LPS dose-dependent reduction of IFNg secretion by T cells in response to OKT3 stimulation.

<p>PBMC were primed overnight with 1 ng/mL, 10 ng/mL or 100 ng/mL LPS. Then PBMC were stimulated with 25 ng/mL OKT3 for 72h before the surpernatant was recovered for IFNγ measurement. Each experiment was performed on <i>n</i> = 4 HV. Technical replicates were summarized by the mean for each individual. For each condition, individual values are summarized on the plots by a horizontal line representing the mean of a given condition.</p

LPS dose-dependently decreases T cell proliferative response to OKT3.

<p>PBMC were primed overnight with LPS concentrations ranging from 0.01 ng/mL to 100 ng/mL (in case of OKT3 stimulation) or from 0.1 ng/mL to 1000 ng/mL (in case of anti-CD2/CD3/CD28-coated beads and PHA stimulations). Then PBMC were stimulated with 4 μg/mL PHA (<b>A</b>), 0.5x10⁶ anti-CD2/CD3/CD28-coated beads/mL (<b>B</b>), or 25 ng/mL OKT3 (<b>C</b>) for 72h. T cell proliferation was measured by flow cytometry and results are expressed as percentages of proliferating cells among total T cells. Each experiment was performed on n = 3 to 4 HV. Technical replicates were summarized by the mean for each individual. For each condition, individual values are summarized on the plots by a horizontal line representing the mean of a given condition.</p

LPS priming alters monocyte T cell accessory cell function but not T cell receptor expression.

<p>PBMC were treated with medium or 10 ng/mL LPS and flow cytometry phenotyping was performed every 2h for 8h. Four markers were studied on monocytes (<b>A</b>): CD64 (<b>A1</b>), HLA-DR (<b>A2</b>), CD86 (<b>A3</b>) and CD80 (<b>A4</b>). For each marker, mean fluorescence intensities (MFI) on CD14⁺ cells were measured. Four markers were studied on T cells (<b>B</b>): CD3 (<b>B1</b>), CD28 (<b>B2</b>), CTLA-4 (<b>B3</b>) and PD-1 (<b>B4</b>). For each marker, mean fluorescence intensities (MFI) on CD3⁺ cells were measured. MFI ratio were calculated as follow = MFI of LPS treated cells divided by the MFI of the medium-treated cells at the same time point. The experiments were performed on <i>n</i> = 4 HV. Technical replicates were summarized by the mean for each individual. For each condition, individual values are summarized on the plots by a horizontal line representing the mean of a given condition.</p

LPS decreases only T cell proliferative response to OKT3.

<p>PBMC were first treated with medium (white points) or primed with 10 ng/mL LPS (black points), then cells were stimulated with 0.1 to 4 μg/mL PHA (<b>A</b>), or 63x10³ to 500x10³ anti-CD2/CD3/CD28-coated beads /mL (<b>B</b>), or 5 to 50 ng/mL OKT3 (<b>C</b>) for 72h. T cell proliferation was measured by flow cytometry and results are expressed as percentages of proliferating cells among total T cells. Each experiment was performed on <i>n</i> = 4 HV. Technical replicates were summarized by the mean for each individual. For each condition, individual values are summarized on the plots by a horizontal line representing the mean of a given condition.</p

Monocytes mediate LPS induced decrease of T cell proliferative response to OKT3.

