Potential Drugable Targets For Immunotherapies

Published Date: 02 Nov 2017

1Department of Clinical Medicine, Institute of Molecular Medicine, Trinity College Dublin, Ireland

2Faculty of Medicine, Imperial College London, London, UK

Abstract

To be able to regulate lymphocyte trafficking, the integrin LFA-1 efficiently coordinates multiple signal transduction pathways and a number of signalling molecules. One of the most important challenging questions is - how an array of biochemical signals linked via LFA-1 is translated to an appropriate migratory response in T-cells. In this context, a class of proteins with multiple modular domains capable of recruiting a number of intracellular molecules collectively known as adaptor proteins, is emerging as key mediators of LFA-1-mediated T-cell motility. They can simultaneously interact with two or more effector molecules and orchestrate the assembly of multi-functional complexes that are important for the propagation of diverse cellular process including LFA-1 associated signal transduction in migrating T-cells. Here, we provide an overview of the involvement of important adaptor proteins in the intracellular signalling cascades by which LFA-1 regulates T-cell migration. The complexity of the LFA-1 associated signalling delineated in this review suggests a series of future research that may be important for the development of immunotherapies.

Introduction

The mobilization and deployment of T-lymphocytes into lymph nodes or inflamed tissues with defined specificity are important for the initiation and maintenance of an effective immune response (Bradley, 2003; Salmi and Jalkanen, 2005; Mora and von Andrian, 2006; Woodland and Kohlmeier, 2009). These complex, well-orchestrated and precisely regulated process of T-cell movements are guided by a concordant series of signalling pathways that are organized in time and space. In order to establish a proper immune response, T-cells require triggering of a set of adhesion receptors and an array of intricate molecular networks. Although several independent but coordinated cellular processes involving an ever increasing number of molecules contribute to T-cell activation, motility and effector functions, integrin family of cell surface receptors plays key roles in mediating these processes. In particular, integrin lymphocyte function associated antigen-1 (LFA-1, also known as αLβ2 integrin) and LFA-1-mediated signal transduction processes are important for T-cell adhesion, polarization, extravasation and migration (Gahmberg, 1997; Smith et al., 2007; Evans et al., 2009; Hogg et al., 2011). LFA-1 mediates adhesive phenomena in T-cells through interaction with its counter receptors or ligands present on the endothelium, of which intercellular adhesion molecule-1 (ICAM-1) plays a major role.

Bidirectional signals via LFA-1:

As a predominant cell adhesion molecule and a transmembrane receptor present on T-cells, integrins including LFA-1 are unique in that they dynamically transmit molecular information through bidirectional signalling i.e. not only information flows from extracellular stimuli to induce intracellular changes (outside-in signalling), but intracellular stimuli can also cause extracellular changes (inside-out signalling). Like other integrins, the functional state of LFA-1 is tightly regulated. LFA-1 receptors are normally expressed on the T-cell surface in an inactive conformation and are unable to efficiently bind to their ligands, such as ICAM-1. This is particularly important for immune cells, which circulate freely through the bloodstream with minimal interactions with the vessel wall, thus avoiding inappropriate adhesion while maintaining the ability to adhere quickly at sites of infection or inflammation. LFA-1 undergoes a series of conformational changes during a partial to full activation (Laudanna, 2005; Smith et al., 2007; Evans et al., 2009; Hogg et al., 2011). In inside-out signaling, integrin-independent signals such as stimuli received by cell surface receptors for cytokines and chemokines, T-cell receptor (TCR) for foreign antigens or phorbol esters initiate intracellular signalling cascades that impinge on cytoplasmic domains of LFA-1 triggering a complex modality of its activation. This activation increases the affinity and adhesiveness of LFA-1 for extracellular ligands, which ultimately facilitates its clustering. Following activation and clustering, LFA-1 receptors are able to transmit an array of biochemical and molecular signals from its extracellular domains across the plasma membrane to the cytoplasm collectively referred to as "outside-in signalling" (Figure 1), all of which are essential for lymphocyte homing.

