Summary of participant characteristics and pre-vaccination Treg values.

<p>Summary of participant characteristics and pre-vaccination Treg values.</p

CONSORT flow diagram.

<p>CONSORT flow diagram.</p

Effect of vaccination of humans with a subunit hepatitis B vaccine on Treg frequency and characteristics.

<p>At day 0, day 28 and day 168, healthy adults were i.m. vaccinated with Engerix-B^® or injected with a placebo. At day 1, 3, and 14 after the third immunization dose (day 169, 171 and 182) changes in Treg frequency and phenotype were determined. The delta Treg percentage per time point was determined per donor (= % Treg day_x—% Treg day₁₆₈). <b>(A)</b> Mean (± SEM) delta percentage of the frequency of rTreg (left) and aTreg (right) in the blood at the different time points after the third dose of Engerix-B^® (n = 20) or placebo (n = 4). <b>(B)</b> Mean (± SEM) delta percentage of CD39 expression on aTreg after vaccination. Means were statistically compared with an unpaired, two-tailed Mann-Whitney test (* p < 0.05, ** p < 0.01, relative to placebo at the same time point).</p

Effect of vaccination of humans with a live attenuated yellow fever vaccine on Treg frequency and characteristics.

<p>At day 0, healthy adults were s.c. vaccinated with Stamaril^® or injected with a placebo. At day 1, 5, 7 and 14 post vaccination changes in Treg frequency and phenotype were determined. The delta Treg percentage per time point was determined per donor (= % Treg day_x—% Treg day₀). <b>(A)</b> Mean (± SEM) delta percentage of the frequency of rTreg (left) and aTreg (right) in the blood at the different time points after vaccination with Stamaril^® (n = 19) or placebo (n = 8). <b>(B)</b> Mean (± SEM) delta percentage of CD25 and CD31 expression on rTreg after vaccination. <b>(C)</b> Mean (± SEM) delta percentage of CD25, CD31 and HLA-DR expression on aTreg after vaccination. Means were statistically compared with an unpaired, two-tailed Mann-Whitney test (* p < 0.05 relative to placebo at the same time point).</p

Effect of vaccination of humans with subunit influenza vaccines on Treg frequency.

<p>At day 0, healthy adults were i.m. vaccinated with a non-adjuvanted (Agrippal^®) or MF59^®-adjuvanted (Fluad^®) trivalent influenza subunit vaccine or injected with a placebo. At day 1, 3, 5, 14 and 21 post vaccination changes in Treg frequency were determined. The delta Treg percentage per time point was determined per donor (= % Treg day_x—% Treg day₀). Mean (± SEM) delta percentage of the frequency of rTreg (left) and aTreg (right) in the blood at the different time points after vaccination with Agrippal^® (n = 12), Fluad^® (n = 8, day 14 n = 2) or placebo (n = 6). Means were statistically compared with a Kruskal-Wallis test.</p

Effect of vaccination on local and systemic Treg frequency in mice.

<p>At day 0, mice were i.m. injected with LPS, CFA or with one of the vaccines (TIV⁺, TIV^- and Engerix-B^®). Mock-injected animals received PBS. At day 3, 7, 14 and 21 post vaccination changes in Treg frequency were determined. The delta Treg percentage per time point was determined by comparing the treatment with the mean of PBS (= % Treg treatment day_x—average % Treg PBS day_x). The mean percentage Treg in PBS-injected mice was 10.9 ± 1.4% (dLN) and 12.0 ± 1.5% (spleen). N = 3–7 mice per group. <b>(A-D)</b> Mean (± SEM) delta percentage of Treg (CD4⁺ CD25⁺ Foxp3⁺) frequency at the different time points after vaccination were analyzed in dLN (A and C) and spleen (B and D) after LPS injection (A and B) and CFA or vaccine injection (C and D). Differences between PBS and LPS (A and B) were determined with an unpaired two-tailed student’s t-test with Welch’s correction. Differences between PBS and vaccines (C and D) were determined with a one-way ANOVA followed by Dunnett’s multiple comparisons test (* p < 0.05, ** p < 0.01, *** p < 0.001 relative to PBS at the same time point). TIV⁺: TIV supplemented with MF59^® adjuvant; TIV^-: TIV only; ENG: Engerix-B^®</p