During innate immune responses, myeloid differentiation primary response 88 (MyD88) functions as a critical signaling adaptor protein integrating stimuli from toll-like receptors (TLR) and the interleukin-1 receptor (IL-1R) family and translates them into specific cellular outcomes. In B cells, somatic mutations in MyD88 trigger oncogenic NF-κB signaling independent of receptor stimulation, which leads to the development of B-cell malignancies. However, the exact molecular mechanisms and downstream signaling targets remain unresolved. We established an inducible system to introduce MyD88 to lymphoma cell lines and performed transcriptomic analysis (RNA-seq) to identify genes differentially expressed by MyD88 bearing the L265P oncogenic mutation. We show that MyD88^L265P activates NF-κB signaling and upregulates genes that might contribute to lymphomagenesis, including CD44, LGALS3 (coding Galectin-3), NFKBIZ (coding IkBƺ), and BATF. Moreover, we demonstrate that CD44 can serve as a marker of the activated B-cell (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) and that CD44 expression is correlated with overall survival in DLBCL patients. Our results shed new light on the downstream outcomes of MyD88^L265P oncogenic signaling that might be involved in cellular transformation and provide novel therapeutical targets.

The adaptive immune response critically hinges on the functionality of T cell receptors, governed by complex molecular mechanisms, including ubiquitination. In this study, we delved into the role of in T cell immunity, focusing on T cell–B cell conjugate formation and T cell activation. Using a CRISPR-Cas9 screening approach targeting deubiquitinases genes in Jurkat T cells, we identified BAP1 as a key positive regulator of T cell-B cell conjugate formation. Subsequent investigations into BAP1 knockout cells revealed impaired T cell activation, evidenced by decreased MAPK and NF-kB signaling pathways and reduced CD69 expression upon T cell receptor stimulation. Flow cytometry and qPCR analyses demonstrated that BAP1 deficiency leads to decreased surface expression of T cell receptor complex components and reduced mRNA levels of the co-stimulatory molecule CD28. Notably, the observed phenotypes associated with BAP1 knockout are specific to T cells and fully dependent on BAP1 catalytic activity. In-depth RNA-seq and mass spectrometry analyses further revealed that BAP1 deficiency induces broad mRNA and protein expression changes. Overall, our findings elucidate the vital role of BAP1 in T cell biology, especially in T cell-B cell conjugate formation and T cell activation, offering new insights and directions for future research in immune regulation.

Lysosomes function as a primary site for catabolism and cellular signaling. These organelles digest a variety of substrates received through endocytosis, secretion and autophagy with the help of resident acid hydrolases. Lysosomal enzymes are folded in the endoplasmic reticulum (ER) and trafficked to lysosomes via Golgi and endocytic route. The inability of hydrolase trafficking due to mutations or mutations in its receptor or cofactor leads to cargo accumulation (storage) in lysosomes, resulting in lysosome storage disorder (LSD). In Gaucher’s disease (GD), the lysosomes accumulate glucosylceramide due to a lack of β-glucocerebrosidase (β-GC) activity that causes lysosome enlargement/dysfunction. We hypothesize that improving the trafficking of mutant β-GC to lysosomes may delay the progression of GD. RNAi screen using high throughput based lysosomal enzyme activity assay followed by reporter trafficking assay utilizing β-GC-mCherry lead to the identification of nine potential phosphatases. Depletion of these phosphatases in HeLa cells enhanced the β-GC activity by increasing the folding and trafficking of Gaucher’s mutants to the lysosomes. Consistently, the lysosomes in primary fibroblasts from GD patients restored their function upon the knockdown of these phosphatases. Thus, these studies provide evidence that altering phosphatome activity possibly delays the GD and forms an alternative therapeutic strategy for this genetic disease. Key points: Phosphatome RNAi screen identified both activators and inhibitors of cellular glucocerebrosidase activity Depletion of selective phosphatases in HeLa cells improved the folding and trafficking of mutant β-glucocerebrosidase to lysosomes Knockdown of selective phosphatases restored the low basal β-glucocerebrosidase activity to that of wild-type in primary cells derived from Gaucher’s disease patients Depletion of selective phosphatases displayed variable β-GC activity in neuropathic and non-neuropathic Gaucher’s disease patient cells