A solution for highly efficient electroporation of primary cytotoxic T lymphocytes.

BACKGROUND: Cytotoxic T lymphocytes (CTLs) are central players in the adaptive immune response. Their functional characterization and clinical research depend on efficient and reliable transfection. Although various methods have been utilized, electroporation remains the preferred technique for transient gene over-expression. However, the efficiency of electroporation is reduced for human and mouse primary CTLs. Lonza offers kits that effectively improve plasmid DNA transfection quality. Unfortunately, the removal of key components of the cell recovery medium considerably reduced the efficiency of their kit for CTLs. Our aim was to develop a new recovery medium to be used with Lonza's Nucleofector system that would significantly enhance transfection rates. RESULTS: We assessed the impact of different media in which the primary CTLs were placed to recover after electroporation on cell survival, transfection rate and their ability to form an immunological synapse and to perform exocytosis. We transfected the cells with pmax-GFP and large constructs encoding for either CD81-super ecliptic pHluorin or granzyme B-pHuji. The comparison of five different media for mouse and two for human CTLs demonstrated that our new recovery medium composed of Opti-MEM-GlutaMAX supplemented with HEPES, DMSO and sodium pyruvate gave the best result in cell survival (> 50%) and transfection rate (> 30 and 20% for mouse and human cells, respectively). More importantly, the functionality of CTLs was at least twice as high as with the original Lonza recovery medium. In addition, our RM significantly improved transfection efficacy of natural killer cells that are notoriously hard to electroporate. CONCLUSION: Our results show that successful transfection depends not only on the electroporation medium and pulse sequence but also on the medium applied for cell recovery. In addition, we have reduced our reliance on proprietary products by designing an effective recovery medium for both mouse and human primary CTLs and other lymphocytes that can be easily implemented by any laboratory. We expect that this recovery medium will have a significant impact on both fundamental and applied research in immunology.

Role of V-ATPase a3-Subunit in Mouse CTL Function.

CTLs release cytotoxic proteins such as granzymes and perforin through fusion of cytotoxic granules (CG) at the target cell interface, the immune synapse, to kill virus-infected and tumorigenic target cells. A characteristic feature of these granules is their acidic pH inside the granule lumen, which is required to process precursors of granzymes and perforin to their mature form. However, the role of acidic pH in CG maturation, transport, and fusion is not understood. We demonstrate in primary murine CTLs that the a3-subunit of the vacuolar-type (H+)-adenosine triphosphatase is required for establishing a luminal pH of 6.1 inside CG using ClopHensorN(Q69M), a newly generated CG-specific pH indicator. Knockdown of the a3-subunit resulted in a significantly reduced killing of target cells and a >50% reduction in CG fusion in total internal reflection fluorescence microscopy, which was caused by a reduced number of CG at the immune synapse. Superresolution microscopy revealed a reduced interaction of CG with the microtubule network upon a3-subunit knockdown. Finally, we find by electron and structured illumination microscopy that knockdown of the a3-subunit altered the diameter and density of individual CG, whereas the number of CG per CTL was unaffected. We conclude that the a3-subunit of the vacuolar adenosine triphosphatase is not only responsible for the acidification of CG, but also contributes to the maturation and efficient transport of the CG to the immune synapse.

An Alternative Exon of CAPS2 Influences Catecholamine Loading into LDCVs of Chromaffin Cells.

The calcium-dependent activator proteins for secretion (CAPS) are priming factors for synaptic and large dense-core vesicles (LDCVs), promoting their entry into and stabilizing the release-ready state. A modulatory role of CAPS in catecholamine loading of vesicles has been suggested. Although an influence of CAPS on monoamine transporter function and on vesicle acidification has been reported, a role of CAPS in vesicle loading is disputed. Using expression of naturally occurring splice variants of CAPS2 into chromaffin cells from CAPS1/CAPS2 double-deficient mice of both sexes, we show that an alternative exon of 40 aa is responsible for enhanced catecholamine loading of LDCVs in mouse chromaffin cells. The presence of this exon leads to increased activity of both vesicular monoamine transporters. Deletion of CAPS does not alter acidification of vesicles. Our results establish a splice-variant-dependent modulatory effect of CAPS on catecholamine content in LDCVs.SIGNIFICANCE STATEMENT The calcium activator protein for secretion (CAPS) promotes and stabilizes the entry of catecholamine-containing vesicles of the adrenal gland into a release-ready state. Expression of an alternatively spliced exon in CAPS leads to enhanced catecholamine content in chromaffin granules. This exon codes for 40 aa with a high proline content, consistent with an unstructured loop present in the portion of the molecule generally thought to be involved in vesicle priming. CAPS variants containing this exon promote serotonin uptake into Chinese hamster ovary cells expressing either vesicular monoamine transporter. Epigenetic tuning of CAPS variants may allow modulation of endocrine adrenaline and noradrenaline release. This mechanism may extend to monoamine release in central neurons or in the enteric nervous system.

