RICERCA ITALIANA     

Interractins between innate and adaptive immunity: from biology to clinic

Università degli Studi di Roma "La Sapienza"

Abstract

The innate immune system not only delivers immediate effectors to control invading pathogens, but also generates regulatory signals to promote adaptive immune responses. Natural killer (NK) and dendritic cells (DC) are among the major players linking innate and adaptive immunity. Although these cells were initially considered unable of specific recognition, accumulating evidence indicate that they use a wide arrays of invariant receptors to recognize molecular patterns demarcating infectious non self as well as normal and abnormal self. These receptors may either induce or inhibit an immune response depending on the meaning of the signals. This proposal is aimed at evaluating:

- The role of activating and inhibitory receptors, and of adhesion molecules in the regulation of NK cell functions and the signalling pathways initiated by these receptors.
- The activity of novel adjuvants and co-stimulatory molecules in the DC-mediated priming of NK and T cell-mediated responses and in the cross-talk between NK and T cells.
- The requirements for optimizing DC cross-presentation of exogenous antigens.
- The relevance in clinical settings such as primary immunodeficiences or bone marrow transplantation of NK or DC-mediated functions.

A deeper knowledge of the molecular and cellular mechanisms linking innate and adaptive immunity will be instrumental for a more rational design of vaccines. <<<

Principal Investigator

Angela SANTONI Universita' degli Studi di ROMA

Research Objectives

The innate immune system not only delivers immediate effectors to control invading pathogens, but also generates regulatory signals to promote adaptive responses. Among the effector cells of innate immunity, natural killer (NK) cells play a pivotal role in the early phase of immune responses against certain intracellular pathogens and tumors by exhibiting cytotoxic functions and secreting a number of cytokines. An essential link between innate and adaptive immunity is provided by dendritic cells (DC) that function as immunological sensors alert to collect dangerous signals and convey them to naive T cells. This function requires DC maturation that is regulated by different microbial products and microenviromental cytokines. DC can also tune the immune response by modulating either the amplitude or the class.
Cells of innate immunity have been considered "non specific", but phagocytes and DC are endowed with a wide array of invariant receptors including Toll-like receptors (TLR) capable of interacting with the classical "microbial" adjuvants. The ability of TLR to recognize "endogenous" adjuvants released by stressed or damaged cells has been less elucidated. NK cell recognition is mainly based on the detection of both self MHC class I molecules and abnormal self. While self MHC class I antigens are mainly coupled to inhibitory pathways, recognition of abnormal self on infected or transformed cells triggers activating receptors. This indicates that the functional outcome of NK cell interaction with its target is determined by the balance of two opposing forces generated by activating and inhibitory receptors. A number of MHC class I inhibitory receptors have been identified in the past few years; less information have been accumulated on activating receptors and in particular on their ligands.
This project represents the continuation of a previous proposal funded by MIUR 2002 and concerns an area of increasing interest in the biomedical research in immunology. Most of the proponent groups have a high level of international competitivity and qualification, and have furnished important research contributions in this field.
This research proposal is aimed at investigating:
1. The regulation of NK cell effector functions by activating and inhibitory receptors, and in particular: - The ligands of orphan non-MHC I recognizing activating receptors (NCR, NTB-A, and NKp80) responsible for the regulation of NK cell cytotoxicity will be identified and characterized by A. Moretta, whereas Lorenzo Moretta's research unit will attempt to identify the receptor for a novel cell surface molecule expressed by tumor cells capable of inhibiting NK cell cytotoxicity. - The signaling pathways leading to NK cell cytotoxic functions triggered by non-MHC I recognizing activating receptors and the relative contribution of adhesion receptors belonging to the integrin family will be analyzed by Santoni's group, also in collaboration with Notarangelo O.U., who will provide rare specimens from patients with specific deficits in the signalling machinery. - The expression and functional role of different inhibitory and activating NK receptors on NK cells undergoing maturation following bone marrow transplantion will be evaluated by Lorenzo Moretta's group in collaboration with the O.U. Velardi.
2. The functional characterization of cells and molecules operating at the interface between innate and adaptive immunity, and in particular: A. Moretta's, and Notarangelo's groups will characterize some of molecular mechanisms involved in the cross-talk between NK and DC, whereas Santoni's research unit will focus on the receptor pairs regulating the cognate interaction between NK and T lymphocytes. Paroli's group will investigate the requirements for optimal cross-presentation of exogenous antigens by the DC to CD8+ T cells. Del Prete's group will analyze the activity of novel "adjuvant" molecules stimulating cells of innate immunity through interaction with the TLR and their influence on adaptive T and B cell immune responses. Piccolella's research unit will focus on the ability of "costimulatory receptor pairs" such as CD28/B7 to deliver pro-inflammatory signals in T cells.
3. In vivo functional relevance of DC and NK cell-mediated functions in human models of disease, and in particular: Notarangelo's group in collaboration with Santoni's and A. Moretta's research units will investigate the possible defects at the interface between innate and adaptive immunity with particular regard to activation and function of NK cells and DC in selected forms of primary immunodeficiences, namely the Wiskott-Aldrich syndrome (WAS) and its allelic variant X-linked thrombocytopenia, the lymphoproliferative X-recessive disease (XLP), and the WHIM syndrome. Velardi's group will provide important information on the role of alloreactive NK cells in bone marrow transplantation and resistance against leukemia, and on the possibility to transfer donor immunity to opportunistic pathogens across MHC I barriers. <<<

