REVIEW doi: 10.1111/sji.12337 ..................................................................................................................................................................
Lysosome-Related Effector Vesicles in T Lymphocytes and NK Cells M. Lettau, D. Kabelitz & O. Janssen
Abstract Institute of Immunology, University Hospital Schleswig-Holstein Campus Kiel, Kiel, Germany
Received 18 May 2015; Accepted in revised form 23 June 2015 Correspondence to: Prof O. Janssen, PhD, Molecular Immunology, Institute of Immunology, UK S-H Campus Kiel, ArnoldHeller-Str. 3 Bldg 17, D-24105 Kiel, Germany. E-mail: [email protected]
Lysosome-related secretory organelles combine metabolic functions of conventional lysosomes with an inducible secretory potential. Specialized variants of such bi-functional organelles are present in several haematopoietic cell types that store, mobilize and/or secrete effector proteins, for example in mast cells, macrophages or cytotoxic effector cells. In the case of T lymphocytes and NK cells, it was believed that secretory lysosomes serve as a common storage and transport compartment for the most relevant cytotoxic effector proteins including FasL, perforin, granzymes and granulysin. However, recent observations suggest that cytotoxic effector cells might be able to mobilize two distinct lysosomal entities in order to react to differential stimulation with either FasL surface appearance or degranulation-associated release of perforin and granzymes. This assumption is supported by the proteomic characterization of enriched organelles from T and NK cells. FasL-associated light lysosomes biochemically segregate from morphologically distinct heavy lysosomes that preferentially contain granzymes, perforin and mature granulysin. Here, we briefly summarize the current knowledge about cargo proteins that are stored and transported in secretory vesicles and how these vesicles might be generated and mobilized. In addition, we describe common features and major differences of the two distinct effector organelles and discuss how these observations might expand existing models of cytotoxic effector function.
Cell types containing ‘secretory lysosomes’ Many of the cells with lysosome-related secretory organelles belong to the haematopoietic lineage. Exceptions are dermal melanocytes which synthesize and store pigment proteins in lysosome-related melanosomes [1] or ocular epithelial cells which contain a secretory lysosomal system that has been implicated in the storage and release of FasL [2]. The extracellular digestion of organic material and the dissolution of the bone matrix are mediated by lysosomal enzymes secreted by osteoclasts [3]. Antigen-presenting cells (APC) such as macrophages, dendritic cells or B cells present antigen complexed with MHC II molecules to T cells. The ‘MHC class II compartment’ for the storage and release of antigen and MHC molecules shares many characteristics with secretory lysosomes [4]. Platelets cause blood coagulation by releasing effector molecules (e.g. serotonin and P-selectin) from secretory vesicles [5]. Basophils and mast cells secrete histamine and serotonin from secretory granules in response to Fc receptor ligation or chemotactic agents [6]. Last but not least, cytotoxic T cells and NK cells lyse target cells by mobilizing cytolytic
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effector molecules from secretory lysosomes [7, 8]. Thus, lysosome-related organelles with secretory properties form highly specialized vehicles for the storage and directed mobilization of cell-type-specific key regulatory or effector molecules.
Secretory lysosomes in T and NK cells Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells form the prominent effector cell populations of the immune system to control virus-infected cells or tumour cells. CTLs are activated via their individual T-cell receptors by professional APC, which present processed viral or tumour-associated antigens in a MHC class I-dependent manner. When CD8+ T cells encounter their cognate antigenic peptide presented on MHC I molecules with their T-cell receptors (TCR), the differentiation into cytotoxic effectors is manifested by the synthesis of cytotoxic granules and their constituent proteins [7–9]. By contrast, in NK cells, cytotoxic granules are presumably formed during NK-cell development and maturation and are released in an antigen-independent manner [10]. NK
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236 Lysosome-Related Effector Vesicles M. Lettau et al. .................................................................................................................................................................. cells do not express antigen-specific receptors but sense either reduced MHC expression (missing self) or stressinduced ligands such as NKG2D ligands on virus-infected or tumour cells by a variety of inhibitory and activating receptors [11–14]. Of note, in both cell types, individual steps of the recognition of a putative target cell, the formation of the cytotoxic synapse and the mode of cytotoxic effector activity are individually integrated on a single cell level. In T cells, tonic signals set basic threshold levels and the TCR signal is further fine-tuned by a variety of activating or inhibiting costimulatory receptors (also including NKG2D) and the local cytokine milieu. The ligation of individual receptors and associated transmembrane or cytosolic enzymes and adapter proteins is integrated into a net signal that determines the cellular response. The same holds true for NK cells, where the signals emerging from the different inhibitory and activating receptors that might be expressed on an individual cell are integrated with signals from adhesion molecules, chemokine or cytokine receptors and respective adapter proteins before cytotoxic granules are mobilized to the cytotoxic synapse [11–14]. Interestingly, although the mechanisms of activation are quite different, CTL and NK cells apparently use a common arsenal of cytotoxic effector proteins which are stored in characteristic lysosome-related granules that have been termed ‘secretory lysosomes’ [8, 15]. In general, secretory lysosomes are membrane-surrounded bi-functional organelles that combine lysosome-associated degrading functions with storage and secretory properties. Thus, secretory lysosomes of cytotoxic T cells and NK cells not only carry typical ‘lysosome-associated membrane proteins’ (LAMPs), but they also display a low pH and contain a battery of hydrolases as do conventional lysosomes [15, 16]. The acidic pH ensures that chondroitin sulphate-rich proteoglycans such as serglycins [17] associate with effector molecules including perforin and granzymes [8] to allow their dense packing in an inactive state [18, 19]. Interestingly, the negative costimulator CTLA-4 (CD152) [20] and also the death factor FasL (CD178) [21] were found as transmembrane molecules of secretory lysosomes. Notably, the expression of most of the lysosome-associated membrane, regulatory or effector proteins is restricted to activated T cells (or NK cells), suggesting that differentiation, effector function and also down-modulation of immune responses are orchestrated by discrete components of secretory lysosomes. Thus, the flow cytometric detection of lysosome-associated membrane proteins such as LAMP-1 (CD107a) or LAMP-2 (CD107b) on the plasma membrane of CTL is employed as a functional assay for cytotoxicity [22]. Of note, LAMP-1 also displays an important function during cytotoxic execution as it is involved in transiently protecting cytotoxic lymphocytes from self-destruction [23]. In a previous review, we described the knowledge about the different effector molecules (e.g. granzymes,
perforin, granulysin and FasL), other lysosome-associated membrane and cargo proteins (e.g. CTLA-4, CD63, Cathepsins) and diseases associated with loss of function mutations in lysosomal transport regulators or cargo proteins at that time [16]. Here, we will thus focus on recent developments and novel aspects regarding the biochemical and functional characterization of lysosomerelated organelles in T and NK cells. Notably, besides classical cytotoxic cells (i.e. CD8+ ab T cells, cd T cells, NK cells), also CD4+ T cells develop secretory granules when activated and expanded in vivo or in vitro [24, 25]. As mentioned, degranulation or exocytosis of secretory lysosomes goes along with the formation of the cytotoxic immunological synapse (IS). In T cells, ligation of the TCR triggers a signalling cascade that leads to Ca2+ influx and polarization of the ‘microtubule-organizing centre’ (MTOC) towards the IS. Vesicular transport via tubulin is mediated by dynein and might also involve actin-dependent movement via myosin IIa. Subsequent fusion with the plasma membrane relies on different addressing factors including ‘soluble N-ethylmaleimidesensitive factor attachment receptors’ (SNAREs) [26]. Membrane fusion and degranulation results in the release of effector proteins (e.g. perforin and granzymes) into the intracellular synaptic space and the appearance of secretory lysosome-associated membrane proteins on the cell surface [21, 27, 28].
