Gabriella Palmieri

Tumor necrosis factor alters synaptic transmission in rat hippocampal slices

The effects of human recombinant tumor necrosis factor (TNF-alpha) on the synaptic transmission were studied in rat hippocampal slices by using extracellular field potential recordings. Population spikes and/or excitatory postsynaptic potentials were extracellularly recorded in hippocampus CA1 region from stratum pyramidale and stratum radiatum, respectively, and synaptic transmission was examined in the Schaffer collateral/commissural-CA1 pathway. Basal neurotransmission slightly and promptly increased in slices acutely exposed to TNF-alpha (1-100 nM). Examination of the long-term potentiation (LTP) revealed that a brief treatment with the cytokine did not influence LTP, while a long-lasting application of TNF-alpha (50 min or more) inhibited LTP in a dose-dependent way in the range of 1-100 nM. A role for TNF-alpha as a peptide of immunological significance belonging to the family of brain neuromodulators is discussed.

CD69-triggered ERK activation and functions are negatively regulated by CD94/NKG2-A inhibitory receptor

CD69 represents a functional triggering molecule on activated NK and T cells, capable of inducing cytotoxic activity and costimulating cytokine production. It belongs to the C‐lectin type superfamily, and its gene maps in the NK gene complex, close to other genes coding for NK receptors. CD94 / NKG2‐A complex is the inhibitory receptor for the non classical MHC class I molecule HLA‐E on human NK cells. To investigate CD69‐initiated signal transduction pathways, and to evaluate CD94 / NKG2‐A interference on CD69 triggering ability, we have generated transfectants expressing both receptors in the RBL cell line. Here we report that CD69 engagement leads to the activation of extracellular signal‐regulated kinase (ERK) enzymes belonging to the MAPK family, and that this event is required for CD69‐mediated cell degranulation. Moreover, we show that the co‐engagement of CD94 / NKG2‐A inhibitory receptor effectively suppresses both CD69‐triggered cell degranulation in RBL transfectants, through the inhibition of ERK activation, and CD69‐induced cytotoxicity in human NK cells. Thus, here we provide new information on the molecular mechanisms initiated by CD69 activation receptor, and show that CD69‐initiated signaling pathways and functional activity are negatively regulated by CD94 / NKG2‐A inhibitory complex.

Src-Dependent Syk Activation Controls CD69-Mediated Signaling and Function on Human NK Cells

CD69 C-type lectin receptor represents a functional triggering molecule on activated NK cells, capable of directing their natural killing function. The receptor-proximal signaling pathways activated by CD69 cross-linking and involved in CD69-mediated cytotoxic activity are still poorly understood. Here we show that CD69 engagement leads to the rapid and selective activation of the tyrosine kinase Syk, but not of the closely related member of the same family, ZAP70, in IL-2-activated human NK cells. Our results indicate the requirement for Src family kinases in the CD69-triggered activation of Syk and suggest a role for Lck in this event. We also demonstrate that Syk and Src family tyrosine kinases control the CD69-triggered tyrosine phosphorylation and activation of phospholipase Cγ2 and the Rho family-specific exchange factor Vav1 and are responsible for CD69-triggered cytotoxicity of activated NK cells. The same CD69-activated signaling pathways are also observed in an RBL transfectant clone, constitutively expressing the receptor. These data demonstrate for the first time that the CD69 receptor functionally couples to the activation of Src family tyrosine kinases, which, by inducing Syk activation, initiate downstream signaling pathways and regulate CD69-triggered functions on human NK cells.

CD16-mediated activation of phosphatidylinositol-3 kinase (Pl-3K) in human NK cells involves tyrosine phosphorylation of Cbl and its association with Grb2, Shc, pp36 and p85 Pl-3K subunit

Phosphatidylinositol 3‐kinase (Pl‐3K) plays a key role in several cellular processes, including mitogenesis, apoptosis, actin reorganization and vesicular trafficking. The molecular events involved in its activation have not been fully elucidated and several reports indicate that a key event for enzyme activation is the interaction of the SH2 domains of the p85 regulatory subunit of Pl‐3K with tyrosine‐phosphorylated proteins. In this study, we investigated the involvement of the product of the proto‐oncogene c‐Cbl in the activation of Pl‐3K triggered by CD16 in human NK cells and the possible mechanisms leading to Pl‐3K recruitment to the plasma membrane. Our results indicate that stimulation of NK cells through CD16 results in a rapid tyrosine phosphorylation of Cbl, which is constitutively associated with Grb2 and forms an activation‐dependent complex with the p85 subunit of Pl‐3K. In addition, we detected the presence of the Grb2‐associated tyrosine‐phosphorylated p36 and Shc proteins in anti‐Cbl and anti‐p85 immunoprecipitates from CD16‐stimulated NK cell lysates. Upon CD16 stimulation, Pl‐3K activity was found associated with Cbl and to a lesser extent with Grb2 and Shc as well as with the ζ chain of the CD16 receptor complex. Overall these results suggest that the formation of a complex containing either Shc or pp36 associated with Grb2, Cbl and the p85 subunit of Pl‐3K is one of the major mechanisms which might couple CD16 to the Pl‐3K pathway in NK cells.

