Different features of tumor-associated NK cells in patients with low-grade or high-grade peritoneal carcinomatosis

Peritoneal carcinomatosis (PC) is a rare disease defined as diffused implantation of neoplastic cells in the peritoneal cavity. This clinical picture occurs during the evolution of peritoneal tumors, and it is the main cause of morbidity and mortality of patients affected by these pathologies, though cytoreductive surgery with heated intra-peritoneal chemotherapy (CRS/HIPEC) is yielding promising results. In the present study, we evaluated whether the tumor microenvironment of low-grade and high-grade PC could affect the phenotypic and functional features and thus the anti-tumor potential of NK cells. We show that while in the peritoneal fluid (PF) of low-grade PC most CD56dim NK cells show a relatively immature phenotype (NKG2A+KIR\u2013CD57\u2013CD16dim), in the PF of high-grade PC NK cells are, in large majority, mature (CD56dimKIR+CD57+CD16bright). Furthermore, in low-grade PC, PF-NK cells are characterized by a sharp down-regulation of some activating receptors, primarily NKp30 and DNAM-1, while, in high-grade PC, PF-NK cells display a higher expression of the PD-1 inhibitory checkpoint. The compromised phenotype observed in low-grade PC patients corresponds to a functional impairment. On the other hand, in the high-grade PC patients PF-NK cells show much more important defects that only partially reflect the compromised phenotype detected. These data suggest that the PC microenvironment may contribute to tumor escape from immune surveillance by inducing different NK cell impaired features leading to altered anti-tumor activity. Notably, after CRS/HIPEC treatment, the altered NK cell phenotype of a patient with a low-grade disease and favorable prognosis was reverted to a normal one. Our present data offer a clue for the development of new immunotherapeutic strategies capable of restoring the NK-mediated anti-tumor responses in association with the CRS/HIPEC treatment to increase the effectiveness of the current therapy

Expression Analysis of the Ligands for the Natural Killer Cell Receptors NKp30 and NKp44

BACKGROUND: The natural cytotoxicity receptors (NCR) are important to stimulate the activity of Natural Killer (NK) cells against transformed cells. Identification of NCR ligands and their level of expression on normal and neoplastic cells has important implications for the rational design of immunotherapy strategies for cancer. METHODOLOGY/PRINCIPAL FINDINGS: Here we analyze the expression of NKp30 ligand and NKp44 ligand on 30 transformed or non-transformed cell lines of different origin. We find intracellular and surface expression of these two ligands on almost all cell lines tested. Expression of NKp30 and NKp44 ligands was variable and did not correlate with the origin of the cell line. Expression of NKp30 and NKp44 ligand correlated with NKp30 and NKp44-mediated NK cell lysis of tumor cells, respectively. The surface expression of NKp30 ligand and NKp44 ligand was sensitive to trypsin treatment and was reduced in cells arrested in G(2)/M phase. CONCLUSION/SIGNIFICANCE: These data demonstrate the ubiquitous expression of the ligands for NKp30 and NKp44 and give an important insight into the regulation of these ligands

Silver sulfadiazine eradicates antibiotic-tolerant Staphylococcus aureus and Pseudomonas aeruginosa biofilms in patients with infected diabetic foot ulcers

Infections are among the most frequent and challenging events in diabetic foot ulcers (DFUs). Pathogenic bacteria growing in biofilms within host tissue are highly tolerant to environmental and chemical agents, including antibiotics. The present study was aimed at assessing the use of silver sulfadiazine (SSD) for wound healing and infection control in 16 patients with DFUs harboring biofilm-growing Staphylococcus aureus and Pseudomonas aeruginosa. All patients received a treatment based on a dressing protocol including disinfection, cleansing, application of SSD, and application of nonadherent gauze, followed by sterile gauze and tibio-breech bandage, in preparation for toilet surgery after 30 days of treatment. Clinical parameters were analyzed by the T.I.M.E. classification system. In addition, the activity of SSD against biofilm-growing S. aureus and P. aeruginosa isolates was assessed in vitro. A total of 16 patients with S. aureus and P. aeruginosa infected DFUs were included in the study. Clinical data showed a statistically significant (p < 0.002) improvement of patients’ DFUs after 30 days of treatment with SSD with significant amelioration of all the parameters analyzed. Notably, after 30 days of treatment, resolution of infection was observed in all DFUs. In vitro analysis showed that both S. aureus and P. aeruginosa isolates developed complex and highly structured biofilms. Antibiotic susceptibility profiles indicated that biofilm cultures were significantly (p ≤ 0.002) more tolerant to all tested antimicrobials than their planktonic counterparts. However, SSD was found to be effective against fully developed biofilms of both S. aureus and P. aeruginosa at concentrations below those normally used in clinical preparations (10 mg/mL). These results strongly suggest that the topical administration of SSD may represent an effective alternative to conventional antibiotics for the successful treatment of DFUs infected by biofilm-growing S. aureus and P. aeruginosa

