CRISPR-TOF Platform: A Single-Cell Approach for Studying Kinase-Dependent Immune-Signaling Dynamics in Human Primary T Cells

Introduction

Chimeric antigen receptor (CAR) T-cell therapies have improved the treatment of hematologic malignancies, but challenges such as activation and persistence limit broader application. Recent insights into the T-cell receptor (TCR) pathway revealed promising strategies for modulating activation kinetics, crucial for enhancing the efficacy of CAR T-cells. Here, we developed a single-cell approach for studying kinase-dependent signaling dynamics in primary human T cells, which unravels critical mechanisms of the T-cell activation cascade to better understand the genetic programs that determine T-cell response to (tumor) antigens.

Methods

We set up a platform called "CRISPR-TOF", in which we combined arrayed CRISPR-Cas9 gene editing, an enhanced mass tag live-cell barcoding approach, and single-cell mass spectrometry (CyTOF). We knocked out nearly 1,000 kinases in primary human T cells and tracked the phosphorylation of 23 different proteins over 30 minutes, at 20 time points, post-stimulation with anti-CD3/CD28.

Results

The expected signaling patterns were observed in the analyzed components of the TCR pathway. Initially, proximal kinases such as SLP-76, ZAP70, and VAV1 exhibited rapid phosphorylation, which decreased after one minute. In contrast, phosphorylation of MEK1/2 and ERK1/2 emerged at 2 minutes, and the S6 ribosomal protein, peaked at nine minutes post-activation. Upon knocking out specific kinases, our study identified a range of novel targets that alter downstream TCR signaling. We categorized genes that either prolong or attenuate activation and identified those that modify signaling relationships or influence individual pathway components through independent mechanisms.

Conclusions

We have generated a dynamic, high-resolution map of T-cell signaling, facilitating targeted investigations of specific pathway components. Our results represent one of the most comprehensive assessments of T-cell signaling dynamics to date. This platform allows focusing on key signaling molecules and is thus a powerful complement to platforms such as Pertub-seq in which RNA changes are measured. It opens avenues for massively multiplexed single-cell assays for drug target discovery, delving deeper into immune signaling by studying entire signaling pathways. In the future, functional analyses of specific gene knockouts in samples will be essential to fully elucidate their roles in TCR activation, signal transduction, and resultant cellular functions. More broadly, these insights could be harnessed to optimize and expand cellular therapies.