Rituximab treatment overcomes reduction of regulatory iNKT cells in patients with rheumatoid arthritis
Introduction
Invariant natural killer T (iNKT) cells are a unique lymphocyte population, restricted by the monomorphic CD1d molecule and characterized by the expression of markers common to NK and T cells. They express a conserved canonical T cell receptor (TCR) α chain (Vα14Jα28 in mouse and Vα24Jα18 in human) paired with a β chain using a selected Vβ segment (Vβ8.2 and 7 in mouse and Vβ11 in human) [1]. Whereas conventional T cells respond to peptide-MHC ligands, the iNKT cells recognize glycolipid antigens such as the α-galactosylceramide (α-GalCer) isolated from marine sponge [2], [3] in association with the CD1d molecule. Because they can produce large amounts of IL-4 and IFN-γ after TCR ligation, iNKT cells are considered to play regulatory roles in a wide range of immune responses [4]. Accumulating evidences showed that iNKT cells are involved in human autoimmune diseases, such as type-1 diabetes and systemic sclerosis, in which decreased numbers of circulating iNKT cells have been reported [5], [6], [7]. Significant therapeutic effects of α-GalCer have been found in animal models of autoimmunity, such as experimental allergic encephalomyelitis [8], [9]. Overexpression of iNKT cells was also demonstrated to delay autoimmune disease in mouse models of autoimmune diabetes and colitis [10], [11], [12].
Alteration of iNKT cells seems to be important in the development of systemic autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Decreased number of circulating iNKT cells was observed in patients with RA and SLE [13], [14], and this diminution was demonstrated to be associated with global dysfunction of these cells [15]. The reduction of iNKT cell numbers in patients with SLE could be due to an increased sensitivity of these cells to apoptosis. Tao et al. [16] described a reduced expression of both the adhesion molecule CD226 and the anti-apoptotic protein survivin on iNKT cells from lupus patients, and proposed that this altered expression is directly linked to the high sensitivity of iNKT cells to apoptosis. This hypothesis is supported by the observation that corticosteroid treatment seems to restore CD226 and survinin expression and is correlated with a significantly decreased apoptosis in iNKT cells [16] in these conditions. Moreover, in SLE patients, the reduced NKT cell frequency seems to be associated with high levels of IgG, suggesting a role for these cells in the regulation of IgG production [17]. A recent study revealed that, compared to controls, the percentage of iNKT cells is lower in the first-degree relatives of lupus patients in correlation with serological and clinical manifestations, indicating that genetically-altered immunoregulation by NKT cells might predispose to autoimmunity [18]. Most of these studies, however, lack a direct, specific method for enumerating iNKT cells. They are often based on the use of a combination of Vα24 and Vβ11 or anti-CD161 monoclonal antibody (mAb) that does not differentiate CD1d-restricted NKT cells bearing the canonical Vα24-Jα18 chain from conventional T cells. When using these mAbs, a large population of non-iNKT cells expressing Vα24 and CD161 are also measured, since CD161 can be detected on up to 20% of circulating T lymphocytes [19], [20]. Moreover, non-invariant and/or non-CD1d reactive Vα24 T cells can also pair with Vβ11 and contribute to the Vα24/Vβ11 population. In previous studies dedicated to the analysis of NKT cell in patients with systemic autoimmune diseases, the reported NKT frequencies occurring at the periphery correspond to 1–10%, whereas only approximately 0.1% are effectively reactive with CD1d tetramers [21], [22], [23], suggesting that NKT cell counts are probably overestimated.
In the present study, we investigated the number of circulating iNKT cells in RA patients and in healthy subjects by flow cytometry, using the mAb 6B11 directed against the canonical Vα24-Jα18 chain. The suitability of the 6B11 mAb was evaluated recently [24]. The authors concluded that this mAb alone or in combination with anti-Vα24, anti-Vβ11 or anti-CD3 is an excellent tool for human iNKT cells analysis, and to our knowledge, the iNKT cell frequency has not yet been evaluated in RA patients using this novel detection system. We also examined the relationship between the iNKT cell frequency and disease parameters. Finally, we performed a longitudinal analysis of iNKT cell frequency in seven RA patients who were given an anti-CD20 therapy.
Section snippets
Patients and healthy individuals
NKT cell characteristics were examined in 36 RA patients (mean age 50 ± 13 years; 8 males and 28 females; mean disease duration 5 ± 4 years; Table 1), 43 SLE patients (mean age 38 ± 12 years; 2 males and 41 females; mean disease duration 8 ± 5 years) who were diagnosed in accordance with the criteria of the American College of Rheumatology [25], [26] and 31 healthy individuals (mean age 41 ± 13 years; 13 males and 18 females).
Disease activity was assessed using the Disease Activity Score (DAS28) for
iNKT cell numbers in patients with RA and SLE and in healthy individuals
The frequency of iNKT cells in the PB from 36 RA patients, 43 SLE patients and 31 healthy individuals was analyzed by flow cytometry by staining total blood cells with the 6B11 mAb, which recognizes T cells expressing the invariant Vα24-Jα18 TCR. We first analyzed the influence of age and gender on the frequency of iNKT cells. The iNKT cell frequency did not significantly differ between females and males (0.33 ± 0.09% vs. 0.17 ± 0.02% in controls, and 0.09 ± 0.02% vs. 0.11 ± 0.04% in RA patients; Fig. 1
Discussion
In the present work, we studied the number of iNKT cells in RA patients and we assessed the evolution of iNKT cell frequency in RA patients who received an anti-CD20 therapy (rituximab). We found that the frequency of iNKT cells in the PB of patients is decreased compared to the one measured in healthy individuals, and that the diminution of the iNKT cell percentage appears to be associated with the disease activity. Taken as a whole, conventional treatments (corticosteroids, immunosuppressive
Acknowledgments
This work was supported by the French Centre National de la Recherche Scientifique (CNRS). VP was supported by a grant from the French “Arthritis Fondation Courtin.”
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2010, Rheumatic Disease Clinics of North AmericaCitation Excerpt :The conflicting roles of NK T cells in arthritis might be explained by differences in the B-cell populations that are presenting antigen to them. A recent study has demonstrated the increased presence of invariant NK T cells with regulatory phenotype following depletion of B cells with anti-CD20 therapy.38 This study suggests a novel mechanism contributing to the success of B-cell depletion therapy, and demonstrates the potential efficacy of targeting the interaction of B cells with iNK T cells.
- 1
Present address: INSERM U951Genethon, Evry, France.
- 2
These authors contributed equally to the study.