Although LFA-1 does not have any catalytic activity, its cytoplasmic tails serve as docking sites for many signalling proteins, which propagate outside-in signals through direct and indirect interactions with several other molecules. The cellular signals initiated by LFA-1 engagement are governed mainly by protein post-translational modifications, recruitment of protein binding partners to specific subcellular domains, and through protein-protein/lipid interactions. These signals are transmitted downstream through a series of signalling cascades by activating/deactivating multiple kinases, phosphatases or proteases influencing their respective substrates (Smith et al., 2003, 2007; Evans et al., 2009, 2011; Hogg et al., 2003, 2011; Abram et al., 2009; Kinashi et al., 2007). We have identified a number of proteins undergoing tyrosine phosphorylation following LFA-1 stimulation (Verma et al., 2011). In addition, we have recently reported the transcriptional activation of numerous genes following LFA-1/ICAM interaction (Verma et al., 2012). Because LFA-1 can often modulate a broad range of cellular responses and there are numerous proteins that either directly of indirectly interact with the LFA-1 cytoplasmic tail, there must exist spatio-temporal regulation of these interactions by different mechanisms. Therefore, it is vitally important to understand the precise spatial and temporal regulation of LFA-1 interaction with its cellular counter-receptors in the various contexts, if this molecule is to be used as a therapeutic target for developing new immunomodulatory drugs.

Role of membrane-rafts:

For a structural organization during the process of migration, T-cells require an orchestrated activation, compartmentalization of membrane receptors and redistribution of several molecules in specific cell locations. Some of these molecules participating in inside-out signalling or facilitating specific downstream responses following LFA-1 engagement have been described as constituents of plasma membrane microdomains enriched in cholestrol, sphingolipids (sphingomyelin and glycosphingolipids) and glycerolipids containing mainly saturated fatty acids residues. These specialized patches of extremely dynamic laterally mobile submicroscopic detergent resistant microdomains that make up to 40% of the membrane of lymphocytes are known as "membrane-rafts". The highly saturated fatty-acid side chains of the phospholipids in membrane rafts enable them to serve as signalling platforms by colocalizing the requisite components and facilitating their interaction (Jeon et al., 2010). Since many important signalling molecules are present in the membrane rafts, they have been implicated in signal transduction through a wide range of receptors, including LFA-1. Membrane-rafts contain proteins and lipids involved in the regulation of cytoskeletal rearrangements. Several reports have now revealed selective recruitment of membrane-rafts to active regions of polarized lymphocytes and demonstrated molecular mechanisms controlling membrane-raft dynamics in T-cells. Key molecules that participate in tethering membrane rafts to the cytoskeletal systems have been identified, suggesting the existence of feedback loops between rafts and the cytoskeleton during T-cell migration and activation (Viola and Gupta, 2007). The lateral mobility of LFA-1 on the plasma membrane also contributes to LFA-1-mediated lymphocyte adhesiveness (Constantin et al., 2000; Laudanna, 2005). In addition, the association of membrane receptors with distinct raft domains dictates their redistribution to the appropriate location during lymphocyte migration. Migrating T-cells acquire polarized shapes by asymmetric redistribution of membrane receptors and other crucial signalling mediators between the L-raft (at the leading edge, which protrudes at the front of cell), and the U-raft (at the uropod of the cell, which retracts). The U-raft contains receptors and signalling molecules that are involved in cell adhesion, such as the ezrin, radixin, and moesin (ERM) proteins, CD44, and intercellular adhesion molecules, whereas the L-raft contains the machinery that senses environment stimuli and induces localized actin polymerization. This segregation of molecules between the two membrane-raft types may enhance directional sensing by increasing integrins (such as LFA-1) and chemokine receptors at the leading edge and adhesion receptors (such as ICAMs) at the rear (Viola and Gupta, 2007). Thus by generating a favourable environment for signal transduction in migrating T-cells, membrane-rafts not only regulate signalling efficiency but also restrict and/or localize, to specific cell areas, the signalling machinery required for gradient sensing and cytoskeletal rearrangements.

The growing understanding of molecular and biochemical basis behind an efficient motility of T-cells to yield an appropriate immune response has uncovered numerous intracellular molecules that are integrated correctly into a signalling complex dictated by spatiotemporal demand. In this context, an emerging class of proteins governing cross-talk and specificity of the signal transduction pathways and playing key roles in lymphocyte motility is ‘adaptor proteins’. A number of adaptor proteins that play integral roles in T-cell migration have been identified in numerous studies published over the past two decades. Spontaneous autoimmunity has also been reported in mice lacking several adaptor proteins of lymphocytes, demonstrating their critical involvement in maintaining T-cell tolerance and homeostasis. In this review, we focus primarily on adaptor proteins that interact with LFA-1 integrin and contribute as regulators of T-cell migration, although we incorporate information gained from other immunoreceptors and leukocytes as appropriate. Detailed list of T-cell adaptors and discussion on their relevance to the regulation of various lymphocyte functioning can be found elsewhere (Ref).

