1 beta efficiently stimulate IL-17 production by gamma/delta T cells**

It is known that a subset of gamma/delta T cells already differentiate to acquire the IL-17 producing function in the thymus (Jensen, et al., 2008), and CCR6 is suggested as a specific marker of Th17 cells (Acosta-Rodriguez, et al., 2007; Annunziato, et al., 2007; Hirota, et al., 2007; Singh, et al., 2008). Therefore, the expression of CCR6 on IL-17 producing gamma/delta T cells in the thymus of naïve DBA1/J mice was evaluated. IL-17-producing, but not IFN-gamma-producing gamma/delta T cells preferentially expressed CCR6 (Fig. 3A). As it has also been reported that small numbers of gamma/delta T cells are present in the normal joints of mice (Arai, et al., 1996), we next examined whether de novo CCR6+ IL-17-producing gamma/delta T cells are present in the normal joints of naïve DBA1/J mice. Cells were collected from the normal joints of naïve mice and intracellular cytokine staining was performed. By analyzing cells from two normal paws and ankles at a time, CCR6+ IL-17-producing gamma/delta T cells could be detected (data not shown). In addition, in CIA mice, 92% of CCR6+ gamma/delta T cells produced IL-17 (Fig. 3B). Next, the signal requirements of IL-17-producing gamma/delta T cells were analyzed. Gamma/delta T cells from naïve DBA1/J mice were stimulated with cytokines in the presence or absence of anti-gamma/delta TCR activating mAb (Fig. 3C). Although small numbers of IL-17-producing gamma/delta T cells were detected by stimulations with anti- gamma/delta TCR mAb, IL-23, or IL-1 beta alone, IL-23 plus IL-1 beta induced IL-17-producing gamma/delta T cells quite efficiently without anti- gamma/delta TCR stimulation. These observations indicated that TCR signaling was not necessary to stimulate IL-17-producing gamma/delta T cells. Furthermore, a combination of IL-23 and IL-1 beta was a much more potent stimulator than TCR signaling. Similar results were obtained with gamma/delta T cells sorted from DLNs of swollen joints at the peak of CIA (Fig. 3C, right panel).

**A**, Thymocytes from naïve mice were stimulated with PMA and ionomycin for 4 h. Gamma/delta TCR+ cells were gated and CCR6+ cells among IL-17-producing or IFN-gamma-producing gamma/delta T cells were detected. The percentages of cells in each quadrant are indicated. **B**, Cells were collected from DLNs of swollen joints of CIA mice. IL-17-producing cells were detected by intracellular cytokine staining. CCR6+ cells were gated and IL-17-producing cells were analyzed. The percentage of IL-17-producing cells among CCR6+ gamma/delta T cells is noted. **C**, Gamma/delta T cells were sorted from peripheral lymph nodes of naïve DBA1/J mice (upper panel) or DLNs of swollen joints of CIA mice at the peak of arthritis (lower panel) and stimulated with cytokines, activating anti-gamma/delta TCR antibodies, and anti-CD28 antibodies for 24 h. By intracellular cytokine staining, the percentages of IL-17-producing cells among gamma/delta T cells were determined. Mean ± SEM is shown from three different mice.

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;

**4. IL-23 and IL-1 beta efficiently stimulate IL-17 production by gamma/delta T** 

It is known that a subset of gamma/delta T cells already differentiate to acquire the IL-17 producing function in the thymus (Jensen, et al., 2008), and CCR6 is suggested as a specific marker of Th17 cells (Acosta-Rodriguez, et al., 2007; Annunziato, et al., 2007; Hirota, et al., 2007; Singh, et al., 2008). Therefore, the expression of CCR6 on IL-17 producing gamma/delta T cells in the thymus of naïve DBA1/J mice was evaluated. IL-17-producing, but not IFN-gamma-producing gamma/delta T cells preferentially expressed CCR6 (Fig. 3A). As it has also been reported that small numbers of gamma/delta T cells are present in the normal joints of mice (Arai, et al., 1996), we next examined whether de novo CCR6+ IL-17-producing gamma/delta T cells are present in the normal joints of naïve DBA1/J mice. Cells were collected from the normal joints of naïve mice and intracellular cytokine staining was performed. By analyzing cells from two normal paws and ankles at a time, CCR6+ IL-17-producing gamma/delta T cells could be detected (data not shown). In addition, in CIA mice, 92% of CCR6+ gamma/delta T cells produced IL-17 (Fig. 3B). Next, the signal requirements of IL-17-producing gamma/delta T cells were analyzed. Gamma/delta T cells from naïve DBA1/J mice were stimulated with cytokines in the presence or absence of anti-gamma/delta TCR activating mAb (Fig. 3C). Although small numbers of IL-17-producing gamma/delta T cells were detected by stimulations with anti- gamma/delta TCR mAb, IL-23, or IL-1 beta alone, IL-23 plus IL-1 beta induced IL-17-producing gamma/delta T cells quite efficiently without anti- gamma/delta TCR stimulation. These observations indicated that TCR signaling was not necessary to stimulate IL-17-producing gamma/delta T cells. Furthermore, a combination of IL-23 and IL-1 beta was a much more potent stimulator than TCR signaling. Similar results were obtained with gamma/delta T cells sorted from DLNs of

**A**, Thymocytes from naïve mice were stimulated with PMA and ionomycin for 4 h. Gamma/delta TCR+ cells were gated and CCR6+ cells among IL-17-producing or IFN-gamma-producing gamma/delta T cells were detected. The percentages of cells in each quadrant are indicated. **B**, Cells were collected from DLNs of swollen joints of CIA mice. IL-17-producing cells were detected by intracellular cytokine staining. CCR6+ cells were gated and IL-17-producing cells were analyzed. The percentage of IL-17-producing cells among CCR6+ gamma/delta T cells is noted. **C**, Gamma/delta T cells were sorted from peripheral lymph nodes of naïve DBA1/J mice (upper panel) or DLNs of swollen joints of CIA mice at the peak of arthritis (lower panel) and stimulated with cytokines, activating anti-gamma/delta TCR antibodies, and anti-CD28 antibodies for 24 h. By intracellular cytokine staining, the percentages of IL-17-producing cells among gamma/delta T cells

swollen joints at the peak of CIA (Fig. 3C, right panel).

were determined. Mean ± SEM is shown from three different mice.

CD4+ T cells).

**cells** 

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

#### **5. IL-17-producing gamma/delta T cells were maintained by IL-23 but not by CII in vitro and induced by IFA in vivo**

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

Cytokine Profile of T Cells in the Joints of Rheumatoid Arthritis and Its Murine Models 9

two weeks before. For naïve mice, CCR6+ gamma/delta T cells, naïve CD4+ T cells, or PBS alone were injected. Mean ± SEM of the arthritis score of affected joints is shown. \* = *P* < 0.05.

