Abstract
The T cell integrin receptor LFA-1 orchestrates adhesion between T cells and antigen-presenting cells (APCs), resulting in formation of a contact zone known as the immune synapse (IS) which is supported by the cytoskeleton. L-plastin is a leukocyte-specific actin bundling protein that rapidly redistributes to the immune synapse following T cell–APC engagement. We used single domain antibodies (nanobodies, derived from camelid heavy-chain only antibodies) directed against functional and structural modules of L-plastin to investigate its contribution to formation of an immune synapse between Raji cells and human peripheral blood mononuclear cells or Jurkat T cells. Nanobodies that interact either with the EF hands or the actin binding domains of L-plastin both trapped L-plastin in an inactive conformation, causing perturbation of IS formation, MTOC docking towards the plasma membrane, T cell proliferation and IL-2 secretion. Both nanobodies delayed Ser5 phosphorylation of L-plastin which is required for enhanced bundling activity. Moreover, one nanobody delayed LFA-1 phosphorylation, reduced the association between LFA-1 and L-plastin and prevented LFA-1 enrichment at the IS. Our findings reveal subtle mechanistic details that are difficult to attain by conventional means and show that L-plastin contributes to immune synapse formation at distinct echelons.
Similar content being viewed by others
Abbreviations
- ABD:
-
Actin binding domain
- Akt:
-
Protein kinase B
- CaM:
-
Calmodulin
- CCD:
-
Charge-coupled device
- CH:
-
Calponin homology
- DDAO-SE:
-
9-H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl)-succinimidyl ester
- GSN:
-
Gelsolin
- IS:
-
Immune synapse
- LCK:
-
Lymphocyte-specific protein tyrosine kinase
- LPL:
-
L-plastin
- mLPL:
-
Monoclonal L-plastin (antibody)
- pLPL:
-
Polyclonal L-plastin (antibody)
- MTOC:
-
Microtubule-organizing center
- Nb:
-
Nanobody
- PKC:
-
Protein kinase C
- R-PE:
-
R-phycoerythrin
- SEE:
-
Staphylococcus enterotoxin E
- SMAC:
-
Supramolecular activation cluster
- pSMAC:
-
Peripheral SMAC
- cSMAC:
-
Central SMAC
References
Grakoui A, Bromley SK, Sumen C, Davis MM, Shaw AS, Allen PM, Dustin ML (1999) The immunological synapse: a molecular machine controlling T cell activation. Science 285(5425):221–227
Monks CR, Freiberg BA, Kupfer H, Sciaky N, Kupfer A (1998) Three-dimensional segregation of supramolecular activation clusters in T cells. Nature 395(6697):82–86
Frauwirth KA, Thompson CB (2002) Activation and inhibition of lymphocytes by costimulation. J Clin Invest 109(3):295–299
Perez OD, Mitchell D, Jager GC, South S, Murriel C, McBride J, Herzenberg LA, Kinoshita S, Nolan GP (2003) Leukocyte functional antigen 1 lowers T cell activation thresholds and signaling through cytohesin-1 and Jun-activating binding protein 1. Nat Immunol 4(11):1083–1092
Nurmi SM, Autero M, Raunio AK, Gahmberg CG, Fagerholm SC (2007) Phosphorylation of the LFA-1 integrin beta2-chain on Thr-758 leads to adhesion, Rac-1/Cdc42 activation, and stimulation of CD69 expression in human T cells. J Biol Chem 282(2):968–975
Burkhardt JK, Carrizosa E, Shaffer MH (2008) The actin cytoskeleton in T cell activation. Annu Rev Immunol 26:233–259
Wang C, Morley SC, Donermeyer D, Peng I, Lee WP, Devoss J, Danilenko DM, Lin Z, Zhang J, Zhou J, Allen PM, Brown EJ (2010) Actin-bundling protein L-plastin regulates T cell activation. J Immunol 185(12):7487–7497
Wabnitz GH, Lohneis P, Kirchgessner H, Jahraus B, Gottwald S, Konstandin M, Klemke M, Samstag Y (2010) Sustained LFA-1 cluster formation in the immune synapse requires the combined activities of L-plastin and calmodulin. Eur J Immunol 40(9):2437–2449
Namba Y, Ito M, Zu Y, Shigesada K, Maruyama K (1992) Human T cell L-plastin bundles actin filaments in a calcium-dependent manner. J Biochem 112(4):503–507
