Chronic Meningococcemia Cutaneous Lesions Involve Meningococcal Perivascular Invasion Through the Remodeling of Endothelial Barriers.

International audienceChronic meningococcemia is a form of sepsis with frequent polymorphous skin lesions. Both in vivo and in vitro data suggest that, in these lesions, meningococci gain access from the capillary lumen to the peripheral extravascular compartment, in the absence of vascular dislocation, through a paraendothelial route

Phenotypic and Functional Characteristics of Blood Natural Killer Cells from Melanoma Patients at Different Clinical Stages

International audienceMelanomas are aggressive skin tumors characterized by high metastatic potential. Immunotherapy is a valuable alternative for metastatic melanoma patients resistant to chemotherapy. Natural Killer (NK) cells are efficient anti-tumor cytotoxic effectors. We previously showed that blood NK cells from stage IV metastatic melanoma patients display decreased NK receptors and that chemotherapy modifies the functional status of blood NK cells. To investigate the role of NK cells along melanoma progression, we have here studied NK cells from patients at different stages of the disease. First, we showed that ex vivo NK cells from certain stage III–IV patients displayed low degranulation potential. Using a dynamic label-free assay, we found that immunoselected IL-2 activated blood NK cells from patients efficiently lysed melanoma cells through NKp46 and NKG2D receptors, independently to the clinical stage. Moreover, the ex vivo phenotype of circulating NK cells from 33 patients (stage I to IV) was extensively analyzed. NK cells from patients displayed higher variability in the percentages of Natural Cytotoxicity Receptors (NCR) and Natural Killer Group 2D (NKG2D) receptor expression compared to donor NK cells. The main defect was the decreased expression of NCR1 (NKp46) by NK cells from metastatic patients. Interestingly, we found a positive correlation between the NK cell percentages of NKp46 and the duration of stage IV in melanoma patients. Finally, we showed that NK cells infiltrated primary melanomas and displayed a predominant peritumoral distribution. These results are new arguments for the development of NK-based therapies in melanoma patients

IL-2-activated NK cells from patients exhibited antitumor capacities.

<p>(A) Dynamic measure of MelC cell index (CI) alone (black dots) and in presence of immunoselected IL-2-activated NK (white dots) from 1 donor and 2 patients are shown. Targets are monitored for cell adhesion for 5 h before and after addition of NK cells (arrow). NK cells (ratio 2/1) induce a rapid decrease of adherent target cell index (dotted line: 120 min after NK addition). In brackets, the clinical stage of each patient was noted. (B) Percentages of melanoma cell lysis by NK cells from 9 patients and 6 donors calculated from CI curves after 120 min of NK/target interactions. (C) Cytolysis experiments (n = 5) were performed in presence of anti-NKp46, anti-NKp30, anti-NKG2D or anti-DNAM-1 mAbs alone or in combination. Cytolysis curves (percentages of lysis) in presence of blocking mAbs are depicted for one representative experiment. (D) The percentages of lysis inhibition induced by the blocking of NK receptors are calculated at 120 min of co-culture (mean values and SD of 5 independent experiments).</p

Patient characteristics.

<p>M: Male; F: Female; NA: Not Available.</p

Prognostic values of NK cell parameters in the natural course of melanoma.

<p>Kaplan Meier curves were designed to analyze the association between NKp46 expression by NK cells and duration of stage IV. Patients (n = 24) were divided into two subsets of 12 patients defined by a cut off of NKp46 expression corresponding to the percentage median value (80.5%; p value<0.5 noted as *).</p

Analysis of blood NK cells from donors and melanoma patients.

<p>(A) Proportions (left) and absolute numbers (right) of NK cells among PBMC from donors and melanoma patients stratified according to their clinical stages. (B) Proportions of CD56^dim and CD56^bright cells among the NK cell population in donors and patients. Statistical analysis was assessed by non-parametric Kruskal-Wallis (K-W) test (n.s = not significant).</p

Characterization of the Microenvironment in Positive and Negative Sentinel Lymph Nodes from Melanoma Patients

<div><p>Melanomas are aggressive skin tumors characterized by high metastatic potential. Our previous results indicate that Natural Killer (NK) cells may control growth of melanoma. The main defect of blood NK cells was a decreased expression of activating NCR1/NKp46 receptor and a positive correlation of NKp46 expression with disease outcome in stage IV melanoma patients was found. In addition, in stage III melanoma patients, we identified a new subset of mature NK cells in macro-metastatic Lymph nodes (LN). In the present studies, we evaluated the numbers of NK cells infiltrating primary cutaneous melanoma and analyzed immune cell subsets in a series of sentinel lymph nodes (SLN). First, we show that NKp46⁺ NK cells infiltrate primary cutaneous melanoma. Their numbers were related to age of patients and not to Breslow thickness. Then, a series of patients with tumor-negative or -positive sentinel lymph nodes matched for Breslow thickness of the cutaneous melanoma was constituted. We investigated the distribution of macrophages (CD68), endothelial cells, NK cells, granzyme B positive (GrzB⁺) cells and CD8⁺ T cells in the SLN. Negative SLN (SLN^-) were characterized by frequent adipose involution and follicular hyperplasia compared to positive SLN (SLN⁺). High densities of macrophages and endothelial cells (CD34), prominent in SLN⁺, infiltrate SLN and may reflect a tumor favorable microenvironment. Few but similar numbers of NK and GrzB⁺ cells were found in SLN^- and SLN⁺: NK cells and GrzB⁺ cells were not correlated. Numerous CD8⁺ T cells infiltrated SLN with a trend for higher numbers in SLN^-. Moreover, CD8⁺ T cells and GrzB⁺ cells correlated in SLN^- not in SLN⁺. We also observed that the numbers of CD8⁺ T cells negatively correlated with endothelial cells in SLN^-. The numbers of NK, GrzB⁺ or CD8⁺ T cells had no significant impact on overall survival. However, we found that the 5 year-relapse rate was higher in SLN with higher numbers of NK cells.</p></div