Natural killer (NK) cells and anti-tumor therapeutic mAb: unexplored interactions

Tumor‐targeting mAb are widely used in the treatment of a variety of solid and hematopoietic tumors and represent the first immunotherapeutic approach successfully arrived to the clinic. Nevertheless, the role of distinct immune mechanisms in contributing to their therapeutic efficacy is not completely understood and may vary depending on tumor‐ or antigen/antibody‐dependent characteristics. Availability of next‐generation, engineered, tumor‐targeting mAb, optimized in their capability to recruit selected immune effectors, re‐enforces the need for a deeper understanding of the mechanisms underlying anti‐tumor mAb functionality. NK cells participate with a major role to innate anti‐tumor responses, by exerting cytotoxic activity and producing a vast array of cytokines. As the CD16 (low‐affinity FcγRIIIA)‐activating receptor is expressed on the majority of NK cells, its effector functions can be ideally recruited against therapeutic mAb‐opsonized tumor cells. The exact role of NK cells in determining therapeutic efficacy of tumor‐targeting mAb is still unclear and much sought after. This knowledge will be instrumental to design innovative combination schemes with newly validated immunomodulatory agents. We will summarize what is known about the role of NK cells in therapeutic anti‐tumor mAb therapy, with particular emphasis on RTX chimeric anti‐CD20 mAb, the first one used in clinical practice for treating B cell malignancies.

Expression of interleukin 15 (IL-15) in human rhabdomyosarcoma, osteosarcoma and Ewing's sarcoma.

Interleukin 15 (IL‐15) is a recently discovered cytokine that stimulates lymphocyte proliferation and migration via a trimeric receptor sharing the β and γ signal transducing chains with the IL‐2 receptor. IL‐15 is typically produced by normal cells that do not release IL‐2, but little information is currently available on human tumors. To assess whether human musculo‐skeletal sarcomas produce IL‐15, we analyzed surgical specimens and cell lines obtained from rhabdomyosarcoma, osteosarcoma and Ewings sarcoma. IL‐15 mRNA was present in 9/9 surgical specimens (3 Ewings sarcomas, 5 osteosarcomas and 1 rhabdomyosarcoma). The analysis of a panel of cell lines (7 derived from Ewings sarcoma, 12 from osteosarcoma and 5 from rhabdomyosarcoma) showed that all rhabdomyosarcoma and osteosarcoma cell lines expressed IL‐15 mRNA at levels ranging from low to high, while Ewings sarcoma cells contained little or no IL‐15 message. ELISA assays showed IL‐15 release in a subset of rhabdomyosarcomas and osteosarcomas, but not in Ewings sarcoma. The highest production of IL‐15, in the picogram/ml range, was found in rhabdomyosarcoma cell lines RH30 and RD. Int. J.Cancer 71:732‐736, 1997.

NKG2D/Ligand dysregulation and functional alteration of innate immunity cell populations in pediatric IBD†

Background: Dysregulated innate immune responses play an important role in inflammatory bowel disease (IBD). NKG2D innate immunity receptor is a major sensor of tissue damage that, by recognizing multiple stress‐induced, cell‐associated ligands (MIC‐A/B and ULBP1‐5), potentiates the effector functions of “innate‐like” (&ggr;/&dgr; TcR+, and natural killer receptor+ [NKR+]) T‐cell populations. We analyzed the representivity, NKG2D/ligand expression pattern, and functional ability of the major innate immunity cell populations in pediatric IBD patients. Methods: We analyzed 41 Crohns disease (CD) patients, 33 ulcerative colitis (UC) patients, and 51 age‐matched non‐IBD controls. The expression of NKG2D and its ligands, interferon‐gamma (IFN‐&ggr;) production, and cytotoxic granule release were assessed by immunostaining and multiparameter cytofluorimetric analysis on circulating and mucosal mononuclear subsets; the inflammatory infiltrate was also characterized by immunohistochemistry. Results: The expression pattern of NKG2D receptor and its ligands on mucosal and circulating innate immunity populations is severely disturbed in IBD; NKG2D and ligands are upregulated on immune infiltrate in both CD and UC active lesions; receptor/ligand upregulation also occurs on circulating leukocyte populations, where it depends on both disease activity and type (UC vs. CD). Finally, the frequency and effector capability of peripheral blood “innate‐like” T‐cell populations are also altered in IBD patients. Conclusions: The circulating and mucosal innate immunity compartment is phenotypically and functionally altered in pediatric IBD; some alterations may represent a distinctive feature of the pediatric disease condition. The disturbance of NKG2D/ligand pathway may play a role in sustaining immune activation which leads to chronic inflammatory tissue damage. (Inflamm Bowel Dis 2012)