Leavitt J (1994) Discovery and characterization of two novel human cancer-related proteins using two-dimensional gel electrophoresis. Electrophoresis 15(3–4):345–357
Park T, Chen ZP, Leavitt J (1994) Activation of the leukocyte plastin gene occurs in most human cancer cells. Cancer Res 54(7):1775–1781
Janji B, Giganti A, De Corte V, Catillon M, Bruyneel E, Lentz D, Plastino J, Gettemans J, Friederich E (2006) Phosphorylation on Ser5 increases the F-actin-binding activity of L-plastin and promotes its targeting to sites of actin assembly in cells. J Cell Sci 119(Pt 9):1947–1960
Grimbert P, Valanciute A, Audard V, Pawlak A, Legouvelo S, Lang P, Niaudet P, Bensman A, Guellaen G, Sahali D (2003) Truncation of C-mip (Tc-mip), a new proximal signaling protein, induces c-maf Th2 transcription factor and cytoskeleton reorganization. J Exp Med 198(5):797–807
Sester U, Wabnitz GH, Kirchgessner H, Samstag Y (2007) Ras/PI3kinase/cofilin-independent activation of human CD45RA+ and CD45RO+ T cells by superagonistic CD28 stimulation. Eur J Immunol 37(10):2881–2891
Harmsen MM, De Haard HJ (2007) Properties, production, and applications of camelid single-domain antibody fragments. Appl Microbiol Biotechnol 77(1):13–22
Van Bockstaele F, Holz JB, Revets H (2009) The development of nanobodies for therapeutic applications. Curr Opin Investig Drugs 10(11):1212–1224
Hmila I, Saerens D, Ben Abderrazek R, Vincke C, Abidi N, Benlasfar Z, Govaert J, El Ayeb M, Bouhaouala-Zahar B, Muyldermans S (2010) A bispecific nanobody to provide full protection against lethal scorpion envenoming. FASEB J 24(9):3479–3489
Rasmussen SG, DeVree BT, Zou Y, Kruse AC, Chung KY, Kobilka TS, Thian FS, Chae PS, Pardon E, Calinski D, Mathiesen JM, Shah ST, Lyons JA, Caffrey M, Gellman SH, Steyaert J, Skiniotis G, Weis WI, Sunahara RK, Kobilka BK (2011) Crystal structure of the beta2 adrenergic receptor-Gs protein complex. Nature 477(7366):549–555
Hultberg A, Temperton NJ, Rosseels V, Koenders M, Gonzalez-Pajuelo M, Schepens B, Ibanez LI, Vanlandschoot P, Schillemans J, Saunders M, Weiss RA, Saelens X, Melero JA, Verrips CT, Van Gucht S, de Haard HJ (2011) Llama-derived single domain antibodies to build multivalent, superpotent and broadened neutralizing anti-viral molecules. PLoS ONE 6(4):e17665
Van den Abbeele A, De Corte V, Van Impe K, Bruyneel E, Boucherie C, Bracke M, Vandekerckhove J, Gettemans J (2007) Downregulation of gelsolin family proteins counteracts cancer cell invasion in vitro. Cancer Lett 255(1):57–70
Irobi J, Van Impe K, Seeman P, Jordanova A, Dierick I, Verpoorten N, Michalik A, De Vriendt E, Jacobs A, Van Gerwen V, Vennekens K, Mazanec R, Tournev I, Hilton-Jones D, Talbot K, Kremensky I, Van Den Bosch L, Robberecht W, Van Vandekerckhove J, Van Broeckhoven C, Gettemans J, De Jonghe P, Timmerman V (2004) Hot-spot residue in small heat-shock protein 22 causes distal motor neuropathy. Nat Genet 36(6):597–601
Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76(9):4350–4354
Delanote V, Vanloo B, Catillon M, Friederich E, Vandekerckhove J, Gettemans J (2010) An alpaca single-domain antibody blocks filopodia formation by obstructing L-plastin-mediated F-actin bundling. FASEB J 24(1):105–118
Van den Abbeele A, De Clercq S, De Ganck A, De Corte V, Van Loo B, Soror SH, Srinivasan V, Steyaert J, Vandekerckhove J, Gettemans J (2010) A llama-derived gelsolin single-domain antibody blocks gelsolin-G-actin interaction. Cell Mol Life Sci 67(9):1519–1535
Tsun A, Qureshi I, Stinchcombe JC, Jenkins MR, de la Roche M, Kleczkowska J, Zamoyska R, Griffiths GM (2011) Centrosome docking at the immunological synapse is controlled by Lck signaling. J Cell Biol 192(4):663–674
