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Clinical and Experimental Immunology

P.I.: Domenico Mavilio, MD PhD

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Domenico Mavilio

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The Unit of Clinical and Experimental Immunology will have access to the clinical facilities of Istituto Clinico Humanitas in order to enroll patients for all programs of translational medicine explained above. In this regard, Dr. Mavilio and Dr. Brunetta will spend a part of their professional duties as physicians within the Department of Internal Medicine of Istituto Clinico Humanitas where a small unit of Clinical Immunology (1 ambulatory and 4 beds to admit patients affected by immunological disorders) has been established. We will submit a detailed research proposal to the Institutional Review Board (IRB) of Istituto Clinico Humanitas, the host organization where patients will be recruited, in order to receive approval to work with human material for our projects of translational medicine. We will educate all patients interested in participating in our program about the aims of our project of clinical research and they will be asked to sign a consent that authorizes us to use their specimens for research purposes only.

This unit will also collaborate with other clinical facilities of Istituto Clinico Humanitas (units of hepatic surgery, gastrointestinal surgery, internal medicine) to enroll patients for programs of translational medicine.

MAIN RESULTS

Dr. Mavilio joined Istituto Clinico Humanitas and Fondazione Humanitas per la Ricerca In September 2008. Before then, Dr. Mavilio’s group investigated on the innate immune system in the physiopathology of viral infection and immunological diseases at Laboratory of Immunoregulation of National Institute of Allergy and Infectious Diseases, national Institutes of Health, Bethesda, Maryland, USA. In particular, the research activity was focused on the role of Natural Killer cells and DCs in the pathogenesis of HIV-1 infection. In our studies performed over the past 6 years, we demonstrated that in chronic and active HIV-1 infection both NK cells and DCs are affected by several phenotypic and functional abnormalities that contribute to the lack of immune-mediated control of HIV-1. In particular, we characterized in HIV-1 infected donors the expansion of a markedly dysfunctional subset of CD56 ^neg/CD56 ^pos NK cells, a population that is rarely represented in uninfected individuals. Even the ability of NK cells to kill tumor cell targets or virally infected cells is markedly impaired in active HIV-1 infection due to the altered expression and functions of several inhibitory and activating NK cell receptors. Moreover, the levels of important antiviral cytokines (IFN-g, TNF-a, GM-CSF)secreted by NK cells is significantly decreased in HIV-1 infected patients with high levels of ongoing viral replication. Finally, we demonstrated that numerous components of the bidirectional NK-DC cross talk are markedly defective in cells purified ex vivo from viremic HIV-1 infected individuals together with aberrancies in DC maturation and function. All these results have been published in several reports on peer reviewed scientific journal.

Dr. Mavilio investigations on HIV-1 pathogenesis were first performed within the Innate Immune Section of the Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases (Director. Anthony S. Fauci), National Institutes of Health, Bethesda, Maryland USA. From January 2008, this research field was transferred at Istituto Clinico Humanitas where Dott. Mavilio established a new Unit of Clinical and Experimental Immunology. Within this new laboratory, Dr. Mavilio and his group started other research projects that will be reported in the following paragraph.

MAIN OBJECTIVES AND RESEARCH LINES

1) Innate Immunity and HIV-1 Infection

On the basis of Dr. Mavilio’s previous research that identified several abnormalities in NK cell phenotype and functions during the course of HIV-1 infection, the Unit of Clinical and Experimental Immunology will keep investigating on the molecular mechanism(s) of these NK cell dysfunctions. In particular, we are currently performing the following experimental approaches: A) Matching the increased surface levels of inhibitory NK cell receptors (iNKRs) on NK cells from HIV-1 infected patients with their MHC-I aplotype: our aim is to understand whether or not HIV-1 is able to escape innate immune control by driving the expansion of NK cell subset expressing high levels of particular iNKRs that limit the NK cell-mediated lysis of HIV-1 infected cells through the binding of specific alleles of MHC-I molecules. B) Performing a microarray analysis on HIV-1 infected and uninfected CD4+ T cells derived ex vivo from infected patients in order to identify the differential genes involved in the expression of ligands for activating NK cell receptors (NCRs) only on uninfected cells and not on infected cells; C) Determining which HIV-1 molecules are responsible for the expression on NKG2D ligands on infected CD4+ T cells using HIV-1 strains selectively deleted of one or another HIV-1 genes; D) On the basis of surface levels of ligands for activating and inhibitory NK cell receptors on HIV-1 infected cells, we plan to select NK cell subsets, mismatched in their iNKRs/MHC-I molecules interactions, expressing high surface levels of those activating NK cell receptors (NCRs and NKG2D) whose ligands are on autologous HIV-1 infected cells. We will activated them ex vivo from patients and test their ability to kill autologous and endogenously HIV-1 infected CD4+ T cells. Our final goal is to enhance NK cell lytic potential by selecting those NK cell subsets that are still able to eliminate HIV-1 infected cells and by depleting dysfunctional and pathologic NK cell populations with an impaired antiviral activity. With the above described experimental approaches, we aim to significantly increase the rate of killing of HIV-1 infected cells, reduce the spreading of infection and limit the establishment of viral reservoirs. E) We also are investigating on the possible direct interaction between HIV-1 and NK cells in order to disclose the receptor-pathways triggering the aberrant activation of these cells during the course of HIV-1 infection. F) Finally, we already reported that the aberrant NK cell phenotype correlate with the clinical stage of HIV-1 infection and with the response to antiretroviral therapy. Therefore, we are currently setting a multicentric clinical trial to find out if NK cells can serve as a novel biological markers that identify clinical phases of diseases and compliance to the therapy.

