Biology subjects

Kotowski, M.

Publications and source records attributed to Kotowski, M..

3 recordsLinked to original sources

bioRxiv2026

Signalome-wide mapping of the NFκB pathway in T-cells reveals novel targets for immunotherapy

Cell signalling networks govern fundamental cellular processes yet remain incompletely defined. Moreover, what is known is biased toward a limited subset of well-characterised components. Phosphoprotein-based interrogation methods, including mass spectrometry and targeted phosphosite panels, have limited utility in physiological settings dependent on cell-cell interactions because the signalling fluxes can be difficult to detect despite producing robust functional responses. Here we developed a perturbation-based experimental framework that infers signalling pathway architecture using quantitative functional outputs rather than direct measurements of effector state, e.g., phosphorylation levels. Using antigen-specific, NF-{kappa}B-GFP reporter-expressing transformed T-cells co-cultured with cellular targets, we performed an arrayed CRISPR-Cas9 screen targeting a curated signalome of kinases, phosphatases, adaptor and scaffolding proteins, totalling 706 genes. Quantitative effect-size profiling recovered canonical T-cell receptor regulators and revealed unequal, family-specific patterns of control over NF-{kappa}B activation. Comparing T-cell stimulation with low- and high-affinity antigen uncovered signal-strength-dependent buffering of proximal signalling nodes, exemplified by reduced sensitivity to perturbation of LCK under high-intensity stimulation. Targeted perturbation in primary human CD8 T-cells validated our findings and identified TRRAP and CTDSPL2 as negative regulators of T-cell effector output, whose disruption enhanced cytotoxicity, degranulation, and cytokine production in both polyclonal and TCR-engineered T cells. Together, these results establish a scalable strategy for mapping signalling pathway architecture in the setting of physiological T-cell activation.

immunology↗

bioRxiv2025

T-cell signaling relies on partial CD45-exclusion at sub-micron sized cellular contacts

How cell contact initiates T-cell activation is uncertain. The local exclusion of the receptor-type protein tyrosine phosphatase CD45 at cell contacts is believed to trigger immune receptor signaling but this is yet to be observed for T cells interacting with authentic cellular targets. Here, quantitative imaging of T cells interacting with tumor cells presenting either native or clinically relevant bi-specific TCR ligands, revealed that they form multiple sub-micron sized close contacts with their targets. The contacts were stabilised by the adhesion protein CD2, but efficient ligand detection required both CD2 and integrin ligation. CD45 was excluded from close contacts at the time of ZAP70 recruitment and signaling, but only partially (30- 40%). A single-cell, mass cytometric analysis showed that this change in kinase/phosphatase activity provoked strong T-cell activation and potent cytotoxicity via very small changes in signaling fluxes. Spatial stochastic simulations suggested that the proximal T-cell signaling network is optimised for efficient antigen discrimination in the setting of partial CD45 exclusion. Our work re-frames early T-cell activation as a process initiated by relatively subtle changes in kinase/phosphatase activity acting on small numbers of signaling effectors at minute cellular contacts.

immunology↗

bioRxiv2024

Cell state and transcription factor modulation during extended ex vivo CD8+ T-cell expansion.

Adoptive cell therapy is becoming a cornerstone of tumour immunotherapy. It relies on the relatively long-term (> 2 week) ex vivo expansion of T cells either in the form of tumour-infiltrating cells, or bulk cells modified with the expression of heterologous signalling proteins, e.g., chimeric antigen receptors. However, relatively little is known about the developmental trajectories of T cells under these conditions at the system level, or whether the pathways governing these trajectories could be manipulated for clinical advantage. Using bulk RNA-seq analysis of T cells expanded and rested over a 17-day period, we produce a resource revealing how gene expression changes as cells transition through distinct cellular states over the course of activation and ex vivo expansion. By integrating this resource with published single-cell RNA-seq data, we identify a member of the AP1 transcription factor (TF) family, FOSL1, that primes CD8+ T-cells towards an effector/killing phenotype. Remarkably, FOSL1 over-expression during T-cell expansion produced super engager-like T-cells, evidenced by their gene-expression signatures and enhanced cancer-cell killing capacity. This establishes proof-of-principle for the rational engineering of T cells via TF modification during ex vivo expansion, offering a route to improving adoptive T-cell therapy.

immunology↗