Can we stay with the ‘one for all’ model for effector organelles? According to a model proposed by Griffiths and coworkers, the FasL molecule has been regarded as a characteristic transmembrane marker protein of secretory lysosomes in T and NK cells and supposedly is associated with the same lysosomal compartment that is used for storage and release of perforin and granzymes [8, 15, 29]. More recent data, however, challenge this model and suggest that it might be oversimplified and not applicable to all types of secretory lysosome-related granules present in different T-cell or NK-cell subpopulations. As an example, Ostergaard and colleagues reported that FasL surface appearance is induced in a Ca2+-independent manner, whereas degranulation and release of granzymes and perforin require Ca2+-dependent signalling [30, 31]. Interestingly, in their system, only a partial colocalization of FasL with the other lytic effector proteins was detected by confocal laser scanning microscopy (cLSM). Moreover, it was shown that signal thresholds and cytoskeletal elements differ for FasL release and perforin/granzyme degranulation [32]. Based on such observations, it was postulated that ‘lytic’ effector proteins might be either recruited via different routes from a common storage compartment or that effector proteins are stored in physically distinct lysosomal entities or compartments.
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To integrate earlier and recent models for a potentially differential mobilization of effector proteins, it was proposed that secretory lysosomal compartments might mature or be included in larger structures termed ‘multivesicular bodies’ (MVB). At some point during maturation or upon activation, MVB might fuse with late endosomes to give rise to either lysosome-derived dense granules (primarily containing granzymes and perforin) or to secretory lysosomes that contain also effector molecules such as FasL [28]. Such a model would be in agreement with a number of laser scanning microscopy analyses showing a colocalization of FasL, perforin and granzymes, and other lysosomal organelle markers [21, 33, 34] and also account for the more recent observations of discrete lysosomal effector organelles. However, if MVB were considered a higher order compartment for the initial storage of different effector proteins, it remains to be elucidated how the mobilization and release of perforin and granzyme-loaded granules might be mechanistically segregated from the recruitment and surface expression of FasL molecules.
Enrichment of lysosome-related organelles Strategies for the enrichment of intact intracellular organelles rely on a mild cell disruption, which is, for example, achieved by Dounce homogenization [35], followed by various steps of differential and density gradient centrifugations. For the analysis of the proteome of lysosome-related organelles, it is crucial to efficiently enrich these compartments from a high but limited number of cells. In 1978, a first density gradient centrifugation protocol was developed to purify organelles from rat hepatocytes using a non-ionic X-ray contrast compound termed metrizamide [36]. Compared to traditional sucrose gradients, the advantage of metrizamide was a much lower osmolarity that enabled adequate separation of lysosomes and mitochondria without artificially altering their density as observed in earlier protocols. In the meantime, several non-ionic iodinated density gradient media such as Nycodenz and its dimer iodixanol have been developed [37], which are often derivatives of metrizoic acid and form iso-osmotic solutions at high densities [38]. In 1994, Graham and colleagues first described the usage of iodixanol in density gradient media and provided a first set of subcellular isolation protocols [39, 40]. We also adapted an iodixanol-based protocol to enrich intact organelles from T lymphocytes and NK cells [41]. Individual fractions were tested by Western blotting using antibodies against an array of putative marker proteins for intracellular compartments. FasL (CD178), LAMP-1 (CD107a) and LAMP-3 (CD63) were selectively enriched in the second fraction of the discontinuous gradient, suggesting that this organelle fraction represented the secretory lysosome compartment. Importantly, as also demonstrated
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by electron microscopy, these vesicles clearly segregated from mitochondria that were enriched in fraction 5 and from dense granules in fraction 6. Moreover, an initial characterization of enriched secretory lysosomes (fraction 2) from expanded T-cell blasts of different blood donors by two-dimensional gel electrophoresis (2D-GE) indicated that this compartment was very stable and consistent in its overall composition [41].
Proteomic characterization of lysosome-related organelles from NK cells As NK cells are characterized by a large content of lysosome-related secretory organelles, we started the proteome profiling of secretory vesicles by comparing enriched organelles from in vitro expanded primary human NK cells and two leukaemic NK-cell lines (YTS and NKL). The individual fractions obtained after iodixanol density gradient centrifugation were analysed by Western blotting, and FasL-containing (fraction 2) vesicles were subjected to 2D difference gel electrophoresis (2D-DIGE) in order to directly compare the vesicular protein content [42]. Moreover, based on mass spectrometric analyses of individual protein spots, we were able to provide a first comprehensive proteome map of ‘secretory lysosomes’ from NK cells. Importantly, more than 75% of the identified proteins were already associated with lysosome-related organelles according to the available database annotations or based on previous studies. Although the overall protein repertoire within the enriched lysosomal fraction was again very similar and biological replicates of individual preparations gave nearly identical results, striking differences were seen in the abundance of several functionally relevant proteins including, for instance, MHC proteins and MHC processing molecules such as cathepsin S, cargo proteins including IL-16 and also cytotoxic effector molecules (e.g. granzymes A and B). These differences were not only detected between the two NK-cell lines, indicating a clonotypic distribution of lysosomal proteins in leukaemic cells, but also compared to primary NK cells [42]. The comparison of the different cell types also indicated that the use of established NK-cell lines instead of primary NK cells for functional assays, for example regarding cytotoxic activity, might have certain limitations. Not only do these cells differ in the surface expression of characteristic NK-cell markers and receptors, but they also contain a partially different protein arsenal in their intracellular organelles. Thus, our results might also help to explain why the different NK-cell types need different stimulatory conditions to exert cytotoxic activity [43, 44]. Furthermore, our results clearly demonstrated that proteome analyses based on individual leukaemic cell lines [45, 46] cannot be simply extrapolated to characterize the lysosomal proteome of their non-transformed counterparts; especially in the case of granzyme B, we were puzzled by the fact that
238 Lysosome-Related Effector Vesicles M. Lettau et al. .................................................................................................................................................................. in isolates from primary cells, the major portion of granzyme B was not associated with the lysosome fraction 2 (although here we detected the highest abundance of FasL), but rather was enriched in heavier fractions and especially in dense granules of fraction 6. In contrast, granzyme B showed a very high abundance and a broad distribution in almost all fractions isolated from YTS cells and was detectable in fractions 1–4 in NKL cells, but again with the highest abundance in fraction 2 [42]. Besides, the differential distribution of FasL and granzyme B in primary NK cells provided a first biochemical evidence for the presence of distinct or at least separable effector organelles in NK cells.
Proteomic characterization of lysosome-related organelles from human T cells Employing a similar experimental strategy, we also analysed the proteome of enriched organelles from PHA-activated human T-cell blasts [33, 34]. Again, individual organelle fractions obtained by iodixanol density gradient centrifugation were tested for the presence of characteristic organelle marker proteins by Western blotting. Cadherin was used as a plasma membrane marker, Bip/Grp78 as a marker for endoplasmatic reticulum and CoxIV as a marker for mitochondria. CD63, LAMP-1 (CD107a), cathepsin D and VTI1B were used to detect lysosomal compartments, and – more specifically – FasL, perforin, granzyme B and granulysin served as markers to identify the subfraction of secretory lysosomes [33]. As expected, FasL and CD63 were enriched in fraction 2 vesicles, which also contained other lysosomal proteins such as LAMP-1 or cathepsin D. The electron microscopic inspection of individual fractions revealed an enrichment of a homogeneous population of membrane-surrounded light-to-medium dense vesicles in fraction 2 and of mitochondria in fraction 5. Therefore, fraction 2 vesicles were used to analyse the luminal proteome of the FasL-associated putative secretory lysosomes from human T cells [33]. The classification of the approximately 400 identified proteins revealed that 70% were annotated to lysosome-related organelle compartments. Some ‘contaminants’ were of cytosolic (11%) or unknown (11%) origin, whereas mitochondrial, nuclear or peroxisomal proteins were largely excluded from fraction 2 preparations. For a detailed list of fraction 2-associated proteins, we refer to the original report [33]. During these studies, it became apparent that also in T-cell blasts, granzyme B was more or less exclusively found in dense granules present in fraction 6. Moreover, precursor granulysin (15 kDa) was detected in fraction 2 lysosomes, whereas the mature form of granulysin (9 kDa) was prominent in fraction 6 granules. Electron microscopic inspection of this fraction revealed that it contained some membrane or vesicular debris, but also an enrichment of
membrane-surrounded granules displaying a characteristic morphology with an electron-dense area and a less electron-dense inclusion [34]. Importantly, also the vesicles/granules contained in fraction 6 are lysosome-related organelles. The proteome analysis revealed that 66.7% of the identified proteins were annotated as ‘lysosomeassociated’ (with 13% of mitochondrial (=fraction 5) ‘contaminants’). Apparently, the fraction of dense granules reflected a second stable lysosomal effector compartment with a clear enrichment of perforin, granzyme B and mature granulysin [34].
