Marcus Lettau

Generation of Soluble NKG2D Ligands: Proteolytic Cleavage, Exosome Secretion and Functional Implications

The activating natural killer group 2 member D (NKG2D) receptor is expressed on NK cells, cytotoxic T cells and additional T cell subsets. Ligands for human NKG2D comprise two groups of MHC class I‐related molecules, the MHC class I chain‐related proteins A and B (MICA/B) and 6 UL16‐binding proteins (ULBP1‐6). While NKG2D ligands are absent from most normal cells, expression is induced upon stress and malignant transformation. In fact, most solid tumours and leukaemia/lymphomas constitutively express at least one NKG2D ligand and thereby are susceptible to NKG2D‐dependent immunosurveillance. However, soluble NKG2D ligands are released from tumour cells and can down‐modulate NKG2D activation as a means of tumour immune escape. In some tumour entities, levels of soluble NKG2D ligands in the serum correlate with tumour progression. NKG2D ligands can be proteolytically shed from the cell surface or liberated from the membrane by phospholipase C in the case of glycosylphosphatidylinositol (GPI)‐anchored molecules. Moreover, NKG2D ligands can be secreted in exosomal microvesicles together with other tumour‐derived molecules. Depending on the specific tumour/immune cell setting, these various forms of soluble and/or exosome‐bound NKG2D ligands can exert multiple effects on NKG2D/NKG2D ligand interactions. In this review, we focus on the role of various proteases in the shedding of human NKG2D ligands from tumour cells and discuss the not completely unanimous reported functional implications of soluble and exosome‐secreted NKG2D ligands for immunosurveillance.

Shedding of endogenous MHC class I-related chain molecules A and B from different human tumor entities: heterogeneous involvement of the "a disintegrin and metalloproteases" 10 and 17.

The interaction of the MHC class I‐related chain molecules A and B (MICA and MICB) with the corresponding natural killer group 2, member D (NKG2D) receptor triggers cytotoxic effector activity of natural killer cells and certain T‐cell subsets and provides a costimulatory signal for cytokine production. Thus, the presence of MICA/B on transformed cells contributes to tumor immunosurveillance. Consequently, the proteolytic cleavage of MICA/B is regarded as an important immune escape mechanism of various cancer cells. To investigate the molecular machinery responsible for the shedding of endogenous MICA/B, we analyzed different human tumor entities including mammary, pancreatic and prostate carcinomas. Flow cytometry and enzyme‐linked immunosorbent assay (ELISA) revealed that all tested tumor cells constitutively expressed MICA and MICB on the cell surface and also released NKG2D ligands into the supernatant. We demonstrate that the “a disintegrin and metalloproteases” (ADAMs) 10 and 17 are largely responsible for the generation of soluble MICA/B. Pharmacological inhibition of metalloproteases reduced the level of released MICA/B and increased cell surface expression. Studies using RNA interference not only revealed a prominent role of ADAM10 and ADAM17 in NKG2D ligand shedding but also a tumor cell‐specific role of ADAM10 and/or ADAM17 in shedding of MICA or MICB. Moreover, we report that in the prostate carcinoma cell line PC‐3, MICA was not shed at all but rather was secreted in exosomes. These data indicate that the release of NKG2D ligands from individual tumor entities is by far more complex than suggested in previously reported MICA/B transfection systems.

Nck adapter proteins: functional versatility in T cells

Nck is a ubiquitously expressed adapter protein that is almost exclusively built of one SH2 domain and three SH3 domains. The two isoproteins of Nck are functionally redundant in many aspects and differ in only few amino acids that are mostly located in the linker regions between the interaction modules. Nck proteins connect receptor and non-receptor tyrosine kinases to the machinery of actin reorganisation. Thereby, Nck regulates activation-dependent processes during cell polarisation and migration and plays a crucial role in the signal transduction of a variety of receptors including for instance PDGF-, HGF-, VEGF- and Ephrin receptors. In most cases, the SH2 domain mediates binding to the phosphorylated receptor or associated phosphoproteins, while SH3 domain interactions lead to the formation of larger protein complexes. In T lymphocytes, Nck plays a pivotal role in the T cell receptor (TCR)-induced reorganisation of the actin cytoskeleton and the formation of the immunological synapse. However, in this context, two different mechanisms and adapter complexes are discussed. In the first scenario, dependent on an activation-induced conformational change in the CD3ε subunits, a direct binding of Nck to components of the TCR/CD3 complex was shown. In the second scenario, Nck is recruited to the TCR complex via phosphorylated Slp76, another central constituent of the membrane proximal activation complex. Over the past years, a large number of putative Nck interactors have been identified in different cellular systems that point to diverse additional functions of the adapter protein, e.g. in the control of gene expression and proliferation.