Kuhn JR, Poenie M (2002) Dynamic polarization of the microtubule cytoskeleton during CTL-mediated killing. Immunity 16(1):111–121
Stinchcombe JC, Majorovits E, Bossi G, Fuller S, Griffiths GM (2006) Centrosome polarization delivers secretory granules to the immunological synapse. Nature 443(7110):462–465
Shinomiya H, Hagi A, Fukuzumi M, Mizobuchi M, Hirata H, Utsumi S (1995) Complete primary structure and phosphorylation site of the 65-kDa macrophage protein phosphorylated by stimulation with bacterial lipopolysaccharide. J Immunol 154(7):3471–3478
Messier JM, Shaw LM, Chafel M, Matsudaira P, Mercurio AM (1993) Fimbrin localized to an insoluble cytoskeletal fraction is constitutively phosphorylated on its headpiece domain in adherent macrophages. Cell Motil Cytoskeleton 25(3):223–233
Morley SC (2012) The actin-bundling protein L-plastin: a critical regulator of immune cell function. Int J Cell Biol 2012:935173
Wabnitz GH, Kocher T, Lohneis P, Stober C, Konstandin MH, Funk B, Sester U, Wilm M, Klemke M, Samstag Y (2007) Costimulation induced phosphorylation of L-plastin facilitates surface transport of the T cell activation molecules CD69 and CD25. Eur J Immunol 37(3):649–662
Kupfer A, Dennert G (1984) Reorientation of the microtubule-organizing center and the Golgi apparatus in cloned cytotoxic lymphocytes triggered by binding to lysable target cells. J Immunol 133(5):2762–2766
Griffiths GM, Tsun A, Stinchcombe JC (2010) The immunological synapse: a focal point for endocytosis and exocytosis. J Cell Biol 189(3):399–406
Lasserre R, Alcover A (2010) Cytoskeletal cross-talk in the control of T cell antigen receptor signaling. FEBS Lett 584(24):4845–4850
Le Goff E, Vallentin A, Harmand PO, Aldrian-Herrada G, Rebiere B, Roy C, Benyamin Y, Lebart MC (2010) Characterization of L-plastin interaction with beta integrin and its regulation by micro-calpain. Cytoskeleton (Hoboken) 67(5):286–296
Baixauli F, Martin-Cofreces NB, Morlino G, Carrasco YR, Calabia-Linares C, Veiga E, Serrador JM, Sanchez-Madrid F (2011) The mitochondrial fission factor dynamin-related protein 1 modulates T-cell receptor signalling at the immune synapse. EMBO J 30(7):1238–1250
Holdorf AD, Lee KH, Burack WR, Allen PM, Shaw AS (2002) Regulation of Lck activity by CD4 and CD28 in the immunological synapse. Nat Immunol 3(3):259–264
Lee KH, Dinner AR, Tu C, Campi G, Raychaudhuri S, Varma R, Sims TN, Burack WR, Wu H, Wang J, Kanagawa O, Markiewicz M, Allen PM, Dustin ML, Chakraborty AK, Shaw AS (2003) The immunological synapse balances T cell receptor signaling and degradation. Science 302(5648):1218–1222
Pazdrak K, Young TW, Straub C, Stafford S, Kurosky A (2011) Priming of eosinophils by GM-CSF is mediated by protein kinase CbetaII-phosphorylated L-plastin. J Immunol 186(11):6485–6496
Jones SL, Wang J, Turck CW, Brown EJ (1998) A role for the actin-bundling protein L-plastin in the regulation of leukocyte integrin function. Proc Natl Acad Sci USA 95(16):9331–9336
Fagerholm S, Morrice N, Gahmberg CG, Cohen P (2002) Phosphorylation of the cytoplasmic domain of the integrin CD18 chain by protein kinase C isoforms in leukocytes. J Biol Chem 277(3):1728–1738
Acknowledgments
We thank Ciska Boucherie for technical support and dr. Leen Van Troys for helpful advice with confocal microscopy. We thank Dr. Evelyne Friedrich and Dr. Elisabeth Schaffner-Reckinger for the polyclonal rabbit IgGs against L-plastin and serine-5 phosphorylated L-plastin. Peter Van den Hemel is acknowledged for help with digital video processing. This work was supported by the Fund for Scientific Research-Flanders (FWO-Vlaanderen), the Stichting tegen Kanker (Belgium), the Concerted Actions Programme of Ghent University (GOA) and the Interuniversity attraction poles (IUAP06). SDC was supported by Ghent University.
