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Nanobody-induced perturbation of LFA-1/L-plastin phosphorylation impairs MTOC docking, immune synapse formation and T cell activation

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.

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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

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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

Correspondence to Jan Gettemans.

Additional information

Aude Guillabert and Jan Gettemans contributed equally to this work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

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)

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)

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)

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)

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)

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)

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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

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Keywords

  • Immune synapse
  • Nanobody
  • LFA-1
  • Integrin
  • L-plastin