The B7 family member B7-H6 is a tumor cell ligand for the activating natural killer cell receptor NKp30 in humans

Cancer development is often associated with the lack of specific and efficient recognition of tumor cells by the immune system. Natural killer (NK) cells are lymphocytes of the innate immune system that participate in the elimination of tumors. We report the identification of a tumor cell surface molecule that binds NKp30, a human receptor which triggers antitumor NK cell cytotoxicity and cytokine secretion. This previously unannotated gene belongs to the B7 family and, hence, was designated B7-H6. B7-H6 triggers NKp30-mediated activation of human NK cells. B7-H6 was not detected in normal human tissues but was expressed on human tumor cells, emphasizing that the expression of stress-induced self-molecules associated with cell transformation serves as a mode of cell recognition in innate immunity

Tumour stromal cells derived from paediatric malignancies display MSC-like properties and impair NK cell cytotoxicity

<p>Abstract</p> <p>Background</p> <p>Tumour growth and metastatic infiltration are favoured by several components of the tumour microenvironment. Bone marrow-derived multipotent mesenchymal stromal cells (MSC) are known to contribute to the tumour stroma. When isolated from healthy bone marrow, MSC exert potent antiproliferative effects on immune effector cells. Due to phenotypic and morphological similarities of MSC and tumour stromal cells (TStrC), we speculated that immunotherapeutic approaches may be hampered if TStrC may still exhibit immunomodulatory properties of MSC.</p> <p>Methods</p> <p>In order to compare immunomodulatory properties of MSC and tumour stromal cells (TStrC), we established and analyzed TStrC cultures from eleven paediatric tumours and MSC preparations from bone marrow aspirates. Immunophenotyping, proliferation assays and NK cell cytotoxicity assays were employed to address the issue.</p> <p>Results</p> <p>While TStrC differed from MSC in terms of plasticity, they shared surface expression of CD105, CD73 and other markers used for MSC characterization. Furthermore, TStrC displayed a strong antiproliferative effect on peripheral blood mononuclear cells (PBMC) in coculture experiments similar to MSC. NK cell cytotoxicity was significantly impaired after co-culture with TStrC and expression of the activating NK cell receptors NKp44 and NKp46 was reduced.</p> <p>Conclusions</p> <p>Our data show that TStrC and MSC share important phenotypic and functional characteristics. The inhibitory effect of TStrC on PBMC and especially on NK cells may facilitate the immune evasion of paediatric tumours.</p

Generation of a Novel Regulatory NK Cell Subset from Peripheral Blood CD34+ Progenitors Promoted by Membrane-Bound IL-15