First Results

Identification of novel ligands for orphan non MHC I activating receptors regulating the cytotoxic function of NK cells. - Identification of novel inhibitory receptors and their ligands regulating the cytotoxic function of NK cells. - Identification of the signalling pathways leading to NK cell lysis of different tumor targets. - Identification of the molecular mechanisms regulating ligand-dependent NK receptor down-modulation and their relevance in the attenuation of NK cell responses. - Identification of surface markers of human NK cell maturation.The characterization of the mechanisms underlying the cross-talk between DC and NK cells, and NK cells and T lymphocytes. - The definition of the role of the endocytic processing machinery in the antigen cross-presentation by dendritic cells. -The identification of novel adjuvants capable of directing specific T cell-mediated immune responses. -The signalling events underlying the ability of co-stimulatory molecules such as CD28 to deliver pro-inflammatory signals.The identification of possible defects of dendritic and NK cells that may be important for development of novel prognostic indicators and patient-specific therapeutic strategies.
- A more accurate selection of NK alloreactive donors for haploidentical mismatched bone marrow transplantation. - The development of novel approaches based on the use of alloreactive NK cells to protect from GvHD and reduce the incidence of post-transplant infectious mortality. <<<

Timescale

24 months

National and international background

Protective immunity results from the interplay of two cardinal components: non-specific innate immunity and antigen-specific adaptive immunity. Localized at the epithelial borders, cells of innate immunity recognize non processed antigens using a variety of invariant receptors. The innate immune system is required to deliver immediate effectors to control the invading pathogen and regulatory signals to promote an adaptive immune response (1). The primary components involved in the innate immune response include humoral mediators such as complement and interferons (IFN) alpha and beta, as well as surveillance and effectors cells such as dendritic cells (DC), natural killer (NK) cells and phagocytic cells.
NK cells are effector cells of innate immunity belonging to a distinct lineage of lymphocytes that play an important role in the early phase of immune responses against certain microbial pathogens and tumors by exhibiting cytotoxic functions and secreting a number of cytokines (2). Unlike T lymphocytes, NK cells mediate effector functions spontaneously without the need for prior sensitization and in a non MHC class I-restricted fashion. NK cell functions are augmented by several cytokines including IFN alpha and beta, IL-12, IL-18, IL-2 and IL-15. IFN-gamma-producing NK cells regulate innate resistance by activating phagocytic cells and shape adaptive immunity toward a T helper-1 (Th1) immune response.
Dendritic cells represent the essential link between innate and adaptive immunity as they are the unique inducers of primary immune responses and are capable of instructing antigen-specific cells in order to generate the right class of immune response (3, 4). As a component of the innate immune system, DC collect and transfer information from the out-side world to the cells of the adaptive immune system. Distributed throughout the body, including the various portals of microbial entry, where they reside in an immature form, DC function as immunological sensors alert to collect potentially dangerous signals. After a process of maturation, they convey this information to naive T cells in the secondary lymphoid organs. Here, DC present the processed antigen to T cells, thereby launching an immune response and immune memory through which the antigenic encounter can be remembered even for a lifetime. DC in vivo are also capable of cross-presenting exogenous antigens on MHC I molecules (5), a process critical for induction of CD8+T cell-mediated immunity against tumors, allografts and certain microbial infections, but still not completely elucidated.
