CD45RA–Foxp3high activated/effector regulatory T cells in the CCR7 + CD45RA–CD27 + CD28 + central memory subset are decreased in peripheral blood from patients with rheumatoid arthritis

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Highlights

  • We classified human CD4+ T cells into six novel subsets based on CD45RA, CCR7, CD27, and CD28.

  • The CD27 + CD28+ TCM subset is significantly decreased in the CD4+ T cells from RA patients.

  • The proportion of TNF-α-producing cells in the CD27 + CD28+ TEM subset was increased in RA.

  • The frequency of activated/effector Treg cells in the CD27 + CD28+ TCM subset was decreased in RA.

  • The percentage of naïve Treg cells negatively correlated with RA disease activity.

Abstract

Human CD4+ T cells can be classified as either naïve, central memory (TCM), or effector memory (TEM) cells. To identify the CD4+ T cell subsets most important in the pathogenesis of rheumatoid arthritis (RA), we phenotypically defined human CD4+ T cells as functionally distinct subsets, and analyzed the distribution and characteristics of each subset in the peripheral blood. We classified CD4+ T cells into six novel subsets based on the expression of CD45RA, CCR7, CD27, and CD28. The CCR7 + CD45RA–CD27 + CD28+ TCM subset comprised a significantly smaller proportion of CD4+ T cells in RA patients compared to healthy controls. The frequency of TNF-α-producing cells in the CCR7–CD45RA–CD27 + CD28+ TEM subset was significantly increased in RA. Furthermore, within the CCR7 + CD45RA–CD27 + CD28+ TCM subset, which was decreased in periperal blood from RA, the proportions of total Foxp3+ Treg cells and CD45RA–Foxp3high activated/effector Treg cells were significantly lower in RA patients. Our findings suggest that the increased proportion of TNF-α-producing cells and the decreased proportion of CD45RA–Foxp3high activated/effector Treg cells in particular subsets may have critical roles in the pathogenesis of RA.

Introduction

Rheumatoid arthritis (RA) is characterized by synovial membrane hyperplasia and infiltration by inflammatory cells, including activated T cells. T cells contribute to the initiation and perpetuation of the inflammation underlying RA, leading to joint destruction and disability. Activated T cells proliferate and induce monocytes, macrophages, and synovial fibroblasts to produce proinflammatory cytokines, such as TNF-α, IL-1, and IL-6, and stimulate osteoclastogenesis, matrix metalloproteinase secretion, and immunoglobulin production by B cells.

Human T cells can be divided into functionally distinct subsets. Two primary categories are T cells that have not been exposed to antigen (naïve) and those that are antigen-experienced (memory). The memory subpopulation is a heterogeneous pool, and previous studies using two surface markers, CCR7 and CD45RA, led to the following proposed phenotypic classification of human T cells: naïve T cells as CCR7 + CD45RA+, central memory T cells (TCM) as CCR7 + CD45RA– and effector memory T cells (TEM) as CCR7–CD45RA– [1], [2], [3]. Recent reports suggest that this phenotypic classification of T cells is useful for investigating human autoimmune diseases such as RA [4], systemic lupus erythematosus (SLE) [5], and granulomatosis with polyangiitis (Wegener’s granulomatosis) [6].

The phenotypic subdivisions of human CD4+ T cell subsets are less studied than those of CD8+ T cells. CD4 + CCR7 + CD45RA– TCM cells have been shown to produce IL-2, and CD4 + CCR7–CD45RA– TEM cells to predominantly produce IFN-γ, IL-4, and TNF-α, although a small population of CD4 + CCR7–CD45RA– TEM cells produces IL-2. In addition to CD45RA and CCR7, CD27 and CD28 have been used as surface markers to characterize CD4+ T cells. Appay et al. established a model of CD4+ T cell differentiation characterized by a sequential down-regulation of CCR7, CD27 and then CD28, accompanied by changes of their functional characteristics [7], [8], [9]. CD27 and CD28 are the main costimulatory molecules required to promote T cell activation, although memory T cells seem to be less dependent on CD27 and CD28 for their reactivation than naive T cells [10], [11]. CD4+ T cells gain cytotoxic potential with the acquisition of lytic granules with granzymes, as they lose CD27 expression, and acquisition of perforin at the CD28 negative stage, so that highly differentiated CD4+ T cells become cytotoxic. With the down-regulation of CD27 and CD28, CD4+ T cells exhibit higher IFN-γ-producing ability and shorter telomere length [12], [13], [14]. Although most studies have used either CD27 or CD28 in conjunction with CD45RA and CCR7 to define the CD4+ T cell subsets, we recently discriminated CD4+ T cells into five major subsets using all four markers, CD45RA, CCR7, CD27, and CD28. We defined the function of each population based on its ability to produce IFN-γ, IL-4, and IL-2, and proposed that this phenotypic characterization would be useful in the study of immunological diseases [15], [16].

