Characterization of the T Cell Response to Lactobacillus casei Cell Wall Extract in Children With Kawasaki Disease and Its Potential Role in Vascular Inflammation

Figures

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  Figure 1. Activation of circulating CD4+ and CD8+ T cells in response to LCWE. PBMC from KD subjects (n = 7) 2 to 4 weeks after disease onset, healthy children (n = 9), and healthy adults (n = 7) were stimulated with 1 and 10 μg/ml of LCWE for 4 days and the percent activated CD4+ HLA-DR+ (A) and activated CD8+ HLA-DR+ (B) T cells were enumerated by flow cytometry with specific monoclonal antibodies. Unstimulated PBMC cultures served as controls in these experiments. A representative FACS plot of HLA-DR expression on CD4+ and CD8+ T cells for each cohort is shown. Percent increase of CD4+ HLA-DR+ (C) and CD8+ HLA-DR+ (D) T cells in response to 1 μg/ml (dark symbols) and 10 μg/ml (light symbols) of LCWE vs. un-stimulated control. The data are shown with median and interquartile range. Each symbol is the result derived from each subject tested. The statistical differences between cohorts were tested by one-way ANOVA. A P < 0.05 was considered significant. The results suggested that LCWE stimulates CD4+ (1 μg/ml, p = 0.5564; 10 μg/ml, p = 0.6553) and CD8+ (1 μg/ml, p = 0.3947; 10 μg/ml, p = 0.3405) T cells in the 3 cohorts with no statistical differences.

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  Figure 2. IL-2 and IFNγ production by T cells in response to LCWE. To validate the antigen-specific response to LCWE, we measured IL-2 (A) and IFNγ (B) secretion in culture supernatants. CD4+ and CD8+ T cells secreted IFNγ, suggesting a pro-inflammatory T helper 1 and cytotoxic functional phenotype in response to LCWE.

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  Figure 3. CD4+ CD25high Treg expansion in response to LCWE. To address peripherally-induced Treg responses to LCWE, we studied CD4+ CD25high Treg expansion in PBMC stimulated with 1 and 10 μg/ml of LCWE for 4 days in 3 subacute KD subjects 3 weeks after disease onset (KD8–10, Table 1), 5 healthy children (HC5–9, Table 1), and 3 healthy adults (ND8–10, Table 1). Unstimulated PBMC served as control (A) Representative CD4+ CD25high T cell enumeration and IL-7R expression is shown. Gray: un-stained control. Culture supernatants derived from the same PBMC cultures were tested for IL-10 (B) and IL-17 (C) secretion by ELISA. The statistical differences of the Treg responses between cohorts were tested by one-way ANOVA. A P < 0.05 was considered significant. The results suggested that LCWE stimulates Treg (1 μg/ml, p = 0.0974; 10 μg/ml, p = 0.1960) in the 3 cohorts with no statistical differences.

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  Figure 4. Enumeration of effector and central memory LCWE-specific CD4+ and CD8+ T cells. PBMC from subacute KD subjects (n = 3; KD 5–7, Table 1), healthy children (n = 5; HC 5–9, Table 1), and healthy adults (n = 2; ND3–4, Table 1) were stimulated with LCWE (1 μg/ml) for 4 days and studied for the expression of IL-15R and CCR7+ on CD4+ HLA-DR+ (A) and CD8+ HLA-DR+ (B) T cells were studied by flow cytometry to enumerate central memory (IL15R+ CCR7+) and effector memory (IL-15+ CCR7-) T cells. A representative FACS plot of IL-15R+ CCR7+ central memory T cells and IL-15R+ CCR7- effector memory T cells from each cohort is shown. In all 3 cohorts, both CD4+ and CD8+ memory T cells were detectable suggesting previous and repeated exposure to the antigen. As expected, effector memory T cells were less numerous than central memory T cells (1.43–22.4% vs. 1.08–12.3% CD4+ T cells and 1.59–43.9% vs. 1.06–19.4% CD8+ T cells, respectively). The data are shown with median and interquartile range. Each symbol is the result derived from each subject tested. The statistical differences between cohorts were tested by one-way ANOVA. A P < 0.05 was considered significant. No difference of CD4+ (central…

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  Figure 5. CCR6 and CD31 expression on activated LCWE-specific CD4+ and CD8+ T cells. To address the ability of LCWE-specific CD4+ and CD8+ T cells to home to the vessels (CCR6) and their potential role in the pathogenesis of vascular inflammation, the expression of CCR6 and CD31 on activated LCWE-specific T cells was studied. PBMC from subacute KD subjects (n = 3; KD 5–7, Table 1), healthy children (n = 5; HC 5–9, Table 1), and healthy adults (n = 2; ND3–4, Table 1) were stimulated with LCWE (1 μg/ml) for 4 days and the expression of CCR6 and CD31 on CD4+ HLA-DR+ (A) and CD8+ HLA-DR+ (B) T cells were studied by flow cytometry. A representative FACS plot of CCR6+ and CCR6+ CD31+ T cells from each cohort is shown. Activated CD4+ T cells showed a higher prevalence of CCR6+ CD31- (4.85–32.3%) than CCR6+ CD31+ (0.36–12.6%) suggesting that if these cells homed to the vessels, they could promote inflammation. Activated CD8+ T cells showed higher expression of CD31+ within the CCR6+ T cells than CD4+ T cells (7.38–39.1%) vs. CD31- (2.87–8.87%). The data are shown as scatter dot plots with median and interquartile range. Each symbol represents the results derived from each subject tested. Closed…