BACKGROUND: NK cells have been long time considered as cytotoxic lymphocytes competent in killing virus-infected cells and tumors. However, NK cells may also play essential immuno-regulatory functions. In this context, the real existence of a defined NK subset with negative regulatory properties has been hypothesized but never clearly demonstrated. METHODOLOGY/PRINCIPAL FINDINGS: Herein, we show the in vitro generation from human peripheral blood haematopoietic progenitors (PB-HP), of a novel subset of non-cytolytic NK cells displaying a mature phenotype and remarkable immuno-regulatory functions (NK-ireg). The main functional hallmark of these NK-ireg cells is represented by the surface expression/release of HLA-G, a major immunosuppressive molecule. In addition, NK-ireg cells secrete two powerful immuno-regulatory factors: IL-10 and IL-21. Through these factors, NK-ireg cells act as effectors of the down-regulation of the immune response: reconverting mature myeloid DC (mDC) into immature/tolerogenic DC, blocking cytolytic functions on conventional NK cells and inducing HLA-G membrane expression on PB-derived monocytes. The generation of "NK-ireg" cells is obtained, by default, in culture conditions favouring cell-to-cell contacts, and it is strictly dependent on reciprocal trans-presentation of membrane-bound IL-15 forms constitutively and selectively expressed by human CD34(+) PB-HP. Finally, a small subset of NKp46(+) HLA-G(+) IL-10(+) is detected within freshly isolated decidual NK cells, suggesting that these cells could represent an in vivo counterpart of the NK-ireg cells. CONCLUSIONS/SIGNIFICANCE: In conclusion, NK-ireg cells represent a novel truly differentiated non-cytolytic NK subset with a self-sustainable phenotype (CD56(+) CD16(+) NKp30(+) NKp44(+) NKp46(+) CD94(+) CD69(+) CCR7(+)) generated from specific pSTAT6(+) GATA3(+) precursors. NK-ireg cells could be employed to develop new immuno-suppressive strategies in autoimmune diseases, transplant rejection or graft versus host diseases. In addition, NK-ireg cells can be easily derived from peripheral blood of the patients and could constitute an autologous biotherapic tool to be used combined or in alternative to other immuno-regulatory cells

The Natural Cytotoxicity Receptor 1 Contribution to Early Clearance of Streptococcus pneumoniae and to Natural Killer-Macrophage Cross Talk

Natural killer (NK) cells serve as a crucial first line of defense against tumors, viral and bacterial infections. We studied the involvement of a principal activating natural killer cell receptor, natural cytotoxicity receptor 1 (NCR1), in the innate immune response to S. pneumoniae infection. Our results demonstrate that the presence of the NCR1 receptor is imperative for the early clearance of S. pneumoniae. We tied the ends in vivo by showing that deficiency in NCR1 resulted in reduced lung NK cell activation and lung IFNγ production at the early stages of S. pneumoniae infection. NCR1 did not mediate direct recognition of S. pneumoniae. Therefore, we studied the involvement of lung macrophages and dendritic cells (DC) as the mediators of NK-expressed NCR1 involvement in response to S. pneumoniae. In vitro, wild type BM-derived macrophages and DC expressed ligands to NCR1 and co-incubation of S. pneumoniae-infected macrophages/DC with NCR1-deficient NK cells resulted in significantly lesser IFNγ levels compared to NCR1-expressing NK cells. In vivo, ablation of lung macrophages and DC was detrimental to the early clearance of S. pneumoniae. NCR1-expressing mice had more potent alveolar macrophages as compared to NCR1-deficient mice. This result correlated with the higher fraction of NCR1-ligandhigh lung macrophages, in NCR1-expressing mice, that had better phagocytic activity compared to NCR1-liganddull macrophages. Overall, our results point to the essential contribution of NK-expressed NCR1 in early response to S. pneumoniae infection and to NCR1-mediated interaction of NK and S. pneumoniae infected-macrophages and -DC

Direct TLR2 Signaling Is Critical for NK Cell Activation and Function in Response to Vaccinia Viral Infection

Natural killer (NK) cells play an essential role in innate immune control of poxviral infections in vivo. However, the mechanism(s) underlying NK cell activation and function in response to poxviruses remains poorly understood. In a mouse model of infection with vaccinia virus (VV), the most studied member of the poxvirus family, we identified that the Toll-like receptor (TLR) 2-myeloid differentiating factor 88 (MyD88) pathway was critical for the activation of NK cells and the control of VV infection in vivo. We further showed that TLR2 signaling on NK cells, but not on accessory cells such as dendritic cells (DCs), was necessary for NK cell activation and that this intrinsic TLR2-MyD88 signaling pathway was required for NK cell activation and played a critical role in the control of VV infection in vivo. In addition, we showed that the activating receptor NKG2D was also important for efficient NK activation and function, as well as recognition of VV-infected targets. We further demonstrated that VV could directly activate NK cells via TLR2 in the presence of cytokines in vitro and TLR2-MyD88-dependent activation of NK cells by VV was mediated through the phosphatidylinositol 3-kinase (PI3K)-extracellular signal-regulated kinase (ERK) pathway. Taken together, these results represent the first evidence that intrinsic TLR signaling is critical for NK cell activation and function in the control of a viral infection in vivo, indicate that multiple pathways are required for efficient NK cell activation and function in response to VV infection, and may provide important insights into the design of effective strategies to combat poxviral infections