<p>PBMC (open diamonds) and T cells (<b>A</b>—black diamonds) or monocyte depleted PBMC (<b>B</b>- black diamonds) were, first, primed overnight by 10 ng/mL LPS, then, stimulated 72h with 25 ng/mL OKT3. T cell proliferation was measured by flow cytometry and results are expressed as percentages of proliferating cells among total T cells. This experiments were performed on <i>n</i> = 2 or 4 HV. Technical replicates were summarized by the mean for each individual. For each condition, individual values are summarized on the plots by a horizontal line representing the mean of a given condition. <b>C-</b> T cells and monocytes were isolated from each HV and were incubated separately overnight in medium alone or with 10 ng/mL LPS. After thorough PBS wash of each fraction, primed or unprimed T cell fractions were mixed with primed or unprimed monocyte fractions (0.5x10⁶ monocytes/mL and 1.5x10⁶ T cells–every combination was tested), and stimulated or not with 25 ng/mL OKT3 for 72h before T cell proliferation measurement. T cell proliferation was measured by flow cytometry and results are expressed as percentages of proliferating cells among total T cells. Similar experiment with total PBMC from each HV was performed in parallel as control. <b>D-</b> Results on purified monocytes and lymphocytes are presented. The different conditions of LPS priming of monocytes and / or T cells are underlined under the graph. Open circles represent results for cell culture conditions with no T cell stimulation. Black circles represent results after 25 ng/mL OKT3 stimulation for 72h. The experiment was performed on <i>n</i> = 5 HV. Technical replicates were summarized by the mean for each individual. For each condition, individual values are summarized on the plots by a horizontal line representing the mean of a given condition.</p

IFNγ partially restores monocyte accessory cell function.

<p>PBMC were incubated for 12h with medium or 10 ng/mL LPS, washed, and treated with medium or 100 ng/mL IFNγ. MFI of either CD64 (<b>A</b>), HLA-DR (<b>B</b>), CD86 (<b>C</b>) or CD80 (<b>D</b>) on CD14⁺ cells were measured, and MFI ratios calculated by dividing the MFI of treated cells by the MFI of control cells (no treatment). The experiment was performed on <i>n</i> = 4 HV. Technical replicates were summarized by the mean for each individual.</p

Additional file 1: Figure S1. of Automated bedside flow cytometer for mHLA-DR expression measurement: a comparison study with reference protocol

Flow chart and additional methods. (DOCX 67 kb

IFNγ partially restores T cell proliferative response to OKT3.

<p>PBMC were treated or not overnight with 10 ng/mL LPS, washed and then treated simultaneously or not with 100 ng/mL IFNγ and 25 ng/mL OKT3 for 72 h before T cell proliferation measurement (Black circles: cells primed with LPS and treated with IFNγ+OKT3; open circles: cells primed with LPS and treated with OKT3 but with no IFNγ treatment). T cell proliferation was measured by flow cytometry and results are expressed as percentages of proliferating cells among total T cells. In addition, results are normalized versus proliferation measured after OKT3 stimulation without any LPS priming or IFNγ treatment (control condition). Results are expressed as percentages to the reference proliferative response. The experiments were performed on <i>n</i> = 6 HV. Technical replicates were summarized by the mean for each individual. For each condition, individual values are summarized on the plots by a horizontal line representing the median of a given condition.</p

Survival after bacterial challenges.

<p>(A) During the first septic assault, after CLP and prior to <i>P</i>. <i>aeruginosa</i> instillation (D5), 28% of CLP-operated mice (22/80) died. During this period, 100% of Sham-operated mice (n = 29) survived. (B) At D5 post CLP, mice were challenged with an intratracheal administration of <i>P</i>. <i>aeruginosa</i>. Since CLP mice died until 5 days, final survival data were given by subtracting lethality induced by CLP during the first assault. So we considered the remaining living mice as 100% for this second survival study. Either PBS (Sham n = 6, CLP n = 10) or <i>Pseudomonas aeruginosa</i> were instilled. For bacterial challenge, three doses were administered: 5.10⁶ CFU (Sham n = 6 and CLP n = 15), 2.10⁷ CFU (Sham n = 10 and CLP n = 17) and 10⁸ CFU (Sham n = 7 and CLP n = 20). Results are expressed as Kaplan Meier survival curves. p < 0.05 were considered statistically significant.</p

Study protocol.

<p>Mice were Sham- or CLP-operated at D0. Five days after surgery (D5), mice were or were not intratracheally instilled with <i>P</i>. <i>aeruginosa</i>. Plasmatic cytokines were measured at 2h, 6h, D1, D2, D3, D5 and D13 and also at D6 and D7 only for instilled mice.</p