T cell receptor Vbeta repertoire of T lymphocytes and T regulatory cells by flow cytometric analysis in healthy children

Abstract

Evaluation of the T cell receptor (TCR) Vbeta repertoire by flow cytometric analysis has been used for studying the T cell compartments for diseases in which T cells are implicated in the pathogenesis. For the interpretation of these studies information is needed about Vbeta usage in healthy individuals and there are few data for normal usage in paediatric populations. We examined the T lymphocyte (sub)populations in 47 healthy controls (age range: 3 months-16 years). We found non-random Vbeta usage with skewed reactivity of some families towards CD4+ or CD4- T cells. Importantly, there appeared to be no significant change in Vbeta usage according to age group. Some controls showed expansions in some Vbeta families, although incidence of such expansions was low. We went on to examine the repertoire of CD4+CD25(Bright) T regulatory cells in 25 healthy controls. We found overlapping quantitative usage for each of the Vbeta families between CD4+CD25- and CD4+CD25(Bright) T cells. However, there was a significant preferential usage for five Vbeta families and decreased usage of two Vbeta families in the CD4+CD25(Bright) T cells, suggesting that although they overlap there may be subtle but important differences in the TCR repertoire of T regulatory cells.

Fig. 1

Forward- and side-scatter (FSC/SSC) pattern of lysed whole blood (a) and gate settings for CD3⁺ (b), CD3⁺CD4⁺ and CD3⁺CD4^– lymphocytes (c). Cell staining for T cell receptor (TCR) families Vβ 5·3 [phycoerythrin (PE)], Vβ 3 [fluorescein isothiocyanate (FITC)] and Vβ 7·1 (PE and FITC) in the CD3⁺CD4⁺ lymphocytes (d).

Fig. 2

(a) To discriminate between CD25^Bright T regulatory and CD25+ activated effector-memory T cells in separated peripheral blood mononuclear cells (PBMCs) we used CD25 expression on CD4^– cells as an internal control. Only CD4⁺ cells expressing CD25 with higher intensities than the CD4^– cells were included in the gate for CD25^Bright cells. (b) To gate for forkhead box P3 (FoxP3) expression in PBMCs we used CD4^– cells as an internal control. Only CD4⁺ cells expressing FoxP3 with higher intensities than the CD4^– cells were considered FoxP3 positive. (c) Percentage of FoxP3 positivity for cells through the CD4⁺CD25^Bright gate. (d) Percentage of FoxP3 positivity for cells through the CD4⁺CD25^– gate.

Fig. 3

Schematic overview showing mean values of all controls for Vβ positivity for T cell receptor (TCR) αβ⁺CD3⁺ T lymphocytes in CD4⁺ and CD4^– subpopulations using a panel of 24 TCR Vβ monoclonal antibodies. *Vβ families with skewed reactivity toward CD4⁺ or CD4^– T cells (P < 0·001).

Fig. 4

Means ± 1 standard deviation for Vβ positivity within the CD3⁺ T cell receptor-αβ⁺ for each of the five age groups. Within each Vβ family there are five sets of values representing (from left to right) 3–9 months, > 9–24 months, > 24 months−5 years, > 5–10 years and > 10–16 years. There were no statistical differences between age groups for each Vβ family.