CSA is involved in IE interaction with human NK cell line but not in primary NK cell activation.

<p>(A) The human NK cell line NK92 was incubated with whole culture of IE in different conditions. After 1 h at 37°C, a sample of the co-culture was placed between slide and cover and analyzed under a microscope. The NK92 cells directly interacts with IE (FCR3-CSA strain) as rosettes, but not with uninfected erythrocytes (x100 original magnification, left panel). RBC infected with FCR3-CSA or 2A5 were co-cultured with NK92 cells alone or in the presence of soluble CSA (+CSA), or with NK92 cells pre-treated with chondroitinase ABC (+Case ABC). At the end of the co-culture, the percentage of NK cells interacting with at least two IE was determined and expressed as % of cytoadhesion. Each dot represents one independent experiment (right panel). (B) Freshly isolated human PBMC were cultured with uninfected RBC (RBC), RBC infected with FCR3-CSA or RBC infected with the <i>var2csa</i> KO parasite 2A5. After 24 h, NK cell activation was analyzed by flow cytometry by gating on CD3⁻CD56⁺ lymphocytes. The CD69 MFI (mean fluorescence intensity) staining on NK cells (left panel), the percentage of CD25⁺ NK cells (middle panel) and the percentage of IFN-γ⁺ NK cells (right panel) were determined for 7 different healthy donors. Means±SEM are represented. Statistical analyses were performed using the Wilcoxon test.</p

Engagement of ICAM-1 with its cellular ligand but not with PfEMP1 is required for NK cell IFN-γ production.

<p>(A) Diagram of an ICAM-1 molecule showing schematic binding sites for LFA-1, Mac-1, CD11c/CD18 and PfEMP1. The epitope map of the anti-ICAM-1 mAb 15.2, My13 and RR1/1 is indicated. (B) Human PBMC were cultured with uninfected RBC (RBC, black bars) or with RBC infected with the 3D7 <i>Pf</i> strain (3D7, grey bars) in presence or absence of antibodies directed against NKG2D (isotype control), ICAM-1 or CD18. Three different clones of anti-ICAM-1 were used: 15.2 blocks the interaction of ICAM-1 with LFA-1 and with PfEMP1, RR1/1 blocks only the interaction with LFA-1 and My13 blocks only the interaction with PfEMP1. After 24 h of co-culture, NK cell activation was analyzed by flow cytometry by gating on CD3⁻CD56⁺ NK cells. The CD69 MFI staining on NK cells (left panel), the percentage of CD25⁺ NK cells (middle panel) and the percentage of IFN-γ⁺ NK cells (right panel) were determined for 24 donors (None, NKG2D and 15.2), 13 donors (CD18), 9 donors (My13) or 5 donors (RR1/1). Means ± SEM are represented. Statistical analyses were performed using the Mann Whitney test.</p

Expression of CSA, CD36 and ICAM-1 on human NK cells.

<p>Primary resting human NK cells (CD56⁺CD3⁻) in total PBMC as well as human NK cell lines, NK92 and NKL were analyzed by flow cytometry to determine the surface expression of three host ligands for PfEMP1: CSA (left panels, dark line), CD36 (middle panels, dark line) and ICAM-1 (right panels, dark line). Stainings with isotype control for each antibody are represented by filled grey histograms. Stainings of primary resting human NK cells are representative of at least 3 donors.</p

Deficiency in CD36 does not alter NK cell response to IE.

<p>(A) The level of CD36 expression was determined on PBMC collected from a healthy donor (Control donor) or from a patient deficient for CD36. Total PBMC protein extracts were prepared and analyzed by Western Blot (left panel). Total PBMC were stained with CD36 antibody or with an isotype control (filled grey histogram, right panel). Histograms for CD3⁻CD56⁺ NK cells from a CD36 deficient donor (dotted line) and a control donor (bold line) are represented. (B) Control or CD36-deficient PBMC were cultured with uninfected RBC (RBC, black bars), or with RBC infected with the 3D7 <i>Pf</i> strain (3D7, grey bars). After 24 h, NK cell activation was analyzed by flow cytometry by gating on CD3⁻CD56⁺ NK cells. The CD69 MFI staining on NK cells (left panel), the percentage of CD25⁺ NK cells (middle panel) and the percentage of IFN-γ⁺ NK cells (right panel) were determined in three independent experiments. Means ± SEM are represented. Statistical analyses were performed using the Mann Whitney test.</p

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

PfEMP1 deficient parasites are potent activators of NK cells.

<p>Human PBMC were cultured with uninfected RBC (RBC, black bars) or with RBC infected with the “PfEMP1 KO” strain DC-J. After 24 h, NK cell activation was analyzed by flow cytometry by gating on CD3⁻CD56⁺ NK cells. The CD69 MFI staining on NK cells (left panel), the percentage of CD25⁺ NK cells (middle panel) and the percentage of IFN-γ⁺ NK cells (right panel) were determined for 5 different healthy donors. Means±SEM are represented. Statistical analyses were performed using the Wilcoxon test.</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

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

Results of the family-based association study between <i>KIR</i> genes and Hodgkin's lymphoma using a dominant model

a<p>informative families are those with at least one heterozygous (+,−) parent for the corresponding KIR gene.</p>b<p>computed using the FBAT software.</p>c<p>computed by conditional logistic regression.</p>d<p>association results for allelic forms of the same KIR locus are provided for the first variant of the pair.</p>e<p>deleted form</p>f<p>wild type</p