To elucidate the pathological differences from other murine arthritis models, the same analysis was performed using SKG mice (Sakaguchi, et al., 2003). SKG mice carry a point mutation of the gene encoding zeta-chain-associated protein kinase 70 (ZAP-70), and homozygous mice show IL-17-dependent arthritis resembling RA. Although the present study could detect only a few IL-17-producing gamma/delta T cells in the DLNs of swollen joints, surprisingly almost all of the IL-17-producing cells were Th17 cells and the numbers of IL-17-producing gamma/delta T cells were negligible in the swollen joints of SKG mice (Fig. 5A). As SKG is a BALB/c-background strain, and SKG arthritis is induced by zymosan as an adjuvant (Sakaguchi, et al., 2003; Yoshitomi, et al., 2005), the possibility that IL-17 producing gamma/delta T cells are absent in the joints of SKG arthritis should be excluded because of the differences in strain or adjuvant from CIA. Thus we counted the absolute numbers of cell subsets from joints immunized with CFA+CII of SKG or BALB/c mice. Even in this condition, IL-17-producing gamma/delta T cells were not detected in SKG mice, whereas IL-17-producing gamma/delta T cells were more abundant than Th17 cells in BALB/c (Fig. 5B). These data suggested that SKG mice have some impairment of

Fig. 5. IL-17-producing gamma/delta T cells were not detected in swollen joints of SKG mice

**7. IL-17-producing gamma/delta T cells were not detected in the swollen** 

development or maintenance in IL-17-producing gamma/delta T cells.

**joints of SKG mice** 

gamma/delta T cells do not specifically respond to CII and may just respond to adjuvant (IFA+solution) or adjuvant-induced IL-23.

#### **6. IL-17-producing gamma/delta T cells did not induce but amplified CIA**

Next, the pathogenic roles of IL-17-producing gamma/delta T cells in CIA were evaluated. Because we found that CCR6 is an exclusive marker for IL-17-producing T cell even in the case of gamma/delta T cells, CCR6+ gamma/delta T cells were sorted from DLN of the swollen joints of CIA mice as the source of IL-17-producing gamma/delta T cells (data not shown). When CCR6+ gamma/delta T cells were transferred to the joints of naïve mice, arthritis was not indiced. However, when it was transferred to the joints of mice immunized with CII+CFA, it significantly worsened the arthritis score compared with PBS (Fig. 4C). The arthritis-exacerbating effect of CCR6+ gamma/delta T cells from swollen joints was equivalent to CCR6+ gamma/delta T cells from DLNs of swollen joints (data not shown).

Fig. 4. IL-17-producing gamma/delta T cells were maintained by IL-23, and did not induce but amplified CIA

**A**, Cells were prepared from the DLNs of swollen joints and cultured for 7 days in the presence of IL-23, CII, or medium alone. IL-17-producing T cells were detected using FACS analysis. The ratio of the numbers of IL-17-producing T cells in the presence of IL-23 or CII to those in medium alone was calculated. Mean ± SEM is shown from at least three different experiments. **B**, Various combinations of substances were administered into the footpads of DBA1/J mice. Ten days later, the absolute numbers of IL-17-producing cells were counted by FACS analysis. Mean ± SEM is shown from at least three different mice. \* = *P* < 0.05. **C**, CCR6+ gamma/delta T cells were sorted from DLNs of swollen joints of CIA mice, then those cells or PBS alone were injected to unimmunized wrists or ankles of mice immunized with CII+CFA

gamma/delta T cells do not specifically respond to CII and may just respond to adjuvant

Next, the pathogenic roles of IL-17-producing gamma/delta T cells in CIA were evaluated. Because we found that CCR6 is an exclusive marker for IL-17-producing T cell even in the case of gamma/delta T cells, CCR6+ gamma/delta T cells were sorted from DLN of the swollen joints of CIA mice as the source of IL-17-producing gamma/delta T cells (data not shown). When CCR6+ gamma/delta T cells were transferred to the joints of naïve mice, arthritis was not indiced. However, when it was transferred to the joints of mice immunized with CII+CFA, it significantly worsened the arthritis score compared with PBS (Fig. 4C). The arthritis-exacerbating effect of CCR6+ gamma/delta T cells from swollen joints was equivalent to CCR6+ gamma/delta T cells from DLNs of swollen joints (data not shown).

Fig. 4. IL-17-producing gamma/delta T cells were maintained by IL-23, and did not induce

**A**, Cells were prepared from the DLNs of swollen joints and cultured for 7 days in the presence of IL-23, CII, or medium alone. IL-17-producing T cells were detected using FACS analysis. The ratio of the numbers of IL-17-producing T cells in the presence of IL-23 or CII to those in medium alone was calculated. Mean ± SEM is shown from at least three different experiments. **B**, Various combinations of substances were administered into the footpads of DBA1/J mice. Ten days later, the absolute numbers of IL-17-producing cells were counted by FACS analysis. Mean ± SEM is shown from at least three different mice. \* = *P* < 0.05. **C**, CCR6+ gamma/delta T cells were sorted from DLNs of swollen joints of CIA mice, then those cells or PBS alone were injected to unimmunized wrists or ankles of mice immunized with CII+CFA

**6. IL-17-producing gamma/delta T cells did not induce but amplified CIA** 

(IFA+solution) or adjuvant-induced IL-23.

but amplified CIA

two weeks before. For naïve mice, CCR6+ gamma/delta T cells, naïve CD4+ T cells, or PBS alone were injected. Mean ± SEM of the arthritis score of affected joints is shown. \* = *P* < 0.05.

#### **7. IL-17-producing gamma/delta T cells were not detected in the swollen joints of SKG mice**

To elucidate the pathological differences from other murine arthritis models, the same analysis was performed using SKG mice (Sakaguchi, et al., 2003). SKG mice carry a point mutation of the gene encoding zeta-chain-associated protein kinase 70 (ZAP-70), and homozygous mice show IL-17-dependent arthritis resembling RA. Although the present study could detect only a few IL-17-producing gamma/delta T cells in the DLNs of swollen joints, surprisingly almost all of the IL-17-producing cells were Th17 cells and the numbers of IL-17-producing gamma/delta T cells were negligible in the swollen joints of SKG mice (Fig. 5A). As SKG is a BALB/c-background strain, and SKG arthritis is induced by zymosan as an adjuvant (Sakaguchi, et al., 2003; Yoshitomi, et al., 2005), the possibility that IL-17 producing gamma/delta T cells are absent in the joints of SKG arthritis should be excluded because of the differences in strain or adjuvant from CIA. Thus we counted the absolute numbers of cell subsets from joints immunized with CFA+CII of SKG or BALB/c mice. Even in this condition, IL-17-producing gamma/delta T cells were not detected in SKG mice, whereas IL-17-producing gamma/delta T cells were more abundant than Th17 cells in BALB/c (Fig. 5B). These data suggested that SKG mice have some impairment of development or maintenance in IL-17-producing gamma/delta T cells.

