Analysis of the CD16, CCR7, and CD57 surface expression on CD56⁺ NK cells.

<p>Flow cytometry relative quantification of CD56⁺ Lin⁻ NK cells (first panels) and of CD16 (second panels), CCR7 (third panels), and CD57 expression (lower panels) on CD56⁺ gated NK cells. HPS2 patients' (Pt1 and Pt2) data are representative of one of the four separate evaluations performed on peripheral blood lymphocytes in a two-year period and are compared with a representative normal subject (CTRL) of the healthy donor group.</p

Prf-positive cells in NLPHL.

<p>Lymph node sections are from Pt1 (a), Pt2 (b) and controls (c and d), stained for anti-Prf (a-d; brown) and anti-CD8 (inserts blue). An increased number of Prf+ CTL co-expressing CD8 (insert) are observed in NLPHL nodules from HPS2 patients compared to controls. Sections are counterstained with Meyer's haematoxylin and secondary antibodies revealed with DAB (Prf) or Ferangi blue (CD8). Original magnification: 200×(a–d) and 1000×(inserts).</p

Impairment of cytolytic activity of IL-2–stimulated NK cells in HPS2 patients.

<p><b>A</b>, Purified polyclonal IL-2–activated NK cells, derived from Pt1 (triangle pointing up) or Pt2 (triangle pointing down) or 5 distinct healthy donors (circle), were tested against NK susceptible target cells (E/T ratio 10∶1): HLA- LCL 721.221 EBV-lymphoblastoid cell lines, HLA+ ALINA EBV-lymphoblastoid cell lines, IGROV ovarian carcinoma, M14 human melanoma, P815 murine mastocytoma, allogeneic PHA blasts, K562 erythroleukemia, SKNEP1 kidney carcinoma, Daudi and Raji Burkitt's lymphoma, A172 human glioblastoma and FO-1 human melanoma. <b>B</b>, Purified polyclonal IL-2-activated NK cells, derived from Pt1 (triangle pointing up), Pt2 (triangle pointing down) or 5 distinct healthy donors (circle), were tested against the Hodgkin's lymphoma derived L428 (HLA-) and L540 (HLA+) cell lines, either in the absence (−) or in the presence (+) of anti-HLA class I mAb at E/T ratio 10∶1. The results shown represent the combination of three independent experiments. Each value represents the mean ± SD of 5 replicates. *  = p<0.01.</p

CD107a expression and IFN-γ production by NK cells of HPS2 patients.

<p><b>A</b>, surface expression of CD107a in resting (upper panels) and IL-2 activated NK cells (lower panels) in a representative healthy donor (CTRL) of six distinct subjects analyzed and in HPS2 patients (Pt1 and Pt2). In each plot the bar defines the percentage of cells that express CD107a. <b>B</b>, IFN-γ–producing IL-2 NK cells were analyzed by flow cytometry. The percentage of IFN-γ producing CD56⁺ NK cells from a representative healthy donor (CTRL) of six distinct subjects analyzed and from patients (Pt1 and Pt2) is shown in un-stimulated conditions (<b>medium</b>), stimulated by PHA (<b>PHA</b>) and stimulated by NK susceptible K562 tumor target cells (<b>K562</b>). The percentages shown in the panels A and B are representative of an experiment repeated three times.</p

DataSheet_1_Neutrophils inhibit γδ T cell functions in the imiquimod-induced mouse model of psoriasis.docx

BackgroundPsoriasis is a chronic skin disease associated with deregulated interplays between immune cells and keratinocytes. Neutrophil accumulation in the skin is a histological feature that characterizes psoriasis. However, the role of neutrophils in psoriasis onset and development remains poorly understood.MethodsIn this study, we utilized the model of psoriasiform dermatitis, caused by the repeated topical application of an imiquimod containing cream, in neutrophil-depleted mice or in mice carrying impairment in neutrophil functions, including p47phox -/- mice (lacking a cytosolic subunit of the phagocyte nicotinamide adenine dinucleotide phosphate - NADPH - oxidase) and Sykfl/fl MRP8-cre+ mice (carrying the specific deletion of the Syk kinase in neutrophils only), to elucidate the specific contribution of neutrophils to psoriasis development.ResultsBy analyzing disease development/progression in neutrophil-depleted mice, we now report that neutrophils act as negative modulators of disease propagation and exacerbation by inhibiting gammadelta T cell effector functions via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-mediated reactive oxygen species (ROS) production. We also report that Syk functions as a crucial molecule in determining the outcome of neutrophil and γδ T cell interactions. Accordingly, we uncover that a selective impairment of Syk-dependent signaling in neutrophils is sufficient to reproduce the enhancement of skin inflammation and γδ T cell infiltration observed in neutrophil-depleted mice.ConclusionsOverall, our findings add new insights into the specific contribution of neutrophils to disease progression in the IMQ-induced mouse model of psoriasis, namely as negative regulatory cells.</p