Biochemical evidence for two lysosome-related effector organelles Of note, also fraction 6 granules isolated from PHAactivated T-cell blasts of different donors were very homogeneous in their overall protein content and regarding the distribution/abundance of individual proteins. By directly comparing fraction 2 and fraction 6 vesicles from individual donors, we were able to provide a preliminary marker profile for the two distinct effector compartments. A selective segregation was verified by Western blotting for the key effector proteins including FasL and granzymes A and B, perforin and granulysin, respectively, but also for other cargo proteins or membrane proteins such as cathepsin W and CD26. Interestingly, the time-dependent processing and the segregation of the precursor and mature forms of granulysin to different subcellular compartments had already been reported when granulysin was identified as an antimicrobial and cytotoxic effector molecule of T and NK cells [47]. While both granulysin forms were found in ‘cytoplasmic granules’, the 9 kDa product, which differs from the 15 kDa precursor in both its amino and carboxyl terminus, was also present in ‘dense, highly cytolytic granules’. Moreover and in agreement with what has been later proposed by Ostergaard and colleagues for FasL [30, 31], it was suggested that the 15 kDa granulysin (which might lack cytotoxic activity [48]) is spontaneously secreted by cytotoxic lymphocytes via a non-exocytotic (calcium-independent) pathway, whereas the 9 kDa cytotoxic form is released by calcium-dependent granule exocytosis during target cell killing [49, 50]. Also the differential association of the two granule types with adapter proteins such as Nck or WASp, cytoskeletal proteins including actin, actinin or myosin and the GTPase dynamin indicates that the two compartments might be linked to or mobilized by distinct cytoskeletal elements (e.g. b-actin for fraction 2 and myosin IIa for fraction 6) (Fig. 1). These findings clearly support the hypothesis that the activation-dependent mobilization of effector granules with defined content follows different cytoskeleton-dependent transport routes. In the following paragraphs, we will thus concentrate on recently unravelled mechanisms that regulate transport and mobilization of lysosome-related organelles.
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Figure 1 Proteomic profiling of T-cell organelles separated by density gradient centrifugation revealed the presence of two species of lysosome-related secretory organelles. The death factor FasL and the lysosomal marker proteins including LAMP-1, CD63 or cathepsin D were enriched in the larger fraction 2 vesicles which we refer to as secretory lysosomes. Classical cytotoxic effector proteins including granzyme A/B, perforin or the mature 9 kDa form of granulysin were more abundant in dense granules in fraction 6. Of note, these granules also share characteristics with lysosomes on the protein level. Besides the different abundance of fraction 2 or fraction 6 specific cargo proteins, the differential association with adapter proteins such as the Nck/WASp/ Arp2/3 module implicated in the formation of branched actin filament networks in the case of fraction 2 vesicles, or with the actin motor protein myosin IIa in the case of dense granules, indicates that the two compartments might be linked to or mobilized by distinct cytoskeletal elements.
Transport and exocytosis of lysosome-related organelles The transport of melanosomes representing the lysosomerelated secretory organelles of melanocytes has been studied in great detail over the past years. It thus served as a model system to better understand the dynamics and molecular characteristics of the transport of other lysosome-related organelles (reviewed in [51, 52]). In mammals, melanin pigments are stored and synthesized in melanosomes. Epidermal melanocytes supply neighbouring keratinocytes with melanosomes thus causing the pigmentation of skin and hair. This relies on the constant transport of melanosomes to the cellular periphery of melanocytes. Melanosomes ‘travel’ bidirectionally along microtubules in melanocytes. Motor proteins of the kinesin family mediate anterograde movement to the (+)-ends of microtubules at the cell periphery, whereas dynein motor proteins move these organelles to the ( )-ends at the cell centre. At the cell periphery, melanosomes associate with a tripartite complex comprising the small GTPase Rab27a, the adapter protein melanophilin and the actin motor protein myosin V that enables tethering to and transport along actin filaments. In this way, melanosomes are captured in the actin-rich periphery near the plasma membrane as a prerequisite for the subsequent transfer to keratinocytes [51, 52]. Importantly, genetic studies of diseases that combine albinism with immunodeficiency like the Chediak–Higashi, Hermansky–Pudlak or Griscelli syndromes revealed that many molecular features of melanosomal
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transport are shared with other lysosome-related organelles in different cell types including T and NK cells [53], although some conceptual and mechanistic differences are evident due to distinct cellular functions.
Exocytosis of T-cell secretory lysosomes at the immunological synapse Upon target cell encounter and ligation of the T-cell receptor, the T cell polarizes towards the target cell and proteins segregate into distinct membrane areas within the intercellular contact. A central area (cSMAC, central supramolecular activation cluster) contains signalling molecules like the TCR or CD8 and is surrounded by a circumferential ring of adhesion molecules such as LFA-1 [54, 55]. Cortical actin initially clusters at the site of TCR engagement but subsequently clears to the periphery to form a ring enveloping the peripheral pSMAC. In cytotoxic T cells, the central area contains a separate secretory domain next to the cSMAC. Lytic granules are exclusively secreted in this defined region into a cleft formed between the cells to assure the target-restricted release of effector proteins [55]. The complex microarchitecture of the intercellular contact area ensures the target-restricted delivery of cytotoxic effector proteins. Nevertheless, immunological synapses are highly dynamic and versatile structures, and it was even shown that a mature synapse with proteins segregated into the cSMAC and pSMAC might be dispensable for efficient target cell elimination [56].
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The transport machinery for secretory organelles in T cells During antigen-induced T-cell polarization, the microtubule-organizing centre (MTOC) translocates to the site of intercellular contact and reorients the entire microtubular network and MTOC-associated organelles like the Golgi apparatus towards the target cell. Secretory lysosomes anchored to microtubules are transported in a ( )-end direction by dynein motor proteins to cluster at the relocalized MTOC [55]. Moreover, the centrosome directly contacts the membrane in the centre of the immunological synapse between the secretory domain and the cSMAC. In this way, the ( )-ends of microtubules are aligned directly under the plasma membrane allowing secretory granules to contact the membrane within the secretory domain as they are transported towards the MTOC [57]. Although the molecular and mechanistic details of this process are still only partially understood, several proteins have been implicated in the transport and exocytosis of secretory lysosomes over the past years (reviewed in [27, 28]).
