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Endosomes Requires Vti1b-Dependent Pairing with CD3 Docking of Lytic Granules at the Immunological Synapse in Human CTL Requires Vti1b-Dependent Pairing with CD3 Endosomes This information is current as of October 2, 2021. Bin Qu, Varsha Pattu, Christian Junker, Eva C. Schwarz, Shruthi S. Bhat, Carsten Kummerow, Misty Marshall, Ulf Matti, Frank Neumann, Michael Pfreundschuh, Ute Becherer, Heiko Rieger, Jens Rettig and Markus Hoth J Immunol published online 11 May 2011 Downloaded from http://www.jimmunol.org/content/early/2011/05/11/jimmun ol.1003471 Supplementary http://www.jimmunol.org/content/suppl/2011/05/11/jimmunol.100347 http://www.jimmunol.org/ Material 1.DC1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on October 2, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published May 11, 2011, doi:10.4049/jimmunol.1003471 The Journal of Immunology Docking of Lytic Granules at the Immunological Synapse in Human CTL Requires Vti1b-Dependent Pairing with CD3 Endosomes Bin Qu,*,1 Varsha Pattu,†,1 Christian Junker,* Eva C. Schwarz,* Shruthi S. Bhat,* Carsten Kummerow,* Misty Marshall,† Ulf Matti,† Frank Neumann,‡ Michael Pfreundschuh,‡ Ute Becherer,† Heiko Rieger,x Jens Rettig,† and Markus Hoth* Lytic granule (LG)-mediated apoptosis is the main mechanism by which CTL kill virus-infected and tumorigenic target cells. CTL form a tight junction with the target cells, which is called the immunological synapse (IS). To avoid unwanted killing of neighboring cells, exocytosis of lytic granules (LG) is tightly controlled and restricted to the IS. In this study, we show that in activated human primary CD8+ T cells, docking of LG at the IS requires tethering LG with CD3-containing endosomes (CD3-endo). Combining Downloaded from total internal reflection fluorescence microscopy and fast deconvolution microscopy (both in living cells) with confocal microscopy (in fixed cells), we found that LG and CD3-endo tether and are cotransported to the IS. Paired but not single LG are accumulated at the IS. The dwell time of LG at the IS is substantially enhanced by tethering with CD3-endo, resulting in a preferential release of paired LG over single LG. The SNARE protein Vti1b is required for tethering of LG and CD3-endo. Downregulation of Vti1b reduces tethering of LG with CD3-endo. This leads to an impaired accumulation and docking of LG at the IS and a reduction of target cell killing. Therefore, Vti1b-dependent tethering of LG and CD3-endo determines accumulation, docking, and efficient http://www.jimmunol.org/ lytic granule secretion at the IS. The Journal of Immunology, 2011, 186: 000–000. ytotoxic T lymphocytes are considered key players in enriched at the IS after its formation (10–12). This is necessary to the immune response to eliminate tumorigenic or virus- recruit effector molecules and initiate downstream signaling that C infected target cells (1–5). CTL cytotoxicity is mainly leads to target cell death (13, 14). mediated by exocytosis of lytic granules (LG) and the Fas path- LG, which contain perforin and granzymes (15), are transported, way (6). To kill cognate target cells efficiently, CTL and target docked, and released at the IS and induce target cell apoptosis (2, cells form a close connection, called the immunological synapse 16, 17). The directed secretion of LG is Ca2+ dependent (18), and (IS), upon TCR engagement by matching peptide–MHC com- it is critical to ensure the selective killing of a cognate target cell. by guest on October 2, 2021 plexes (4, 7, 8). Formation of the IS involves drastic morpholog- Upon target cell recognition, CTL undergo large-scale rear- ical changes and cell polarization, which facilitates the stable rangements of the cytoskeleton. In NK cells, actin polymerization physical contact between the two cells (4, 9) leading to CTL ac- is required for receptor clustering, granule polarization (19), and tivation and target cell death (2). TCR are constitutively in- cytotoxic function (20). The convergence of LG to the microtu- ternalized and recycled back to the cell surface and are quickly bule organizing center (MTOC) depends on dynein (21), and myosin IIA is required for interaction of LG and F-actin as well as *Department of Biophysics, Saarland University, 66421 Homburg, Germany; the subsequent release of LG (22). In mouse CTL, the MTOC has † Department of Physiology, Saarland University, 66421 Homburg, Germany; even been shown to deliver LG to the IS by touching the plasma ‡Department of Internal Medicine I, Saarland University, 66421 Homburg, Germany; x and Department of Theoretical Physics, Saarland University, 66041 Saarbru¨cken, membrane (23). There is currently substantial