Role of the frequency of blood CD4+ CXCR5+ CCR6+ T cells in autoimmunity in patients with Sjögren’s syndrome

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Abstract

The blood CD4+ CXCR5+ T cells, known as “circulating” Tfh, have been shown to efficiently induce naïve B cells to produce immunoglobulin. They play an important role in certain autoimmune diseases. In the present study, we show for the first time that the frequency of CD4+ CXCR5+ T cells is increased in pSS patients and positively correlated with autoantibodies in the blood. The concentration of Th17-like subsets (CD4+ CXCR5+ CCR6+) in pSS patients was found to be significantly higher than in healthy controls. Functional assays showed that activated Th17-like subtypes in the blood display the key features of Tfh cells, including invariably coexpressed PD-1, ICOS, CD40L and IL-21. Th17 subsets were found to highly express Bcl-6 protein and Th1 and Th2 were not. Bcl-6 is believed to be a master transforming factor for Tfh cell differentiation and facilitate B cell proliferation and somatic hypermutation within the germinal center. These data indicate that Th17 subsets of CD4+ CXCR5+ T cells in the blood may participate in the antibody-related immune responses and that high frequency of CD4+ CXCR5+ CCR6+ Tfh cells in blood may be suitable biomarkers for the evaluation of the active immune stage of pSS patients. It might provide insights into the pathogenesis and perhaps help researchers identify novel therapeutic targets for pSS.

Highlights

► The frequency of CD4+ CXCR5+ CCR6+ T cells increased in pSS patients and positively correlated with autoantibodies in the blood. ► CD4+ CXCR5+ CCR6+ T cells in blood invariably coexpressed PD-1, ICOS, CD40L, Bcl-6 and secreted IL-21 after stimulated by PHA. ► CD4+ CXCR5+ CCR6+ Tfh cells in blood may be suitable biomarkers for the evaluation of the active immune stage of pSS patients.

Introduction

Primary Sjögren’s syndrome (pSS) affects almost 0.5% of the general population and is second in prevalence only to rheumatoid arthritis among systemic autoimmune diseases [1]. One of the serologic hallmarks of the disease is the presence of variety of characteristic circulating autoantibodies, such as antinuclear antibodies, anti-Ro/SSA, and anti-La/SSB antibodies that mark the decline of the salivary glands, most of which relate to the autoimmunity-mediated destruction of acinar cells [2]. Accordingly, B cell hyper-activation is a predominant feature of pSS related to hypergammaglobulinemia and to the production of autoantibodies [3]. Most studies on the subject have shown that autoantibodies play a decisive role in the pathogenesis of pSS but their creation and regulation in pSS remains unclear [4], [5], [6]. Thus, elucidating the mechanisms of autoantibody generation and regulation is crucial for the study of autoimmune diseases.

Physiologically, antibody responses including autoantibody are largely dependent on the help provided by CD4+ T cells, which are fundamental for the generation of germinal centers (GC), a discrete structure in secondary lymphoid organs where selection of high-affinity B cells and development of B cell memory occur [7]. T follicular helper cells (Tfh) have recently emerged as a separate CD4+ T helper lineage specialized in the assistance of B cells during GC reactions in secondary lymphoid tissue [8]. Tfh cells arise from activated T cells that express Bcl-6, a master transcription factor for Tfh cell differentiation [9]. The distinguishing features of Tfh cells are the expression of CXCR5, PD-1, ICOS, and CD40L and the secretion of IL-21.These molecules promote growth, differentiation, and class switching of B cells in the absence of Blimp-1 [10], [11], [12]. Recently, several studies have shown that over representation of Tfh cells is associated with the development of systemic autoimmunity, including RA and systemic lupus erythematosus (SLE) in human and animal models [13], [14]. However, their association with pSS remains largely unknown.

In humans, most of the Tfh cells are located in the light zone of GC in secondary lymph nodules [15]. One problem that has arisen in studies of human Tfh cells, particularly in the investigation of patients suffering from autoimmune conditions, is that, the lymphoid tissue of these patients cannot be accessed. This makes it very difficult to identify Tfh cells and determine whether the generation or function of these cells is dysregulated. This has created a strong need to establish surrogate strategies to assess the quality of Tfh cell responses in humans. For this reason, researchers have used enumeration of circulating CD4+ CXCR5+ T cells in blood as a counterpart to measure Tfh cell disorders. Some types of CD4+ CXCR5+ T cells are known as “circulating” Tfh, which share functional properties with Tfh cells and appear to represent their circulating memory compartment [16]. Several studies have reported increased numbers of CD4+ CXCR5+ cells in the blood of patients with autoimmunity and decreased numbers in patients with antibody deficiencies [16], [17], [18], [19]. This suggests that the levels of these cells may be a correlated with Tfh cells. However, little is known about the frequency of circulating Tfh cells in pSS patients [20]. In this study, we explored the frequency of circulating Tfh cells in human peripheral blood from pSS patients and examined the potential association of the frequency of CD4+ CXCR5+ T cells with laboratory measures. Progress in this field would be useful not only to defining the mechanisms that control autoimmune disease but also to identifying key pathogenic steps that could be targets of therapeutic action.

Section snippets

Patients and samples

Participants in this study were selected from patients seen at the Department of Clinical Immunology, Xi-jing Hospital, Fourth Military Medical University. Twenty-five pSS patients fulfilling the 2002 American-European consensus group criteria for pSS (55.6 ± 6.2 years old, mean ± s.d. n = 25) provided written informed consent and blood samples. The details of the clinical characteristics, and clinical lab data such as immunoglobulin, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) are

High frequency of CD4+ CXCR5+ CCR6+ T cells in the peripheral blood of pSS patients

To determine the frequency of CD4+ CXCR5+ T cell in peripheral blood, 25 pSS patients and 25 healthy subjects were recruited. As expected, the levels of serum anti- Ro/SSA, anti-La/SSB and RF in pSS patients were significantly higher than in healthy subjects(data not shown). To determine the potential role of peripheral Tfh cells in pSS patients, the frequency of peripheral blood CD4+ CXCR5+ in CD4+ T cell and the percentages of Th1(CXCR3+ CCR6) Th2 (CXCR3 CCR6) and Th17 (CXCR3 CCR6+) in CD4

Discussion

Although pSS has appeared to be a T-cell-mediated autoimmune disease over the past few decades, it is unclear which types of T cells are involved and how the dysregulation of these T cells triggers the development of pSS. Several studies show that patients with autoimmune diseases such as pSS or lupus display high-affinity somatically mutated auto antibodies in sera [27], [28]. This suggests that Tfh cells are crucial regulators and are associated with the pathogenic processes of many

Funding

This work supported by grants from the National Natural Science Foundation of China (No. 81172849) and the National Basic Research Program (No. 2009CB521705).

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    These authors contributed equally to this work.

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