Insights into the transport machineries based on diseases As mentioned, the Griscelli syndrome type 2 is characterized by albinism and immunodeficiency due to the defective release of melanin from melanocytes and of cytotoxic effector proteins from T and NK cells. This disease and the respective phenotype in mice (ashen) have been attributed to biallelic mutations of the small GTPase Rab27a [58, 59]. In Rab27a-deficient T and NK cells, the cytotoxic granules polarize to the MTOC, but fail to dock at the plasma membrane and subsequent exocytosis is thus impaired [58, 60, 61]. Whereas in melanocytes Rab27a interacts with melanophilin that associates with the actin motor protein myosin V and thus enables actin filament transport of melanosomes in the periphery [62], no effector of Rab27a involved in the transport of secretory granules along actin filaments has been identified in CTLs so far [63]. However, the synaptotagmin-like proteins SLP1 and SLP2 were identified as targets of active Rab27a. As these proteins exhibit C2 domains that can associate with plasma membrane phospholipids, a role in tethering Rab27a to the membrane was suggested [63, 64]. Familial haemophagocytic lymphohistiocytosis type 3 is caused by the absence of the protein Munc13-4 [65]. Although cytotoxic granules dock at the immunological synapse, no fusion occurs [65]. It was postulated that Munc13-4 might mediate the priming of cytotoxic granule fusion by regulating the interaction of respective SNARE proteins required for membrane fusion. More recently, Munc13-4 was shown to directly interact with Rab27a and that this complex tethers secretory lysosomes at the plasma
membrane [66]. In a more complex scenario, Munc13-4 mediates the merging of Rab7/Rab27a-positive late endosomal vesicles and recycling vesicles containing Rab11 and Munc13-4 to a so-called exocytic vesicle. Upon target cell recognition, exocytic vesicles and secretory granules containing cytotoxic effector molecules independently polarize towards the site of intercellular contact. Finally, the fusion of these vesicles generates a structure with both exocytotic and lytic potential [67] (reviewed in [68]). Notably, also the small GTPase Rab7 was implicated in the transport of cytolytic granules to the MTOC via an indirect interaction with dynein [69]. As mentioned above, the fusion of membranes is mediated by the selective pairing of respective SNARE proteins [26]. Therefore, the loss of the SNARE family member syntaxin 11 impairs granule exocytosis but not polarization and has been identified as the cause of familial haemophagocytic lymphohistiocytosis type 4 [70]. The interaction of SNARE proteins is modulated by regulatory proteins including the SEC1/Munc18 family of proteins. Thus, mutations of the gene encoding for Munc18-2 were identified as the cause of familial haemophagocytic lymphohistiocytosis type 5 [71]. Munc18-2-deficient T cells exhibited a markedly reduced expression of syntaxin 11 and thus showed the same exocytosis defects as in FLH4 [72]. The interaction of Munc18-2 and syntaxin 11 at the immunological synapse may thus regulate SNARE complex formation. Importantly, also the lipid composition of vesicular membranes is relevant for fission and fusion processes and vesicular transport. As an example, the lipid-modifying enzyme acid sphingomyelinase (ASMase) was shown to regulate the exocytosis of cytotoxic granules after fusion with the immunological synapse. Here, it was suggested that the ASMase-mediated cleavage of sphingolipids might increase the contractile force that facilitates the extrusion of the granule content [73]. Overall, the role of the actin cytoskeleton in the transport of secretory lysosomes is still a matter of debate. Whereas confocal imaging suggested that actin is cleared to the dSMAC of the IS [74], super-resolution imaging revealed a residual actin network with focal actin hypodensities [75]. Functionally, these granule-sized actin holes were identified as the sites of degranulation. Interestingly, the actin motor protein myosin IIA is associated with NK-cell lytic granules and facilitates the interaction of granules with F-actin at the IS for exocytosis [76].
Arising questions and perspectives Although the knowledge about the content and mobilization of lysosome-related cytotoxic effector compartments has increased significantly over the past years (see model in Fig. 2), several issues still remain a matter of debate and need further exploration. For instance, it has not been
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Figure 2 Cytotoxic effector molecules and other constituent soluble proteins are transported from the trans-Golgi network to early endosomes that mature into late endosomes. This maturation includes the formation of luminal vesicles by the ESCRT (endosomal sorting complex required for transport) machinery and the accumulation of cytolytic effector proteins within electron-dense cores. Secretory lysosomes that combine the degradative properties of conventional lysosomes with the ability to store and release cytotoxic effector proteins might directly emanate from late endosomes upon further maturation and are thus characterized by the presence of lysosomal marker proteins including LAMP-1 and CD63 and cell-specific effector proteins including perforin and granzymes. Upon target cell encounter, these compartments are transported to the immunological synapse to fuse with the plasma membrane thus releasing soluble effector proteins while exposing LAMP-1 and the death factor FasL locally on the cell surface. In another scenario, late endosomes or secretory lysosomes might give rise to different species of effector granules that are characterized either by the presence of FasL, LAMP-1 and CD63 or soluble effector proteins including granzymes, perforin and the mature form of granulysin. Upon recognition of a target cell, these vesicles might be independently transported to and released at the site of intercellular contact thus allowing for an independent regulation of FasL surface expression and granzyme/perforin exocytosis. In a more complex scenario, the merger of Rab7-/Rab27a-positive late endosomal vesicles and recycling vesicles containing Rab11 and Munc13-4 gives rise to the so-called exocytic vesicles. After T-cell activation, these exocytic vesicles and secretory granules containing cytotoxic effector molecules independently polarize towards the site of intercellular contact. The subsequent fusion of these vesicles generates a structure with both exocytotic and lytic potential and might represent the last maturation step of secretory lysosomes that enables the fusion with the plasma membrane. As the exact molecular composition of the so-called exocytic vesicles is presently unknown, future analyses have to clarify whether these structures might resemble the LAMP-1-/FasL-positive vesicles described above.
systematically addressed whether all subpopulations of cytotoxic effector T or NK cells contain similar or even identical sets of effector proteins in one or the other lysosomal compartment. As mentioned, besides the classical cytotoxic immune cell populations including CD8+ ab T cells, cd T cells or NK cells, many CD4+ T cells acquire cytotoxic potential during in vitro expansion [24, 25]. Thus, it will be interesting to address whether individual lysosomal compartments in lymphocyte subpopulations differ with respect to their effector, cargo or associated cytoskeletal proteins. Although preliminary results of our group showed that the overall protein content of enriched lysosome-related organelles is very similar in most effector cells, we detected several unique
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features regarding cargo or transport proteins in ab T cells as compared to cd T cells or in CD4+ T cells as compared to CD8+ T cells that seem worthwhile to be addressed in more detail. In terms of maturation, organelle loading with different effector molecules or in terms of organelle segregation with respect to differential storage and mobilization, there is an apparent need to verify the culminating biochemical evidence morphologically, for example, by advanced high-resolution microscopic inspection, or biologically by applying different cell-type-specific stimuli including differential TCR ligation in the presence or absence of classical costimulation or simultaneous ligation of TLRs or inhibitory or activatory receptors such as NKG2D.
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Acknowledgment Cited work from the authors’ laboratories was supported by the Deutsche Forschungsgemeinschaft (grant JA 610 7/1), the Medical Faculty of the University of Kiel and the Cluster of Excellence ‘Inflammation-at-Interfaces’.
