Possible involvement of regulatory T cell abnormalities and variational usage of TCR repertoire in children with autoimmune neutropenia

Abstract

Autoimmune neutropenia (AIN) in childhood is characterized by chronic neutropenia and positivity for anti-neutrophil antibodies, resulting in the excessive destruction of neutrophils. In this study, we investigated the involvement of regulatory T cells (T_regs ) in the pathogenesis of AIN in childhood. T_regs have been classified into three subpopulations based on the expressions of CD45RA and forkhead box protein 3 (FoxP3): resting T_regs , activated T_regs and non-suppressive T_regs . The frequency of activated T_regs (CD4⁺ CD25⁺ FoxP3^high CD45RA^- T cells) as well as that of total T_regs (CD4⁺ CD25⁺ FoxP3⁺ T cells) in peripheral blood was significantly decreased in patients with AIN. Analysis of the T cell receptor (TCR)-Vβ repertoire of CD4⁺ T cells revealed skewed usages in patients with AIN compared with that observed in age-matched control subjects. Regarding T cell subsets, the use of four of 24 TCR-Vβ families in T_regs and one in conventional T cells were increased in patients with AIN. The number of patients with AIN who showed skewed usages of TCR-Vβ family in conventional and T_regs was significantly higher than that reported in control subjects. When the preference between T_regs and conventional T cells in each TCR-Vβ family was individually compared, different use was prominently observed in the TCR-Vβ 9 family in patients with AIN. These results suggest that the quantitative abnormalities of T_regs and the skew of the TCR-Vβ repertoire in CD4⁺ T cells, including T_regs and conventional T cells, may be related to autoantibody production through a human neutrophil antigen-reactive T cell clone.

Keywords: autoimmunity; children; neutropenia; regulatory T cell; repertoire.

Fig. 1

Flow cytometric analysis of regulatory T cells (T_regs) and T_reg subpopulations. CD4⁺CD25⁺forkhead box protein 3 (FoxP3)^high T cells are counted as total T_regs and CD4⁺CD25⁺FoxP3^highCD45RA⁻ T cells in CD4⁺ T cells (Fraction II in the T_reg subpopulations) are counted as activated T_regs (a). Data are presented in box‐plots which display the minimum value, 25th, 50^th and 75th maximum values and describe means as X (b). For the evaluation of T cell receptor (TCR)‐Vβ usage of each T cell subsets by flow cytometry, CD4⁺CD25⁻ T cells in CD4⁺ T cell are classified as conventional T cells (T_cons) and CD4⁺CD25⁺CD127^low T cells are classified as T_regs. Each T cell subset is sorted with fluorescein isothiocyanate (FITC) or phycoerythrin (PE)‐conjugated anti‐TCR‐Vβ family antibodies (c). The data for the percentage of each T cell subset (T_cons in CD4⁺ T cell, T_regs in CD4⁺ T cell) are presented as box‐plots (d). *P = 0·0123, **P = 0·0123, ***P = 0·0113; n.s. = not significant (Wilcoxon’s rank‐sum test).

Fig. 2

The usages of 24 T cell receptor (TCR)‐Vβ families in CD4⁺ T cells. Data are represented as box‐plots. Analysis was performed in the control group (control, n = 22) and autoimmune neutropenia (AIN) group (AIN, n = 17). *Vβ 9, P = 0·0252, **Vβ 17, P = 0·0361 (Wilcoxon’s rank‐sum test).

Fig. 3

The usages of 24 T cell receptor (TCR)‐Vβ families in regulatory T cells (T_regs) (CD4⁺CD25⁺CD127^low T cells) and conventional T cells (T_cons) (CD4⁺CD25⁻ T cells). Data are represented as box‐plots. Analysis was performed in the control group (control, n = 22) and autoimmune neutropenia (AIN) group (AIN, n = 17). Upper clonograph: TCR in T_regs. Lower clonograph; TCR in T_cons. *Vβ9‐T_reg; P = 0·0031, **Vβ17‐T_reg; P = 0·0234, ***Vβ20‐T_reg; P = 0·0218, ****Vβ21·3‐T_reg; P = 0·0262, *****Vβ17‐T_con; P = 0·0325 (Wilcoxon’s rank‐sum test).

Fig. 4

The increased/decreased usage of T cell receptor (TCR)‐Vβ families in CD4⁺CD25⁻ T cells and CD4⁺CD25⁺CD127^low T cells of patients with autoimmune neutropenia (AIN) and control subjects. Means and standard deviations (SDs) of TCR‐Vβ family usage of conventional T cells (CD4⁺CD25⁻ T cells) and regulatory T cells (CD4⁺CD25⁺CD127^low T cells) were calculated in the control group and used as normal values. Heat‐map classifies the degrees of each usage of TCR‐Vβ families using five colors; within normal range: −2 standard deviation (s.d.) – +2 s.d. (white), > +2 s.d. (pink), > +3 s.d. (red), < −2 s.d. (pale green), < −3 s.d. (green) (a). The increased and decreased numbers of CD4⁺CD25⁻ T cell and CD4⁺CD25⁺CD127^low T cells per person were compared. Data are represented as box‐plots (b). *P = 0·0022, **P = 0·0001 (Wilcoxon’s rank‐sum test); n.s. = not significant.

Fig. 5

The different usage of T cell receptor (TCR)‐Vβ families in individual conventional T cells (T_cons) and regulatory T cells (T_regs). Each line presents individual usage of TCR‐Vβ families in T_con and T_reg. *P < 0·05, **P < 0·01, ***P < 0·001, ****P < 0·0001 (Wilcoxon’s signed‐rank test).

Fig. 6

Preferential T cell subsets with higher usage of T cell receptor (TCR) Vβ families. Usage of TCR Vβ families was compared between conventional and regulatory T cells, and the T cell subsets with higher usage of TCR Vβ families were defined as preferential T cell subsets. Red color or pink color indicates a preferential subset, while blue color or light blue color indicates a decreased subset. White color indicates no preference between conventional and regulatory T cells. Con T, conventional T cell; Reg T, regulatory T cell (Wilcoxon’s signed‐rank test: pink‐light blue, P < 0·05; red‐blue, P < 0·001).