Fig. 5. IL-17-producing gamma/delta T cells were not detected in swollen joints of SKG mice

Cytokine Profile of T Cells in the Joints of Rheumatoid Arthritis and Its Murine Models 11

This study initially focused on IL-17-producing T cells in the swollen joints of CIA. We found that gamma/delta T cells were the predominant source of IL-17 and were more abundant than Th17 cells, and DX5+ NK cells did not secrete IL-17 in swollen joints of CIA mice. Although it is known that gamma/delta T cells are dispensable for the induction of CIA, because gamma/delta TCR deficient mice can mount CIA (Corthay, et al., 1999), the present findings in the kinetic study and adoptive transfer experiments, together with previous reports (Arai, et al., 1996; Peterman, et al., 1993; Roark, et al., 2007), suggest that not only Th17 cells but also IL-17-producing gamma/delta T cells contribute to the exacerbation of CIA. On the other hand, alpha/beta T cells, especially Th17 cells, are essential for the induction of CIA because alpha/beta TCR deficient mice cannot mount CIA (Corthay, et al., 1999). In addition, IL-17-producing iNKT cells in CIA have been reported

The origin and functions of IL-17-producing gamma/delta T cells in physiological and pathological conditions have been elucidated recently. It was reported that a subset of gamma/delta T cells acquired IL-17-produing function in the thymus (Jensen, et al., 2008) and produced cytokines immediately responding to the initial stimulation. In various murine infectious disease models, these gamma/delta T cells predominantly produce IL-17 and eradicate pathogens (Lockhart, et al., 2006; Romani, et al., 2008; Shibata, et al., 2007; Umemura, et al., 2007). Although IL-23 is known to be a sufficient stimulant of IL-17 production by gamma/delta T cells in naïve mice (Shibata, et al., 2007), the precise requirements of IL-17 production by gamma/delta T cells especially in CIA are unknown. We herein demonstrated that the combination of IL-23 and IL-1 beta synergistically stimulated IL-17 production, but stimulation via gamma/delta TCR had a limited effect. Given the enhanced expression of IL-1 beta and IL-23 in inflamed joints of CIA (Kim, et al., 2007; Weiss, et al., 2005), these findings suggest that IL-17-production by gamma/delta T cells in CIA might mainly be an inflammatory cytokine-driven, and not a TCR-signal-driven

The present study showed that IL-17-producing gamma/delta T cells were CCR6 positive, and CCR6 was already expressed on IL-17-producing gamma/delta T cells in the thymus of naïve mice. CC chemokine ligand 20 (CCL20), the only chemokine known to interact with CCR6, is physiologically expressed at epithelial surfaces (Schutyser, et al., 2003) and fibroblast-like synoviocytes (Hirota, et al., 2007), and is upregulated in inflammatory conditions (Hirota, et al., 2007; Schutyser, et al., 2003). These findings suggest that CCR6 might have some role in determining the physiological distribution of IL-17-producing gamma/delta T cells. In fact, it was found that a small number of CCR6+ IL-17-producing

Next, the differences between IL-17-producing gamma/delta T cells and Th17 cells were focused upon. IL-17-producing gamma/delta T cells are maintained by IL-23 but not by a specific antigen (CII, in this case). In contrast, Th17 cells responded to both CII and IL-23. Furthermore, IL-17-producing gamma/delta T cells were induced equivalently in response to stimulation by IFA+solution in the absence of CII. Together with the previous study demonstrating that IL-17-producing gamma/delta T cells are induced equally by CFA+CII and CFA (Roark, et al., 2007), the present data suggest that IL-17-producing

gamma/delta T cells were present in the joints of naïve mice.

recently (Yoshiga, et al., 2008), but these cells were not analyzed in this study.

**9. Conclusion** 

process.

**A**, Cells were collected from an ankle with maximum arthritis and its DLNs of SKG mice treated with zymosan 7 weeks previously. IL-17-producing cells and IFN-gamma-producing cells were detected by intracellular cytokine staining (left column). IL-17-producing IFNgamma-negative cells (middle column) or IFN-gamma-producing IL-17-negative cells (right column) were gated and plotted by their expressions of gamma/delta TCR and CD4. The absolute numbers and percentages of CD4+ cells and gamma/delta TCR+ cells are indicated. **B**, SKG or BALB/c mice were immunized with CFA+CII and cells in their immunized joints were collected 10 days later. Absolute numbers of cells were counted by FACS analysis. Mean ± SEM is shown from three different mice.

#### **8. In RA affected joints, IL-17-producing gamma/delta T cells were not detected, and IFN-gamma-producing T cells were dominant**

Finally, cells in synovial tissues or fluids with RA patients were analyzed to determine the presence of IL-17-producing gamma/delta T cells, Th17 and Th1 cells. In contrast to CIA, IL-17-producing gamma/delta T cells could not be detected in either synovial tissues or synovial fluids of the affected joints with RA, whereas a small number of IFN-gammaproducing gamma/delta T cells were present in synovial tissues (Fig. 6A). Among the CD4+ T cells in both synovial tissues and synovial fluids, IL-17-producing cells were rarely present, and IFN-gamma-producing T cells were clearly dominant in the affected joints of RA patients (Fig. 6A and B). Although this finding is consistent with a previous report (Yamada, et al., 2007), we confirmed it in both synovial tissues and fluids of RA patients.

Fig. 6. IL-17-producing gamma/delta T cells were not detected, and IFN-gamma-producing T cells were dominant in the affected joints of RA

Cells in synovial tissues (n=4) or synovial fluids (n=7) of RA were isolated and stained with antibodies against CD4, gamma/delta TCR, IL-17, and IFN-gamma after stimulation by PMA/ionomycin for 6 hours. The percentages of cells out of the total of gamma/delta T cells plus CD4+ T cells were determined. Mean ± SEM is shown.

### **9. Conclusion**

10 Rheumatoid Arthritis – Etiology, Consequences and Co-Morbidities

**A**, Cells were collected from an ankle with maximum arthritis and its DLNs of SKG mice treated with zymosan 7 weeks previously. IL-17-producing cells and IFN-gamma-producing cells were detected by intracellular cytokine staining (left column). IL-17-producing IFNgamma-negative cells (middle column) or IFN-gamma-producing IL-17-negative cells (right column) were gated and plotted by their expressions of gamma/delta TCR and CD4. The absolute numbers and percentages of CD4+ cells and gamma/delta TCR+ cells are indicated. **B**, SKG or BALB/c mice were immunized with CFA+CII and cells in their immunized joints were collected 10 days later. Absolute numbers of cells were counted by FACS analysis.

