T cell activation, key in the unfolding of the adaptive immune response, depends on ligand recognition by the TCR. Although this interaction is of low affinity, T cells are remarkably sensitive and exquisitely specific. Our previous work, in close collaboration with the group of Schamel/Minguet has shown that T cells can achieve and regulate this sensitivity by formation of TCR nanoclusters (Kumar et al., 2011). The project aims to define molecular interactions that mediate TCR nanoclustering in human T cell lines, which can then be applied to therapeutically relevant TCR‐ and CAR‐expressing primary human T cells to achieve increased sensitivity.

Objectives:

1. Generation of human T cell lines lacking the endogenous CD3 genes via CRISPR/Cas9‐mediated gene editing;
2. reconstitution of these cells with mutant CD3 chains and test TCR nanocluster formation via FRET, EM and/or plasmon coupling techniques (in collaboration with MUWIEN);
3. determine sensitivity of the functional response of these cells;
4. over‐expression of proven mutants in primary T cells and test functional consequences (in collaboration with LUMC and CTH);
5. generate a model of inter‐TCR interactions in TCR nanoclusters (in collaboration with UNIVBRIS)

We will be able to get an improved molecular understanding of the domains and modifications that promote or impede TCR nanocluster formation. Overexpression of mutant CD3 chains, peptide‐based interaction‐interfering reagents, or knock‐down of particular CD3 components may provide a means to improve sensitivity of therapeutically relevant T cells.

References:

Kumar R, Ferez M, Swamy M, Arechaga I, Rejas MT, Valpuesta JM, Schamel WW, Alarcon B*, van Santen HM* (2011). Increased Sensitivity of Antigen-Experienced T Cells through the Enrichment of Oligomeric T Cell Receptor Complexes. Immunity 35, 375-387 (*: co-corresponding authors).

Castro M*, van Santen HM*, Ferez M, Alarcón B, Lythe GD, Molina-Paris C (2014). Receptor Pre-Clustering and T cell Responses: Insights into Molecular Mechanisms. Front. Immunol. 5, 132; doi: 10.3389/fimmu.2014.00132 (*: co-corresponding authors).

Alarcón B, van Santen HM (2016). T Cell Receptor Triggering. In: Ralph A Bradshaw and Philip D Stahl (Editors-in-Chief), Encyclopedia of Cell Biology, Vol 3, Functional Cell Biology, pp. 650-659. Waltham, MA: Academic Press; doi:10.1016/B978-0-12-394447-4.30097-9.

Schamel WW, Arechaga, I, Risueño RM, van Santen HM, Cabezas P, Risco C, Valpuesta JM, Alarcón B. (2005). Co-existence of multivalent and monovalent TCRs: a potential mechanism to explain high sensitivity and wide range of response. J. Exp. Med. 202(4):493-503.