Gaetana Pezzino

Role of natural killer cells in the pathogenesis and progression of multiple sclerosis

Natural killer (NK) cells are a subset of lymphocytes which have long been alleged to play an immunoregulatory role in the prevention of autoimmune diseases. Here, we briefly review NK cell features and the major findings from studies on NK cells in human and animals susceptible to multiple sclerosis (MS). Although most studies in human seem to suggest an association between disease and deficiencies in NK cells, it is also clear that NK cells can be both protective and pathogenic in MS models. These contrasting observations could result from differences in experimental procedures as well as from differences in NK cell subset targeted. Whatever the case, the functional features of these cells and their potential role in regulation of autoimmunity suggest that NK cell-based therapies might be an interesting approach for the treatment of multiple sclerosis.

Susceptibility of Human Melanoma Cells to Autologous Natural Killer (NK) Cell Killing: HLA-Related Effector Mechanisms and Role of Unlicensed NK Cells

Background Despite Natural Killer (NK) cells were originally defined as effectors of spontaneous cytotoxicity against tumors, extremely limited information is so far available in humans on their capability of killing cancer cells in an autologous setting. Methodology/Principal Findings We have established a series of primary melanoma cell lines from surgically resected specimens and here showed that human melanoma cells were highly susceptible to lysis by activated autologous NK cells. A variety of NK cell activating receptors were involved in killing: particularly, DNAM-1 and NKp46 were the most frequently involved. Since self HLA class I molecules normally play a protective role from NK cell-mediated attack, we analyzed HLA class I expression on melanomas in comparison to autologous lymphocytes. We found that melanoma cells presented specific allelic losses in 50% of the patients analyzed. In addition, CD107a degranulation assays applied to NK cells expressing a single inhibitory receptor, revealed that, even when expressed, specific HLA class I molecules are present on melanoma cell surface in amount often insufficient to inhibit NK cell cytotoxicity. Remarkably, upon activation, also the so called “unlicensed” NK cells, i.e. NK cells not expressing inhibitory receptor specific for self HLA class I molecules, acquired the capability of efficiently killing autologous melanoma cells, thus additionally contributing to the lysis by a mechanism independent of HLA class I expression on melanoma cells. Conclusions/Significance We have investigated in details the mechanisms controlling the recognition and lysis of melanoma cells by autologous NK cells. In these autologous settings, we demonstrated an efficient in vitro killing upon NK cell activation by mechanisms that may be related or not to abnormalities of HLA class I expression on melanoma cells. These findings should be taken into account in the design of novel immunotherapy approaches against melanoma.

Seroma fluid subsequent to axillary lymph node dissection for breast cancer derives from an accumulation of afferent lymph

Seroma is a frequent complication of breast cancer surgery, the etiology of which remains indefinite. It represents a subcutaneous accumulation of fluid frequently reported after surgical procedures such as axillary lymph node dissection. Despite previous studies have associated seroma fluid to an inflammatory exudate, the surgical removal of draining lymph nodes may indicate that seroma might not represent a mere exudate but rather an accrual of lymph drained from tributary tissues. To verify this hypothesis, seromas were collected at different intervals of time in patients operated upon for axillary lymph node removal. Fluids were analyzed in details by flow cytometry and biochemical assays for their cellular content and for their molecular features and relevant cytokine content. Lymphocytes and other peculiar blood mononuclear cells were present, while erythrocytes, platelets and granulocytes were absent or extremely rare. The protein concentration resulted lower (median 64%) than in peripheral blood. However, specific proteins related to locoregional tissues resulted highly concentrated (e.g. up to 500% for ferritin and 300% for lactate deydrogenase and exclusive presence of interleukin-6) whereas all enzymes and proteins synthesized in the liver or other organs (e.g. alkaline phosphatase, ALT, gammaGT, prealbumin, transferrin, ceruloplasmin, C3 and C4, alpha2 macroglobulin from liver; apolipoproteins from liver and gut; amylase and lipase from pancreas) were represented in reduced concentrations, thus ruling out that seroma proteins derive directly from blood serum. As a whole, this comprehensive cytological and molecular analysis provided evidences that seroma is constituted by serum ultrafiltrated-derived extracellular fluid of regions located upstream of removed lymph nodes. This fluid is then enriched by proteins and cells collected in the drained regions. Remarkably, seroma fluids collected in the same patient at different time points (up to 50 days following surgery) displayed similar biochemical features, clearly indicating that fluid composition was not significantly affected by post-surgical locoregional flogosis. Finally, the period of seroma formation indicates that lymph accumulates in the axillary region during the interval of time needed for afferent lymphatic vessels to re-anastomose with the efferent ducts. Therefore, seroma fluid represents a font of biological material suitable for investigating the biology of breast cancer, healing tissues and lymph.

