Phenotype and Functions of Natural Killer Cells in Critically-Ill Septic Patients

<div><h3>Rationale</h3><p>Natural killer cells, as a major source of interferon-γ, contribute to the amplification of the inflammatory response as well as to mortality during severe sepsis in animal models.</p> <h3>Objective</h3><p>We studied the phenotype and functions of circulating NK cells in critically-ill septic patients.</p> <h3>Methods</h3><p>Blood samples were taken <48 hours after admission from 42 ICU patients with severe sepsis (<em>n</em> = 15) or septic shock (<em>n</em> = 14) (Sepsis group), non-septic SIRS (<em>n</em> = 13) (SIRS group), as well as 21 healthy controls. The immuno-phenotype and functions of NK cells were studied by flow cytometry.</p> <h3>Results</h3><p>The absolute number of peripheral blood CD3–CD56⁺ NK cells was similarly reduced in all groups of ICU patients, but with a normal percentage of NK cells. When NK cell cytotoxicity was evaluated with degranulation assays (CD107 expression), no difference was observed between Sepsis patients and healthy controls. Under antibody-dependent cell cytotoxicity (ADCC) conditions, SIRS patients exhibited increased CD107 surface expression on NK cells (62.9[61.3–70]%) compared to healthy controls (43.5[32.1–53.1]%) or Sepsis patients (49.2[37.3–62.9]%) (p = 0.002). Compared to healthy (10.2[6.3–13.1]%), reduced interferon-γ production by NK cells (K562 stimulation) was observed in Sepsis group (6.2[2.2–9.9]%, p<0.01), and especially in patients with septic shock. Conversely, SIRS patients exhibited increased interferon-γ production (42.9[30.1–54.7]%) compared to Sepsis patients (18.4[11.7–35.7]%, p<0.01) or healthy controls (26.8[19.3–44.9]%, p = 0.09) in ADCC condition.</p> <h3>Conclusions</h3><p>Extensive monitoring of the NK-cell phenotype and function in critically-ill septic patients revealed early decreased NK-cell function with impaired interferon-γ production. These results may aid future NK-based immuno-interventions.</p> <h3>Trial Registration</h3><p>NTC00699868.</p> </div

Evaluation of NK cell functions in ICU septic patients.

<p>NK degranulation (<b>A</b>) and intracellular production of IFN-γ (<b>B</b>) of ICU patients with Sepsis, SIRS, and healthy controls. <b>A:</b> Degranulation responses by CD107a cell-surface expression (% of positive NK cells) against K562 target cells (natural cytotoxicity) or P815 mouse mastocytoma cells coated with rabbit anti-mouse lymphocyte antibodies (ADCC). <b>B:</b> Intracellular IFN-γ expression (percentage of positive NK cells), against K562 target cells or P815 (ADCC). Number of samples from each group: Sepsis group (<i>n</i> = 29), SIRS group (<i>n</i> = 13), and healthy controls (<i>n</i> = 21). A black bar inside the box-and-whiskers plots indicates the median. <i>p(kw)</i>: Comparison between healthy, SIRS and Sepsis groups by Kruskal-Wallis test. <i>p</i>: pairwise comparisons between groups (healthy, SIRS, Sepsis) by Kruskal-Wallis post–hoc methods for multiple comparisons adjusted by step-up Simes method.</p

Evaluation of cytotoxic functions of NK cells in ICU patients.

<p>Correlation between the direct cytotoxicity CFSE-based assay and the degranulation CD107a expression assay to evaluate cytotoxic functions of NK cells in ICU patients (<i>n</i> = 14). Results are expressed as % lysis of target cell for the CFSE-assay, and as % NK-cell expressing CD107a for the degranulation assay. Effector–target ratio is 50/1 (PBMC/K562) for the CFSE-assay, and 2.5/1 (NK/K562) for the CD107a expression assay.</p

Characteristics on admission and outcome of ICU patients.

<p>Sepsis group includes patients with severe sepsis and septic shock. CMV: cytomegalovirus; ICU: intensive care unit; MV: mechanical ventilation; SIRS: systemic inflammatory response syndrome; SOFA: sepsis-related organ-failure assessment.</p><p><i>p:</i> Comparison between SIRS and Sepsis groups using Mann-Whitney U test or Pearson Chi-Square test. <i>p*:</i> Comparison between severe sepsis and septic shock using Mann-Whitney U test or Pearson Chi-Square test.</p

Range, mean, median and standard deviation according to assays (POC and Lab).

<p>Range, mean, median and standard deviation according to assays (POC and Lab).</p

Between-day imprecision of the Point-of-Care Siemens RAPIDPoint 500 blood gas system (POC).

<p>Between-day imprecision of the Point-of-Care Siemens RAPIDPoint 500 blood gas system (POC).</p

Between-day imprecision of central laboratory automated analyzers (Lab).

<p>Between-day imprecision of central laboratory automated analyzers (Lab).</p