Endocytosis of Cytotoxic Granules Is Essential for Multiple Killing of Target Cells by T Lymphocytes.

CTLs are serial killers that kill multiple target cells via exocytosis of cytotoxic granules (CGs). CG exocytosis is tightly regulated and has been investigated in great detail; however, whether CG proteins are endocytosed following exocytosis and contribute to serial killing remains unknown. By using primary CTLs derived from a knock-in mouse of the CG membrane protein Synaptobrevin2, we show that CGs are endocytosed in a clathrin- and dynamin-dependent manner. Following acidification, endocytosed CGs are recycled through early and late, but not recycling endosomes. CGs are refilled with granzyme B at the late endosome stage and polarize to subsequent synapses formed between the CTL and new target cells. Importantly, inhibiting CG endocytosis in CTLs results in a significant reduction of their cytotoxic activity. Thus, our data demonstrate that continuous endocytosis of CG membrane proteins is a prerequisite for efficient serial killing of CTLs and identify key events in this process.

Different Munc13 isoforms function as priming factors in lytic granule release from murine cytotoxic T lymphocytes.

In order to fuse lytic granules (LGs) with the plasma membrane at the immunological synapse, cytotoxic T lymphocytes (CTLs) have to render these LGs fusion-competent through the priming process. In secretory tissues such as brain and neuroendocrine glands, this process is mediated by members of the Munc13 protein family. In human CTLs, mutations in the Munc13-4 gene cause a severe loss in killing efficiency, resulting in familial hemophagocytic lymphohistiocytosis type 3, suggesting a similar role of other Munc13 isoforms in the immune system. Here, we investigate the contribution of different Munc13 isoforms to the priming process of murine CTLs at both the mRNA and protein level. We demonstrate that Munc13-1 and Munc13-4 are the only Munc13 isoforms present in mouse CTLs. Both isoforms rescue the drastical secretion defect of CTLs derived from Munc13-4-deficient Jinx mice. Mobility studies using total internal reflection fluorescence microscopy indicate that Munc13-4 and Munc13-1 are responsible for the priming process of LGs. Furthermore, the domains of the Munc13 protein, which is responsible for functional fusion, could be identified. We conclude from these data that both isoforms of the Munc13 family, Munc13-1 and Munc13-4, are functionally redundant in murine CTLs.

Different effects of Sec61α, Sec62 and Sec63 depletion on transport of polypeptides into the endoplasmic reticulum of mammalian cells.

Co-translational transport of polypeptides into the endoplasmic reticulum (ER) involves the Sec61 channel and additional components such as the ER lumenal Hsp70 BiP and its membrane-resident co-chaperone Sec63p in yeast. We investigated whether silencing the SEC61A1 gene in human cells affects co- and post-translational transport of presecretory proteins into the ER and post-translational membrane integration of tail-anchored proteins. Although silencing the SEC61A1 gene in HeLa cells inhibited co- and post-translational transport of signal-peptide-containing precursor proteins into the ER of semi-permeabilized cells, silencing the SEC61A1 gene did not affect transport of various types of tail-anchored protein. Furthermore, we demonstrated, with a similar knockdown approach, a precursor-specific involvement of mammalian Sec63 in the initial phase of co-translational protein transport into the ER. By contrast, silencing the SEC62 gene inhibited only post-translational transport of a signal-peptide-containing precursor protein.

Separation of Single Core and Multicore Lytic Granules by Subcellular Fractionation and Immunoisolation.