Binding of the Intracellular Fas Ligand (FasL) Domain to the Adaptor Protein PSTPIP Results in a Cytoplasmic Localization of FasL

The tumor necrosis factor family member Fas ligand (FasL) induces apoptosis in Fas receptor-expressing target cells and is an important cytotoxic effector molecule used by CTL- and NK-cells. In these hematopoietic cells, newly synthesized FasL is stored in specialized secretory lysosomes and only delivered to the cell surface upon activation and target cell recognition. FasL contains an 80-amino acid-long cytoplasmic tail, which includes a proline-rich domain as a bona fide Src homology 3 domain-binding site. This proline-rich domain has been implicated in FasL sorting to secretory lysosomes, and it may also be important for reverse signaling via FasL, which has been described to influence T-cell activation. Here we report the identification of the Src homology 3 domain-containing adaptor protein PSTPIP as a FasL-interacting partner, which binds to the proline-rich domain. PSTPIP co-expression leads to an increased intracellular localization of Fas ligand, thereby regulating extracellular availability and cytotoxic activity of the molecule. In addition, we demonstrate recruitment of the tyrosine phosphatase PTP-PEST by PSTPIP into FasL·PSTPIP·PTP-PEST complexes which may contribute to FasL reverse signaling.

Insights into the molecular regulation of FasL (CD178) biology

Fas ligand (FasL, CD95L, APO-1L, CD178, TNFSF6, APT1LG1) is the key death factor of receptor-triggered programmed cell death in immune cells. FasL/Fas-dependent apoptosis plays a pivotal role in activation-induced cell death, termination of immune responses, elimination of autoreactive cells, cytotoxic effector function of T and NK cells, and the establishment of immune privilege. Deregulation or functional impairment of FasL threatens the maintenance of immune homeostasis and defense and results in severe autoimmunity. In addition, FasL has been implicated as an accessory or costimulatory receptor in T cell activation. The molecular mechanisms underlying this reverse signaling capacity are, however, poorly understood and still controversially discussed. Many aspects of FasL biology have been ascribed to selective protein-protein interactions mediated by a unique polyproline region located in the membrane-proximal intracellular part of FasL. Over the past decade, we and others identified a large number of putative FasL-interacting molecules that bind to this polyproline stretch via Src homology 3 or WW domains. Individual interactions were analyzed in more detail and turned out to be crucial for the lysosomal storage, the transport and the surface appearance of the death factor and potentially also for reverse signaling. This review summarizes the work in the framework of the Collaborative Research Consortium 415 (CRC 415) and provides facts and hypotheses about FasL-interacting proteins and their potential role in FasL biology.

Posttranslational regulation of Fas ligand function

The TNF superfamily member Fas ligand acts as a prototypic death factor. Due to its ability to induce apoptosis in Fas (APO-1, CD95) expressing cells, Fas ligand participates in essential effector functions of the immune system. It is involved in natural killer cell- and T cell-mediated cytotoxicity, the establishment of immune privilege, and in termination of immune responses by induction of activation-induced cell death. In addition, Fas ligand-positive tumours may evade immune surveillance by killing Fas-positive tumour-infiltrating cells. Given these strong cytotoxic capabilities of Fas ligand, it is obvious that its function has to be strictly regulated to avoid uncontrolled damage. In hematopoietic cells, the death factor is stored in secretory lysosomes and is mobilised to the immunological synapse only upon activation. The selective sorting to and the release from this specific lysosomal compartment requires interactions of the Fas ligand cytosolic moiety, which mediates binding to various adapter proteins involved in trafficking and cytoskeletal reorganisation. In addition, Fas ligand surface expression is further regulated by posttranslational ectodomain shedding and subsequent regulated intramembrane proteolysis, releasing a soluble ectodomain cytokine into the extracellular space and an N-terminal fragment with a potential role in intracellular signalling processes. Moreover, other posttranslational modifications of the cytosolic domain, including phosphorylation and ubiquitylation, have been described to affect various aspects of Fas ligand biology. Since FasL is regarded as a potential target for immunotherapy, the further characterisation of its biological regulation and function will be of great importance for the development and evaluation of future therapeutic strategies.

Considering Fas ligand as a target for therapy.

About a decade ago, the death factor Fas ligand (FasL) was identified as the natural trigger of Fas/CD95-dependent apoptosis and as an inducer of Fas-dependent activation-induced cell death. Meanwhile, it is known that this molecule not only contributes to target cell lysis in the immune system but also to the establishment of immune privilege and tumour survival. Because delivering a specific antiproliferative signal to T lymphocytes is of major biomedical interest, the FasL/Fas system has gained much attention over the last few years. However, only recently it became evident that the biology of FasL is more complex than initially anticipated. FasL displays a complex pattern of inducible and constitutive expression associated with a number of different functions as a death factor or a co-stimulatory/accessory molecule in lymphocyte activation. Thus, side effects are likely to occur following systemic administration of, for example, anti-FasL medication, not only because of the constitutive FasL expression on cells within immune privileged tissues and vascular endothelium. In addition, FasL comes in different forms: as a surface molecule, as a protease-shed soluble variant or secreted in vesicles. Because increased levels of soluble FasL (sFasL) have been determined in various immunological and non-immunological diseases, it has been suggested that sFasL might serve as a prognostic or diagnostic marker even though the pathophysiological cause for its enhanced production is hardly known in most cases. This review summarises the current facts and ideas about the clinical and pharmacological potential of FasL and sFasL as targets for therapeutic interventions.