**8. In RA affected joints, IL-17-producing gamma/delta T cells were not** 

Finally, cells in synovial tissues or fluids with RA patients were analyzed to determine the presence of IL-17-producing gamma/delta T cells, Th17 and Th1 cells. In contrast to CIA, IL-17-producing gamma/delta T cells could not be detected in either synovial tissues or synovial fluids of the affected joints with RA, whereas a small number of IFN-gammaproducing gamma/delta T cells were present in synovial tissues (Fig. 6A). Among the CD4+ T cells in both synovial tissues and synovial fluids, IL-17-producing cells were rarely present, and IFN-gamma-producing T cells were clearly dominant in the affected joints of RA patients (Fig. 6A and B). Although this finding is consistent with a previous report (Yamada, et al., 2007), we confirmed it in both synovial tissues and fluids of RA patients.

Fig. 6. IL-17-producing gamma/delta T cells were not detected, and IFN-gamma-producing

Cells in synovial tissues (n=4) or synovial fluids (n=7) of RA were isolated and stained with antibodies against CD4, gamma/delta TCR, IL-17, and IFN-gamma after stimulation by PMA/ionomycin for 6 hours. The percentages of cells out of the total of gamma/delta T

**detected, and IFN-gamma-producing T cells were dominant** 

Mean ± SEM is shown from three different mice.

T cells were dominant in the affected joints of RA

cells plus CD4+ T cells were determined. Mean ± SEM is shown.

This study initially focused on IL-17-producing T cells in the swollen joints of CIA. We found that gamma/delta T cells were the predominant source of IL-17 and were more abundant than Th17 cells, and DX5+ NK cells did not secrete IL-17 in swollen joints of CIA mice. Although it is known that gamma/delta T cells are dispensable for the induction of CIA, because gamma/delta TCR deficient mice can mount CIA (Corthay, et al., 1999), the present findings in the kinetic study and adoptive transfer experiments, together with previous reports (Arai, et al., 1996; Peterman, et al., 1993; Roark, et al., 2007), suggest that not only Th17 cells but also IL-17-producing gamma/delta T cells contribute to the exacerbation of CIA. On the other hand, alpha/beta T cells, especially Th17 cells, are essential for the induction of CIA because alpha/beta TCR deficient mice cannot mount CIA (Corthay, et al., 1999). In addition, IL-17-producing iNKT cells in CIA have been reported recently (Yoshiga, et al., 2008), but these cells were not analyzed in this study.

The origin and functions of IL-17-producing gamma/delta T cells in physiological and pathological conditions have been elucidated recently. It was reported that a subset of gamma/delta T cells acquired IL-17-produing function in the thymus (Jensen, et al., 2008) and produced cytokines immediately responding to the initial stimulation. In various murine infectious disease models, these gamma/delta T cells predominantly produce IL-17 and eradicate pathogens (Lockhart, et al., 2006; Romani, et al., 2008; Shibata, et al., 2007; Umemura, et al., 2007). Although IL-23 is known to be a sufficient stimulant of IL-17 production by gamma/delta T cells in naïve mice (Shibata, et al., 2007), the precise requirements of IL-17 production by gamma/delta T cells especially in CIA are unknown. We herein demonstrated that the combination of IL-23 and IL-1 beta synergistically stimulated IL-17 production, but stimulation via gamma/delta TCR had a limited effect. Given the enhanced expression of IL-1 beta and IL-23 in inflamed joints of CIA (Kim, et al., 2007; Weiss, et al., 2005), these findings suggest that IL-17-production by gamma/delta T cells in CIA might mainly be an inflammatory cytokine-driven, and not a TCR-signal-driven process.

The present study showed that IL-17-producing gamma/delta T cells were CCR6 positive, and CCR6 was already expressed on IL-17-producing gamma/delta T cells in the thymus of naïve mice. CC chemokine ligand 20 (CCL20), the only chemokine known to interact with CCR6, is physiologically expressed at epithelial surfaces (Schutyser, et al., 2003) and fibroblast-like synoviocytes (Hirota, et al., 2007), and is upregulated in inflammatory conditions (Hirota, et al., 2007; Schutyser, et al., 2003). These findings suggest that CCR6 might have some role in determining the physiological distribution of IL-17-producing gamma/delta T cells. In fact, it was found that a small number of CCR6+ IL-17-producing gamma/delta T cells were present in the joints of naïve mice.

Next, the differences between IL-17-producing gamma/delta T cells and Th17 cells were focused upon. IL-17-producing gamma/delta T cells are maintained by IL-23 but not by a specific antigen (CII, in this case). In contrast, Th17 cells responded to both CII and IL-23. Furthermore, IL-17-producing gamma/delta T cells were induced equivalently in response to stimulation by IFA+solution in the absence of CII. Together with the previous study demonstrating that IL-17-producing gamma/delta T cells are induced equally by CFA+CII and CFA (Roark, et al., 2007), the present data suggest that IL-17-producing

Cytokine Profile of T Cells in the Joints of Rheumatoid Arthritis and Its Murine Models 13

most notable difference was predominance of Th1 cells (IFN-gamma-producing Th cell) rather than Th17 cells in the jointd of RA, and this observation is consistent with a previous report (Yamada, et al., 2007). This discrepancy could be explained by some artifacts. For example, the synovium samples of RA available are not from an early phase but a relatively late phase. In fact, Leipe et al. (2011) reported recently that the percentages of Th17 cells correlate strongly with the activity of RA using very early active and naïve RA patients, and concluded that Th17 cells play an important role in human RA. However, their data also indicated that IFN-gamma-producing T cells were dominant even in those cases. This discrepancy may also be explained by the differences between mice and humans. Alternatively, IL-17-producing T cells may play important roles in RA as well, but are suppressed by the effects of medicines. In conclusion, we have to wait for the results of clinical trials of IL-17 blocking agents in RA patients to further understand the true roles of

Fig. 7. A schema of cytokine profiles and T cell phenotype of two murine arthritis models

Acosta-Rodriguez, E. V., Rivino, L., Geginat, J., Jarrossay, D., Gattorno, M., Lanzavecchia,

A., Sallusto, F. & Napolitani, G. (2007). Surface phenotype and antigenic specificity of human interleukin 17-producing T helper memory cells. *Nat Immunol*, Vol.8,

This work was mainly conducted by Dr. Ito Yoshinaga (Ito, et al., 2009).

No.6, pp.639-646, ISSN 1529-2908

Th17 cells in RA.

and RA

**10. Acknowledgment** 

**11. References** 

gamma/delta T cells do not recognize the specific antigen (CII) but rather proliferate in response to IL-23, which may be produced locally by synovial cells (Kim, et al., 2007). The ligands of gamma/delta T cells are largely unknown and further analysis of possible antigens of IL-17-producing gamma/delta T cells in CIA could be difficult (Konigshofer & Chien, 2006). However, we confirmed the diverse usage of gamma/delta TCR in IL-17 producing gamma/delta T cells in CIA (Ito, et al., 2009), which supported the present conclusion that IL-17-producing gamma/delta T cells are antigen-independently induced by inflammatory cytokines.

