**3.1.1 Peripheral blood compartment**

In the earliest published study on NKT cells in RA, Yanagihara et al. (1999) looked at CD3+NKR-P1A+(CD161+) NKT cells in 60 patients with established RA compared with 36 healthy controls. They found a 5.8 fold difference in NKT cells but no difference in NK cells. Although patients and controls were mismatched for age, no correlation with age was found in either group. There was no apparent correlation with disease duration, clinical disease activity, inflammatory markers, RF status or drug treatment.

Recent studies of iNKT cell frequency using more specific detection reagents have confirmed results from earlier studies. Parietti et al. (2010) detected iNKT cells with a monoclonal antibody (mAb) against the canonical Vα24Jα18 invariant TCR chain in 36 RA, 43 SLE and 31 healthy subjects. The investigators confirmed the lower frequencies and percentages of iNKT in RA and SLE vs controls (0.09% and 0.01% vs 0.26%, respectively). They found no effect of age, gender or treatment on iNKT cell frequency.

Our own group analysed the frequency of Vα24+Vϐ11+ NKT cells among 46 RA and 22 healthy controls, taking care to use a statistically robust minimum number of lymphocytegated events set at 500,000 in order to reliably measure the infrequent iNKT cells. Our results showed that RA patients have a 15-fold lower iNKT cell relative frequency compared to healthy controls (0.001% vs 0.21%, respectively), either before or after commencing immunosuppressive treatment (Tudhope et al., 2010).

#### **3.1.2 Synovial compartment**

In a comparative analysis of NKT cell frequency in different compartments, Spadaro et al. (2004) studied 29 patients with psoriasis and psoriatic arthritis (PsA), 27 patients with RA and 27 healthy controls. Blood and synovial fluid (SF) lymphocyte subsets, including CD3+CD16+CD56+ NKT cells, were measured and compared. In peripheral blood, there was no statistically significant difference in NKT cell absolute or relative numbers between PsA, RA and healthy control subjects (61 cells/μL or 3.6%, 93 cells/μL or 5% and 89 cells/μL or 3.9%, respectively). SF NKT cells however were significantly reduced in both absolute and relative numbers as compared to peripheral blood in PsA and RA (2% vs 3.2% and 1.6% vs 4.1%, respectively) (Spadaro et al., 2004).

Linsen et al. (2005) studied 23 RA and 22 healthy control patients using peripheral blood and, when available, synovial fluid and tissue specimens. They found that Vα24+Vϐ11+CD3+ NKT cells were significantly reduced in relative frequency in RA as compared to control

Invariant Natural Killer T Cells in Rheumatoid Arthritis and Other Inflammatory Arthritides 25

(PBMC) stimulated with α-GalCer for 10 days resulted in expansion of iNKT cells in just 3 out of 10 RA patients (from 0-10 to 61-1480 cells/105 lymphocytes) as compared to all 7 healthy controls (from 6-123 to 350-3169 cells/105 lymphocytes). The proportion of responders was 50% in patients with SLE (n=10) and SSc (n=8). No clear relationship was

In further experiments, Linsen et al. (2005) also characterised the phenotype of Vα24+Vϐ11+CD3+ iNKT cells in RA compared to healthy controls. The capacity of NKT cells to respond to α-GalCer was tested in peripheral blood of 7 healthy and 13 RA patients as well as the SF from 5 RA patients. PBMC or synovial mononuclear cells (SFMC) were stimulated with α-GalCer and re-stimulated at day 7 with pulsed, irradiated PBMCs then analysed by flow cytometry at day 14. The number of NKT cells from RA peripheral blood (PB) and SF remained lower than that of healthy controls (8.4 and 4.4 vs 15.8%, respectively). Like Kojo et al. (2001), the investigators noted two separate RA populations comprised of non-responders (6/13 patients) and responders (7/13 patients). In fact, RA responders showed stronger responses than healthy controls (294 vs 149 fold, respectively). They too did not find any correlation between response and clinical or treatment phenotype. SF iNKT

In keeping with these earlier results, our own experiments showed that peripheral iNKT cells stimulated with α-GalCer for 12 days display impaired expansion in RA patients compared to controls (31 vs 121 fold, respectively). Using the 25th percentile of foldexpansion in healthy controls, 75% of RA vs 20% of healthy controls are non-responders

While investigating the mechanism underlying non-response to α-GalCer in RA patients, Kojo et al. (2001) found that non-responder APCs could expand responder iNKT cells in the presence of α-GalCer and IL-2, albeit with a lower response than responder APCs. In contrast, non-responder iNKT cells failed to expand in the presence of responder APCs under the same culture conditions. This suggested that in non-responders, the defect lay