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61 Haddad EK, Wu X, Hammer JA III, Henkart PA. Defective granule exocytosis in Rab27a-deficient lymphocytes from Ashen mice. J Cell Biol 2001;152:835–42. 62 Wu X, Wang F, Rao K, Sellers JR, Hammer JA. Rab27a is an essential component of melanosome receptor for myosin Va. Mol Biol Cell 2003;13:1735–49. 63 Menasche G, Menager MM, Lefebvre JM et al. A newly identified isoform of Slp2a associates with Rab27a in cytotoxic T cells and participates to cytotoxic granule secretion. Blood 2008;112:5052–62. 64 Holt O, Kanno E, Bossi G et al. Slp1 and Slp2-a localize to the plasma membrane of CTL and contribute to secretion from the immunological synapse. Traffic 2008;9:446–57. 65 Feldmann J, Callebaut I, Raposo G et al. Munc13-4 is essential for cytolytic granules fusion and is mutated in a form of familial hemophagocytic lymphohistiocytosis (FHL3). Cell 2003;115:461–73. 66 Elstak ED, Neeft M, Nehme NT et al. The munc13-4-rab27 complex is specifically required for tethering secretory lysosomes at the plasma membrane. Blood 2011;118:1570–8. 67 Menager MM, Menasche G, Romao M et al. Secretory cytotoxic granule maturation and exocytosis require the effector protein hMunc13-4. Nat Immunol 2007;8:257–67. 68 van der Sluijs P, Zibouche M, van Kerkhof P. Late steps in secretory lysosome exocytosis in cytotoxic lymphocytes. Front Immunol 2013;4:359. 69 Daniele T, Hackmann Y, Ritter AT et al. A role for Rab7 in the movement of secretory granules in cytotoxic T lymphocytes. Traffic 2011;12:902–11. 70 zur Stadt U, Schmidt S, Kasper B et al. Linkage of familial hemophagocytic lymphohistiocytosis (FHL) type-4 to chromosome 6q24 and identification of mutations in syntaxin 11. Hum Mol Genet 2005;14:827–34. 71 zur Stadt U, Rohr J, Seifert W et al. Familial hemophagocytic lymphohistiocytosis type 5 (FHL-5) is caused by mutations in Munc18-2 and impaired binding to syntaxin 11. Am J Hum Genet 2009;85:482–92. 72 Cote M, Menager MM, Burgess A et al. Munc18-2 deficiency causes familial hemophagocytic lymphohistiocytosis type 5 and impairs cytotoxic granule exocytosis in patient NK cells. J Clin Invest 2009;119:3765–73. 73 Herz J, Pardo J, Kashkar H et al. Acid sphingomyelinase is a key regulator of cytotoxic granule secretion by primary T lymphocytes. Nat Immunol 2009;10:761–8. 74 Orange JS, Ramesh N, Remold-O’Donnell E et al. Wiskott-Aldrich syndrome protein is required for NK cell cytotoxicity and colocalizes with actin to NK cell-activating immunologic synapses. Proc Natl Acad Sci USA 2002;99:11351–6. 75 Rak GD, Mace EM, Banerjee PP, Svitkina T, Orange JS. Natural killer cell lytic granule secretion occurs through a pervasive actin network at the immune synapse. PLoS Biol 2011;9:e1001151. 76 Sanborn KB, Rak GD, Maru SY et al. Myosin IIA associates with NK cell lytic granules to enable their interaction with F-actin and function at the immunological synapse. J Immunol 2009;182:6969–84.
Lysosome-Related Effector Vesicles in T Lymphocytes and NK Cells M. Lettau, D. Kabelitz & O. Janssen
Abstract Institute of Immunology, University Hospital Schleswig-Holstein Campus Kiel, Kiel, Germany
Received 18 May 2015; Accepted in revised form 23 June 2015 Correspondence to: Prof O. Janssen, PhD, Molecular Immunology, Institute of Immunology, UK S-H Campus Kiel, ArnoldHeller-Str. 3 Bldg 17, D-24105 Kiel, Germany. E-mail: [email protected]
Lysosome-related secretory organelles combine metabolic functions of conventional lysosomes with an inducible secretory potential. Specialized variants of such bi-functional organelles are present in several haematopoietic cell types that store, mobilize and/or secrete effector proteins, for example in mast cells, macrophages or cytotoxic effector cells. In the case of T lymphocytes and NK cells, it was believed that secretory lysosomes serve as a common storage and transport compartment for the most relevant cytotoxic effector proteins including FasL, perforin, granzymes and granulysin. However, recent observations suggest that cytotoxic effector cells might be able to mobilize two distinct lysosomal entities in order to react to differential stimulation with either FasL surface appearance or degranulation-associated release of perforin and granzymes. This assumption is supported by the proteomic characterization of enriched organelles from T and NK cells. FasL-associated light lysosomes biochemically segregate from morphologically distinct heavy lysosomes that preferentially contain granzymes, perforin and mature granulysin. Here, we briefly summarize the current knowledge about cargo proteins that are stored and transported in secretory vesicles and how these vesicles might be generated and mobilized. In addition, we describe common features and major differences of the two distinct effector organelles and discuss how these observations might expand existing models of cytotoxic effector function.
Cell types containing ‘secretory lysosomes’ Many of the cells with lysosome-related secretory organelles belong to the haematopoietic lineage. Exceptions are dermal melanocytes which synthesize and store pigment proteins in lysosome-related melanosomes [1] or ocular epithelial cells which contain a secretory lysosomal system that has been implicated in the storage and release of FasL [2]. The extracellular digestion of organic material and the dissolution of the bone matrix are mediated by lysosomal enzymes secreted by osteoclasts [3]. Antigen-presenting cells (APC) such as macrophages, dendritic cells or B cells present antigen complexed with MHC II molecules to T cells. The ‘MHC class II compartment’ for the storage and release of antigen and MHC molecules shares many characteristics with secretory lysosomes [4]. Platelets cause blood coagulation by releasing effector molecules (e.g. serotonin and P-selectin) from secretory vesicles [5]. Basophils and mast cells secrete histamine and serotonin from secretory granules in response to Fc receptor ligation or chemotactic agents [6]. Last but not least, cytotoxic T cells and NK cells lyse target cells by mobilizing cytolytic
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effector molecules from secretory lysosomes [7, 8]. Thus, lysosome-related organelles with secretory properties form highly specialized vehicles for the storage and directed mobilization of cell-type-specific key regulatory or effector molecules.
Secretory lysosomes in T and NK cells Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells form the prominent effector cell populations of the immune system to control virus-infected cells or tumour cells. CTLs are activated via their individual T-cell receptors by professional APC, which present processed viral or tumour-associated antigens in a MHC class I-dependent manner. When CD8+ T cells encounter their cognate antigenic peptide presented on MHC I molecules with their T-cell receptors (TCR), the differentiation into cytotoxic effectors is manifested by the synthesis of cytotoxic granules and their constituent proteins [7–9]. By contrast, in NK cells, cytotoxic granules are presumably formed during NK-cell development and maturation and are released in an antigen-independent manner [10]. NK
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236 Lysosome-Related Effector Vesicles M. Lettau et al. .................................................................................................................................................................. cells do not express antigen-specific receptors but sense either reduced MHC expression (missing self) or stressinduced ligands such as NKG2D ligands on virus-infected or tumour cells by a variety of inhibitory and activating receptors [11–14]. Of note, in both cell types, individual steps of the recognition of a putative target cell, the formation of the cytotoxic synapse and the mode of cytotoxic effector activity are individually integrated on a single cell level. In T cells, tonic signals set basic threshold levels and the TCR signal is further fine-tuned by a variety of activating or inhibiting costimulatory receptors (also including NKG2D) and the local cytokine milieu. The ligation of individual receptors and associated transmembrane or cytosolic enzymes and adapter proteins is integrated into a net signal that determines the cellular response. The same holds true for NK cells, where the signals emerging from the different inhibitory and activating receptors that might be expressed on an individual cell are integrated with signals from adhesion molecules, chemokine or cytokine receptors and respective adapter proteins before cytotoxic granules are mobilized to the cytotoxic synapse [11–14]. Interestingly, although the mechanisms of activation are quite different, CTL and NK cells apparently use a common arsenal of cytotoxic effector proteins which are stored in characteristic lysosome-related granules that have been termed ‘secretory lysosomes’ [8, 15]. In general, secretory lysosomes are membrane-surrounded bi-functional organelles that combine lysosome-associated degrading functions with storage and secretory properties. Thus, secretory lysosomes of cytotoxic T cells and NK cells not only carry typical ‘lysosome-associated membrane proteins’ (LAMPs), but they also display a low pH and contain a battery of hydrolases as do conventional lysosomes [15, 16]. The acidic pH ensures that chondroitin sulphate-rich proteoglycans such as serglycins [17] associate with effector molecules including perforin and granzymes [8] to allow their dense packing in an inactive state [18, 19]. Interestingly, the negative costimulator CTLA-4 (CD152) [20] and also the death factor FasL (CD178) [21] were found as transmembrane molecules of secretory lysosomes. Notably, the expression of most of the lysosome-associated membrane, regulatory or effector proteins is restricted to activated T cells (or NK cells), suggesting that differentiation, effector function and also down-modulation of immune responses are orchestrated by discrete components of secretory lysosomes. Thus, the flow cytometric detection of lysosome-associated membrane proteins such as LAMP-1 (CD107a) or LAMP-2 (CD107b) on the plasma membrane of CTL is employed as a functional assay for cytotoxicity [22]. Of note, LAMP-1 also displays an important function during cytotoxic execution as it is involved in transiently protecting cytotoxic lymphocytes from self-destruction [23]. In a previous review, we described the knowledge about the different effector molecules (e.g. granzymes,
perforin, granulysin and FasL), other lysosome-associated membrane and cargo proteins (e.g. CTLA-4, CD63, Cathepsins) and diseases associated with loss of function mutations in lysosomal transport regulators or cargo proteins at that time [16]. Here, we will thus focus on recent developments and novel aspects regarding the biochemical and functional characterization of lysosomerelated organelles in T and NK cells. Notably, besides classical cytotoxic cells (i.e. CD8+ ab T cells, cd T cells, NK cells), also CD4+ T cells develop secretory granules when activated and expanded in vivo or in vitro [24, 25]. As mentioned, degranulation or exocytosis of secretory lysosomes goes along with the formation of the cytotoxic immunological synapse (IS). In T cells, ligation of the TCR triggers a signalling cascade that leads to Ca2+ influx and polarization of the ‘microtubule-organizing centre’ (MTOC) towards the IS. Vesicular transport via tubulin is mediated by dynein and might also involve actin-dependent movement via myosin IIa. Subsequent fusion with the plasma membrane relies on different addressing factors including ‘soluble N-ethylmaleimidesensitive factor attachment receptors’ (SNAREs) [26]. Membrane fusion and degranulation results in the release of effector proteins (e.g. perforin and granzymes) into the intracellular synaptic space and the appearance of secretory lysosome-associated membrane proteins on the cell surface [21, 27, 28].