In summary, the sequence of pathology of CIA is speculated to be as follows. First, CIIspecific Th17 cells are induced by CII+CFA, which then infiltrate into the joints and cause primary inflammation. Although antigen-independent IL-17-producing gamma/delta T cells could be induced simultaneously by CFA, they are dispensable for the induction of arthritis. Next, primary inflammation induces local production of IL-23 from synoviocytes and increases the expression of IL-1 beta in the joint cartilage and pannus (Weiss, et al., 2005). Locally-produced IL-23 induces the proliferation of resident IL-17-producing gamma/delta T cells. These gamma/delta T cells, stimulated by IL-1 beta and IL-23, produce enhanced amounts of IL-17 and exacerbate the arthritis of CIA. Another, but not mutually exclusive, possibility is that primary inflammation enhances CCL20 expression in vascular endothelial cells and fibroblast-like synoviocytes (Hirota, et al., 2007) in inflamed joints, and recruits CCR6+ IL-17-producing cells. In the ankylosing phase, the burned out tissue does not produce inflammatory cytokines, and then the activities and the number of IL-17-producing gamma/delta T cells decrease to the basal level.

The cytokine profiles of T cells were compared in the inflamed joints of SKG mice and RA with those of CIA mice. In contrast to CIA, IL-17-producing gamma/delta T cells were not detected in the swollen joints of SKG mice. A lack of IL-17-producing gamma/delta T cells in SKG mice was not caused by the differences in strain or adjuvant. It was also found that IL-17-producing gamma/delta T cells are hardly induced, not only in immunized joints but also in their DLNs and the spleens of SKG mice by immunization with CFA+CII (data not shown). Given that the TCR signals in SKG mice are attenuated because of a point mutation in ZAP-70 (Sakaguchi, et al., 2003) and differentiation of gamma/delta T cells requires a strong signal via TCR (Haks, et al., 2005; Hayes, et al., 2005), there may be some defects in the gamma/delta T cell differentiation in SKG mice. This speculation was supported by the data of impaired development of specific subsets of gamma/delta T cells in ZAP-70 knockout mice (Kadlecek, et al., 1998). Therefore, it is true that both murine RA models share the property of IL-17-driven arthritis, and IFNgamma-producing Th1 cells seem dispensable in these models. However, there is also a significant dissimilarity in their pathogeneses. CIA mice appear to be an arthritis model that is driven by innate factor, because the major population of swollen joints was antigen-independent gamma/delta T cell. In contrast, SKG arthritis seems to be a pure Th17-driven arthritis (Fig 7). However, the target antigen of Th17 cells in the affected joint of SKG mice is still unknown.

Contrary to the findings of murine RA models, there are big differences in the phenotypes and the cytokine profiles in the affected joints of RA patients. IL-17-production from gamma/delta T cells in synovial tissues of RA was negligible, and a small number of IFNgamma-producing gamma/delta T cells were present in RA synovium. Furthermore, the most notable difference was predominance of Th1 cells (IFN-gamma-producing Th cell) rather than Th17 cells in the jointd of RA, and this observation is consistent with a previous report (Yamada, et al., 2007). This discrepancy could be explained by some artifacts. For example, the synovium samples of RA available are not from an early phase but a relatively late phase. In fact, Leipe et al. (2011) reported recently that the percentages of Th17 cells correlate strongly with the activity of RA using very early active and naïve RA patients, and concluded that Th17 cells play an important role in human RA. However, their data also indicated that IFN-gamma-producing T cells were dominant even in those cases. This discrepancy may also be explained by the differences between mice and humans. Alternatively, IL-17-producing T cells may play important roles in RA as well, but are suppressed by the effects of medicines. In conclusion, we have to wait for the results of clinical trials of IL-17 blocking agents in RA patients to further understand the true roles of Th17 cells in RA.

Fig. 7. A schema of cytokine profiles and T cell phenotype of two murine arthritis models and RA

#### **10. Acknowledgment**

This work was mainly conducted by Dr. Ito Yoshinaga (Ito, et al., 2009).

#### **11. References**

12 Rheumatoid Arthritis – Etiology, Consequences and Co-Morbidities

gamma/delta T cells do not recognize the specific antigen (CII) but rather proliferate in response to IL-23, which may be produced locally by synovial cells (Kim, et al., 2007). The ligands of gamma/delta T cells are largely unknown and further analysis of possible antigens of IL-17-producing gamma/delta T cells in CIA could be difficult (Konigshofer & Chien, 2006). However, we confirmed the diverse usage of gamma/delta TCR in IL-17 producing gamma/delta T cells in CIA (Ito, et al., 2009), which supported the present conclusion that IL-17-producing gamma/delta T cells are antigen-independently induced

In summary, the sequence of pathology of CIA is speculated to be as follows. First, CIIspecific Th17 cells are induced by CII+CFA, which then infiltrate into the joints and cause primary inflammation. Although antigen-independent IL-17-producing gamma/delta T cells could be induced simultaneously by CFA, they are dispensable for the induction of arthritis. Next, primary inflammation induces local production of IL-23 from synoviocytes and increases the expression of IL-1 beta in the joint cartilage and pannus (Weiss, et al., 2005). Locally-produced IL-23 induces the proliferation of resident IL-17-producing gamma/delta T cells. These gamma/delta T cells, stimulated by IL-1 beta and IL-23, produce enhanced amounts of IL-17 and exacerbate the arthritis of CIA. Another, but not mutually exclusive, possibility is that primary inflammation enhances CCL20 expression in vascular endothelial cells and fibroblast-like synoviocytes (Hirota, et al., 2007) in inflamed joints, and recruits CCR6+ IL-17-producing cells. In the ankylosing phase, the burned out tissue does not produce inflammatory cytokines, and then the activities and the number of

The cytokine profiles of T cells were compared in the inflamed joints of SKG mice and RA with those of CIA mice. In contrast to CIA, IL-17-producing gamma/delta T cells were not detected in the swollen joints of SKG mice. A lack of IL-17-producing gamma/delta T cells in SKG mice was not caused by the differences in strain or adjuvant. It was also found that IL-17-producing gamma/delta T cells are hardly induced, not only in immunized joints but also in their DLNs and the spleens of SKG mice by immunization with CFA+CII (data not shown). Given that the TCR signals in SKG mice are attenuated because of a point mutation in ZAP-70 (Sakaguchi, et al., 2003) and differentiation of gamma/delta T cells requires a strong signal via TCR (Haks, et al., 2005; Hayes, et al., 2005), there may be some defects in the gamma/delta T cell differentiation in SKG mice. This speculation was supported by the data of impaired development of specific subsets of gamma/delta T cells in ZAP-70 knockout mice (Kadlecek, et al., 1998). Therefore, it is true that both murine RA models share the property of IL-17-driven arthritis, and IFNgamma-producing Th1 cells seem dispensable in these models. However, there is also a significant dissimilarity in their pathogeneses. CIA mice appear to be an arthritis model that is driven by innate factor, because the major population of swollen joints was antigen-independent gamma/delta T cell. In contrast, SKG arthritis seems to be a pure Th17-driven arthritis (Fig 7). However, the target antigen of Th17 cells in the affected joint

Contrary to the findings of murine RA models, there are big differences in the phenotypes and the cytokine profiles in the affected joints of RA patients. IL-17-production from gamma/delta T cells in synovial tissues of RA was negligible, and a small number of IFNgamma-producing gamma/delta T cells were present in RA synovium. Furthermore, the

IL-17-producing gamma/delta T cells decrease to the basal level.

by inflammatory cytokines.

of SKG mice is still unknown.