Novel perspectives on dendritic cell-based immunotherapy of cancer.

Advances in immunobiology knowledge as well as in cell culture processes that generate large numbers of purified and functionally mature dendritic cells (DCs) have raised the possibility that DCs might represent promising clinical agents to generate effective immune responses against cancer. Here, we discuss the present pitfalls of dendritic cell vaccines for the treatment of human cancer with regard to the most recent knowledge in the biology of DCs. In particular, we highlight the relevance of improving our current understanding of DC trafficking, functions and interactions with other cells of innate immunity for the development of more effective cancer vaccines.

Membrane Transfer from Tumor Cells Overcomes Deficient Phagocytic Ability of Plasmacytoid Dendritic Cells for the Acquisition and Presentation of Tumor Antigens

The potential contribution of plasmacytoid dendritic cells (pDCs) in the presentation of tumor cell Ags remains unclear, and some controversies exist with regard to the ability of pDCs to phagocytose cell-derived particulate Ags and cross-present them to MHC class I–restricted T lymphocytes. In this study, we show that human pDCs, although inefficient in the internalization of cell membrane fragments by phagocytosis, can efficiently acquire membrane patches and associated molecules from cancer cells of different histotypes. The transfer of membrane patches to pDCs occurred in a very short time and required cell-to-cell contact. Membrane transfer also included intact HLA complexes, and the acquired Ags could be efficiently recognized on pDCs by tumor-specific CD8+ T cells. Remarkably, pDCs isolated from human colon cancer tissues displayed a strong surface expression of epithelial cell adhesion molecule, indicating that the exchange of exogenous Ags between pDCs and tumor cells also can occur in vivo. These data demonstrate that pDCs are well suited to acquire membrane patches from contiguous tumor cells by a cell-to-cell contact–dependent mechanism that closely resembles “trogocytosis.” This phenomenon may allow pDCs to proficiently present tumor cell–derived Ags, despite limited properties of endophagocytosis.

Cognate HLA absence in trans diminishes human NK cell education

NK cells are innate lymphocytes with protective functions against viral infections and tumor formation. Human NK cells carry inhibitory killer cell Ig-like receptors (KIRs), which recognize distinct HLAs. NK cells with KIRs for self-HLA molecules acquire superior cytotoxicity against HLA- tumor cells during education for improved missing-self recognition. Here, we reconstituted mice with human hematopoietic cells from donors with homozygous KIR ligands or with a mix of hematopoietic cells from these homozygous donors, allowing assessment of the resulting KIR repertoire and NK cell education. We found that co-reconstitution with 2 KIR ligand-mismatched compartments did not alter the frequency of KIR-expressing NK cells. However, NK cell education was diminished in mice reconstituted with parallel HLA compartments due to a lack of cognate HLA molecules on leukocytes for the corresponding KIRs. This change in NK cell education in mixed human donor-reconstituted mice improved NK cell-mediated immune control of EBV infection, indicating that mixed hematopoietic cell populations could be exploited to improve NK cell reactivity against leukotropic pathogens. Taken together, these findings indicate that leukocytes lacking cognate HLA ligands can disarm KIR+ NK cells in a manner that may decrease HLA- tumor cell recognition but allows for improved NK cell-mediated immune control of a human γ-herpesvirus.

Divergent signaling pathways regulate IL-12 production induced by different species of Lactobacilli in human dendritic cells.