T-cell adaptor proteins

Adaptor proteins are critical regulator of immune response as they play important roles in the integration and propagation of signals for lymphocyte migration processes. They bring effector molecules into close proximity to their targets and control cellular functions by mediating constitutive or inducible protein-protein/lipid interactions via specific modular domains. These domains, often multiple, bind and recruit other signalling proteins that link protein-binding partners together facilitating creation of larger signalling complexes. Important modular domains for such protein-protein interactions include Src homology 2 (SH2) and phosphotyrosine-binding (PTB) domains that recognize short phosphotyrosine motifs, Src homology 3 (SH3) domain that bind proline-rich regions (PxxP), pleckstrin homology (PH) domain that associate with phospholipids, and tyrosine-based signaling motifs (TBSMs) comprising short peptide sequences containing a core tyrosine residue, which upon phosphorylation, mediates a high-affinity interaction with SH2 or PTB domains (Figure 2) (Sudol, 1998). Many adaptors contain multiple domains and act as scaffolds to facilitate the formation of macromolecular signalling complexes. The particular type of protein binding domains within adaptor proteins dictates specificity, sub-cellular localization, its proximity to the binding partners and its potential modification and regulation by enzymes, such as protein tyrosine kinases (Isakov, 2008). By linking specific proteins together in the organization of signalling complexes for lymphocyte trafficking, adaptors mediate propagation of cellular signals that prompt an appropriate response from the cell to the environment or vice versa. Adaptor proteins appear to connect either common or distinct molecules to downstream pathways and function in different ways depending on the lineage of the specific cell. Hence, these proteins regulate cell signalling in both a spatial and a temporal fashion to specifically regulate the interaction between the cell and its environments. In the recent past, biochemical and molecular studies have greatly helped to understand the biological role of adaptor proteins and on the basis of their both localization and functions they have been classified into two major groups: transmembrane and cytosolic adaptor proteins.

T-cell transmembrane adaptor proteins:

The integral membrane protein (transmembrane) adaptors, also known as transmembrane adaptor proteins or TRAPs, form the backbone of signalling complexes at the membrane and are responsible for the recruitment of additional adapters and enzymes. This subgroup of adaptors contains short extracellular domanin, single transmembrane domain, and intracellular domain with multiple tyrosine motifs, which can rapidly become phosphorylated after specific triggering. The activated intracellular domains then associate with SH2-domain containing effector molecules such as Grb2, PLC, SLP-76, PI3K, and SHP2. The membrane anchor, which is usually in the amino terminus, can consist of a transmembrane region, fatty acid modification or a PH domain, which mediates membrane association by binding to phosphatidylinositol phosphates. The primary function of these proteins is to redirect signalling molecules to required locations in the membrane rather than to directly arrange protein-protein interactions, although these functions are closely associated.

A number of TRAP families expressed in lymphocytes have been identified during the last two decades, and are listed in Table 1. Four of the known TRAPs reside in lipid-rafts: LAT (The linker for activation of T-cells), NTAL (non T-cell activation liner; also known as linker for activation of B-cells, LAB), PAG (phosphoprotein associated with glycosphingolipid-enriched microdomain; also known as carboxy-terminal Src-kinase (Csk)-binding protein, CBP) and LIME (Lck-interacting membrane protein). The other three known non-raft TRAPs include SIT (SH2-domain-containing protein tyrosine phosphatase SHP2-interacting TRAP), TRIM (TCR-interacting molecule) and LAX (linker for activation if X cells, where X denotes an as yet unidentified cell) (Sommers et al., 2004; Horejsí et al., 2004). All raft-associated TRAPs have a juxtamembrane CxxC motif (where ‘x’ denotes any amino acid), which becomes palmitoylated and is required to target these proteins to rafts. The cytoplasmic tails of all these TRAPs are more or less similar if not identical (Figure 3).

T-cell cytosolic adaptor proteins:

The cytosolic adaptor proteins, also known as CAPs, primarily mediate protein-protein interactions in the cell cytoplasms.They contain a variety of protein-binding modules that link multiple partners together and facilitate the creation of large signalling complexes. Specificity in signalling is achieved by the type of protein binding modules encoded by CAPs, the sequence of domains or motifs that dictates specificity in binding, as well as the sub-cellular localization and proximity of binding partners. T-cell cytosolic adaptor proteins associated with LFA-1-mediated signal transduction fall into three broad categories: 1) adaptors that have a mainly structural function, 2) adaptors that fulfill a scaffolding function by providing binding sites for additional focal-adhesion proteins, and 3) adaptors that have catalytic activity.