Acosta-Rodriguez, E. V., Rivino, L., Geginat, J., Jarrossay, D., Gattorno, M., Lanzavecchia, A., Sallusto, F. & Napolitani, G. (2007). Surface phenotype and antigenic specificity of human interleukin 17-producing T helper memory cells. *Nat Immunol*, Vol.8, No.6, pp.639-646, ISSN 1529-2908

Cytokine Profile of T Cells in the Joints of Rheumatoid Arthritis and Its Murine Models 15

Hirota, K., Yoshitomi, H., Hashimoto, M., Maeda, S., Teradaira, S., Sugimoto, N.,

Infante-Duarte, C., Horton, H. F., Byrne, M. C. & Kamradt, T. (2000). Microbial lipopeptides

Ito, Y., Usui, T., Kobayashi, S., Iguchi-Hashimoto, M., Ito, H., Yoshitomi, H., Nakamura, T.,

Jensen, K. D., Su, X., Shin, S., Li, L., Youssef, S., Yamasaki, S., Steinman, L., Saito, T.,

Kadlecek, T. A., van Oers, N. S., Lefrancois, L., Olson, S., Finlay, D., Chu, D. H., Connolly,

Kim, H. R., Cho, M. L., Kim, K. W., Juhn, J. Y., Hwang, S. Y., Yoon, C. H., Park, S. H., Lee, S.

Konigshofer, Y. & Chien, Y. H. (2006). Gammadelta T cells - innate immune lymphocytes?

Leipe, J., Grunke, M., Dechant, C., Reindl, C., Kerzendorf, U., Schulze-Koops, H. &

Lockhart, E., Green, A. M. & Flynn, J. L. (2006). IL-17 production is dominated by

Lubberts, E., Joosten, L. A., Oppers, B., van den Bersselaar, L., Coenen-de Roo, C. J., Kolls,

Lubberts, E., Koenders, M. I., Oppers-Walgreen, B., van den Bersselaar, L., Coenen-de Roo,

*Curr Opin Immunol*, Vol.18, No.5, pp.527-533, ISSN 0952-7915

infection. *J Immunol*, Vol.177, No.7, pp.4662-4669, ISSN 0022-1767

*Rheum*, Vol.62, No.10, pp.2876-2885, ISSN 1529-0131

pp.2803-2812, ISSN 1540-9538

Vol.60, No.8, pp.2294-2303, ISSN 0004-3591

Vol.29, No.1, pp.90-100, ISSN 1074-7613

ISSN 0022-1767

0022-1767

1767

64, ISSN 1462-0324

Yamaguchi, T., Nomura, T., Ito, H., Nakamura, T., Sakaguchi, N. & Sakaguchi, S. (2007). Preferential recruitment of CCR6-expressing Th17 cells to inflamed joints via CCL20 in rheumatoid arthritis and its animal model. *J Exp Med*, Vol.204, No.12,

induce the production of IL-17 in Th cells. *J Immunol*, Vol.165, No.11, pp.6107-6115,

Shimizu, M., Kawabata, D., Yukawa, N., Hashimoto, M., Sakaguchi, N., Sakaguchi, S., Yoshifuji, H., Nojima, T., Ohmura, K., Fujii, T. & Mimori, T. (2009). Gamma/delta T cells are the predominant source of interleukin-17 in affected joints in collagen-induced arthritis, but not in rheumatoid arthritis. *Arthritis Rheum*,

Locksley, R. M., Davis, M. M., Baumgarth, N. & Chien, Y. H. (2008). Thymic selection determines gammadelta T cell effector fate: antigen-naive cells make interleukin-17 and antigen-experienced cells make interferon gamma. *Immunity*,

K., Killeen, N. & Weiss, A. (1998). Differential requirements for ZAP-70 in TCR signaling and T cell development. *J Immunol*, Vol.161, No.9, pp.4688-4694, ISSN

H. & Kim, H. Y. (2007). Up-regulation of IL-23p19 expression in rheumatoid arthritis synovial fibroblasts by IL-17 through PI3-kinase-, NF-kappaB- and p38 MAPK-dependent signalling pathways. *Rheumatology (Oxford)*, Vol.46, No.1, pp.57-

Skapenko, A. (2011). Role of Th17 cells in human autoimmune arthritis. *Arthritis* 

gammadelta T cells rather than CD4 T cells during Mycobacterium tuberculosis

J. K., Schwarzenberger, P., van de Loo, F. A. & van den Berg, W. B. (2001). IL-1 independent role of IL-17 in synovial inflammation and joint destruction during collagen-induced arthritis. *J Immunol*, Vol.167, No.2, pp.1004-1013, ISSN 0022-

C. J., Joosten, L. A. & van den Berg, W. B. (2004). Treatment with a neutralizing anti-murine interleukin-17 antibody after the onset of collagen-induced arthritis


Annunziato, F., Cosmi, L., Santarlasci, V., Maggi, L., Liotta, F., Mazzinghi, B., Parente, E.,

Arai, K., Yamamura, S., Hanyu, T., Takahashi, H. E., Umezu, H., Watanabe, H. & Abo, T.

Bettelli, E., Carrier, Y., Gao, W., Korn, T., Strom, T. B., Oukka, M., Weiner, H. L. & Kuchroo,

Clague, R. B. & Moore, L. J. (1984). IgG and IgM antibody to native type II collagen in

Cook, A. D., Rowley, M. J., Mackay, I. R., Gough, A. & Emery, P. (1996). Antibodies to type

Corthay, A., Johansson, A., Vestberg, M. & Holmdahl, R. (1999). Collagen-induced arthritis

Courtenay, J. S., Dallman, M. J., Dayan, A. D., Martin, A. & Mosedale, B. (1980).

