See accompanying article: http://dx.doi/10.1002/eji.201142343
Deciphering the syntax of cytotoxic lymphocyte degranulation
Article first published online: 16 JAN 2013
© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
European Journal of Immunology
Volume 43, Issue 1, pages 46–49, January 2013
How to Cite
Lopez, J. A. and Voskoboinik, I. (2013), Deciphering the syntax of cytotoxic lymphocyte degranulation. Eur. J. Immunol., 43: 46–49. doi: 10.1002/eji.201243221
- Issue published online: 16 JAN 2013
- Article first published online: 16 JAN 2013
- Manuscript Accepted: 10 DEC 2012
- Manuscript Received: 3 DEC 2012
- Manuscript Revised: 3 DEC 2012
- NHM&RC of Australia
- CD8+ T cells;
- NK cells;
- Protein trafficking
In the killer lymphocyte, the targeted delivery of perforin- and granzyme-containing cytotoxic granules to the immunological synapse is crucial for the eradication of pathogen-infected or transformed targets. This process is achieved through a tightly controlled and highly efficient granule exocytosis pathway. Mutations in the granule trafficking proteins Munc13–4, syntaxin 11, Munc18–2 or Rab27 leads to a fatal lapse of immune surveillance and can be manifested as haemophagocytic lymphohistiocytosis in humans. Elucidation of the role of these proteins in exocytic trafficking is pivotal for our understanding of their role in health and disease. In this issue of the European Journal of Immunology, D'Orlando et al. [Eur. J. Immunol. 2013. 43: 194-208] make an important step in this direction, as they generate and characterise syntaxin 11 deficient mice. Herein, we discuss the role of syntaxin-11 in soluble NSF (N-ethylmaleimide sensitive fusion) attachment protein receptors complex formation leading to cytotoxic lymphocyte degranulation and its importance in maintaining immune homeostasis.
In this issue of the European Journal of Immunology, D'Orlando and et al.  report the generation and characterisation of syntaxin 11 deficient (stx11−/−) mice and provide new data for a role of syntaxin 11 in cytotoxic lymphocyte (CL) and neutrophil degranulation. This study demonstrates that syntaxin 11 deficiency leads to severely impaired CL function, thus corroborating strong human genetic data demonstrating a critical role for syntaxin 11 in immune homoeostasis.
Intracellular membrane trafficking is an essential homoeostatic biological process that regulates the formation of cellular compartments, protein trafficking, the uptake of extracellular milieu and the secretion of intracellular material. In multicellular organisms, specialised secretory cells have evolved tightly controlled trafficking machinery that responds to specific extracellular stimuli and recruits exocytic vesicles to the plasma membrane, facilitating the release of soluble cargo from the cell. Regulated exocytosis in many immune cells facilitates the secretion of cytokines and chemokines into the extracellular environment. In CLs, secretory granules, which are derived from the lysosome, store and release cytotoxic effector molecules into the immunological synapse formed between the CL and its cognate target cell . These effector molecules act synergistically to deliver the lethal hit to the target: perforin forms large transmembrane pores in the target cell membrane allowing the diffusion into the cytosol of pro-apoptotic serine proteases, granzymes, where they cleave and activate specific protein targets in various apoptotic pathways . Mutations in genes encoding perforin or regulators of the degranulation machinery are detrimental to an organism and lead to fatal immune dysregulation, manifested as haemophagocytic lymphohistiocytosis [4-8].
As with all exocytic events, the transport of individual cargo proteins destined for release at the cell surface follows pre-determined trafficking routes, often requiring movement between multiple intracellular compartments. One set of proteins that are critical for membrane transport are the soluble N-ethylmaleimide sensitive fusion (NSF)-attachment protein receptors (SNAREs), a family of coiled-coil proteins that interact with each other on opposing membranes to facilitate membrane docking and fusion . Syntaxins termed target cell membrane SNAREs (t-SNAREs) pair with other t-SNAREs (e.g. SNAP23) and with v-SNAREs on the opposing vesicle membrane (e.g. VAMP8) to form a ternary complex. The high affinity SNARE ternary complex interaction is thought to bring the two membrane bilayers closer, making it energetically favourable for them to fuse. Each membrane terminal utilises distinct SNARE complexes to regulate individual membrane fusion reactions. Partner SNAREs have likely coevolved to ensure transport specificity.
Ascribing a functional role for individual SNAREs in secretory systems has been difficult due to isoform redundancies and the fact that many SNARE knockout models result in embryonic lethality . Genetic ablation of the ubiquitously expressed SNAREs, syntaxin 4  and SNAP23 , for example, results in early embryonic lethality, highlighting their essential role in tissue development, and only tissue-specific knockout mouse models have helped overcome some of these issues . Given the importance of these proteins in embryonic development, it is hardly surprising that no congenital loss of SNAREs has been reported in humans.
In 2005, zur Stadt et al. discovered bi-allelic mutations in the STX11 gene (encoding syntaxin 11) in patients with familial haemophagocytic lymphohistiocytosis (FHL) . Unlike many of its homologues, syntaxin 11 lacks a transmembrane domain [14, 15]. Although syntaxin 11 has high levels of expression in various haematopoietic cells, in general, it has a more restricted tissue distribution than many of its homologues, and therefore, it is not surprising that even a complete loss of syntaxin 11 has no apparent effect on human embryonic development. The most profound effect of syntaxin 11 deficiency in humans is a loss of CL function due to impaired exocytosis of cytotoxic granules . More recently, two independent reports identified mutations in the STXBP2 gene encoding syntaxin 11's partner protein Munc18–2, as another cause of FHL [7, 8]. These studies [7, 8] demonstrated that syntaxin 11 and Munc18–2 biochemically interact within the cell and thus it has been hypothesised that they are likely to function at the same step in secretory granule exocytosis.