Can we stay with the ‘one for all’ model for effector organelles? According to a model proposed by Griffiths and coworkers, the FasL molecule has been regarded as a characteristic transmembrane marker protein of secretory lysosomes in T and NK cells and supposedly is associated with the same lysosomal compartment that is used for storage and release of perforin and granzymes [8, 15, 29]. More recent data, however, challenge this model and suggest that it might be oversimplified and not applicable to all types of secretory lysosome-related granules present in different T-cell or NK-cell subpopulations. As an example, Ostergaard and colleagues reported that FasL surface appearance is induced in a Ca2+-independent manner, whereas degranulation and release of granzymes and perforin require Ca2+-dependent signalling [30, 31]. Interestingly, in their system, only a partial colocalization of FasL with the other lytic effector proteins was detected by confocal laser scanning microscopy (cLSM). Moreover, it was shown that signal thresholds and cytoskeletal elements differ for FasL release and perforin/granzyme degranulation [32]. Based on such observations, it was postulated that ‘lytic’ effector proteins might be either recruited via different routes from a common storage compartment or that effector proteins are stored in physically distinct lysosomal entities or compartments.
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To integrate earlier and recent models for a potentially differential mobilization of effector proteins, it was proposed that secretory lysosomal compartments might mature or be included in larger structures termed ‘multivesicular bodies’ (MVB). At some point during maturation or upon activation, MVB might fuse with late endosomes to give rise to either lysosome-derived dense granules (primarily containing granzymes and perforin) or to secretory lysosomes that contain also effector molecules such as FasL [28]. Such a model would be in agreement with a number of laser scanning microscopy analyses showing a colocalization of FasL, perforin and granzymes, and other lysosomal organelle markers [21, 33, 34] and also account for the more recent observations of discrete lysosomal effector organelles. However, if MVB were considered a higher order compartment for the initial storage of different effector proteins, it remains to be elucidated how the mobilization and release of perforin and granzyme-loaded granules might be mechanistically segregated from the recruitment and surface expression of FasL molecules.
Enrichment of lysosome-related organelles Strategies for the enrichment of intact intracellular organelles rely on a mild cell disruption, which is, for example, achieved by Dounce homogenization [35], followed by various steps of differential and density gradient centrifugations. For the analysis of the proteome of lysosome-related organelles, it is crucial to efficiently enrich these compartments from a high but limited number of cells. In 1978, a first density gradient centrifugation protocol was developed to purify organelles from rat hepatocytes using a non-ionic X-ray contrast compound termed metrizamide [36]. Compared to traditional sucrose gradients, the advantage of metrizamide was a much lower osmolarity that enabled adequate separation of lysosomes and mitochondria without artificially altering their density as observed in earlier protocols. In the meantime, several non-ionic iodinated density gradient media such as Nycodenz and its dimer iodixanol have been developed [37], which are often derivatives of metrizoic acid and form iso-osmotic solutions at high densities [38]. In 1994, Graham and colleagues first described the usage of iodixanol in density gradient media and provided a first set of subcellular isolation protocols [39, 40]. We also adapted an iodixanol-based protocol to enrich intact organelles from T lymphocytes and NK cells [41]. Individual fractions were tested by Western blotting using antibodies against an array of putative marker proteins for intracellular compartments. FasL (CD178), LAMP-1 (CD107a) and LAMP-3 (CD63) were selectively enriched in the second fraction of the discontinuous gradient, suggesting that this organelle fraction represented the secretory lysosome compartment. Importantly, as also demonstrated
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by electron microscopy, these vesicles clearly segregated from mitochondria that were enriched in fraction 5 and from dense granules in fraction 6. Moreover, an initial characterization of enriched secretory lysosomes (fraction 2) from expanded T-cell blasts of different blood donors by two-dimensional gel electrophoresis (2D-GE) indicated that this compartment was very stable and consistent in its overall composition [41].
Proteomic characterization of lysosome-related organelles from NK cells As NK cells are characterized by a large content of lysosome-related secretory organelles, we started the proteome profiling of secretory vesicles by comparing enriched organelles from in vitro expanded primary human NK cells and two leukaemic NK-cell lines (YTS and NKL). The individual fractions obtained after iodixanol density gradient centrifugation were analysed by Western blotting, and FasL-containing (fraction 2) vesicles were subjected to 2D difference gel electrophoresis (2D-DIGE) in order to directly compare the vesicular protein content [42]. Moreover, based on mass spectrometric analyses of individual protein spots, we were able to provide a first comprehensive proteome map of ‘secretory lysosomes’ from NK cells. Importantly, more than 75% of the identified proteins were already associated with lysosome-related organelles according to the available database annotations or based on previous studies. Although the overall protein repertoire within the enriched lysosomal fraction was again very similar and biological replicates of individual preparations gave nearly identical results, striking differences were seen in the abundance of several functionally relevant proteins including, for instance, MHC proteins and MHC processing molecules such as cathepsin S, cargo proteins including IL-16 and also cytotoxic effector molecules (e.g. granzymes A and B). These differences were not only detected between the two NK-cell lines, indicating a clonotypic distribution of lysosomal proteins in leukaemic cells, but also compared to primary NK cells [42]. The comparison of the different cell types also indicated that the use of established NK-cell lines instead of primary NK cells for functional assays, for example regarding cytotoxic activity, might have certain limitations. Not only do these cells differ in the surface expression of characteristic NK-cell markers and receptors, but they also contain a partially different protein arsenal in their intracellular organelles. Thus, our results might also help to explain why the different NK-cell types need different stimulatory conditions to exert cytotoxic activity [43, 44]. Furthermore, our results clearly demonstrated that proteome analyses based on individual leukaemic cell lines [45, 46] cannot be simply extrapolated to characterize the lysosomal proteome of their non-transformed counterparts; especially in the case of granzyme B, we were puzzled by the fact that
238 Lysosome-Related Effector Vesicles M. Lettau et al. .................................................................................................................................................................. in isolates from primary cells, the major portion of granzyme B was not associated with the lysosome fraction 2 (although here we detected the highest abundance of FasL), but rather was enriched in heavier fractions and especially in dense granules of fraction 6. In contrast, granzyme B showed a very high abundance and a broad distribution in almost all fractions isolated from YTS cells and was detectable in fractions 1–4 in NKL cells, but again with the highest abundance in fraction 2 [42]. Besides, the differential distribution of FasL and granzyme B in primary NK cells provided a first biochemical evidence for the presence of distinct or at least separable effector organelles in NK cells.