In keeping with iNKT cell frequency as measured by flow cytometry, linsen et al. performed ELISPOT analysis of cytokine production by isolated PBMC stimulated with α-GalCer and found a decreased frequency of reactive cells producing IFN-γ (2.3 vs 24.3, respectively) and IL-4 (0.2 vs 3.9, respectively) in RA patients compared to healthy controls. The IL-4/IFN-γ ratio was also reduced (0.07 vs 0.30, respectively), suggesting a Th1-like phenotypic bias in RA which could not be explained by any differences in CD4+ and CD4- subsets, or selective clonal expansion as shown by Vα24 and Vϐ11 TCR CDR3 fragment length spectra analysis

Intracellular cytokine staining of iNKT cells in PB revealed marked differences between RA patients and healthy controls for IFN-γ+ (92.5 vs 64.5%, respectively), IL-4+ (1.4 vs 15.7%, respectively) and IFN-γ+IL-4+ iNKT cells (6.1 vs 19.7%, respectively), confirming the Th1 bias in PBMC-derived iNKT cells. In contrast, cytokine profiles for SF iNKT cells were more similar to that of healthy control PB iNKT cells, with smaller proportion of IL-4+ and higher proportion of IFN-γ+IL-4+ iNKT cells (5.3 and 28.4%, respectively), suggesting a Th0-profile in SF-derived iNKT cells. No correlation with response to α-GalCer, or disease and

with no detectable differences between early and late RA (Tudhope et al., 2010).

noted to disease activity.

cells however responded in all tested patients.

within iNKT cells rather than APCs.

treatment parameters was noted (Linsen et al., 2005).

**3.2.2 Cytokine production** 

(Linsen et al., 2005).

peripheral blood (0.03% vs 0.11%, respectively) but unlike Spadaro et al. (2004), their synovial fluid samples showed an inconsistent trend toward higher percentage of NKT cells in SF from seven patients as compared to matched peripheral blood (0.08% vs 0.05%, respectively).

#### **3.1.3 iNKT cell subsets**

iNKT cell subsets include CD4- (DN), CD4+ and CD8+ cells. iNKT cells from these subsets have been shown to be functionally distinct and therefore individual subset frequency may be immunologically more relevant than global iNKT cell numbers.

In a study of patients with a range of autoimmune diseases including 20 patients with RA, Vα24Jα18+ DN T cells were found to express polymorphic Vϐ11 and CD161 almost universally, suggesting that these were likely to be true iNKT cells. The frequency of these DN NKT cells was markedly reduced in RA patients who had a mean of 48.8 cells/ml as compared to 290 cells/ml in healthy controls. Similar findings applied to patients with SLE, systemic sclerosis (SSc) and Sjogren's syndrome (SS) but not Behcet's disease (BD), and CD4+ NKT cells were similarly reduced in frequency (Kojo et al., 2001).

Vα24+CD8+ NKT cells have been shown to consist mainly of CD161+, CD1d-restricted NKT cells that have an immunoregulatory phenotype (Ho et al. 2004; Takahashi 2002). Mitsuo et al. (2006) examined the frequency of this NKT cell subset in patient with RA (n=24), SLE (n=54), SSc (n=14), mixed connective tissue disease (MCTD) (n=15) and polymyositis/ dermatomyositis (PM/DM) (n=13) compared to healthy controls (n=18). The absolute frequency of CD161+CD8+ T cells was reduced in all patients compared to healthy donors, although in RA patients the relative frequency was not statistically significantly lower. No correlation with age, gender or treatment was noted (Mitsuo et al., 2006).

#### **3.1.4 Correlation with disease activity and treatment response**

Whilst Yanagihara et al. (1999) found no correlation between iNKT cell frequency and clinical disease activity, Parietti et al. (2010) noted a trend toward lower iNKT cell numbers with higher disease activity (DAS28), ESR and CRP. Our group also found no correlation with disease activity although a moderate but statistically significant inverse relationship with CRP could be demonstrated (Tudhope et al., 2010).

Parietti et al. (2010) measured iNKT cell frequencies in seven patients before and after treatment with rituximab, an anti-CD20 monoclonal antibody targeting B cells. They found a significant increase in NKT frequency and percentage from baseline at days 45 and 120 post-infusion (1.7, 3.4 and 4.1 cells/μL, or 0.1%, 0.32% and 0.3%, respectively). There was also a clear correlation between clinical outcome and NKT cell frequency change with nonresponders showing no change whilst responders saw a 600% increase in frequency. In our study, we measured iNKT cell frequencies in seven patients before and after initiating methotrexate therapy and found that iNKT frequency increased as early as two weeks after the start of treatment. Unlike Parietti et al.'s results however, we found no obvious link to clinical response (Tudhope et al., 2010).
