The expression of ZAP-70 in a subset of CLL patients strongly correlates with a more aggressive clinical course, though the exact underlying mechanisms remain elusive. The ability of ZAP-70 to enhance B cell receptor (BCR) signaling, independently of its kinase function, is considered to contribute. Here we employed RNA-sequencing and proteomic analyses of primary cells differing only in their expression of ZAP-70 to further define how ZAP-70 increases aggressiveness of CLL. We identified that ZAP-70 is directly required for cell survival in the absence of an overt BCR signal, which can compensate for ZAP-70 deficiency as an anti-apoptotic signal. In addition, the expression of ZAP-70 regulates the transcription of factors regulating recruitment and activation of T cells, such as CCL3, CCL4 and IL4I1. Quantitative mass spectrometry of double-cross linked ZAP-70 complexes further demonstrated constitutive and direct protein-protein interactions between ZAP-70 and BCR-signaling components. Unexpectedly, ZAP-70 also binds to ribosomal proteins, which is not dependent on, but further increased by BCR-stimulation. Importantly, decreased expression of ZAP-70 significantly reduced MYC-expression and global protein synthesis, providing evidence that ZAP-70 contributes to translational dysregulation in CLL. In conclusion, ZAP-70 constitutively promotes cell survival, microenvironment-interactions and protein synthesis in CLL cells, likely to improve cellular fitness and to further drive disease progression.

Platelet deficiency, known as thrombocytopenia, can cause hemorrhage and is treated with platelet transfusions. We developed a system for the production of platelet precursor cells, megakaryocytes, from pluripotent stem cells. These cultures can be maintained for >100 days, implying culture renewal by megakaryocyte progenitors (MKPs). However, it is unclear whether the MKP state in vitro mirrors the state in vivo, and MKPs cannot be purified using conventional surface markers. We performed single-cell RNA sequencing throughout in vitro differentiation and mapped each state to its equivalent in vivo. This enabled the identification of five surface markers that reproducibly purify MKPs, allowing us insight into their transcriptional and epigenetic profiles. Last, we performed culture optimization, increasing MKP production. Together, this study has mapped parallels between the MKP states in vivo and in vitro and allowed the purification of MKPs, accelerating the progress of in vitro–derived transfusion products toward the clinic.

The transition from bulk to single-cell analyses refocused the computational challenges for high-throughput sequencing data-processing. The core of single-cell pipelines is partitioning cells and assigning cell-identities; extensive consequences derive from this step; generating robust and reproducible outputs is essential. From benchmarking established single-cell pipelines, we observed that clustering results critically depend on algorithmic choices (e.g. method, parameters) and technical details (e.g. random seeds). We present ClustAssess, a suite of tools for quantifying clustering robustness both within and across methods. The tools provide fine-grained information enabling (a) the detection of optimal number of clusters, (b) identification of regions of similarity (and divergence) across methods, (c) a data driven assessment of optimal parameter ranges. The aim is to assist practitioners in evaluating the robustness of cell-identity inference based on the partitioning, and provide information for choosing robust clustering methods and parameters. We illustrate its use on three case studies: a single-cell dataset of in-vivo hematopoietic stem and progenitors (10x Genomics scRNA-seq), in-vitro endoderm differentiation (SMART-seq), and multimodal in-vivo peripheral blood (10x RNA+ATAC). The additional checks offer novel viewpoints on clustering stability, and provide a framework for consistent decision-making on preprocessing, method choice, and parameters for clustering.