TCR ITAM multiplicity is required for the generation of follicular helper T-cells.

Sujin Hwang,Noah Tubo,Ekaterina Zvezdova,Paul E Love,Jan Lee,Marc K Jenkins,Liqi Li,Amy C Palin,Renaud Lesourne,Ki-Duk Song,Dorian Mcgavern,Hamlet Chu,Marion Pepper,Julia Pinkhasov,Jasmin Herz

doi:10.1038/ncomms7982

Abstract

The T-cell antigen receptor (TCR) complex contains 10 copies of a di-tyrosine Immunoreceptor-Tyrosine-based-Activation-Motif (ITAM) that initiates TCR signalling by recruiting protein tyrosine kinases. ITAM multiplicity amplifies TCR signals, but the importance of this capability for T-cell responses remains undefined. Most TCR ITAMs (6 of 10) are contributed by the CD3ζ subunits. We generated ‘knock-in' mice that express non-signalling CD3ζ chains in lieu of wild-type CD3ζ. Here we demonstrate that ITAM multiplicity is important for the development of innate-like T-cells and follicular helper T-cells, events that are known to require strong/sustained TCR–ligand interactions, but is not essential for ‘general' T-cell responses including proliferation and cytokine production or for the generation of a diverse antigen-reactive TCR repertoire.

Highlights

The T-cell antigen receptor (TCR) complex contains 10 copies of a di-tyrosine Immunoreceptor-Tyrosine-based-Activation-Motif (ITAM) that initiates TCR signalling by recruiting protein tyrosine kinases
Since the mature TCR complex (TCRab/ CD3ge/CD3de/CD3zz) contains a total of 10 ITAM signalling motifs (6 contributed by the CD3zz homodimer and 4 contributed by the CD3ge and CD3de dimers) the signalling potential of the TCR is theoretically reduced by 60% (6/10 ITAMs) in 6F/6F mice relative to ‘wild-type’ 6Y/6Y mice
Positive selection of 6F/6F thymocytes expressing a transgenic MHC Class I (MHC I)restricted (H-Y) or an MHC Class II (MHC II)-restricted (AND) abTCR transgene was almost completely blocked at the immature CD4 þ CD8 þ (double positive (DP)) stage (Fig. 1b,c)

Summary

Introduction

The T-cell antigen receptor (TCR) complex contains 10 copies of a di-tyrosine Immunoreceptor-Tyrosine-based-Activation-Motif (ITAM) that initiates TCR signalling by recruiting protein tyrosine kinases. To address outstanding and unresolved questions concerning the role and importance of ITAM multiplicity for TCR-mediated signalling, we analysed two lines of ‘knock-in’ mice generated by gene targeting in embryonic stem cells: 6Y/6Y, which encodes a ‘wild-type’ CD3z chain, and 6F/6F, which encodes a CD3z protein where each of the 6 ITAM Y residues was mutated to Phenylalanine (F), rendering these ITAMs non-functional for signal transduction[20] (Fig. 1a). Both ‘knock-in’ alleles were placed under the control of endogenous CD3z regulatory sequences so that expression of the 6Y and 6F CD3z proteins mimics that of endogenous CD3z, both developmentally and quantitatively[20]. These results reveal that a key function of ITAM multiplicity is to facilitate developmental and functional responses that are dependent on strong or sustained TCR/ligand interactions

Methods

Results

Conclusion