Structural adaptors connect LFA-1 to cellular cytoskeletal systems via multiple routes and specific adhesions. For example, talin and filamin bind to F-actin and therefore couple LFA-1 integrin to the cytoskeleton (Critchley, 2009; Critchley and Gingras, 2008; Nurmi et al., 2006). Both talin and α-actinin bind to integrin cytoplasmic domains and actin (Hemmings et al., 1996). In addition, talin recruits the actin nucleating protein complex Arp2/3 via constitutive association of vinculin with talin and Arp2/3, thus facilitates cytoskeletal reorganization (Mace et al., 2010). Vav serves as an adaptor that links cytoskeletal and signaling molecules to actin by physically associating with talin, vinculin and SLP-76 (Fischer et al., 1988; Tuostoet al., 1996; Bustelo, 2001). Another adaptor protein SH3P7 also links signal transduction from lymphocyte integrins to components of the cytoskeleton (Larbolette et al., 1999). Catalytic adaptors can place signalling enzymes from multiple pathways in close proximity to a subset of substrates, modulate the specificity and the affinity of the bound enzymes to these substrates and thus effectively increase the repertoire of their functions. In these cases, catalytic adaptors coordinate physical association between enzymes and substrates and thus facilitates the catalysis of specific reactions. For example, paxillin binds to FAK, GTPase activating proteins (GAPs), ILK etc. and facilitates their activities (Rose et al., 2003; Tabassam et al., 1999; Wang, 2009). A recently identified signal transducing adaptor protein-2 (STAP-2) containing PH and SH2-like domains as well as a YXXQ motif in its C-terminal region associates with FAK and enhances its degradation (Sekine et a., 2009). T-Cell Specific Adapter Protein (TSAd) through its interaction with Tec kinase ITK and Lck and promotes T-cell migration (Berge et al., 2010). Scaffolding adaptors serve important roles in LFA-1-mediated signal transduction by acting as a scaffold and coordinating interactions between signalling intermediates. These include SLP-76, Gads, Gab, ADAP, 14-3-3, Nck etc. Although there is a clear distinction between the three classes of CAPs, there is significant functional crossover because of the other interactions of these proteins. For example, FAK can interface with the actin cytoskeleton through an interaction with the actin-regulatory Arp2-Arp3 (Arp2/3) complex (Serrels et al., 2007). FAK can also form a complex with talin and both interact with integrins (Serrels and Frame, 2012; Lawson et al., 2012). The interconnectedness of protein-protein interactions that are mediated by adaptors is key to the assembly of the complex structural and signalling platform required by T-lymphocytes in the migratory processes. Table 2 presents a list of cytoplasmic adaptor proteins identified to be involved in LFA-1-mediated signalling in T-cells.

Adaptors regulation of inside-out signalling

More than two decades of research has now expanded the understanding of intracellular signalling pathways that cooperate in T-cells to bring about the dramatic conversion of the integrin LFA-1 from bent conformation to the extended high-affinity form. The signalling pathways involved in LFA-1 stimulation via inside-out signalling in lymphocytes have been investigated using three different modes of triggering: 1) TCR activation, 2) cytokines or chemokine signalling, and 3) selectin binding to the ligands P-selectin glycoprotein ligand 1 (PSGL1), CD44 (Koopman et al., 1990), and CD43 (Hogg et al., 2011; Matsumoto et al., 2005; Fuhlbrigge et al., 2006). Additionally, ligation of CD2, CD45, CD9, CD73, or IL-2 receptor can also induce LFA-1-mediated T-cell adhesion (Dustin and Springer, 1989; van Kooyk et al., 1989; Spertini et al., 1991; Lorenz et al., 1994; Sugaa et al., 2001; Airas et al., 2000; Nielsen et al., 1996). In this context, adaptor proteins play key role in the integration and propagation of signalling cascade initiated via these molecules that leads to transient activation of LFA-1. In this section, we discuss adaptors involved in inside-out signalling mediated primarily by TCR leading to LFA-1 stimulation on T-cells, as this has been most thoroughly investigated. While the transmembrane adaptor proteins organize signalling complexes close to the plasma membrane connecting TCR or other immune receptors with intracellular signalling pathways, cytosolic adaptors facilitates propagation of downstream signals, which ultimately trigger LFA-1 and facilitate the induction of appropriate cellular response. Extensive molecular and biochemical studies and concomitant analysis have identified a number of proteins that form a macromolecular signalling complex following T-cell activation involved in LFA-1 stimulation. As an example, key roles of a transmembrane adaptor protein LAT in facilitating inside-out signalling pathway is discussed in the following section.