Author information
Authors and Affiliations
Corresponding author
Additional information
Aude Guillabert and Jan Gettemans contributed equally to this work.
Electronic supplementary material
Below is the link to the electronic supplementary material.
18_2012_1169_MOESM1_ESM.jpg
Suppl. Figure S1. L-plastin is a highly and specifically expressed protein in hematopoietic cells. Lysates (10 µg) of PBMCs (monocytes and lymphocytes) from whole blood were analyzed by immunoblotting with gelsolin, fascin, L-plastin and CapG antibodies. (JPEG 274 kb)
18_2012_1169_MOESM2_ESM.jpg
Suppl. Figure S2. Actin and LPL localization in Jurkat T cells, conjugated with CD3/CD28 Dynabeads ®. (a, b) Jurkat T cells transfected with EGFP, LPL Nb5-EGFP or LPL Nb9-EGFP were incubated for 15 minutes with CD3/CD28 Dynabeads® and stained for actin (phalloidin-alexa 594, red) (a) or L-plastin (Alexa-594, red) (b). Transfected Jurkat cells are depicted in green (EGFP) and the beads can be seen (in black) in the DIC and merged images. (Note the autofluorescence of the beads when stained for LPL.) The pictures were acquired with a laser scanning confocal microscope. Bar: 5 µm. (JPEG 1638 kb)
18_2012_1169_MOESM3_ESM.jpg
Suppl. Figure S3. Actin and LPL localization in PBMCs, conjugated with CD3/CD28 Dynabeads ®. (a, b) PBMCs transfected with EGFP, LPL Nb5-EGFP or LPL Nb9-EGFP were incubated for 15 minutes with CD3/CD28 Dynabeads® and stained for actin (phalloidin-alexa 594, red) (a) or L-plastin (Alexa-594, red) (b). Transfected PBMCs are depicted in green (EGFP) and the beads can be seen (in black) in the DIC and merged images. (Note the autofluorescence of the beads when stained for LPL.) The pictures were acquired with a laser scanning confocal microscope. Bar: 5 µm. (JPEG 1269 kb)
18_2012_1169_MOESM4_ESM.jpg
Suppl. Figure S4. Phospho-LFA-1 interacts with phospho-LPL in stimulated Jurkat T cells. Lysates from unstimulated and CD3/CD28 stimulated Jurkat cells were incubated with IgG, monoclonal LPL (mLPL), LFA-1 (CD18) and phospho-LFA-1 antibodies (P-LFA-1) and LPL Nb5/9 in an immunoprecipitation experiment. The blots were stained for LFA-1, phospho-LFA-1, phospho-LPL (P-LPL) and LPL. Blots are representative for 2 independent experiments. (JPEG 412 kb)
18_2012_1169_MOESM5_ESM.jpg
Suppl. Figure S5. LPL Nbs do not affect the phosphorylation of cofilin and MEK1/2. EGFP, LPL Nb5 and LPL Nb9 transfected T cells were stimulated for different periods of time (indicated on top) with coated anti-CD3 (MEM92) and soluble anti-CD28. The blots were stained for phospho-cofilin and phospho-MEK1/2. The data are representative of three independent experiments. (JPEG 440 kb)
Suppl. Video S1. EGFP or LPL Nb5-EGFP transfected Jurkat cells were incubated for 45 min with SEE-pulsed Raji cells which were prelabeled with a fluorescent dye. Acquisition of the conjugated cells (stained for alpha-tubulin (alexa 594 (red) and DAPI (blue)) was done along the z-axis. A 3D reconstruction of the cell-cell interaction for EGFP (A) and LPL Nb5 (B) transfected cells is shown. Note: Jurkat cells are the polarized, elongated cells directly in front of the Raji cells. (MPEG 1408 kb)
Supplementary material 7 (MPEG 1404 kb)
Rights and permissions
About this article
Cite this article
De Clercq, S., Zwaenepoel, O., Martens, E. et al. Nanobody-induced perturbation of LFA-1/L-plastin phosphorylation impairs MTOC docking, immune synapse formation and T cell activation. Cell. Mol. Life Sci. 70, 909–922 (2013). https://doi.org/10.1007/s00018-012-1169-0
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00018-012-1169-0