Tumor-associated and immunochemotherapy-dependent long-term alterations of the peripheral blood NK cell compartment in DLBCL patients

Natural Killer (NK) cells are a key component of tumor immunosurveillance and thus play an important role in rituximab-dependent killing of lymphoma cells via an antibody-dependent cellular cytotoxicity (ADCC) mechanism. We evaluated the phenotypic and functional assets of peripheral blood NK cell subsets in 32 newly-diagnosed diffuse large B-cell lymphoma (DLBCL) patients and in 27 healthy controls. We further monitored long-term modifications of patient NK cells for up to 12 months after rituximab-based immunochemotherapy. At diagnosis, patients showed a higher percentage of CD56dim and CD16+ NK cells, and a higher frequency of GrzB+ cells in CD56dim, CD56bright, and CD16+ NK cell subsets than healthy controls. Conversely, DLBCL NK cell killing and interferon γ (IFNγ) production capability were comparable to those derived from healthy subjects. Notably, NK cells from refractory/relapsed patients exhibited a lower “natural” cytotoxicity. A marked and prolonged therapy-induced reduction of both “natural” and CD16-dependent NK cytotoxic activities was accompanied by the down-modulation of CD16 and NKG2D activating receptors, particularly in the CD56dim subset. However, reduced NK cell killing was not associated with defective lytic granule content or IFNγ production capability. This study firstly describes tumor-associated and therapy-induced alterations of the systemic NK cell compartment in DLBCL patients. As these alterations may negatively impact rituximab-based therapy efficacy, our work may provide useful information for improving immunochemotherapeutic strategies.

Obinutuzumab-mediated high-affinity ligation of FcγRIIIA/CD16 primes NK cells for IFNγ production

ABSTRACT Natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC), based on the recognition of IgG-opsonized targets by the low-affinity receptor for IgG FcγRIIIA/CD16, represents one of the main mechanisms by which therapeutic antibodies (mAbs) mediate their antitumor effects. Besides ADCC, CD16 ligation also results in cytokine production, in particular, NK-derived IFNγ is endowed with a well-recognized role in the shaping of adaptive immune responses. Obinutuzumab is a glycoengineered anti-CD20 mAb with a modified crystallizable fragment (Fc) domain designed to increase the affinity for CD16 and consequently the killing of mAb-opsonized targets. However, the impact of CD16 ligation in optimized affinity conditions on NK functional program is not completely understood. Herein, we demonstrate that the interaction of NK cells with obinutuzumab-opsonized cells results in enhanced IFNγ production as compared with parental non-glycoengineered mAb or the reference molecule rituximab. We observed that affinity ligation conditions strictly correlate with the ability to induce CD16 down-modulation and lysosomal targeting of receptor-associated signaling elements. Indeed, a preferential degradation of FcϵRIγ chain and Syk kinase was observed upon obinutuzumab stimulation independently from CD16-V158F polymorphism. Although the downregulation of FcϵRIγ/Syk module leads to the impairment of cytotoxic function induced by NKp46 and NKp30 receptors, obinutuzumab-experienced cells exhibit an increased ability to produce IFNγ in response to different stimuli. These data highlight a relationship between CD16 aggregation conditions and the ability to promote a degradative pathway of CD16-coupled signaling elements associated to the shift of NK functional program.

T lymphocytes engineered to express a CD16-chimeric antigen receptor redirect T-cell immune responses against immunoglobulin G-opsonized target cells.

BACKGROUND AIMS Chimeric antigen receptors (CARs) designed for adoptive immunotherapy need to achieve two functions: antigen recognition and triggering of the lytic machinery of reprogrammed effector cells. Cytotoxic T cells have been engineered with FcγRIII (CD16) chimeric molecules to be redirected against malignant cells by monoclonal antibodies (mAbs). These cells have been proven to mediate granule-dependent cellular cytotoxicity, but it is not clear whether they can also kill malignant cells by a granule-independent mechanism of cell cytotoxicity. METHODS We engineered a CD16A-CAR equipped with the extracellular CD16A, the hinge spacer and the transmembrane region of CD8, and the ζ-chain of the T-cell receptor/CD3 complex in tandem with the CD28 co-stimulatory signal transducer module. The CD16A-CAR was expressed and functionally tested in the MD45 cell line, a murine T-cell hybridoma with a defective granular exocytosis pathway but capable of killing target cells by a Fas ligand-mediated lysis. RESULTS Our results indicate that in vitro cross-linking of CD16A-CAR on MD45 cells by the Fc fragment of mAb opsonized tumor cells induced interleukin-2 release and granule-independent cellular cytotoxicity. CONCLUSIONS We conclude that strategies aimed to implement the therapeutic functions of mAbs used in the clinic with T-dependent immune responses driven by engineered T cells expressing FcγR-CAR can boost the antitumor efficacy of mAbs used in the clinic.