Increased circulating regulatory T cells (CD4(+)CD25 (+)CD127 (-)) contribute to lymphocyte anergy in septic shock patients

Abstract

Purpose: Sepsis syndrome represents the leading cause of death in intensive care unit. Patients present features consistent with a decline in immune responsiveness potentially contributing to mortality. We investigated whether CD4(+)CD25(+) regulatory T cells (Treg) participate in the induction of lymphocyte anergy after sepsis.

Method: Observational study in septic shock patients and experimental study in mice.

Results: We took advantage of the recently described flow cytometric gating strategy using the measurement of CD25 and CD127 expressions for monitoring Treg (CD4(+)CD25(+)CD127(-)Foxp3(+)). In patients the increased circulating Treg percentage significantly correlated with a decreased lympho-proliferative response. In a murine model of sepsis mimicking these observations, the ex vivo downregulation of Foxp3 expression using siRNA was associated with a restoration of this response.

Conclusion: The relative increase in circulating Treg might play a role in lymphocyte anergy described after septic shock and represent a standardizable surrogate marker of declining proliferative capacity after sepsis.

Fig. 1

Flow cytometry phenotyping of whole blood lymphocytes from healthy individuals and septic shock patients. EDTA-anticoagulated blood was harvested from healthy individuals and septic shock patients. Cells were stained using 3-color flow cytometry staining (CD4-ECD–CD25-PC5–CD127-PE) or 4-color flow cytometry staining (CD4-ECD–CD25-PC5–CD127-PE–Foxp3-FITC). Red blood cells were lysed using the automated TQ-Prep lysing system or Versalyse reagent. After gating on CD4⁺ lymphocytes selected based on a SSC/CD4 dot-plot, CD4⁺CD25⁺CD127⁻ cells were selected based on a CD25/CD127 2-color dot-plot (left panel). The percentage of Foxp3 expressing cells among this population was subsequently measured (right panel). One representative experiment for healthy individuals (a) and septic shock patients (b) are presented

Fig. 2

The percentage of CD4⁺CD25⁺CD127⁻ cells is increased in whole blood of septic patients in comparison with healthy individuals. EDTA-anticoagulated blood was harvested from 17 healthy individuals (HI, open boxes and circles) and 30 septic shock patients (SS, gray boxes and circles). Cells were stained using 3-color flow cytometry staining (CD4-ECD–CD25-PC5–CD127-PE). Red blood cells were lysed using automated TQ-Prep lysing system. Absolute count measurements were performed using Flow-count fluorospheres™. Results are presented as individual values and box-plots with 5th/95th percentiles. Non-parametric Mann Whitney U test was used for comparison between groups (*P < 0.05, **P < 0.001). a Percentage of CD4⁺CD25⁺CD127⁻ lymphocytes measured among CD4⁺ cells. b Absolute count of peripheral CD4⁺ T lymphocytes (cells/µl of whole blood). c Absolute count of peripheral CD4⁺CD25⁺CD127⁻ T lymphocytes (cells/µl of whole blood). d Absolute count of peripheral CD4⁺CD25⁻CD127⁺ T lymphocytes (cells/µl of whole blood)

Fig. 3

A decreased cell proliferation is associated with an increased circulating percentage of CD4⁺CD25⁺CD127⁻ cells in septic shock patients. EDTA-anticoagulated blood was harvested from healthy individuals (n = 2) and septic shock patients (n = 4). Flow cytometry: Whole blood cells were stained using 3-color flow cytometry staining (CD4-ECD–CD25-PC5–CD127-PE). Red blood cells were lysed using automated TQ-Prep lysing system. Results are expressed as percentages of CD4⁺CD25⁺CD127⁻ lymphocytes measured among CD4⁺ cells (y-axis, gray bars: septic patients, open bars: healthy individuals). Cell proliferation: Peripheral blood mononuclear cells (PBMCs) were harvested using Ficoll-Paque Plus gradient centrifugation and cultured in complete RPMI medium (2 × 10⁵ cells/200 µl) with phytohaemagglutinin (PHA 10 µg/ml, black circles) or concanavalin A (ConA, 0.1 mg/ml, black triangles) or pokeweed (PWD, 20 µg/ml, black crosses). Proliferation was measured by the increased incorporation of [methyl-³H]-Thymidine. The assays were carried out in triplicate. Results are expressed as counts per minute (cpm, z-axis). Normal values from the laboratory: PHA and ConA > 15,000 cpm, PWD > 5,000 cpm

Fig. 4

Transfection of CD4⁺CD25⁺Foxp3⁺CD127⁻ regulatory T cells with Foxp3 siRNA is associated with recovery of splenocytes proliferative capacity in septic mice. Murine splenocytes were harvested 24 h after the induction of a polymicrobial septic challenge (cecal ligation and puncture model: CLP, n = 17) or from sham/control animals (Sh, n = 17). Immediately after harvesting, 2 × 10⁶ cells were transfected with 2 µM Foxp3 siRNA or control siRNA or left untouched. a Cells were stained using 3-color flow cytometry staining (CD4-PECy7–CD25-PE–Foxp3-APC) 16 h after transfection or not with siRNA. Results are presented as mean ± SEM [black bar: septic mice, light gray bar: septic mice after transfection with Foxp3 siRNA, dark gray bar: septic mice after transfection with control (CTRL) siRNA]. b After overnight incubation, cells were stained with PKH26 and cultured in 96-well plates in RPMI with or without concanavalin A (0.1 mg/ml, 1 × 10⁶ cells/ml) for 5–6 days. Proliferation was assessed by flow cytometric measurement of the decrease in PKH26 (yellow) fluorescence on viable cells selected based on SSC/FCS characteristics. Results are presented as mean ± SEM of proliferation ratio [i.e. (percentage of cells with decreased fluorescence/percentage of highly fluorescent cells) × 100] measured on splenocytes from sham animals (open bar) or from mice after CLP transfected or not (black bar) with Foxp3 (light-gray bar) or control (CTRL) siRNA (medium-gray bar). The Student t test was used for comparison between groups (*P < 0.05) and the Wilcoxon Signed Ranked test was used for comparison between paired values (^#P < 0.05) 682