2) Role of innate immunity in the pathogenesis of autoimmune diseases

Even though several important aspects of the pathogenesis of autoimmune diseases have been already clarified or are currently under investigation (T and B cell tolerance, nature of auto-antigens, characteristics of auto-antibodies, genetic aspects of auto-reactivity, apoptosis, cytokines involved, etc.), very little is known about the role of NK cells and DCs in such pathological conditions. On the basis of our previous experience focused on HIV-1 infection, we sought to study the contribution of innate immune responses in the physiopathology of other human pathologic conditions. In this regard, we plan to set up clinical trials to enroll patients affected by the following systemic autoimmune diseases: 1) Systemic Lupus Erythematosus (SLE); 2) Rheumatoid Arthritis RA; 3) Systemic Sclerosis; 4) Bechet Syndrome; 5) Sarcoidosis 6) Sjogren’s Syndrome. The aims of the proposed projects will focus on the following:

A) To extend the analysis of the role of innate immune system to other important human diseases that, similarly to HIV-1 infection, also feature a chronic and aberrant activation of the immune system. Our goal is to establish whether or not the several NK cell and DC abnormalities observed in the course of active and chronic HIV-1 infection are restricted to this specific and pathologic human condition. Our hypothesis is based on the evidence that, during the course of HIV-1 infection, it is not HIV-1 that directly trigger NK cell and DC abnormalities, but is rather the presence of a chronic and aberrant immune activation. Therefore, we want to investigate whether or not other model of human diseases sharing chronic inflammation have similar abnormalities in the NK cell and Dc compartments.

B) To specifically investigate on the role of NK cells and DCs in the pathogenesis of several systemic autoimmune diseases. Using techniques of cellular and molecular biology, we are analyzing all phenotypic and functional aspects of freshly purified NK cells and DCs from patients affected by auto-reactive disorders in order to characterize any potential and harmful aberrancy associated with auto-immunity. In human diseases that lack an effective and optimal immunologic tolerance, we think it is extremely important to understand whether NK cells are still capable of sparing normal and autologous self-cells or are still able to eliminate transformed and potential hazardous non-self target cells.

C) To evaluate the effect of chronic and aberrant immune activation on NK and DCs in untreated naïve patients in active phases of diseases and to establish the effect of immunosuppressive and/or biologic therapy in subjects with diseases in remission either in cross-sectional or longitudinal studies.

D) To analyze the interactions between NK cells and DCs in systemic autoimmune disorders. The ability of DCs to achieve an optimal maturation after antigen uptake is essential for the antigen-specific priming of autologous T cells. This process will guarantee an effective link between innate and adaptive immune responses. In this regard, it is essential to understand whether DCs, either alone or in their interactions with autologous NK cells, are still able to properly undergo maturation.

E) To develop new diagnostic tools that, through the characterization of the status of innate immune competent cells, could better characterize the clinical stages of autoimmune disorders and the efficacy of the immunosuppressive agents and biological therapies in reducing inflammation.

3) Trafficking of Natural Killer cells in human liver, intestine and cervix/uterus.

The main aim of the present project is to exactly characterize the phenotype, functions and trafficking of NK cells in the human tissues under physiological condition. The majority of the studies that analyzed NK cell characteristics and trafficking have been performed preferentially in experimental model rather than human given the difficulties in obtaining large specimens of hepatic tissue from healthy individuals and the technical limitation in processing and analyzing a sufficient number of NK cells. In this regard, it has been described that an average human liver contains a high amount of lymphocytes in its infiltrate (1 x 10 ¹⁰) including a large fraction of NK cells simply defined as CD56 ^pos cells. We are currently collecting specimens from healthy livers from human donors in order to investigate on NK cell characteristics and fill this important chapter of physiology on innate immune system in peripheral tissues. In particular, we will study: A) A possible local development and differentiation of NK cells within the liver, independent from NK cell ontogenesis occurring in thymus or bone marrow, that might be responsible of the composition, distribution and compartmentalization of intrahepatic NK cells; B) If the migration and recruitment of intra-hepatic NK cell subsets from peripheral blood occurs through specific pathway(s) driven by chemokines or other adhesion molecules; C) The baseline activation state of NK cells in the absence of a direct inflammatory insult of human liver. It has been shown that intrahepatic lymphocytes generally have a more activated phenotype in comparison to peripheral cells. These observations are consistent with the role of the liver in filtering intestinal antigens. The liver receives the portal circulation, which drains much of the gastrointestinal tract. It remains to be investigated whether NK cells are affected by this phenomenon and if they respond directly to the chronic antigen stimulation occurring in the liver. It will be also important to highlight the differences in NK cells cell phenotype and functions in the presence of specific inflammatory insult of the liver such as hepatitis B, hepatitis C and cytomegalovirus infections, cancer and other pathologic condition.

