Thymus transplantation restores the repertoires of forkhead box protein 3 (FoxP3)+ and FoxP3- T cells in complete DiGeorge anomaly

Abstract

The development of T cells with a regulatory phenotype after thymus transplantation has not been examined previously in complete DiGeorge anomaly (cDGA). Seven athymic infants with cDGA and non-maternal pretransplantation T cell clones were assessed. Pretransplantation forkhead box protein 3 (Foxp3)(+) T cells were detected in five of the subjects. Two subjects were studied in greater depth. T cell receptor variable β chain (TCR-Vβ) expression was assessed by flow cytometry. In both subjects, pretransplantation FoxP3(+) and total CD4(+) T cells showed restricted TCR-Vβ expression. The development of naive T cells and diverse CD4(+) TCR-Vβ repertoires following thymic transplantation indicated successful thymopoiesis from the thymic tissue grafts. Infants with atypical cDGA develop rashes and autoimmune phenomena before transplantation, requiring treatment with immunosuppression, which was discontinued successfully subsequent to the observed thymopoiesis. Post-transplantation, diverse TCR-Vβ family expression was also observed in FoxP3(+) CD4(+) T cells. Interestingly, the percentages of each of the TCR-Vβ families expressed on FoxP3(+) and total CD4(+) T cells differed significantly between these T lymphocyte subpopulations before transplantation. By 16 months post-transplantation, however, the percentages of expression of each TCR-Vβ family became significantly similar between FoxP3(+) and total CD4(+) T cells. Sequencing of TCRBV DNA confirmed the presence of clonally amplified pretransplantation FoxP3(+) and FoxP3(-) T cells. After thymus transplantation, increased polyclonality was observed for both FoxP3(+) and FoxP3(-) cells, and pretransplantation FoxP3(+) and FoxP3(-) clonotypes essentially disappeared. Thus, post-transplantation thymic function was associated with the development of a diverse repertoire of FoxP3(+) T cells in cDGA, corresponding with immunological and clinical recovery.

© 2013 British Society for Immunology.

Figures

Figure 1

T cells in complete DiGeorge anomaly subjects. (a) T cell percentages (median values with ranges) are shown for complete DiGeorge anomaly subjects DIG405, DIG406, DIG409, DIG411, DIG413 and DIG416 (white bars) and six simultaneously assessed healthy adult controls (black bars). Historical median percentages [with 10th and 90th percentiles] for total and CD4+ T cells are provided for 3–6-month-old and 6–12-month-old infants . The percentages of total T cells, CD4+ T cells, naive (CD45RA+CD62L+) T cells and forkhead box protein 3 (FoxP3)+ CD4+ T cells were obtained using the first pretransplantation samples tested for FoxP3 expression after starting immunosuppression. Subject DIG113 was not assessed for FoxP3+ T cells by flow cytometry but had 79% CD3+ lymphocytes (< 3% expressing CD45RA and CD62L) when examined for FoxP3 expression by quantitative real-time polymerase chain reaction (PCR) analysis. (b) Absolute numbers of naive T cells in the peripheral blood are shown for all seven subjects before and after allogeneic thymus transplantation. (c) Flow cytometry results are displayed gated on CD3+ cells. Scatterplots for subjects DIG405, DIG406, DIG409, DIG411, DIG413 and DIG416 are shown in the top row. The examples given are the first pretransplantation samples tested for FoxP3 expression after starting immunosuppression (at 1·2, 6·5, 2·0, 2·6, 0·2 and 14·3 months pretransplantation, respectively). The FoxP3 expression analyses for the healthy adult controls tested simultaneously with the subject samples are displayed in the bottom row.

Figure 2

T cell receptor variable β chain (TCR-Vβ) repertoires in forkhead box protein 3 (FoxP3)+ and total CD4+ T cells. (a) TCR-Vβ repertoires of FoxP3+ CD4+ T cells (white bars) and total CD4+ T cells (black bars) were assessed by flow cytometry. The various TCR-Vβ families tested are shown along the horizontal axes. Data are shown for adult controls (means with standard error bars), subject DIG411 and subject DIG416. The times before (negative values) or after (positive values) thymus transplantation at which the samples were obtained are shown above each panel. (b) TCR-Vβ repertoires of FoxP3+ CD4+ T cells were compared to the repertoires of total CD4+ T cells. Ratios of the percentages of FoxP3+ to total CD4+ T cells expressing a given TCR-Vβ chain are displayed for various TCR-Vβ families (shown along the x-axes). The dotted lines demarcate 2 standard deviations above and below the mean (1·0) as determined from healthy adult controls. Data are shown for subjects DIG411 and DIG416 (open circles) and concurrently tested healthy adult controls (triangles). The times before or after thymus transplantation at which the samples were obtained are shown above each panel. Significant differences in TCR-Vβ expression between the FoxP3+ CD4+ and total T cell populations for each subject or control, defined as ratios outside the dotted lines, are designated (*P < 0·025; **P < 0·0001).