The interaction between syntaxin 11 and Munc18–2, like other cognate syntaxin/Munc18 interactions, controls the assembly of SNARE complexes, with considerable evidence suggesting that the Munc18 proteins positively regulate the SNARE reaction [16, 17]. Loss of Munc18–2-protein expression in FHL patients also results in a reduction in the steady state level of syntaxin 11 protein , which has made it difficult to assess the contribution of Munc18–2 alone in the manifestation of FHL. However, as the loss of syntaxin 11 does not affect Munc18–2 levels, stx11−/− mice make an excellent experimental model for investigating the role of SNAREs in FHL and CL biology.
As expected, the Stx11−/− mice reported by D'Orlando et al.  appear to develop normally, and their immune cell counts are indistinguishable from those of WT mice. However, the secretory function of NK cells and cytotoxic T lymphocytes is severely affected, highlighting an essential role of syntaxin 11 in that process. Importantly, the authors demonstrated significant restoration of lytic function when Stx11−/– NK cells were reconstituted with WT syntaxin 11. Intriguingly, stx11−/− deficiency appears to affect NK-cell function more than cytotoxic T cells . Whether this is a consequence of different cell activation protocols is yet to be understood, but these results are, to some extent, consistent with those noted in syntaxin 11 deficient FHL patients. In contrast to the observed defects in CL degranulation upon loss of syntaxin 11 expression, the secretion of IFN-γ and TNF cytokines is not impaired, but rather the constitutive secretion of these cytokines appears to be enhanced, indicating differential trafficking routes to the plasma membrane (Fig. 1).
Previous reports demonstrated that defects in CL degranulation and cytotoxicity, which were associated with syntaxin 11 and Munc18–2 mutations in FHL patients, could be partially or fully rescued by in vitro treatment with IL-2 [7, 8, 18]. These data suggested that IL-2 may upregulate a compensatory exocytosis pathway that bypasses the requirement for syntaxin 11 and Munc18–2. In contrast, in the current study , IL-2 stimulation could not overcome the cytolytic defects in stx11−/– CLs. Similar observations were reported in two other independent, concurrently published studies [19, 20]. These data argue that the earlier findings of IL-2 compensation in human NK cells may have occurred through potentiation (increased protein expression) of partially functioning Syntaxin 11 mutants. Therefore, while it is tempting to postulate that syntaxin 11 plays a rate-limiting step in CL degranulation, future complementation studies using stx11−/– CLs are required to establish genotype/phenotype relationships, similar to those described for perforin mutants [21, 22].
Do stx11−/− mice faithfully recapitulate the phenotype of syntaxin 11 deficient FHL patients? Two recent studies [19, 20] (one of which used the mice generated by D'Orlando et al.) addressed this issue by challenging stx11−/− mice with lymphocytic chorio-meningitis virus, which has been used in the past to investigate FHL-like disease in perforin-deficient mice. The results clearly demonstrated that syntaxin 11 is required for CL function and clearance of the virus. However, unlike prf1−/− mice, which present with severe fatal haemophagocytic lymphohistio-cytosis, stx11−/− mice remain viable despite having a similar viral titre. Although the exact mechanism of such resistance is currently unknown, there are indications that it may be at least partly due to T-cell exhaustion . In comparison, human clinical data clearly demonstrate that FHL caused by a complete loss of syntaxin 11 leads to delayed onset of FHL, compared to perforin-deficient patients . This may be attributed to the same mechanism(s) identified in mice. The eventual development of FHL in syntaxin 11 deficient patients may be a result of sequential and diverse antigenic challenges, which could not be replicated in these initial animal studies, where mice were tested under strictly controlled conditions. There is little doubt that subsequent studies will aim at elucidating this apparent discrepancy and will provide a novel framework for understanding the biology of CLs and immune homoeostasis in general.
Even though there is now a growing body of evidence supporting a role for syntaxin 11 in CL degranulation, it still remains unclear where syntaxin 11 is functioning within the cell. Early studies characterising syntaxin 11 localised it to the TGN/endosome , cytosol  or late endosome  compartments. More recently syntaxin 11 has been shown to regulate late-endosome to lysosome fusion in macrophages . If the syntaxin 11/Munc18–2 complex is functional prior to granule delivery to the cell surface, then what SNARE players are working at the cell surface? Possibly this step is controlled by the more ubiquitous isoforms syntaxins 2, 3 or 4. In fact, a recent characterisation of SNARE proteins within CD8+ lymphocytes  showed accumulation of syntaxins 2 and 3 at the immunological synapse. Nevertheless, real-time fluorescence-tracking of syntaxin containing compartments during engagement of killer cells with their targets may prove useful in identifying their point of action and deciphering the events leading to killer cell degranulation.
Overall, recent studies addressing novel and complex questions in cell biology of CLs and the generation of essential experimental in vivo models lead the field towards better understanding of the role of these cells in immune-mediated diseases.
J.A.L. is supported by a NH&MRC Postdoctoral Training Fellowship, I.V., is supported by a fellowship and grants from the NHM&RC of Australia. We thank Vivien Sutton for critical reading of the manuscript.
Conflict of interest
The authors declare no financial or commercial conflict of interest.
- 19Distinct severity of HLH in both human and murine mutants with complete loss of cytotoxic effector PRF1, RAB27A and STX11. Blood 2012. DOI: 10.1182/blood-2012-07-440339., , , , , , et al.,
familial haemophagocytic lymphohistiocytosis
soluble NSF-attachment protein receptor