Subcellular fractionation is an important tool used to separate intracellular organelles, structures or proteins. Here, we describe a stepwise protocol to isolate two types of lytic granules, multicore (MCG), and single core (SCG), from primary murine CTLs. We used cell disruption by nitrogen cavitation followed by separation of organelles via discontinuous sucrose density gradient centrifugation. Immunoisolation with a Synaptobrevin 2 antibody attached to magnetic beads was then used to harvest Synaptobrevin 2 positive granules for immunoblotting, mass spectrometry, electron, and light microscopy.

Lytic granule exocytosis at immune synapses: lessons from neuronal synapses.

Regulated exocytosis is a central mechanism of cellular communication. It is not only the basis for neurotransmission and hormone release, but also plays an important role in the immune system for the release of cytokines and cytotoxic molecules. In cytotoxic T lymphocytes (CTLs), the formation of the immunological synapse is required for the delivery of the cytotoxic substances such as granzymes and perforin, which are stored in lytic granules and released via exocytosis. The molecular mechanisms of their fusion with the plasma membrane are only partially understood. In this review, we discuss the molecular players involved in the regulated exocytosis of CTL, highlighting the parallels and differences to neuronal synaptic transmission. Additionally, we examine the strengths and weaknesses of both systems to study exocytosis.

Identification of distinct cytotoxic granules as the origin of supramolecular attack particles in T lymphocytes.

Cytotoxic T lymphocytes (CTL) kill malignant and infected cells through the directed release of cytotoxic proteins into the immunological synapse (IS). The cytotoxic protein granzyme B (GzmB) is released in its soluble form or in supramolecular attack particles (SMAP). We utilize synaptobrevin2-mRFP knock-in mice to isolate fusogenic cytotoxic granules in an unbiased manner and visualize them alone or in degranulating CTLs. We identified two classes of fusion-competent granules, single core granules (SCG) and multi core granules (MCG), with different diameter, morphology and protein composition. Functional analyses demonstrate that both classes of granules fuse with the plasma membrane at the IS. SCG fusion releases soluble GzmB. MCGs can be labelled with the SMAP marker thrombospondin-1 and their fusion releases intact SMAPs. We propose that CTLs use SCG fusion to fill the synaptic cleft with active cytotoxic proteins instantly and parallel MCG fusion to deliver latent SMAPs for delayed killing of refractory targets.

Remodelling of Ca2+ homeostasis is linked to enlarged endoplasmic reticulum in secretory cells.

The endoplasmic reticulum (ER) is extensively remodelled during the development of professional secretory cells to cope with high protein production. Since ER is the principal Ca2+ store in the cell, we characterised the Ca2+ homeostasis in NALM-6 and RPMI 8226 cells, which are commonly used as human pre-B and antibody secreting plasma cell models, respectively. Expression levels of Sec61 translocons and the corresponding Sec61-mediated Ca2+ leak from ER, Ca2+ storage capacity and store-operated Ca2+ entry were significantly enlarged in the secretory RPMI 8226 cell line. Using an immunoglobulin M heavy chain producing HeLa cell model, we found that the enlarged Ca2+ storage capacity and Ca2+ leak from ER are linked to ER expansion. Our data delineates a developmental remodelling of Ca2+ homeostasis in professional secretory cells in which a high Sec61-mediated Ca2+ leak and, thus, a high Ca2+ turnover in the ER is backed up by enhanced store-operated Ca2+ entry.

Studying the biology of cytotoxic T lymphocytes in vivo with a fluorescent granzyme B-mTFP knock-in mouse.

Understanding T cell function in vivo is of key importance for basic and translational immunology alike. To study T cells in vivo, we developed a new knock-in mouse line, which expresses a fusion protein of granzyme B, a key component of cytotoxic granules involved in T cell-mediated target cell-killing, and monomeric teal fluorescent protein from the endogenous Gzmb locus. Homozygous knock-ins, which are viable and fertile, have cytotoxic T lymphocytes with endogeneously fluorescent cytotoxic granules but wild-type-like killing capacity. Expression of the fluorescent fusion protein allows quantitative analyses of cytotoxic granule maturation, transport and fusion in vitro with super-resolution imaging techniques, and two-photon microscopy in living knock-ins enables the visualization of tissue rejection through individual target cell-killing events in vivo. Thus, the new mouse line is an ideal tool to study cytotoxic T lymphocyte biology and to optimize personalized immunotherapy in cancer treatment.