As secondary objective of this project, we are investigating on the interactions between NK cells and Dendritic Cells (DCs) in human liver. Indeed, the liver contains several types of cells that can function as antigen presenting cells (APCs), including resident dendritic cells, sinusoidal endothelial cells, Kupffer cells and, under some conditions, even hepatocytes and cholangiocytes.

We are currently setting up a similar project to investigate the trafficking of NK cells in human guts and cervix/uterus, in order to study the physiology of NK cells in this tissue compartments and understand whether NK cells have any role in the pathogenesis of inflammatory bowel diseases and tumor of the cervix.

4) Neuro-immune modulation of inflammatory responses: role of Natural Killer cells

The origin of neuroimmunology dates back to early ‘70s. Nevertheless, only recently, unpredicted potential therapeutic applications have been identified. In particular, an emerging issue is the role of the catecholamines (CAs) dopamine (DA), norepinephrine (NE) and epinephrine (E) as both neuroendocrine and immune cell-derived mediators linking neural and immune networks. Until a few years ago, DA, NE and E were just considered conventional neurotransmitters. However there are now robust evidences demonstrating that also immune cells produce CAs exerting significant modulations on the function and homeostasis of immune system in humans.

Very little is known on the ability of NK cell to produce CAs and on modulation of NK cell effector-functions given by DA, NE and E. We have undertaken a multidisciplinary project that will exploit the role of NK cells in the interactions between nervous system and innate immunity. The first aim of this project is to understand whether NK cells respond to stimuli given by important neurotransmitters either through paracrine or autocrine mechanisms, as described in other human lymphocytes. Given the fact that NK cells serve as effector cell as well as regulatory cells in the links between innate and adaptive immune responses and that they express on their surface some of the receptors for catecholamine, we think is important to understand if DA, NE and E are able to influence NK cell cytolytic ability, NK cell production of cytochines and chemokines, NK cell homing and migration to peripheral tissues and the NK-DC crosstalk.

As second task of the present project, we plan to translate our basic research ex vivo on a model of human diseases. In this regard, it has already been showed that catecholamine markedly affect the functional maturation of DCs and enhance the replication of HIV-1, Moreover, we and others recently demonstrated that in course of HIV-1 infection high levels of chronic viremia are associated with several pathologic aberrancies of NK cell and DC phenotype and function. Whether DA, NE and E have a direct role in the highly defective responses of innate immune compartment in HIV-1 infected patients is still remain to be determined. Understanding the role of catecholamine in the pathogenesis of abnormal response of NK cells and DCs in HIV-1 infection could give new insights on new therapeutic HIV-1 strategies, considering also that different agonist and antagonist of catecholamine are commercially available and already used for the cure of several neurological human diseases.

The Unit of Clinical and Experimental Immunology will have access to the clinical facilities of Istituto Clinico Humanitas in order to enroll patients for all programs of translational immunology explained below. In this regard, Dr. Mavilio will spend a part of their professional duties as physicians within the Department of Internal Medicine of Istituto Clinico Humanitas where a small unit of Clinical Immunology (2 ambulatory and 5 beds within the department of Internal Medicin to admit patients affected by immunological disorders) has been established. This unit will also collaborate with other clinical facilities of Istituto Clinico Humanitas (units of hepatic surgery, gastrointestinal surgery, gynecological surgery and internal medicine) to enroll patients for programs of translational medicine.

Moreover, in order to implement and make it possible all these research programs, Dr, Mavilio started several scientific collaboration with several Italian universities (Milano, Genova, Pavia, Napoli, Padova, Varese, Udine) ed research institues in Italy, (Istituto Giannina Gaslini, Genova; Istituto San Raffaele Pisana di Roma), Europe (Istituto di Ricerca in Biomedicina, Bellinzona, Svizzera; Instituto de Medicina Molecular, Faculty of Medicine, University of Lisbon, Portugal) e USA (The Wistar Institute, Philadelphia, Pennsylvania; Rush University Medical Center, Chicago, Illinois; National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD).

FINANCIAL SUPPORT (COMPLETED AND ACTIVE)

09/2003 – 09/2007: Intramural Program of NIAID/NIH, USA

Project Number Z01 AI000958-01 LIR ($ 130.000,00 per anno) (Completed).