Memory T-cells and characterization of peripheral T-cell clones in acute Kawasaki disease

Abstract

Kawasaki disease (KD) is a pediatric self-limited vasculitis characterized by immune-mediated destruction of the arterial wall and myocardium. Neither the trigger that incites the inflammation nor the switch that turns it off is known. To further our understanding of KD pathogenesis and the role of regulatory T-cells in modulating the inflammatory response, we studied circulating effector memory T-cells (CCR7- and IL-15+ T(em)) and central memory T-cells (CCR7+ and IL-15+ T(cm)) in six KD subjects. In two of the subjects, we cloned the remaining T-cell population by limiting dilution. TaqMan analysis of T(em) studied in two KD subjects suggested that T(em) are pro-inflammatory CD4+T-helper 1 cells and CD8+ cytotoxic T-cells. Following memory T-cells over time, we defined that circulating T(em) and T(cm) are detectable during the acute phase in some KD subjects before treatment with intravenous immunoglobulin. Both T(em) and T(cm) expand rapidly within 2 weeks of treatment. The circulating T(em) pool contracts, while T(cm) further proliferate in the convalescent phase. Following depletion of memory T-cells, numerous T-cell clones were derived from two acute KD subjects. The large majority of these T-cells displayed the functional phenotype of peripherally induced regulatory T-cells (T(reg)). These findings provide insight into the nature and kinetics of the adaptive immune response in KD.

Figure 1

T^em isolation and characterization in two acute KD subjects. (A) CCR7-negative cells were FACS sorted to obtain CD4+ and CD8+ IL-15r+ T-cells. (B) mRNA levels of TGF-β, IL-4, IL-10, FoxP3, IFN-γ, and IL-17 measured after 6 h stimulation with anti-CD3 using TaqMan 5′-nuclease gene expression assays.

Figure 2

T^em and T^cm in four acute, subacute, and convalescent KD subjects. (A) Analysis of the IL-15+ T-cells within the CCR7− population. (B) Analysis of IL-15+ T-cells within the CCR7+ population.

Figure 3

Characterization of Treg clones in acute KD. Lymphokine profile and FoxP3 mRNA transcript levels of three CD4+ T-cell clones derived from subject 1 and six CD4+ T-cell clones derived from subject 2 demonstrating a Treg phenotype. Cytokine levels (TGF-β, IL-10, and IL-4) in culture supernatants were determined using ELISA and are shown in (A), (B), and (C). FoxP3 mRNA transcript levels are shown in (D).

Figure 4

TGF-β secretion by a Th-1 clone derived from subject 1. Lymphokine profiles measured by ELISA and FoxP3 RNA transcript levels of a CD4+ T-cell clone derived from subject 1 on day 4 of illness which produced both TGF-β and IFN-γ.

Figure 5

Functional characterization of two CD8+ Treg clones derived from subject 2. Lymphokine profiles measured by ELISA (panel A) and FoxP3 RNA transcript levels (B) of two CD8+ T-cell clones, derived from subject 2 on day 8 of illness which produced IFN-γ and TGF-β.

Figure 6

Ex vivo Treg frequency in bulk PBMC. TGF-β, IL-10, and FoxP3 RNA transcript levels have been measured in bulk PBMC derived from subject 1 and subject 1 before T-cell cloning.