Discussion
Tregs play the most important role in the tolerance of immune response in the peripheral environment (10). Tregs express biomarkers CD4, CD25, and FOXP3, and maintain tolerance to self-antigens, resulting in the prevention of autoimmune diseases. Several studies have found depletion of Tregs in autoimmune diseases producing autoantibodies, such as SLE, idiopathic thrombocytopenic purpura, and autoimmune thyroid disease (22–24). This study demonstrated low frequencies of activated Tregs (CD4+CD25+FOXP3highCD45RAT cells) and decrease in total Tregs (CD4+CD25+FOXP3highT cells) of peripheral blood in patients with AIN. Previously, we reported low levels of Tregs (CD4+CD25+FOXP3+) in peripheral blood during the neutropenic period and natural restoration after the recovery of neutropenia in patients with AIN (9). Activated Tregs exhibited the most suppressive function among Tregs (12). Thus, the significant decrease in activated Tregs in patients with AIN may play an important role in autoimmune response to HNA due to the deficiency of suppressive function in children with AIN.
It has been reported that the percentage of Tregs in peripheral blood during the period of infancy are the lowest across the lifetime of an individual, despite the abundance noted in the neonatal period. After infancy, the frequency of Tregs gradually increases to the adult levels (25, 26). Infants with a low frequency of Tregs encounter various external antigens, such as foods, microorganisms, vaccinations, and/or other environmental factors, for the first time in their life. Furthermore, maternal antibodies gradually disappear during that period (27). When infants receive stimulations from various antigens, the activation of the immune system may lead to the development of self-reactive T cells. In addition to the decrease in activated Tregs, patients with AIN showed significantly skewed usage of several TCR-Vβ families in CD4+, conventional T cells, and Tregs compared with those noted in age-matched control subjects. Furthermore, the number of patients with AIN who displayed expanded usage of TCR-Vβ families was significantly higher than that reported in control subjects in both conventional T cells and Tregs. The mouse model study showed that depletion of Tregs led to the expression of a more diverse TCR repertoire in CD4+ T cells. TCR-Vβ transgenic mice with depletion of Tregs tended to produce some undesired self-reactive clonotypes (28). Taken together, the undesired T cell clonotype that reacts against HNA could expand to develop a self-reactive immune response under the low level of Tregs in patients with AIN.
TCR repertoire skewing has been studied in several immune and inflammatory diseases in children, such as SLE, type 1 diabetes mellitus, systemic vasculitis, immune thrombocytopenia, and Kawasaki disease (17, 18, 29). Although the consistent usage of TCR-Vβ subfamilies in CD4 cells was not noted among autoimmune disorders, skewed usage was observed in patients with several immune disorders. Our study also presented the skewed usage of two TCR-Vβ families in CD4+ cells of patients with AIN. Furthermore, skewed usages of four and one TCR-Vβ families in Tregs and conventional T cells, respectively, were observed when CD4 T cells were divided into conventional T cells and Tregs differentiated based on the expression of CD127 on the cell surface. The high frequency in the expansion of TCR-Vβ families appeared in Tregs of patients with AIN, as shown inFigure 4b . The several skewed usages of TCR-Vβ families in regulatory and conventional T cells of patients with AIN may affect the development of autoreactive clones against HNA. A previous study on the TCR-Vβ repertoire using CDR3 spectratyping in patients with immune thrombocytopenia reported that less expansion of the TCR-Vβ repertoire was associated with good response to splenectomy; in contrast, patients with more expansion exhibited poor response (20). It is likely that the frequency of expansion of the TCR-Vβ repertoire is associated with tolerance to immune response in autoimmune diseases. Collectively, these findings suggest that skewed usages of TCR-Vβ families of Tregs and low frequencies of activated Tregs in patients with AIN may be involved in the development of antineutrophil antibodies in children with AIN. However, the precise mechanism between the skewed usages of TCR Vβ families and low frequency of activated Tregs remains unclear.
Human Tregs have shown a very high TCR diversity compared with other T cell subsets including naïve T cells. This evidence suggested an important role in the immune-regulatory function of Tregs (30, 31). These results led us to individually study the difference in the usage of the TCR repertoire between regulatory and conventional T cells. The different usage of TCR-Vβ families in conventional T cells and Tregs has been reported in healthy children. The results indicated a significant preferential usage for five Vβ families and decreased usage for two Vβ families in Tregs (32). A summary of the results of our current study is shown in Figure 6 . Preferential and decreased usages were similarly observed in several TCR Vβ families. Collectively, these results implied that reactivity to self-antigens is an important feature of the TCR repertoire in Tregs.
Quantitative differences in the usage of TCR Vβ repertoire of Tregs were more observed in patients with AIN than in control subjects (Figure 4b ). Among the different usages of the TCR Vβ repertoire in conventional T cells and Tregs, the usage of the TCR-Vβ 9 family observed in patients with AIN was prominently different from that in control subjects (Figure 6 ). Animal models of autoimmunity and immunodeficiency demonstrated that a diverse Treg repertoire is essential to maintain Treg function (28). Thus, the findings of TCR Vβ diversity in Tregs may be associated with abnormalities of Tregs observed in patients with AIN. Recently, abnormalities in the Treg repertoire have been reported in juvenile idiopathic arthritis (33). The restricted and clonotypic expansion of the Treg repertoire engendered antigenic triggers for disease pathogenesis in juvenile idiopathic arthritis. Furthermore, hematopoietic stem cell transplantation ameliorated the autoimmune diseases through the functional renewal and TCR diversification of Tregs (34).
This study had some limitations. First, this analysis with flow cytometry informed us the abnormal usage of TCR- Vβ families of T cells in patients with AIN, which do not indicate expansion of autoreactive clone directly. Considering that many Tregs have TCRs against self-antigen, the change of repertoire of Treg in patients with AIN could indicate disruption of homeostasis in repertoire of Tregs. The results of this study suggest that future research for the pathogenesis of AIN also require the deeper analysis using high throughput sequencing. Second, we could not follow up most patients from disease onset to recovery. There is no study on the individual and longitudinal change in the TCR-Vβ repertoire in children, although data on the cross-sectional frequency of TCR-Vβ in each age of healthy children or atopic children are available (17, 32, 35). We could not precisely conclude whether the expansions of the TCR-Vβ repertoire in patients with AIN has individually continued during the course of the disease. Therefore, longitudinal Tregs and TCR repertoire analyses in each patient with AIN are warranted because neutropenia spontaneously resolves within several years in the majority of patients with AIN.
In conclusion, this study showed low frequencies of total and activated Tregs in patients with AIN. Furthermore, it is the first investigation of the skewed uses of the TCR-Vβ repertoire in patients with AIN. The low frequencies of total and activated Tregs and the skew of TCR-Vβ families would allow the development of HNA-reactive T cell clones. Further studies are necessary to clarify the involvement of Tregs and the TCR-Vβ repertoire in the pathogenesis of AIN in children.