Mechanism of LAT facilitated inside-out signalling:

Among several transmembrane adaptor proteins involved in recruiting other adaptors and cytosolic signalling proteins to the plasma membrane following T-cell activation, LAT is recognized as functionally most important leukocyte raft molecule. Initially identified as a phosphoprotein (June et al., 1990), the 36/38 kDa type III (leaderless) raft-associated transmembrane protein LAT is of great interest as it is rapidly tyrosine-phosphorylated protein upon TCR engagement (Wonerow and Watson, 2001). LAT contains a short extracellular domain (four amino acids), one membrane spanning domain and a long cytosolic sequence. The cytoplasmic tail posses nine TBSMs, which following immunoreceptor engagement (most notably TCR) becomes phosphorylated. At least five of its tyrosine residues can be phosphorylated by ZAP-70 or Syk kinases (Zhang et al., 1998). It can also be phosphorylated by IL2-inducible T-cell kinase (Itk) upon CD28 ligation (Pullar et al., 2003), and possibly Lck (Jiang and Cheng, 2007). The essential role of LAT in TCR-mediated LFA-1 activation was first revealed by analyzing LAT-deficient variants of the Jurkat T-cell line (Zhang et al., 1999). In these cells, it was found that key elements of TCR-mediated signalling cascades that lead to IL-2-gene expression were completely abolished. LAT deficiency impaired a whole spectrum of TCR-induced downstream signalling including phosphorylation of PLCγ1, SLP-76 and Vav, Ca2+ mobilization, activation of Ras/MAPK pathway, expression of CD69, and activation of the transcription factor NFAT (Finco et al., 1998; Zhang et al., 1999; Wonerow and Watson, 2001). All of these defects could be reconstituted by re-expressing wild-type LAT. Most importantly, LAT-/- mice revealed critical functions of LAT in thymocyte development, presumably due to a failure of the pre-TCR to transduce downstream signalling (Park and Yun, 2009; Shen et al., 2009). Further, the involvement of LAT-containing rafts in TCR-initiated activation was also confirmed using T-cell transfectants expressing LAT-GFP (Tanimura et al., 2003). Following stimulation via CD3, LAT-GFP translocated to the area where T-cells were in contact with the immobilized anti-CD3 beads. The mobility increased after raft disruption by cholesterol depletion, and was also dependent on the integrity of critical binding sites in the cytoplasmic domain of LAT.

TCR ligation initially activates tyrosine kinases, including the Src kinase Lck. Once activated, Lck phosphorylates ITAMs of the CD3ζ chain as well as a second kinase Fyn (Friedl et al., 2005). The phosphorylated TCR CD3ζ chain provides a binding site for SH2 domain of the kinase ZAP70, which is then activated by Lck and Fyn (Horejsi et al., 2004). Activated ZAP70 then rapidly phosphorylates ITAMs of many substrates including multiple tyrosine residues present in the cytoplasmic domains of LAT. Following phosphorylation, LAT produce a number of attachment sites for SH2-domain-containing molecules and recruits a number of cytoplasmic adaptor and downstream signalling proteins. These include PLC1, Grb2, Gads, Grap, p85, PI3K, Vav, 3Bp2, and Shb (Gilliland et al., 1992; Sieh et al., 1994; Zhang et al., 2000; Lin and Weiss, 2001; Paz et al., 2001), which form signalosomes needed for the initiation of several intracellular signaling pathways. A key cytoplasmic adaptor SLP-76 is recruited to phospho-LAT through its constitutive interaction of Gads (Liu et al., 1999), which serves as a platform for several molecules such as Vav, NCK and Itk (Motto et al., 1996) (discussed in details in the following paragraph). A haematopoitietic cell-specific cytosolic adaptor protein ADAP [adhesion- and degranulation-promoting adaptor protein, also known as Fyn-T binding protein (FYB) or SLP-76 associated phospoprotein of 130 kDa (SLAP130)], SKAP-55 (Src kinase associated protein of 55 kDa, also termed Skap1), SKAP-HOM (SKAP-homologue, also known as SKAP55-related or SKAP55-R) also take part in this complex. This signalling scaffold in turn mobilizes intracellular Ca2+ influx, propagates of Ras/MAPK pathway, reorganizes T-cell cytoskeleton systems and ultimately activates integrins including LFA-1 (Bunnell et al., 2001) (Figure 4).