Introduction
Autoimmune neutropenia (AIN) in children is characterized by a low absolute neutrophil count caused by the excessive destruction of neutrophils through antibodies against human neutrophil antigens (anti-HNA Abs) (1). The median patient age at diagnosis of AIN is 7–9 months (2, 3). During the period of neutropenia, many patients tend to present bacterial, but not severe infections, because their bone marrow is intact and immediately produces neutrophils after receiving an infectious signal. Some patients receive prophylactic medication, such as sulfamethoxazole-trimethoprim combination to avoid recurrent infections (4). Unlike other autoimmune diseases with autoantibodies, AIN is a self-limited disease which does not require any special therapies (e.g., steroid medications). Because anti-HNA Abs disappear gradually in many cases, almost all patients recover without treatment in 2–3 years (3, 5).
Although AIN is not a rare neutropenia of childhood, its etiopathogenesis remains unclear. Several researchers have attributed causes of this disease to the modification of antigens after exposure to drugs, molecular mimicry of microbial antigens, post-infectious autoantibodies, and differences in human leukocyte antigen types (5–8). Previously, we reported a deficiency of regulatory T cells (Tregs; CD4+CD25+ FOXP3+ T cells) in children with AIN as another cause of this disease (9). Tregs play a key role in suppressing the immune response based on the control of autoimmunity in peripheral tissue (10). In fact, the deficiency of Tregs has been shown in several autoimmune diseases (11). Furthermore, Tregs could separate subpopulations such as resting Tregs, activated Tregs, and non-suppressive Tregs according to the extent of expression of CD45RA and FOXP3. Activated Tregs, defined as CD4+CD25+FOXPhighCD45RAT cells, have the most suppressive function in these subpopulations (12), and the fluctuation of the aforementioned three subpopulations is associated with autoimmune diseases (13, 14).
T cells, in combination with B cells, play an important role in antibody production. For the recognition of various antigens, each T cell has a specific T cell receptor (TCR) on its surface (a heterodimer comprised of α- and β-chains). TCR is formed by random re-combinations of TCR gene elements termed V, D, J-segments (15). The random re-combination causes diversity of the TCR repertoire. The complementarity determining region on the β-chain made from the V-region is the most important region for diversity (16). Recently, associations with repertoire of TCR-Vβ and autoimmune diseases, such as systemic lupus erythematosus (SLE), type 1 diabetes mellitus, autoimmune thyroiditis, and idiopathic thrombocytopenic purpura, have been reported in numerous research studies (17–20). The results have shown several different usages or expansions of the TCR-Vβ family in each disease and indicated an association between disease and an unusual repertoire of the TCR-Vβ family. However, there has been no study analyzing the repertoire of the TCR-Vβ family in patients with AIN. Although the high throughput sequencing is often used for TCR-Vβ repertoire analysis nowadays, it requires much cost. The analysis using flow cytometry could give us less information than high throughput sequencing, but this method is easy and useful to get overview of the repertoire of the TCR-Vβ family in each T cell subset.
In this study, we analyzed the frequency of total Tregs and activated Tregs in CD4+ T cells. Subsequently, we investigated the repertoire of the TCR-Vβ family in patients with AIN using flow cytometry to obtain information on the usages of the TCR-Vβ family in T cells.