Ferretti, S., Bonneau, O., Dubois, G. R., Jones, C. E. & Trifilieff, A. (2003). IL-17, produced by

Gregersen, P. K., Silver, J. & Winchester, R. J. (1987). The shared epitope hypothesis. An

Harrington, L. E., Hatton, R. D., Mangan, P. R., Turner, H., Murphy, T. L., Murphy, K. M. &

Hayes, S. M., Li, L. & Love, P. E. (2005). TCR signal strength influences

arthritis. *Arthritis Rheum*, Vol.30, No.11, pp.1205-1213, ISSN 0004-3591 Haks, M. C., Lefebvre, J. M., Lauritsen, J. P., Carleton, M., Rhodes, M., Miyazaki, T., Kappes,

*Arthritis Rheum*, Vol.39, No.10, pp.1720-1727, ISSN 0004-3591

*Nature*, Vol.283, No.5748, pp.666-668, ISSN 0028-0836

Th17 cells. *J Exp Med*, Vol.204, No.8, pp.1849-1861, ISSN 0022-1007

1767

1476-4687

0953-8178

ISSN 0022-1767

ISSN 1074-7613

7613

pp.1123-1132, ISSN 1529-2908

No.12, pp.1370-1377, ISSN 0004-3591

Fili, L., Ferri, S., Frosali, F., Giudici, F., Romagnani, P., Parronchi, P., Tonelli, F., Maggi, E. & Romagnani, S. (2007). Phenotypic and functional features of human

(1996). Extrathymic differentiation of resident T cells in the joints of mice with collagen-induced arthritis. *J Immunol*, Vol.157, No.11, pp.5170-5177, ISSN 0022-

V. K. (2006). Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. *Nature*, Vol.441, No.7090, pp.235-238, ISSN

rheumatoid arthritis serum and synovial fluid. Evidence for the presence of collagen-anticollagen immune complexes in synovial fluid. *Arthritis Rheum*, Vol.27,

II collagen in early rheumatoid arthritis. Correlation with disease progression.

development requires alpha beta T cells but not gamma delta T cells: studies with T cell-deficient (TCR mutant) mice. *Int Immunol*, Vol.11, No.7, pp.1065-1073, ISSN

Immunisation against heterologous type II collagen induces arthritis in mice.

lymphocytes and neutrophils, is necessary for lipopolysaccharide-induced airway neutrophilia: IL-15 as a possible trigger. *J Immunol*, Vol.170, No.4, pp.2106-2112,

approach to understanding the molecular genetics of susceptibility to rheumatoid

D. J. & Wiest, D. L. (2005). Attenuation of gammadeltaTCR signaling efficiently diverts thymocytes to the alphabeta lineage. *Immunity*, Vol.22, No.5, pp.595-606,

Weaver, C. T. (2005). Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. *Nat Immunol*, Vol.6, No.11,

alphabeta/gammadelta lineage fate. *Immunity*, Vol.22, No.5, pp.583-593, ISSN 1074-


Cytokine Profile of T Cells in the Joints of Rheumatoid Arthritis and Its Murine Models 17

Singh, S. P., Zhang, H. H., Foley, J. F., Hedrick, M. N. & Farber, J. M. (2008). Human T cells

Tarkowski, A., Klareskog, L., Carlsten, H., Herberts, P. & Koopman, W. J. (1989). Secretion

Umemura, M., Yahagi, A., Hamada, S., Begum, M. D., Watanabe, H., Kawakami, K., Suda,

Veldhoen, M., Hocking, R. J., Atkins, C. J., Locksley, R. M. & Stockinger, B. (2006).

Weaver, C. T., Hatton, R. D., Mangan, P. R. & Harrington, L. E. (2007). IL-17 family

Weiss, R. J., Erlandsson Harris, H., Wick, M. C., Wretenberg, P., Stark, A. & Palmblad, K.

Weyand, C. M. & Goronzy, J. J. (1999). T-cell responses in rheumatoid arthritis: systemic

Weyand, C. M., Klimiuk, P. A. & Goronzy, J. J. (1998). Heterogeneity of rheumatoid arthritis:

Wooley, P. H., Whalen, J. D. & Chapdelaine, J. M. (1989). Collagen-induced arthritis in mice.

Yamada, H., Nakashima, Y., Okazaki, K., Mawatari, T., Fukushi, J. I., Kaibara, N., Hori, A.,

Yoshitomi, H., Sakaguchi, N., Kobayashi, K., Brown, G. D., Tagami, T., Sakihama, T., Hirota,

of patients with rheumatoid arthritis. *Ann Rheum Dis*, ISSN 1468-2060 Yoshiga, Y., Goto, D., Segawa, S., Ohnishi, Y., Matsumoto, I., Ito, S., Tsutsumi, A., Taniguchi,

vitro. *Cell Immunol*, Vol.124, No.2, pp.227-238, ISSN 0008-8749

Vol.180, No.1, pp.214-221, ISSN 0022-1767

*Immunol*, Vol.25, pp.821-852, ISSN 0732-0582

*Immunol*, Vol.62, No.1, pp.55-62, ISSN

pp.3786-3796, ISSN 0022-1767

ISSN 1074-7613

1040-8711

1107-3756

22, ISSN 0344-4325

1767

3591

infection via IL-17 production. *J Immunol*, Vol.178, No.7, pp.4466-4472, ISSN 0022-

that are able to produce IL-17 express the chemokine receptor CCR6. *J Immunol*,

of antibodies to types I and II collagen by synovial tissue cells in patients with rheumatoid arthritis. *Arthritis Rheum*, Vol.32, No.9, pp.1087-1092, ISSN 0004-

T., Sudo, K., Nakae, S., Iwakura, Y. & Matsuzaki, G. (2007). IL-17-mediated regulation of innate and acquired immune response against pulmonary Mycobacterium bovis bacille Calmette-Guerin infection. *J Immunol*, Vol.178, No.6,

TGFbeta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells. *Immunity*, Vol.24, No.2, pp.179-189,

cytokines and the expanding diversity of effector T cell lineages. *Annu Rev* 

(2005). Morphological characterization of receptor activator of NFkappaB ligand (RANKL) and IL-1beta expression in rodent collagen-induced arthritis. *Scand J* 

abnormalities-local disease. *Curr Opin Rheumatol*, Vol.11, No.3, pp.210-217, ISSN

from phenotypes to genotypes. *Springer Semin Immunopathol*, Vol.20, No.1-2, pp.5-

VI. Synovial cells from collagen arthritic mice activate autologous lymphocytes in

Iwamoto, Y. & Yoshikai, Y. (2007). Th1 but not Th17 cells predominate in the joints

M. & Sumida, T. (2008). Invariant NKT cells produce IL-17 through IL-23 dependent and -independent pathways with potential modulation of Th17 response in collagen-induced arthritis. *Int J Mol Med*, Vol.22, No.3, pp.369-374, ISSN

K., Tanaka, S., Nomura, T., Miki, I., Gordon, S., Akira, S., Nakamura, T. &

reduces joint inflammation, cartilage destruction, and bone erosion. *Arthritis Rheum*, Vol.50, No.2, pp.650-659, ISSN 0004-3591


Luross, J. A. & Williams, N. A. (2001). The genetic and immunopathological processes

Mangan, P. R., Harrington, L. E., O'Quinn, D. B., Helms, W. S., Bullard, D. C., Elson, C. O.,

Mullazehi, M., Mathsson, L., Lampa, J. & Ronnelid, J. (2007). High anti-collagen type-II

Murphy, C. A., Langrish, C. L., Chen, Y., Blumenschein, W., McClanahan, T., Kastelein, R.

