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+FOXP3highCD45RA−T 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.