DC can also tune the immune response by modulating either the amplitude or the class of the response through their capacity of producing different cytokines including IL-12, IL-18, IFN alpha or IL-10. Different populations of DC (myeloid vs plasmacytoid) appear to be capable of producing distinct cytokines, and this function can be also modulated by microbes and cytokine microenviroment.
Recent reports indicate that DC can also act on the priming arm of innate immunity by triggering NK cell effector functions (6-9). In turn, activated NK cells can induce either maturation of DC or negatively control their activation by displaying cytotoxicity against immature DC. Thus the cognate interaction between NK and DC provides a further coordinated mechanism for regulating the initiation and amplification of immune responses. Emerging evidence indicate that NK cells may also shape adaptive immune responses by interacting with T lymphocytes, but the receptor pairs involved and the functional relevance of this novel cellular cross-talk have not been explored yet.
An essential difference between innate and adaptive immunity is the means by which they recognize self and nonself.
Innate immune systems use proteins encoded in the germ line to identify potentially noxious substances. Recognition of microbial products is mostly based on the detection of conserved molecular patterns (PAMP) that are unique to microbes and shared by a large group of microrganisms. One major class of surface receptors evolved to recognize PAMP is the Toll-like receptor (TLR) family (10, 11). TLRs activate signalling pathways that induce anti-microbial effector responses and inflammation upon recognition of PAMP. In addition, activation of TLRs on dendritic cells plays a critical role in the initiation of adaptive immune responses. TLR triggering on these cells induces the expression of co-stimulatory molecules on the cell surface and pro-inflammatory cytokines such as IL-12, IL-18, IFN alpha/beta, needed for the activation of naive T cells. In this regard, TLRs are considered to be adjuvant receptors, in that most of the effects of microbial T cell adjuvants have been assigned to members of TLR family (12). Also nonmicrobial endogenous products, such as those released by necrotic cell death and stressed or damaged tissues, can act as powerful adjuvant and intersect at the level of the DC (12). These evidence support the idea that mechanisms enabling the detection of both indirect (damage) and direct (pathogen structures) signatures of infection might be co-evolved. However, only little evidence are available on the ability of TLRs to recognize endogenous ligands released from damaged or stressed mammalian cells. Moreover, the information on the expression and functions of TLRs on cells other than monocytes/macrophages and DC are still rather poor. Elucidating the effects of adjuvants and their receptors on NK cells and on different lymphocyte subsets of adaptive immune response will undoubtedly bring novel insights into the mechanisms underlying immune response to stress and damage and its possible involvement in autoimmunity.
Self/nonself discrimination by NK cells is based on the recognition of missing self and abnormal self. Missing self recognition is coupled with inhibitory pathways that block initiation of immune responses against self, whereas recognition of abnormal self on infected or transformed cells triggers activating receptors (13).