Proteomic characterization of lysosome-related organelles from human T cells Employing a similar experimental strategy, we also analysed the proteome of enriched organelles from PHA-activated human T-cell blasts [33, 34]. Again, individual organelle fractions obtained by iodixanol density gradient centrifugation were tested for the presence of characteristic organelle marker proteins by Western blotting. Cadherin was used as a plasma membrane marker, Bip/Grp78 as a marker for endoplasmatic reticulum and CoxIV as a marker for mitochondria. CD63, LAMP-1 (CD107a), cathepsin D and VTI1B were used to detect lysosomal compartments, and – more specifically – FasL, perforin, granzyme B and granulysin served as markers to identify the subfraction of secretory lysosomes [33]. As expected, FasL and CD63 were enriched in fraction 2 vesicles, which also contained other lysosomal proteins such as LAMP-1 or cathepsin D. The electron microscopic inspection of individual fractions revealed an enrichment of a homogeneous population of membrane-surrounded light-to-medium dense vesicles in fraction 2 and of mitochondria in fraction 5. Therefore, fraction 2 vesicles were used to analyse the luminal proteome of the FasL-associated putative secretory lysosomes from human T cells [33]. The classification of the approximately 400 identified proteins revealed that 70% were annotated to lysosome-related organelle compartments. Some ‘contaminants’ were of cytosolic (11%) or unknown (11%) origin, whereas mitochondrial, nuclear or peroxisomal proteins were largely excluded from fraction 2 preparations. For a detailed list of fraction 2-associated proteins, we refer to the original report [33]. During these studies, it became apparent that also in T-cell blasts, granzyme B was more or less exclusively found in dense granules present in fraction 6. Moreover, precursor granulysin (15 kDa) was detected in fraction 2 lysosomes, whereas the mature form of granulysin (9 kDa) was prominent in fraction 6 granules. Electron microscopic inspection of this fraction revealed that it contained some membrane or vesicular debris, but also an enrichment of
membrane-surrounded granules displaying a characteristic morphology with an electron-dense area and a less electron-dense inclusion [34]. Importantly, also the vesicles/granules contained in fraction 6 are lysosome-related organelles. The proteome analysis revealed that 66.7% of the identified proteins were annotated as ‘lysosomeassociated’ (with 13% of mitochondrial (=fraction 5) ‘contaminants’). Apparently, the fraction of dense granules reflected a second stable lysosomal effector compartment with a clear enrichment of perforin, granzyme B and mature granulysin [34].
Biochemical evidence for two lysosome-related effector organelles Of note, also fraction 6 granules isolated from PHAactivated T-cell blasts of different donors were very homogeneous in their overall protein content and regarding the distribution/abundance of individual proteins. By directly comparing fraction 2 and fraction 6 vesicles from individual donors, we were able to provide a preliminary marker profile for the two distinct effector compartments. A selective segregation was verified by Western blotting for the key effector proteins including FasL and granzymes A and B, perforin and granulysin, respectively, but also for other cargo proteins or membrane proteins such as cathepsin W and CD26. Interestingly, the time-dependent processing and the segregation of the precursor and mature forms of granulysin to different subcellular compartments had already been reported when granulysin was identified as an antimicrobial and cytotoxic effector molecule of T and NK cells [47]. While both granulysin forms were found in ‘cytoplasmic granules’, the 9 kDa product, which differs from the 15 kDa precursor in both its amino and carboxyl terminus, was also present in ‘dense, highly cytolytic granules’. Moreover and in agreement with what has been later proposed by Ostergaard and colleagues for FasL [30, 31], it was suggested that the 15 kDa granulysin (which might lack cytotoxic activity [48]) is spontaneously secreted by cytotoxic lymphocytes via a non-exocytotic (calcium-independent) pathway, whereas the 9 kDa cytotoxic form is released by calcium-dependent granule exocytosis during target cell killing [49, 50]. Also the differential association of the two granule types with adapter proteins such as Nck or WASp, cytoskeletal proteins including actin, actinin or myosin and the GTPase dynamin indicates that the two compartments might be linked to or mobilized by distinct cytoskeletal elements (e.g. b-actin for fraction 2 and myosin IIa for fraction 6) (Fig. 1). These findings clearly support the hypothesis that the activation-dependent mobilization of effector granules with defined content follows different cytoskeleton-dependent transport routes. In the following paragraphs, we will thus concentrate on recently unravelled mechanisms that regulate transport and mobilization of lysosome-related organelles.
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Figure 1 Proteomic profiling of T-cell organelles separated by density gradient centrifugation revealed the presence of two species of lysosome-related secretory organelles. The death factor FasL and the lysosomal marker proteins including LAMP-1, CD63 or cathepsin D were enriched in the larger fraction 2 vesicles which we refer to as secretory lysosomes. Classical cytotoxic effector proteins including granzyme A/B, perforin or the mature 9 kDa form of granulysin were more abundant in dense granules in fraction 6. Of note, these granules also share characteristics with lysosomes on the protein level. Besides the different abundance of fraction 2 or fraction 6 specific cargo proteins, the differential association with adapter proteins such as the Nck/WASp/ Arp2/3 module implicated in the formation of branched actin filament networks in the case of fraction 2 vesicles, or with the actin motor protein myosin IIa in the case of dense granules, indicates that the two compartments might be linked to or mobilized by distinct cytoskeletal elements.
Transport and exocytosis of lysosome-related organelles The transport of melanosomes representing the lysosomerelated secretory organelles of melanocytes has been studied in great detail over the past years. It thus served as a model system to better understand the dynamics and molecular characteristics of the transport of other lysosome-related organelles (reviewed in [51, 52]). In mammals, melanin pigments are stored and synthesized in melanosomes. Epidermal melanocytes supply neighbouring keratinocytes with melanosomes thus causing the pigmentation of skin and hair. This relies on the constant transport of melanosomes to the cellular periphery of melanocytes. Melanosomes ‘travel’ bidirectionally along microtubules in melanocytes. Motor proteins of the kinesin family mediate anterograde movement to the (+)-ends of microtubules at the cell periphery, whereas dynein motor proteins move these organelles to the ( )-ends at the cell centre. At the cell periphery, melanosomes associate with a tripartite complex comprising the small GTPase Rab27a, the adapter protein melanophilin and the actin motor protein myosin V that enables tethering to and transport along actin filaments. In this way, melanosomes are captured in the actin-rich periphery near the plasma membrane as a prerequisite for the subsequent transfer to keratinocytes [51, 52]. Importantly, genetic studies of diseases that combine albinism with immunodeficiency like the Chediak–Higashi, Hermansky–Pudlak or Griscelli syndromes revealed that many molecular features of melanosomal
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transport are shared with other lysosome-related organelles in different cell types including T and NK cells [53], although some conceptual and mechanistic differences are evident due to distinct cellular functions.
Exocytosis of T-cell secretory lysosomes at the immunological synapse Upon target cell encounter and ligation of the T-cell receptor, the T cell polarizes towards the target cell and proteins segregate into distinct membrane areas within the intercellular contact. A central area (cSMAC, central supramolecular activation cluster) contains signalling molecules like the TCR or CD8 and is surrounded by a circumferential ring of adhesion molecules such as LFA-1 [54, 55]. Cortical actin initially clusters at the site of TCR engagement but subsequently clears to the periphery to form a ring enveloping the peripheral pSMAC. In cytotoxic T cells, the central area contains a separate secretory domain next to the cSMAC. Lytic granules are exclusively secreted in this defined region into a cleft formed between the cells to assure the target-restricted release of effector proteins [55]. The complex microarchitecture of the intercellular contact area ensures the target-restricted delivery of cytotoxic effector proteins. Nevertheless, immunological synapses are highly dynamic and versatile structures, and it was even shown that a mature synapse with proteins segregated into the cSMAC and pSMAC might be dispensable for efficient target cell elimination [56].
