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IntroductionAntigen binding to the T cell antigen receptor (TCR) leads to the phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3 complex, and thereby to T cell activation. The CD3ε subunit plays a unique role in TCR activation by recruiting the kinase LCK and the adaptor protein NCK prior to ITAM phosphorylation. Here, we aimed to investigate how phosphorylation of the individual CD3ε ITAM tyrosines impacts the CD3ε signalosome.MethodsWe mimicked irreversible tyrosine phosphorylation by substituting glutamic acid for the tyrosine residues in the CD3ε ITAM.ResultsIntegrating CD3ε phospho-mimetic variants into the complete TCR-CD3 complex resulted in reduced TCR signal transduction, which was partially compensated by the involvement of the other TCR-CD3 ITAMs. By using novel CD3ε phospho-mimetic Chimeric Antigen Receptor (CAR) variants, we avoided any compensatory effects of other ITAMs in the TCR-CD3 complex. We demonstrated that irreversible CD3ε phosphorylation prevented signal transduction upon CAR engagement. Mechanistically, we demonstrated that glutamic acid substitution at the N-terminal tyrosine residue of the CD3ε ITAM (Y39E) significantly reduces NCK binding to the TCR. In contrast, mutation at the C-terminal tyrosine of the CD3ε ITAM (Y50E) abolished LCK recruitment to the TCR, while increasing NCK binding. Double mutation at the C- and N-terminal tyrosines (Y39/50E) allowed ZAP70 to bind, but reduced the interaction with LCK and NCK.ConclusionsThe data demonstrate that the dynamic phosphorylation of the CD3ε ITAM tyrosines is essential for CD3ε to orchestrate optimal TCR and CAR signaling and highlights the key role of CD3ε signalosome to tune signal transduction.

Abstract Coral reefs represent some of the most biodiverse ecosystems in the world but are currently undergoing large‐scale degradation due to anthropogenic stressors. Such degradation usually begins with coral bleaching, and if the stress condition is inflicted for too long may eventually result in loss of structural complexity (or “flattening”) of the reef, dramatically changing habitat availability for reef‐associated fauna. Despite having been linked to important ecosystem functions, cryptobenthic organisms are often overlooked in ecological monitoring programs, and their microhabitat dependencies are poorly understood. Here, we combined collection‐based biodiversity monitoring techniques with five different environmental DNA (eDNA) sampling techniques (Reef water, sediment, crevice water, slurp gun, and bulk sediment) to survey cryptobenthic fishes and annelids on a Maldivian fringing coral reef. Collectively, 176 fish and 140 annelid taxa were detected with eDNA across 14 surveyed sites, more than doubling the reported annelid taxa in the region with 88 new occurrences. Water filtered near the reef structure revealed the highest species richness out of the five eDNA sampling techniques tested. Furthermore, we found correlations between fish species richness and topographic complexity for both collection‐ and eDNA‐based techniques. This suggests that detection by eDNA may be linked to site‐specific predictors and reveal community differences across small spatial scales (tens of meters). We also report that reef flattening (going from structural complex to less complex sites) can cause a 50% reduction in fish diversity and that cryptobenthic fish species richness was highly associated with branching corals. In contrast, annelid communities showed no clear correlations with environmental predictors, but co‐amplification of non‐target, non‐annelid taxa may have distorted such correlations if present. This suggest that the predictive powers of eDNA for environmental gradients may be dependent on the targeted taxa.

Dipolar recoupling is a central concept in the nuclear magnetic resonance spectroscopy of powdered solids and is used to establish correlations between different nuclei by magnetization transfer. The efficiency of conventional cross-polarization methods is low because of the inherent radio frequency (rf) field inhomogeneity present in the magic angle spinning (MAS) experiments and the large chemical shift anisotropies at high magnetic fields. Very high transfer efficiencies can be obtained using optimal control–derived experiments. These sequences had to be optimized individually for a particular MAS frequency. We show that by adjusting the length and the rf field amplitude of the shaped pulse synchronously with sample rotation, optimal control sequences can be successfully applied over a range of MAS frequencies without the need of reoptimization. This feature greatly enhances their applicability on spectrometers operating at differing external fields where the MAS frequency needs to be adjusted to avoid detrimental resonance effects.