Cytotoxic, or killer, T cells are a key part of the immune system. They carry a lethal mixture of toxic chemicals, stored in packages called cytotoxic granules. Killer T cells inject the contents of these granules into infected, cancerous or otherwise foreign cells, forcing them to safely self-destruct. In test tubes, T cells are highly efficient serial killers, moving from one infected cell to the next at high speed. But, inside the body, their killing rate slows down. Researchers think that this has something to do with how killer T cells interact with other immune cells, but the details remain unclear. To get to grips with how killer T cells work in their natural environment, researchers need a way to follow them inside the body. One approach could be to use genetic engineering to attach a fluorescent tag to a protein found inside killer T cells. That tag then acts as a beacon, lighting the cells up and allowing researchers to track their movements. Tagging a protein inside the cytotoxic granules would allow close monitoring of T cells as they encounter, recognize and kill their targets. But fluorescent tags are bulky, and they can stop certain proteins from working as they should. To find out whether it is possible to track killer T cells with fluorescent tags, Chitirala, Chang et al. developed a new type of genetically modified mouse. The modification added a teal-colored tag to a protein inside the granules of the killer T cells. Chitirala, Chang et al. then used a combination of microscopy techniques inside and outside of the body to find out if the T cells still worked. This analysis showed that, not only were the tagged T cells able to kill diseased cells as normal, the tags made it possible to watch it happening in real time. Super-resolution microscopy outside of the body allowed Chitirala, Chang et al. to watch the killer T cells release their toxic granule content. It was also possible to follow individual T cells as they moved into, and destroyed, foreign tissue that had been transplanted inside the mice. These new mice provide a tool to understand how killer T cells really work. They could allow study not only of the cells themselves, but also their interactions with other immune cells inside the body. This could help to answer open questions in T cell research, such as why T cells seem to be so much more efficient at killing in test tubes than they are inside the body. Understanding this better could support the development of new treatments for viruses and cancer.

CAPS facilitates filling of the rapidly releasable pool of large dense-core vesicles.

Calcium-activator protein for secretion (CAPS) is a cytosolic protein that associates with large dense-core vesicles and is involved in their secretion. Mammals express two CAPS isoforms, which share a similar domain structure including a Munc13 homology domain that is believed to be involved in the priming of secretory vesicles. A variety of studies designed to perturb CAPS function indicate that CAPS is involved in the secretion of large dense-core vesicles, but where in the secretory pathway CAPS acts is still under debate. Mice in which one allele of the CAPS-1 gene is deleted exhibit a deficit in catecholamine secretion from chromaffin cells. We have examined catecholamine secretion from chromaffin cells in which both CAPS genes were deleted and show that the deletion of both CAPS isoforms causes a strong reduction in the pool of rapidly releasable chromaffin granules and of sustained release during ongoing stimulation. We conclude that CAPS is required for the adequate refilling and/or maintenance of a rapidly releasable granule pool.

Identification of the minimal protein domain required for priming activity of Munc13-1.

Most nerve cells communicate with each other through synaptic transmission at chemical synapses. The regulated exocytosis of neurotransmitters, hormones, and peptides occurs at specialized membrane areas through Ca2+-triggered fusion of secretory vesicles with the plasma membrane . Prior to fusion, vesicles are docked at the plasma membrane and must then be rendered fusion-competent through a process called priming. The molecular mechanism underlying this priming process is most likely the formation of the SNARE complex consisting of Syntaxin 1, SNAP-25, and Synaptobrevin 2. Members of the Munc13 protein family consisting of Munc13-1, -2, -3, and -4 were found to be absolutely required for this priming process . In the present study, we identified the minimal Munc13-1 domain that is responsible for its priming activity. Using Munc13-1 deletion constructs in an electrophysiological gain-of-function assay of chromaffin-granule secretion, we show that priming activity is mediated by the C-terminal residues 1100-1735 of Munc13-1, which contains both Munc13-homology domains and the C-terminal C2 domain. Priming by Munc13-1 appears to require its interaction with Syntaxin 1 because point mutants that do not bind Syntaxin 1 do not prime chromaffin granules.