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The transport machinery for secretory organelles in T cells During antigen-induced T-cell polarization, the microtubule-organizing centre (MTOC) translocates to the site of intercellular contact and reorients the entire microtubular network and MTOC-associated organelles like the Golgi apparatus towards the target cell. Secretory lysosomes anchored to microtubules are transported in a ( )-end direction by dynein motor proteins to cluster at the relocalized MTOC [55]. Moreover, the centrosome directly contacts the membrane in the centre of the immunological synapse between the secretory domain and the cSMAC. In this way, the ( )-ends of microtubules are aligned directly under the plasma membrane allowing secretory granules to contact the membrane within the secretory domain as they are transported towards the MTOC [57]. Although the molecular and mechanistic details of this process are still only partially understood, several proteins have been implicated in the transport and exocytosis of secretory lysosomes over the past years (reviewed in [27, 28]).
Insights into the transport machineries based on diseases As mentioned, the Griscelli syndrome type 2 is characterized by albinism and immunodeficiency due to the defective release of melanin from melanocytes and of cytotoxic effector proteins from T and NK cells. This disease and the respective phenotype in mice (ashen) have been attributed to biallelic mutations of the small GTPase Rab27a [58, 59]. In Rab27a-deficient T and NK cells, the cytotoxic granules polarize to the MTOC, but fail to dock at the plasma membrane and subsequent exocytosis is thus impaired [58, 60, 61]. Whereas in melanocytes Rab27a interacts with melanophilin that associates with the actin motor protein myosin V and thus enables actin filament transport of melanosomes in the periphery [62], no effector of Rab27a involved in the transport of secretory granules along actin filaments has been identified in CTLs so far [63]. However, the synaptotagmin-like proteins SLP1 and SLP2 were identified as targets of active Rab27a. As these proteins exhibit C2 domains that can associate with plasma membrane phospholipids, a role in tethering Rab27a to the membrane was suggested [63, 64]. Familial haemophagocytic lymphohistiocytosis type 3 is caused by the absence of the protein Munc13-4 [65]. Although cytotoxic granules dock at the immunological synapse, no fusion occurs [65]. It was postulated that Munc13-4 might mediate the priming of cytotoxic granule fusion by regulating the interaction of respective SNARE proteins required for membrane fusion. More recently, Munc13-4 was shown to directly interact with Rab27a and that this complex tethers secretory lysosomes at the plasma
membrane [66]. In a more complex scenario, Munc13-4 mediates the merging of Rab7/Rab27a-positive late endosomal vesicles and recycling vesicles containing Rab11 and Munc13-4 to a so-called exocytic vesicle. Upon target cell recognition, exocytic vesicles and secretory granules containing cytotoxic effector molecules independently polarize towards the site of intercellular contact. Finally, the fusion of these vesicles generates a structure with both exocytotic and lytic potential [67] (reviewed in [68]). Notably, also the small GTPase Rab7 was implicated in the transport of cytolytic granules to the MTOC via an indirect interaction with dynein [69]. As mentioned above, the fusion of membranes is mediated by the selective pairing of respective SNARE proteins [26]. Therefore, the loss of the SNARE family member syntaxin 11 impairs granule exocytosis but not polarization and has been identified as the cause of familial haemophagocytic lymphohistiocytosis type 4 [70]. The interaction of SNARE proteins is modulated by regulatory proteins including the SEC1/Munc18 family of proteins. Thus, mutations of the gene encoding for Munc18-2 were identified as the cause of familial haemophagocytic lymphohistiocytosis type 5 [71]. Munc18-2-deficient T cells exhibited a markedly reduced expression of syntaxin 11 and thus showed the same exocytosis defects as in FLH4 [72]. The interaction of Munc18-2 and syntaxin 11 at the immunological synapse may thus regulate SNARE complex formation. Importantly, also the lipid composition of vesicular membranes is relevant for fission and fusion processes and vesicular transport. As an example, the lipid-modifying enzyme acid sphingomyelinase (ASMase) was shown to regulate the exocytosis of cytotoxic granules after fusion with the immunological synapse. Here, it was suggested that the ASMase-mediated cleavage of sphingolipids might increase the contractile force that facilitates the extrusion of the granule content [73]. Overall, the role of the actin cytoskeleton in the transport of secretory lysosomes is still a matter of debate. Whereas confocal imaging suggested that actin is cleared to the dSMAC of the IS [74], super-resolution imaging revealed a residual actin network with focal actin hypodensities [75]. Functionally, these granule-sized actin holes were identified as the sites of degranulation. Interestingly, the actin motor protein myosin IIA is associated with NK-cell lytic granules and facilitates the interaction of granules with F-actin at the IS for exocytosis [76].
Arising questions and perspectives Although the knowledge about the content and mobilization of lysosome-related cytotoxic effector compartments has increased significantly over the past years (see model in Fig. 2), several issues still remain a matter of debate and need further exploration. For instance, it has not been
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Figure 2 Cytotoxic effector molecules and other constituent soluble proteins are transported from the trans-Golgi network to early endosomes that mature into late endosomes. This maturation includes the formation of luminal vesicles by the ESCRT (endosomal sorting complex required for transport) machinery and the accumulation of cytolytic effector proteins within electron-dense cores. Secretory lysosomes that combine the degradative properties of conventional lysosomes with the ability to store and release cytotoxic effector proteins might directly emanate from late endosomes upon further maturation and are thus characterized by the presence of lysosomal marker proteins including LAMP-1 and CD63 and cell-specific effector proteins including perforin and granzymes. Upon target cell encounter, these compartments are transported to the immunological synapse to fuse with the plasma membrane thus releasing soluble effector proteins while exposing LAMP-1 and the death factor FasL locally on the cell surface. In another scenario, late endosomes or secretory lysosomes might give rise to different species of effector granules that are characterized either by the presence of FasL, LAMP-1 and CD63 or soluble effector proteins including granzymes, perforin and the mature form of granulysin. Upon recognition of a target cell, these vesicles might be independently transported to and released at the site of intercellular contact thus allowing for an independent regulation of FasL surface expression and granzyme/perforin exocytosis. In a more complex scenario, the merger of Rab7-/Rab27a-positive late endosomal vesicles and recycling vesicles containing Rab11 and Munc13-4 gives rise to the so-called exocytic vesicles. After T-cell activation, these exocytic vesicles and secretory granules containing cytotoxic effector molecules independently polarize towards the site of intercellular contact. The subsequent fusion of these vesicles generates a structure with both exocytotic and lytic potential and might represent the last maturation step of secretory lysosomes that enables the fusion with the plasma membrane. As the exact molecular composition of the so-called exocytic vesicles is presently unknown, future analyses have to clarify whether these structures might resemble the LAMP-1-/FasL-positive vesicles described above.
systematically addressed whether all subpopulations of cytotoxic effector T or NK cells contain similar or even identical sets of effector proteins in one or the other lysosomal compartment. As mentioned, besides the classical cytotoxic immune cell populations including CD8+ ab T cells, cd T cells or NK cells, many CD4+ T cells acquire cytotoxic potential during in vitro expansion [24, 25]. Thus, it will be interesting to address whether individual lysosomal compartments in lymphocyte subpopulations differ with respect to their effector, cargo or associated cytoskeletal proteins. Although preliminary results of our group showed that the overall protein content of enriched lysosome-related organelles is very similar in most effector cells, we detected several unique
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features regarding cargo or transport proteins in ab T cells as compared to cd T cells or in CD4+ T cells as compared to CD8+ T cells that seem worthwhile to be addressed in more detail. In terms of maturation, organelle loading with different effector molecules or in terms of organelle segregation with respect to differential storage and mobilization, there is an apparent need to verify the culminating biochemical evidence morphologically, for example, by advanced high-resolution microscopic inspection, or biologically by applying different cell-type-specific stimuli including differential TCR ligation in the presence or absence of classical costimulation or simultaneous ligation of TLRs or inhibitory or activatory receptors such as NKG2D.
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Acknowledgment Cited work from the authors’ laboratories was supported by the Deutsche Forschungsgemeinschaft (grant JA 610 7/1), the Medical Faculty of the University of Kiel and the Cluster of Excellence ‘Inflammation-at-Interfaces’.
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