Nakae, S., Nambu, A., Sudo, K. & Iwakura, Y. (2003). Suppression of immune induction of

Park, H., Li, Z., Yang, X. O., Chang, S. H., Nurieva, R., Wang, Y. H., Wang, Y., Hood, L.,

Peterman, G. M., Spencer, C., Sperling, A. I. & Bluestone, J. A. (1993). Role of gamma delta T

Roark, C. L., French, J. D., Taylor, M. A., Bendele, A. M., Born, W. K. & O'Brien, R. L. (2007).

delta T cells. *J Immunol*, Vol.179, No.8, pp.5576-5583, ISSN 0022-1767 Romani, L., Fallarino, F., De Luca, A., Montagnoli, C., D'Angelo, C., Zelante, T., Vacca, C.,

mice. *Nature*, Vol.426, No.6965, pp.454-460, ISSN 1476-4687

*Ann Rheum Dis*, Vol.66, No.4, pp.537-541, ISSN 0003-4967

*Rheum*, Vol.50, No.2, pp.650-659, ISSN 0004-3591

No.7090, pp.231-234, ISSN 1476-4687

pp.1951-1957, ISSN 0022-1007

pp.6173-6177, ISSN 0022-1767

pp.1133-1141, ISSN 1529-2908

ISSN 0022-1767

6101

0019-2805

reduces joint inflammation, cartilage destruction, and bone erosion. *Arthritis* 

underlying collagen-induced arthritis. *Immunology*, Vol.103, No.4, pp.407-416, ISSN

Hatton, R. D., Wahl, S. M., Schoeb, T. R. & Weaver, C. T. (2006). Transforming growth factor-beta induces development of the T(H)17 lineage. *Nature*, Vol.441,

antibody levels and induction of proinflammatory cytokines by anti-collagen antibody-containing immune complexes in vitro characterise a distinct rheumatoid arthritis phenotype associated with acute inflammation at the time of disease onset.

A., Sedgwick, J. D. & Cua, D. J. (2003). Divergent pro- and antiinflammatory roles for IL-23 and IL-12 in joint autoimmune inflammation. *J Exp Med*, Vol.198, No.12,

collagen-induced arthritis in IL-17-deficient mice. *J Immunol*, Vol.171, No.11,

Zhu, Z., Tian, Q. & Dong, C. (2005). A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17. *Nat Immunol*, Vol.6, No.11,

cells in murine collagen-induced arthritis. *J Immunol*, Vol.151, No.11, pp.6546-6558,

Exacerbation of collagen-induced arthritis by oligoclonal, IL-17-producing gamma

Bistoni, F., Fioretti, M. C., Grohmann, U., Segal, B. H. & Puccetti, P. (2008). Defective tryptophan catabolism underlies inflammation in mouse chronic granulomatous disease. *Nature*, Vol.451, No.7175, pp.211-215, ISSN 1476-4687 Sakaguchi, N., Takahashi, T., Hata, H., Nomura, T., Tagami, T., Yamazaki, S., Sakihama, T.,

Matsutani, T., Negishi, I., Nakatsuru, S. & Sakaguchi, S. (2003). Altered thymic Tcell selection due to a mutation of the ZAP-70 gene causes autoimmune arthritis in

receptor CCR6. *Cytokine Growth Factor Rev*, Vol.14, No.5, pp.409-426, ISSN 1359-

gammadelta T cells control early infiltration of neutrophils after Escherichia coli

Schutyser, E., Struyf, S. & Van Damme, J. (2003). The CC chemokine CCL20 and its

Shibata, K., Yamada, H., Hara, H., Kishihara, K. & Yoshikai, Y. (2007). Resident Vdelta1+

infection via IL-17 production. *J Immunol*, Vol.178, No.7, pp.4466-4472, ISSN 0022- 1767


**2** 

Evin Sowden and Wan-Fai Ng

*Newcastle University United Kingdom* 

double negative (DN)

**Invariant Natural Killer T Cells in Rheumatoid** 

Like cells of the adaptive immune system, natural killer T (NKT) cells possess immune recognition receptors formed by germline DNA rearrangement. However, in common with cells of the innate immune system, the repertoire of NKT cell receptors is limited and NKT cells can mount a robust effector response with little capacity for immunological memory, sharing some of the characteristics of other innate-like lymphocytes such as γδ T cells, marginal zone B cells, B1 B cells and NK cells. NKT cells have been shown to play a staggering array of roles in infection, cancer and autoimmunity. Autoimmune diseases in which NKT cells have been implicated include type I diabetes, multiple sclerosis, systemic lupus erythematosus and graft-versus-host disease. In rheumatoid arthritis, much work has been done to characterise the frequency and phenotype of NKT cells. Animal models such as collagen-induced arthritis or the antibody-mediated arthritis in the K/BxN serum transfer model have provided valuable insight into the multi-faceted potential of these remarkable cells and in time, pharmacological manipulation of their immune function may

thymocytes bearing the murine natural killer (NK) cell marker NK1.1 or the orthologous CD161 in humans (Ballas & Rasmussen, 1990). Since their discovery, the unexpected ontological complexity, development, function and pathophysiological roles of NKT cells have begun to unravel. Here, we briefly review the biology of NKT cells and focus on a subset known as invariant NKT (iNKT) cells by virtue of their CD1d-restricted, semiinvariant T cell receptor in order to better understand their significance in autoimmune

The first mouse anti-human monoclonal antibody recognised an antigen found on human thymocytes and certain B cell lymphoma lines subsequently termed the first cluster of differentiation or CD1. Five CD1 protein isoforms encoded on chromosome 1 bear resemblance to the α chains of MHC class I encoded on chromosome 6. CD1 molecules associate non-covalently with ϐ2-microglobulin, but unlike MHC class I molecules they

provide us with the prospect of novel therapeutic tools.

**2.1 CD1 molecules & the ontogeny of the NKT cell** 

Natural killer T cells were first recognised in 1990 as CD4-CD8-

**2. Biology of natural killer T cells** 

diseases such as rheumatoid arthritis.

**1. Introduction** 

**Arthritis and Other Inflammatory Arthritides** 

Sakaguchi, S. (2005). A role for fungal {beta}-glucans and their receptor Dectin-1 in the induction of autoimmune arthritis in genetically susceptible mice. *J Exp Med*, Vol.201, No.6, pp.949-960, ISSN 0022-1007
