IL-7 complexes treatment induced both conventional and regulatory T cell expansion.

<p>C57Bl/6 mice were i.p. injected with PBS or IL-7/anti-IL-7 complexes (1 µg rmIL-7 plus 5 µg M25) three times at 2 day intervals. LN and SP cells were analyzed 7 days after the first injection. (A) Representative dot plots of CD25 vs FOXP3 expression on CD4⁺TCRβ⁺ cells recovered from PBS or IL-7 complexes treated mice. (B) Total number of CD4⁺TCRβFOXP3⁻ conventional T cells (left panels) and CD4⁺ TCRβ FOXP3⁺ regulatory T cells (right panels) from lymph nodes and spleen.</p

IL-7 induced expansion of both conventional and regulatory T cells is thymic independent.

<p>CFSE labeled total CD4 T cells from CD45.1 mice were transferred into CD45.2 C57Bl/6 hosts. Mice were then treated with IL-7/anti-IL-7 complexes as in Fig. 2. (A) Representative dot plots of CD45.1 vs FOXP3 expression on CD4⁺ TCRβ cells recovered from lymph nodes and spleens of PBS or IL-7 complexes treated mice. (B) Total numbers of adoptively transferred CD45.1 FOXP3⁻ conventional T cells (left panels) and CD45.1 FOXP3⁺ regulatory T cells (right panels) from lymph nodes and spleen.</p

Baseline Characteristics and outcome of newborns.

<p>Continuous data are expressed as median and interquartile range [IQR] and categorical data as number (%).</p><p>ELBW, extremely low birth weight born before 28 wks of gestational age; VLBW, very low birth weight born between 28 and 32 wks of gestational age, WBC, white blood cell; IVH, intraventricular haemorrhage. †, WBC count was performed in term newborn if there was an infectious risk at delivery (WBC count performed in ELBW, N = 13; VLBW, N = 24; and in term newborns, N = 8). Four premature infants (ELBW, N = 3; VLBW, N = 1) did not receive pulmonary maturation with antenatal corticosteroid therapy.</p

Phagocytosis of bacteria by newborn and adult neutrophils, and plasma opsonic activity.

<p>(A) Phagocytosis of fluorescent <i>E.coli</i>, <i>S.aureus</i>, and <i>S.epidermidis</i> by neutrophils from one representative subject from each group (ELBW, extremely low birth weight premature infants born before 28 wks of gestational age; VLBW, very low birth weight premature infants born between 28–32 wks of gestational age; TN, term newborns; and CA, control adults). Phagocytosis was measured by flow cytometry after 10 min (red lines) and 20 min (green lines) incubation with bacteria opsonised with autologous plasma. Neutrophils pre-incubated with the phagocytosis blocker cytochalasin D are presented with filled curves (negative control). (B) The opsonic capacity of patient's plasma using neutrophil-like human HL-60 cells is tested. Fluorescent <i>E.coli</i>, <i>S.aureus</i>, and <i>S.epidermidis</i> were opsonised with autologous plasma and incubated 40 min with DMSO-differentiated HL-60 cells. Intracellular fluorescence (phagocytosis of bacteria) was measured by flow cytometry and expressed as mean phagocytic index (± SEM) of the four groups (ELBW, extremely low birth weight premature infants born before 28 wks of gestational age N = 21; VLBW, very low birth weight premature infants born between 28–32 wks of gestational age, N = 24; TN, term newborns, N = 25; CA, control adults, N = 20). On each line, a reference group (*) is compared to other groups and respective P value expressed using Mann-Whitney <i>U</i> test.</p

LPS responsiveness of leukocytes and opsonic capacity of plasma from premature infants with and without the addition of interferon-γ.

<p>(A) Whole blood from 7 extremely premature infants (born before 28 wks of gestational age) was treated <i>ex vivo</i> or not for 12 hrs with interferon (IFN)-γ. LPS was then added for an additional 12 hrs and IL-6, TNF-α, and IL-10 were measured (mean ± SEM) in conditioned plasma. (B) The plasma from whole blood obtained in seven extremely premature infants (born before 28 wks of gestational age) treated <i>ex vivo</i> or not with for 12 hrs with IFN-γ was incubated with fluorescent <i>E.coli</i>, <i>S.aureus</i> and <i>S.epidermidis</i> (opsonisation). Opsonised bacteria were then incubated with neutrophil-like HL-60 cells for 40 min; intracellular fluorescence was measured by flow cytometry, and expressed as mean phagocytic index (± SEM). Statistical significance was tested with Wilcoxon matched-pairs signed rank test.</p

TLR2, TLR4, CD14, and MD-2 surface expression of blood leukocytes and plasma soluble MD-2 activity from premature infants, term newborns, and control adults.

<p>(A) Representative flow cytometry plot showing forward and side-scatter characteristics gating used to identify neutrophils (R1), and monocytes (R2). (B) Surface expression of TLR4, MD-2, CD14 and TLR2 of phagocytes (neutrophils and monocytes) from extremely low birth weight infants (ELBW, born before 28 wks of gestational age, N = 18), very low birth weight infants (VLBW, born between 28–32 wks of gestational age, N = 17), term newborn (TN, N = 25), and control adult (CA, N = 20). In addition the expression of the Fcγ receptor CD16 on neutrophils and the major histocompatibility HLA-DR on monocytes are shown. Receptor expression was measured by flow cytometry, and expressed as mean fluorescence index (MFI, geo mean receptor/geo mean IgG control). Errors bars are means ± SEM, On each line, a reference group (*) is compared to other groups and respective P value expressed using Mann-Whitney <i>U</i> test. (C) Plasma soluble MD-2 activity was measured as the capacity of plasma to support TLR4-HEK293 cell activation after a 30 ng/mL LPS challenge <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032863#pone.0032863-Pugin3" target="_blank">[50]</a>. Human recombinant soluble MD-2 (1 µg/mL) was used as a positive control. (ELBW, extremely low birth weight premature infants born before 28 wks of gestational age N = 20; VLBW, very low birth weight premature infants born between 28–32 wks of gestational age, N = 20; TN, term newborns, N = 20; CA, control adults, N = 20). Errors bars are means ± SEM.</p