CD4+CD25+ regulatory T cells (T_REG) in Systemic Lupus Erythematosus (SLE) patients: The possible influence of treatment with corticosteroids

Abstract

Systemic Lupus Erythematosus (SLE) is a chronic, systemic autoimmune disease characterized by loss of tolerance to self-antigens. Regulatory T cells (T_REG) are those CD4+ T cells that constitutively express high levels of CD25 and exhibit powerful suppressive properties. The aim of this work was to quantify CD4+CD25+ (T_REG) cells and the Mean Fluorescence Index (MFI) of T_REG in the peripheral blood of patients with SLE and to correlate these findings with their disease activity scores and drug therapy. This study included 24 SLE patients with various disease activity scores (SLEDAI) and 24 healthy age and sex matched controls. Flow cytometry was used to examine the frequency of CD4+CD25+ T cells and the MFI of CD4+CD25+^high T cells (T_REG). CD4+CD25+ T cells % and MFI of CD4+CD25+^high T cells were higher in SLE patients than controls (p value = 0.62 and = 0.037 respectively) and both CD4+CD25+ T cell % and the MFI of CD4+CD25+^high T cells showed highly significant correlation with SLEDAI scores (both with a p value < 0.001) and were higher in patients taking glucocorticoids than those not on glucocorticoids (p =  0.023, 0.048 respectively). We conclude that the increase in T_REG cells in our patients may be due to corticosteroid treatment.

Introduction

Growing evidence suggests that naturally acquired immunological self-tolerance, in addition to clonal deletion, anergy and ignorance, are accounted for by a population of CD4 (+) T cells called natural regulatory T (T_REG) cells, which actively suppress the activation and expansion of self-reactive T cells. Those are produced in the normal thymus as functionally mature cells – hence they are called natural regulatory T cells – and seed into the periphery, creating a distinct subpopulation of CD4 (+) T lymphocytes with immunosuppressive properties [1]. Other regulatory cells have been described including Tr1 and Th3 regulatory T cells [2], [3] and CD8+ regulatory T cells [4]. The CD4+CD25+ T_REG cells are currently the best defined and characterized among the various subgroups of regulatory T cells [1].

Within the population of CD4+ CD25+ T cells from healthy volunteers, there exists a minor subset of in-vivo activated anergic regulatory T cells (T_REG) which mediate contact-dependent,cytokine-independent suppression of T cells activation [5], [6].

T regulatory cells that are positive for CD4 and CD25 surface expression have generated the highest level of interest amongst immunologists. It is well documented that the breakdown of mechanisms assuring the recognition of self and non-self antigens is a hallmark of autoimmune diseases, which adds to the importance to those natural regulatory T cells (T_REG) [7].

Systemic Lupus Erythematosus (SLE) is a chronic inflammatory autoimmune disease. In those patients, there is loss of tolerance to self-antigens with polyclonal activation of B lymphocytes, production of different auto-antibodies, and an altered function of T cells [8]. The CD4+CD25+ T regulatory cells not only play a major role in the maintenance of self-tolerance and the control of various autoimmune diseases [9], [10] but they are also involved in the regulation of T cell homeostasis [11], [12] and in the modulation of immune responses to allergens, cancer cells, and pathogens [13], [14]. Those findings have opened new prospects in immunotherapeutic interventions for several diseases. Such T regulatory cells represent a master player in the immune system, and their manipulation could be used in new therapeutics. Recently, Scherer et al. [15] successfully treated a case of scleroderma with anti-CD25 monoclonal antibody and Nieuwhof et al. [16] reported successful treatment of cerebral vasculitis with anti CD25 monoclonal antibody.

T regulatory cells (T_REG) can be classified into two major categories; thymus-derived natural T_REG cells and those induced in the periphery [3]. There are some signals that facilitate peripheral T_REG formation which may involve co-stimulatory molecules such as cytotoxic T lymphocytes-associated antigen (CTLA-4) and cytokines such as IL2, IL10, TNF-α and TGF-B.[1], [17]. T_REG lymphocytes are characterized by the expression of CD4 and by high levels of the α chain of IL-2 receptor (CD25) as well as by their lack of responsiveness to antigenic stimulation [18], [19].

FOXP3, which is the transcription factor whose expression confers the regulatory phenotype on both murine and human T cells, is now thought to represent the most specific T_REG cell marker [20]. However, its intracellular expression doesn't yet allow live sorting of cells for functional assays [21]. Also, Valencia et al. [22] on their work on T_REG reported that the regulation of the suppressive activity of T_REG is more complex because in vitro activation of CD4+CD25+ T cells results in transient expression of FOXP3 but no regulatory activity. However, FOXP3 has been shown to be essential for both the development and function of regulatory T cells [20]. Other surface markers have been reported to be expressed on human CD4+CD25+^high T cells, including cytotoxic T lymphocytes — associated antigen (CTLA-4), CD 69 legend, glucocorticoid-induced tumor necrosis factor receptor (GITR), membrane bound TGF-β and α4β7/α4β1 integrin [23]. Upon activation of T cells – independent of their regulatory capacity – most of their markers become up-regulated and have therefore limited capacity to identify T_REG[6]. However, only cells expressing the highest levels of CD25 (termed CD25+^high) demonstrate potent regulatory function [5], [24]. So, gating of CD4+CD25+^high T cells is usually preferred to define T_REG and the most suppressor function purportedly attributed to T_REG comprises the bright population (CD4+CD25+^high) [5].

Phenotypic identification and quantification and suppressor functional studies of the CD4+CD25+ regulatory T cell population have been investigated in several autoimmune diseases, with conflicting results in most studies [25], [26]. In contrast to strong suspicions, few data yet exist as to whether the uncontrolled T and B cell activation and pathogenesis in SLE can be attributed to a deficiency in CD4+CD25+ regulatory T cells.

This study was carried out aiming to quantify CD4+CD25+ T cells population and CD4+CD25+^high (T_REG) T lymphocytes in SLE patients and to elucidate if there is a possible influence of disease activity scores and drug therapy.