Paralogs of the Calcium-Dependent Activator Protein for Secretion Differentially Regulate Synaptic Transmission and Peptide Secretion in Sensory Neurons.

The two paralogs of the calcium-dependent activator protein for secretion (CAPS) are priming factors for synaptic vesicles (SVs) and neuropeptide containing large dense-core vesicles (LDCVs). Yet, it is unclear whether CAPS1 and CAPS2 regulate exocytosis of these two vesicle types differentially in dorsal root ganglion (DRG) neurons, wherein synaptic transmission and neuropeptide release are of equal importance. These sensory neurons transfer information from the periphery to the spinal cord (SC), releasing glutamate as the primary neurotransmitter, with co-transmission via neuropeptides in a subset of so called peptidergic neurons. Neuropeptides are key components of the information-processing machinery of pain perception and neuropathic pain generation. Here, we compared the ability of CAPS1 and CAPS2 to support priming of both vesicle types in single and double knock-out mouse (DRG) neurons using a variety of high-resolution live cell imaging methods. While CAPS1 was localized to synapses of all DRG neurons and promoted synaptic transmission, CAPS2 was found exclusively in peptidergic neurons and mediated LDCV exocytosis. Intriguingly, ectopic expression of CAPS2 empowered non-peptidergic neurons to drive LDCV fusion, thereby identifying CAPS2 as an essential molecular determinant for peptidergic signaling. Our results reveal that these distinct functions of both CAPS paralogs are based on their differential subcellular localization in DRG neurons. Our data suggest a major role for CAPS2 in neuropathic pain via control of neuropeptide release.

Cytotoxic granule endocytosis depends on the Flower protein.

Cytotoxic T lymphocytes (CTLs) kill target cells by the regulated release of cytotoxic substances from granules at the immunological synapse. To kill multiple target cells, CTLs use endocytosis of membrane components of cytotoxic granules. We studied the potential calcium dependence of endocytosis in mouse CTLs on Flower, which mediates the calcium dependence of synaptic vesicle endocytosis in Drosophila melanogaster Flower is predominantly localized on intracellular vesicles that move to the synapse on target cell contact. Endocytosis is entirely blocked at an early stage in Flower-deficient CTLs and is rescued to wild-type level by reintroducing Flower or by raising extracellular calcium. A Flower mutant lacking binding sites for the endocytic adaptor AP-2 proteins fails to rescue endocytosis, indicating that Flower interacts with proteins of the endocytic machinery to mediate granule endocytosis. Thus, our data identify Flower as a key protein mediating granule endocytosis.

The role of Snapin in neurosecretion: snapin knock-out mice exhibit impaired calcium-dependent exocytosis of large dense-core vesicles in chromaffin cells.

Identification of the molecules that regulate the priming of synaptic vesicles for fusion and the structural coupling of the calcium sensor with the soluble N-ethyl maleimide sensitive factor adaptor protein receptor (SNARE)-based fusion machinery is critical for understanding the mechanisms underlying calcium-dependent neurosecretion. Snapin binds to synaptosomal-associated protein 25 kDa (SNAP-25) and enhances the association of the SNARE complex with synaptotagmin. In the present study, we abolished snapin expression in mice and functionally evaluated the role of Snapin in neuroexocytosis. We found that the association of synaptotagmin-1 with SNAP-25 in brain homogenates of snapin mutant mice is impaired. Consequently, the absence of Snapin in embryonic chromaffin cells leads to a significant reduction of calcium-dependent exocytosis resulting from a decreased number of vesicles in releasable pools. Overexpression of Snapin fully rescued this inhibitory effect in the mutant cells. Furthermore, Snapin is relatively enriched in the purified large dense-core vesicles of chromaffin cells and associated with synaptotagmin-1. Thus, our biochemical and electrophysiological studies using snapin knock-out mice demonstrate that Snapin plays a critical role in modulating neurosecretion by stabilizing the release-ready vesicles.

Behavior and Properties of Mature Lytic Granules at the Immunological Synapse of Human Cytotoxic T Lymphocytes.