Human regulatory T cells (Treg cells) play an indispensable role for the maintenance of self tolerance and immune homeostasis [17]. Quantitative and/or qualitative deficiencies in Treg cells could lead to the development of autoimmune diseases [18], [19], [20]. Foxp3 is a key transcription factor for the development and function of natural CD4+ Treg cells. However, recent studies have shown that human Foxp3 + CD4+ T cells are not homogeneous in gene expression, phenotype and suppressive function. Miyara et al. revealed that human Foxp3 + CD4+ T cells can be separated into three functionally and phenotypically unique subpopulations, based on the expression of Foxp3 and their cell surface phenotype. The three distinct subpopulations are as follows: (fraction (Fr.) I) CD45RA + Foxp3low naïve Treg cells; (Fr. II) CD45RA–Foxp3high activated/effector Treg cells, both of which are suppressive in vitro; and (Fr. III) non-suppressive cytokine-secreting CD45RA–Foxp3low non-Treg cells. Terminally differentiated CD45RA–Foxp3high activated/effector Treg cells rapidly died whereas CD45RA + Foxp3low naïve Treg cells proliferated and converted into activated/effector Treg cells in vitro and in vivo. Recent studies report the proportion of the three subpopulations differed among patients with immunological diseases such as SLE and sarcoidosis [21], [22], [23].

In the present study, we phenotypically classified human peripheral blood CD4+ T cells into six novel functionally distinct major subsets using the four cell-surface markers CD45RA, CCR7, CD27 and CD28, and characterized the pro-inflammatory and regulatory characteristics of each subset. In addition, we classified Foxp3 + CD4+ T cells into three functionally subpopulations and studied their distribution in peripheral blood from RA patients.

Section snippets

Sample collection

This study was approved by the ethics committee at the Kobe University Graduate School of Medicine. The human samples were used in accordance with the guidelines of Kobe University Hospital, and written informed consent was obtained from all subjects. The blood samples were obtained from patients with RA (n = 32) and age- and sex-matched healthy volunteers (n = 19) at Kobe University Hospital. The clinical characteristics of the RA patients are summarized in Table 1. All the patients met the 2010

The CCR7+CD45RA–CD27+CD28+ TCM subset is significantly decreased in the peripheral blood CD4+ T cells from RA patients

We classified the peripheral blood CD4+ T cells from healthy subjects and RA patients into the six subsets by four cell-surface markers, CD45RA, CCR7, CD27, and CD28. Flow cytometry analysis revealed six major populations of human CD4+ T cells (Fig. 1A). The CCR7 + CD45RA–CD27–CD28+ TCM subset was not studied previously, because it was considered as a minor population containing few cells [15]. However, we found that 5% of the CD4+ T cells belonged to this subset (Fig. 1A). We therefore

Discussion

The impaired function of Treg cells has been implicated in the development of autoimmune diseases, including RA [25]. Numerous studies dealt with Treg population in RA with various conclusions regarding the frequency of circulating Treg cells. Most studies reported decreased [26], [27] or normal proportions [28], [29] of Treg cells, whereas some other groups reported an increase [30], [31]. Maldonado et al. reported that the CD45RA–CD62L + CD4+ TCM subset was significantly increased in RA

Acknowledgments

We thank Drs. Seiji Kawano, Takeshi Sugimoto and Goichi Kageyama for their helpful comments and suggestions. We are also grateful to Dr. Keisuke Nishimura for collecting the patient data, and Mses. Shino Tanaka and Chinami Oyabu for help with collecting blood samples.

This work was supported the BioLegend/Tomy Digital Biology Research Grant Program (F.M.) and JSPS KAKENHI Grant Number 22790929 (J.S.).

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