The adapter protein Nck: Role of individual SH3 and SH2 binding modules for protein interactions in T lymphocytes

Nck is a ubiquitously expressed, primarily cytosolic adapter protein consisting of one SH2 domain and three SH3 domains. It links receptor and nonreceptor tyrosine kinases to actin cytoskeleton reorganizing proteins. In T lymphocytes, Nck is a crucial component of signaling pathways for T cell activation and effector function. It recruits actin remodeling proteins to T cell receptor (TCR)‐associated activation clusters and thereby initiates changes in cell polarity and morphology. Moreover, Nck is crucial for the TCR‐induced mobilization of secretory vesicles to the cytotoxic immunological synapse. To identify the interactome of Nck in human T cells, we performed a systematic screen for interaction partners in untreated or pervanadate‐treated cells. We used GST fusion proteins containing full length Nck, the combined SH3 domains or the individual SH3 and SH2 domains to precipitate putative Nck interactors from cellular lysates. Protein bands were excised from gels, processed by tryptic in‐gel digestion and analyzed by mass spectrometry. Using this approach, we confirmed previously established interactions (e.g., with Slp76, CD3ε, WASP, and WIPF1) and identified several novel putative Nck‐binding proteins. We subsequently verified the SH2 domain binding to the actin‐binding protein HIP55 and to FYB/ADAP, and the SH3‐mediated binding to the nuclear proteins SFPQ/NONO. Using laser scanning microscopy, we provide new evidence for a nuclear localization of Nck in human T cells. Our data highlight the fundamental role of Nck in the TCR‐to‐cytoskeleton crosstalk and point to yet unknown nuclear functions of Nck also in T lymphocytes.

2‐D DIGE analyses of enriched secretory lysosomes reveal heterogeneous profiles of functionally relevant proteins in leukemic and activated human NK cells

As part of the innate immune system, natural killer (NK) cells detect and lyse tumor and virus‐infected cells without prior antigen‐dependent recognition and expansion. To this end, they utilize dual‐function organelles that combine properties of conventional lysosomes and exocytotic vesicles. Upon stimulation, these secretory lysosomes (SLs) release their cytotoxic molecules into the immunological synapse. In addition, several molecules associated with secretory vesicles become exposed on the plasma membrane. Recent studies often took advantage of the few established NK cell lines, for instance to analyze the exocytotic machinery associated with NK cell vesicles. NK cell lines and primary NK cells differ, however, substantially in the expression of “typical” surface receptors and their requirements to induce target cell lysis. Here, we directly compared the lysosomal compartments of different NK cell populations. We enriched SLs of two leukemic cell lines (YTS and NKL) and IL‐2‐expanded NK cells by subcellular fractionation and characterized their proteome by 2‐D difference gel electrophoresis and MS. Although the overall protein composition of the lysosomal preparations was very similar and more than 90% of the proteins were present at comparable levels, we define a cell line‐specific setup of functionally relevant proteins involved in antigen presentation and cytotoxic effector function.

Effector granules in human T lymphocytes: proteomic evidence for two distinct species of cytotoxic effector vesicles.

Cytotoxic T cells mobilize effector proteins from prestored lysosomal compartments. Since different activation signals result in alternative routes of target cell killing, utilizing either FasL or the granzyme B/perforin pathway, the existence of distinct forms of effector granules was recently suggested. Applying a protocol for the separation of intact organelles from activated T lymphoblasts, we noticed that FasL-associated secretory lysosomes (SL) segregate from vesicles containing larger amounts of granzymes and granulysin. We previously analyzed the proteome of secretory lysosomes from NK and T cells and now describe the proteome of granzyme-containing vesicles. Moreover, intact FasL-associated SL and granzyme-containing vesicles were compared by electron microscopy and respective extracts were characterized by Western blotting. With the present report, we provide a comprehensive proteome map of granzyme-containing granules and unequivocally demonstrate that T lymphoblasts contain at least two distinct types of effector vesicles. Moreover, the overall protein content of the two vesicle populations was compared by 2D difference gel electrophoresis. Interestingly, the observed differences in protein distribution were not restricted to effector proteins but also applied to cytoskeleton-associated elements that could argue for a differential transport or initiation of degranulation. To our knowledge, this is the first comprehensive description of distinct effector granules in T cells.