Killing of virally infected cells or tumor cells by cytotoxic T lymphocytes requires targeting of lytic granules to the junction between the CTL and its target. We used whole-cell patch clamp to measure the cell capacitance at fixed intracellular [Ca2+] to study fusion of lytic granules in human CTLs. Expression of a fluorescently labeled human granzyme B construct allowed identification of lytic granule fusion using total internal reflection fluorescence microscopy. In this way capacitance steps due to lytic granule fusion were identified. Our goal was to determine the size of fusing lytic granules and to describe their behavior at the plasma membrane. On average, 5.02 ± 3.09 (mean ± s.d.) lytic granules were released per CTL. The amplitude of lytic granule fusion events was ~ 3.3 fF consistent with a diameter of about 325 nm. Fusion latency was biphasic with time constants of 15.9 and 106 seconds. The dwell time of fusing lytic granules was exponentially distributed with a mean dwell time of 28.5 seconds. Fusion ended in spite of the continued presence of granules at the immune synapse. The mobility of fusing granules at the membrane was indistinguishable from that of lytic granules which failed to fuse. While dwelling at the plasma membrane lytic granules exhibit mobility consistent with docking interspersed with short periods of greater mobility. The failure of lytic granules to fuse when visible in TIRF at the membrane may indicate that a membrane-confined reaction is rate limiting.

Secretory vesicle priming by CAPS is independent of its SNARE-binding MUN domain.

Priming of secretory vesicles is a prerequisite for their Ca(2+)-dependent fusion with the plasma membrane. The key vesicle priming proteins, Munc13s and CAPSs, are thought to mediate vesicle priming by regulating the conformation of the t-SNARE syntaxin, thereby facilitating SNARE complex assembly. Munc13s execute their priming function through their MUN domain. Given that the MUN domain of Ca(2+)-dependent activator protein for secretion (CAPS) also binds syntaxin, it was assumed that CAPSs prime vesicles through the same mechanism as Munc13s. We studied naturally occurring splice variants of CAPS2 in CAPS1/CAPS2-deficient cells and found that CAPS2 primes vesicles independently of its MUN domain. Instead, the pleckstrin homology domain of CAPS2 seemingly is essential for its priming function. Our findings indicate a priming mode for secretory vesicles. This process apparently requires membrane phospholipids, does not involve the binding or direct conformational regulation of syntaxin by MUN domains of CAPSs, and is therefore not redundant with Munc13 action.

Complexin synchronizes primed vesicle exocytosis and regulates fusion pore dynamics.

ComplexinII (CpxII) and SynaptotagminI (SytI) have been implicated in regulating the function of SNARE proteins in exocytosis, but their precise mode of action and potential interplay have remained unknown. In this paper, we show that CpxII increases Ca(2+)-triggered vesicle exocytosis and accelerates its secretory rates, providing two independent, but synergistic, functions to enhance synchronous secretion. Specifically, we demonstrate that the C-terminal domain of CpxII increases the pool of primed vesicles by hindering premature exocytosis at submicromolar Ca(2+) concentrations, whereas the N-terminal domain shortens the secretory delay and accelerates the kinetics of Ca(2+)-triggered exocytosis by increasing the Ca(2+) affinity of synchronous secretion. With its C terminus, CpxII attenuates fluctuations of the early fusion pore and slows its expansion but is functionally antagonized by SytI, enabling rapid transmitter discharge from single vesicles. Thus, our results illustrate how key features of CpxII, SytI, and their interplay transform the constitutively active SNARE-mediated fusion mechanism into a highly synchronized, Ca(2+)-triggered release apparatus.

Super-resolution microscopy in studying neuroendocrine cell function.

The last two decades have seen a tremendous development in high resolution microscopy techniques giving rise to acronyms such as TIRFM, SIM, PALM, STORM, and STED. The goal of all these techniques is to overcome the physical resolution barrier of light microscopy in order to resolve precise protein localization and possibly their interaction in cells. Neuroendocrine cell function is to secrete hormones and peptides on demand. This fine-tuned multi-step process is mediated by a large array of proteins. Here, we review the new microscopy techniques used to obtain high resolution and how they have been applied to increase our knowledge of the molecular mechanisms involved in neuroendocrine cell secretion. Further the limitations of these methods are discussed and insights in possible new applications are provided.