The role of endosomes in receptor signal transduction is a long-standing question, which remains largely unanswered. The T cell Ag receptor and various components of its proximal signaling machinery are associated with distinct endosomal compartments, but how endosomal traffic affects T cell signaling remains ill-defined. In this article, we demonstrate in human T cells that the subcellular localization and function of the protein tyrosine kinase Lck depends on the Rab11 effector FIP3 (Rab11 family interacting protein-3). FIP3 overexpression or silencing and its ability to interact with Rab11 modify Lck subcellular localization and its delivery to the immunological synapse. Importantly, FIP3-dependent Lck localization controls early TCR signaling events, such as tyrosine phosphorylation of TCRζ, ZAP70, and LAT and intracellular calcium concentration, as well as IL-2 gene expression. Interestingly, FIP3 controls both steady-state and poststimulation phosphotyrosine and calcium levels. Finally, our findings indicate that FIP3 modulates TCR-CD3 cell surface expression via the regulation of steady-state Lck-mediated TCRζ phosphorylation, which in turn controls TCRζ protein levels. This may influence long-term T cell activation in response to TCR-CD3 stimulation. Therefore, our data underscore the importance of finely regulated endosomal traffic in TCR signal transduction and T cell activation leading to IL-2 production.
J.B. designed the project, designed and performed the experiments, analyzed the data, and contributed to writing the manuscript; I.d.R.-I. performed experiments, analyzed and discussed data, and critically commented on the manuscript; E.V.-C. designed, performed, analyzed and discussed experiments, and contributed to writing the manuscript; R.L., S.A.-G., and V.D.B. provided expertise, contributed to data analyses and discussions, and critically commented on the manuscript; C.C. provided technical and organizational support; M.W.M. provided expression vectors, previous expertise, and critical reading of the manuscript; A.A. conceived the project, contributed to data analyses and discussions, and wrote the manuscript.
This work was supported by grants from the Agence Nationale de Recherche (ANR; Grant 11 BSV3 025 01), the ANR sur le SIDA et les Hepatitis Virales (Grant AO 2013-02 CSS1 No 1339/14673), the Institut Pasteur, CNRS, INSERM, and the People Programme (Marie Sklodowska-Curie Actions) of the European Union’s Seventh Framework Programme (Grant FP7/2007-2013) under the Research Executive Agency Grant agreement 317057 HOMIN-ITN (to A.A.), and by Science Foundation Ireland Principal Investigator Award 09/IN.1/B2629 (to M.W.M.). The Imagopole is part of the France BioImaging infrastructure supported by Grant ANR-10-INSB-04-01, “Investments for the Future.” Personnel funding was as follows: ANR sur le SIDA et les Hepatitis Virales and Roux-Institut Pasteur postdoctoral funding to J.B.; European Union Marie Curie Actions HOMIN-ITN (cited earlier) predoctoral funding to I.d.R.-I., who is a scholar in the Pasteur-Paris University International Ph.D. program; Roux-Institut Pasteur and ANR postdoctoral funding to R.L.; Fondation ARC pour la Recherche sur le Cancer and ANR postdoctoral funding to S.A.-G.; and ANR and Sidaction postdoctoral funding to E.V.-C.
Abbreviations used in this article:
- Agence Nationale de Recherche
- endosomal recycling compartment
- Rab11 family interacting protein-3
- Institut Pasteur Clinical Investigation and Access to Biological Resources
- intraflagellar transport protein
- phorbol 12,13-dibutyrate
- retrotranscription quantitative PCR
- small interfering RNA
- wild type.
- Received April 18, 2016.
- Accepted January 27, 2017.
- Copyright © 2017 by The American Association of Immunologists, Inc.