excitement in elu- Germany cidating the molecular mechanisms of LG polarization to the se- 1B.Q. and V.P. contributed equally to this work. cretory domain of the CTL–target contact zone (4, 24). Jenkins Received for publication October 20, 2010. Accepted for publication April 11, 2011. et al. (25) have shown that the strength of the TCR signal is ex- This work was supported by the Deutsche Forschungsgemeinschaft (Graduate Pro- ceptionally important for LG accumulation at the IS, and Beal gram on Calcium Signalling and Cellular Nanodomains, Grants RE 1092/6-1 and et al. (26) unmasked the kinetics of a short and long path of LG to SFB 894 to J.R. and M.H.) by the Deutsche Krebshilfe (to M.P.), and by a Homburger Forschungsfo¨rderung grant from the Medical Faculty, Saarland University, Homburg, the synapse. Germany (to J.R., M.H., and E.C.S.). The importance of CTL for the immune response is highlighted Address correspondence and reprint requests to Prof. Markus Hoth or Prof. Jens by life-threatening diseases: dysfunction of LG release is associ- Rettig, Institute of Biophysics, Saarland University, Building 58, 66421 Homburg, ated with familial hemophagocytic lymphohistiocytosis (FHL) or Germany (M.H.) or Institute of Physiology, Saarland University, Building 59, 66421 Homburg, Germany (J.R.). E-mail addresses: [email protected] (M.H.) acquired hemophagocytic lymphohistiocytosis (27–29). Among and [email protected] (J.R.) the known causes of FHL are mutations in perforin (FHL2) (27), The online version of this article contains supplemental material. Munc13-4 (FHL3) (30), syntaxin11 (FHL4) (31), and Munc18-2 Abbreviations used in this article: CD3-endo, CD3-containing endosomes; 4D, four- (FHL5) (32). In addition, Vti1b and Vamp8 are important for the dimensional; FHL, familial hemophagocytic lymphohistiocytosis; LG, lytic granule; MTOC, microtubule organizing center; qRT-PCR, quantitative real-time PCR; SEA, killing capacity of CTL (33). staphylococcal enterotoxin A; siRNA, small interfering RNA; SNARE, soluble N- These diseases illustrate the importance of directed transport ethylmaleimide–sensitive factor attachment receptor; Stx11, syntaxin 11; TfR, trans- regulated by soluble N-ethylmaleimide–sensitive factor attach- ferrin receptors; TIRF, total internal reflection fluorescence. ment receptor (SNARE) and/or SNARE-related proteins in tar- Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 geting LG to the IS. SNARE proteins play a central role in www.jimmunol.org/cgi/doi/10.4049/jimmunol.1003471 2 LG DOCKING REQUIRES Vti1b-DEPENDENT LG/CD3-ENDO PAIRING budding, target selection, and fusion of intracellular compartments 2mML-glutamine (Life Technologies, Invitrogen, Karlsruhe, Germany) (34–36). It has been reported that SNARE proteins are also in- and 1% penicillin/streptomycin (Life Technologies) and pulsed with pep- volved in focal and multidimensional pathways of cytokine re- tide p495–503 (2 mM, 37˚C, 1.5 h). Thereafter cells were washed, irra- diated with 30 Gy, and resuspended in X-Vivo medium with 10% human lease in T cells (37). The 36 mammalian SNAREs can be defined AB serum and served as APC. The same number of CD8+ T cells were as either Q- (Qa, Qb, Qc or Qbc) or R-SNAREs, depending on coincubated as prospective effector cells. At days 7 and 14, the CD8+ 2 which conservative residue the SNARE protein contributes at effector cells were restimulated using peptide-pulsed CD8 APC under layer 0 within the four-helix bundle of SNARE complexes. identical conditions. At day 14, specificity of the stimulated effector cells was assessed by IFN-g segregation using an ELISPOT assay as described SNARE proteins mediate membrane fusion in all trafficking steps previously (41). of the secretory pathway. Prior to fusion, SNAREs on opposing membranes are able to form four helix bundles that lead to a tight Establishment of epitope-specific CD8+ T cell clones connection of vesicular and target membranes (36). Responding T cells were isolated out of the bulk population by IFN-g–based We report that tethering (or pairing) of LG and CD3-containing magnetic cell enrichment using a cytokine-secretion assay (Miltenyi Bio- endosomes (CD3-endo) is required for the enrichment, docking, tec) and cloned by limiting-dilution culture, following a protocol de- + and release of LG at the IS, which is needed for CTL-dependent scribed by zum Bu¨schenfelde et al. (42). Specifically reacting CD8 clones were expanded by restimulation performed after 14 d under identical target cell killing. In addition, we unmask that LG/CD3-endo + conditions. Cytotoxicity of CD8 T cells from CTL clone KL-1/#35 tethering requires the Qb-SNARE protein Vti1b.
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