Recent studies have indicated that different strains of Lactobacilli differ in their ability to regulate IL-12 production by dendritic cells (DCs), as some strains are stronger inducer of IL-12 while other are not and can even inhibit IL-12 production stimulated by IL-12-inducer Lactobacilli. In this report we demonstrate that Lactobacillus reuteri 5289, as previously described for other strains of L. reuteri, can inhibit DC production of IL-12 induced by Lactobacilllus acidophilus NCFM. Remarkably, L. reuteri 5289 was able to inhibit IL-12 production induced not only by Lactobacilli, as so far reported, but also by bacteria of different genera, including pathogens. We investigated in human DCs the signal transduction pathways involved in the inhibition of IL-12 production induced by L. reuteri 5289, showing that this potential anti-inflammatory activity, which is also accompanied by an elevated IL-10 production, is associated to a prolonged phosphorilation of ERK1/2 MAP kinase pathway. Improved understanding of the immune regulatory mechanisms exerted by Lactobacilli is crucial for a more precise employment of these commensal bacteria as probiotics in human immune-mediated pathologies, such as allergies or inflammatory bowel diseases.

HUMAN LEUKOCYTE ANTIGEN FREQUENCY IN HUMAN HIGH-GRADE GLIOMAS: A CASE-CONTROL STUDY IN SICILY

OBJECTIVEHuman leukocyte antigens (HLAs) are widely expressed cell surface molecules that present antigenic peptides to T lymphocytes and modulate immune response against inflammatory and malignant diseases. The aim of this study was to compare HLA distribution in patients with newly diagnosed high-grade gliomas (HGGs) and 2 control groups from a restricted geographic area (eastern Sicily). METHODSHLA allele frequency, as determined from peripheral blood of 56 adult patients with HGGs, was compared with that of 2 different control groups: 140 healthy bone marrow donors (group A) and 69 virtually brain tumor–free patients (group B). HLA expression was evaluated using a reverse transcriptase polymerase chain reaction–sequence-specific oligonucleotide probe. RESULTSThere was significant expression of HLA-A*11 in patients with HGGs compared with control groups A and B (P < 0.003 and P < 0.018, respectively). Significant expression of HLA genotypes in patients with HGGs was also identified for HLA-DQB1*06 (P = 0.005), HLA-DRB1*14 (P = 0.001), and HLA-DRB3*01 (P = 0.007) compared with control group B. In HGG patients, there was statistically significantly decreased expression, compared with control groups A and B, of HLA-B*07 (P = 0.002 and P = 0.03, respectively) and HLA-C*04 (P = 0.007 and P = 0.016, respectively). There was statistically significant lower expression of HLA-C*05 in the HGG group compared with group B (P < 0.03). CONCLUSIONThis is the first study to describe the frequency of distribution of HLAs in a population from a restricted geographic area. The findings suggest a possible correlation between HLA allele distribution and the occurrence of newly diagnosed malignant astroglial brain tumors.

Drag cells in immunity: Plasmacytoid DCs dress up as cancer cells

Cross-dressing, in immunology, is a term originally coined to indicate the transfer of peptide-MHC complexes belonging to neighboring cells on antigen presenting cells. We have recently shown that plasmacytoid dendritic cells (pDCs) are particularly suited to be cross-dressed by tumor cells and that this phenomenon provides a unique pathway for abundant presentation of tumor antigens by pDCs.

MiRNA expression profiling in human gliomas: upregulated miR-363 increases cell survival and proliferation

The role of microRNAs (miRNAs) in glioma biology is increasingly recognized. To investigate the regulatory mechanisms governing the malignant signature of gliomas with different grades of malignancy, we analyzed miRNA expression profiles in human grade I–IV tumor samples and primary glioma cell cultures. Multiplex real-time PCR was used to profile miRNA expression in a set of World Health Organization (WHO) grade I (pilocytic astrocytoma), II (diffuse fibrillary astrocytoma), and IV (glioblastoma multiforme) astrocytic tumors and primary glioma cell cultures. Primary glioma cell cultures were used to evaluate the effect of transfection of specific miRNAs and miRNA inhibitors. miRNA microarray showed that a set of miRNAs was consistently upregulated in all glioma samples. miR-363 was upregulated in all tumor specimens and cell lines, and its expression correlated with tumor grading. The transfection of glioma cells with the specific inhibitor of miR-363 increased the expression level of tumor suppressor growth-associated protein 43 (GAP-43). Transfection of miR-363 induced cell survival, while inhibition of miR-363 significantly reduced glioma cell viability. Furthermore, miRNA-363 inhibition induced the downregulation of AKT, cyclin-D1, matrix metalloproteinase (MMP)-2, MMP-9, and Bcl-2 and upregulation of caspase 3. Together, these data suggest that the upregulation of miR-363 may play a role in malignant glioma signature.