The "missing-self" hypothesis was initially proposed to explain the protective effect of target cell MHC class I on NK lysis (14). When tissues undergo partial or complete loss of MHC class I, such as during viral infections and neoplastic transformation, this inhibitory influence is released permitting target lysis mediated by NK cells. In the last few years, much attention has been given to the characterization of inhibitory receptors recognizing MHC class I molecules. They comprise molecules belonging to immunoglobulin-like (KIR) and C-type lectin (CD94/NKG2A) families characterized by the presence in the cytoplasmic tail of a sequence called ITIM (immunoreceptor tyrosine-based inhibitory motif) (15). When the tyrosine in the ITIM is phosphorylated, it recruits the protein tyrosine phosphatases SHP-1 and SHP-2, which in turn inhibit NK cytotoxic activity and cytokine release by dephosphorylating tyrosine-phosphorylated cellular substrates critical for activating NK cells.
The knowledge about NK receptors delivering positive signals is less advanced (16, 17). Evidence is emerging that NK cells may use a combination of several receptors and signalling pathways for the recognition of stressed, unhealthy and abnormal cells (13). Most of the activating receptors are transmembrane molecules with short intracellular domains that lack intrinsic signalling activity. They function by coupling to ITAM (immunoreceptor tyrosine-based activating motif)-containing signal-transducing transmembrane adaptor molecules through charged amino acids in their transmembrane regions. Human DAP-12-associated receptors include KIR2DS, CD94/NKG2C and NKp44. CD16, the low-affinity receptor for the Fc fragment of IgG, NKp30 and NKp46 receptors couple to the FcepsilsonRI gamma and CD3 zeta adaptor proteins. KIR2DS and CD94/NKG2C receptors bind to MHC class I molecules, but with lower affinity as compared to the inhibitory receptors This suggests that the ligands for these activating receptors may be of different nature, namely foreign or microbial antigens that resemble MHC class I molecules. NKp44 and NKp46 receptors have been reported to bind to the hemoagglutinin of the influenza virus due to the presence of sialic acid on these receptors. Moreover, the existence of ligands for these receptors on tumors is suggested by the ability of specific antibodies against these receptors to block killing of selected tumor targets. Another activating NK cell receptor is NKG2D, a C-type lectin disulphide linked dimer that forms a complex with DAP 10, an adaptor molecule containing in the cytoplasmic domain a specific aminoacid sequence capable of recruiting phosphoinositide-3-kinase (PI-3K). NKG2D recognizes several MHC-class-I-related ligands that are preferentially expressed on tumor or virus-infected cells (18). Recent evidence demonstrate that NKG2D ligands are also expressed by activated T cells (19), thus suggesting a role for NKG2D-mediated NK cell lysis in the negative regulation of T cell responses. Finally, several surface molecules (beta 1 and beta 2 integrins, CD44, CD28, CD244, TNF family) may be involved in the regulation of NK cell activation displaying either stimulatory or co-stimulatory function (20-23). Of interest, CD244 that recognizes the CD48 adhesion molecules associates with the signalling lymphocyte activation molecule (SLAM)-associated protein SAP, a cytoplasmic adaptor that enables the receptor to activate NK cells. Patients who have a recessive genetic X-linked lymphoproliferative syndrome lack a functional SAP protein and the CD244 receptor fails to trigger NK cells in these individuals (23, 24). EBV infection in these patients results in fatal infectious mononucleosis and those who survive can develop B cell lymphomas, indicating that SAP-associated receptors might be critical in the control of EBV infection. In addition to CD244, SAP associates with other receptors including NTB-A, a novel molecule belonging to the CD2 subfamily that contributes to the inability of NK cells to kill EBV-infected B cells in X-linked lymphoproliferative disease (25). Overall, the ability of NK cells to lyse specific target cells or to secrete regulatory cytokines depends on a combination of structurally distinct activating and inhibitory receptors and co-receptors. <<<