Rab8a is a small GTPase with a wide range of reported functions in different cell types, including vesicle recycling, vesicle traffic to cilia, cell ruffling, migration, neurite outgrowth, Toll-like receptor signalling and T cell receptor docking at the immune synapse. However, the role of Rab8a in B lymphocytes has not been described to date. Here, using a conditional B cell-specific Rab8a knockout mouse model, we investigate the role of Rab8a both in vivo and in vitro . Rab8a KO mice present enhanced antibody responses to both T-dependent and T-independent immunisations. Rab8a KO cells showed normal BCR trafficking and antigen processing and presentation but however, increased class-switch recombination. While the early BCR signalling responses, such as proximal kinase activation and calcium-flux, were normal, the signalling via AKT and ERK1/2 was decreased. We propose that the lack of Rab8a alters cellular signalling leading to enhanced antibody responses and increased class-switch recombination potentially via downmodulation of the PI3K/AKT/mTOR pathway.

In order to fulfil the special requirements of antigen-specific activation and communication with other immune cells, B lymphocytes require finely regulated endosomal vesicle trafficking. How the endosomal machinery is regulated in B cells remains largely unexplored. In our previous proximity proteomic screen, we identified the SNARE protein Vti1b as one of the strongest candidates getting accumulated to the sites of early BCR activation. In this report, we follow up on this finding and investigate the localisation and function of Vti1b in B cells. We found that GFP-fused Vti1b was concentrated at the Golgi complex, around the MTOC, as well as in the Rab7+ lysosomal vesicles in the cell periphery. Upon BCR activation with soluble antigen, Vti1b showed partial localization to the internalized antigen vesicles, especially in the periphery of the cell. Moreover, upon BCR activation using surface-bound antigen, Vti1b polarised to the immunological synapse, colocalising with the Golgi complex, and with lysosomes at actin foci. To test for a functional role of Vti1b in early B cell activation, we used primary B cells isolated from Vit1b-deficient mouse. However, we found no functional defects in BCR signalling, immunological synapse formation, or processing and presentation of the internalized antigen, suggesting that the loss of Vti1b in B cells could be compensated by its close homologue Vti1a or other SNAREs.
Copyright © 2022 Music, Tejeda-González, Cunha, Fischer von Mollard, Hernández-Pérez and Mattila.

We report three new cases of a germline heterozygous gain-of-function missense (p.(Met1141Lys)) mutation in the C2 domain of phospholipase C gamma 2 (PLCG2) associated with symptoms consistent with previously described auto-inflammation and phospholipase Cγ2 (PLCγ2)-associated antibody deficiency and immune dysregulation (APLAID) syndrome and pediatric common variable immunodeficiency (CVID). Functional evaluation showed platelet hyper-reactivity, increased B cell receptor-triggered calcium influx and ERK phosphorylation. Expression of the altered p.(Met1141Lys) variant in a PLCγ2-knockout DT40 cell line showed clearly enhanced BCR-triggered influx of external calcium when compared to control-transfected cells. Our results further expand the molecular basis of pediatric CVID and phenotypic spectrum of PLCγ2-related defects.

In cancer biology, the functional interpretation of genomic alterations is critical to achieve the promise of genomic profiling in the clinic. For chronic lymphocytic leukemia (CLL), a heterogeneous disease of B-lymphocytes maturing under constitutive B cell receptor (BCR) stimulation, the functional role of diverse clonal mutations remains largely unknown. Here, we demonstrate that alterations in BCR signaling dynamics underlie the progression of B cells toward malignancy. We reveal emergent dynamic features-bimodality, hypersensitivity, and hysteresis-in the BCR signaling pathway of primary CLL B cells. These signaling abnormalities in CLL quantitatively derive from BCR clustering and constitutive signaling with positive feedback reinforcement, as demonstrated through single-cell analysis of phospho-responses, computational modeling, and super-resolution imaging. Such dysregulated signaling segregates CLL patients by disease severity and clinical presentation. These findings provide a quantitative framework and methodology to assess complex and heterogeneous leukemia pathology and to inform therapeutic strategies in parallel with genomic profiling.
Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.

The aim was to determine the effect of the Bruton tyrosine kinase (Btk)-selective inhibitor PCI-32765, currently in Phase I/II studies in lymphoma trials, in arthritis and immune-complex (IC) based animal models and describe the underlying cellular mechanisms.
PCI-32765 was administered in a series of murine IC disease models including collagen-induced arthritis (CIA), collagen antibody-induced arthritis (CAIA), reversed passive anaphylactic reaction (RPA), and passive cutaneous anaphylaxis (PCA). Clinical and pathologic features characteristic of each model were examined following treatment. PCI-32765 was then examined in assays using immune cells relevant to the pathogenesis of arthritis, and where Btk is thought to play a functional role. These included proliferation and calcium mobilization in B cells, cytokine and chemokine production in monocytes/macrophages, degranulation of mast cells and its subsequent cytokine/chemokine production.
PCI-32765 dose-dependently and potently reversed arthritic inflammation in a therapeutic CIA model with an ED(50) of 2.6 mg/kg/day. PCI-32765 also prevented clinical arthritis in CAIA models. In both models, infiltration of monocytes and macrophages into the synovium was completely inhibited and importantly, the bone and cartilage integrity of the joints were preserved. PCI-32765 reduced inflammation in the Arthus and PCA assays. In vitro, PCI-32765 inhibited BCR-activated primary B cell proliferation (IC(50) = 8 nM). Following FcγR stimulation, PCI-32765 inhibited TNFα, IL-1β and IL-6 production in primary monocytes (IC(50) = 2.6, 0.5, 3.9 nM, respectively). Following FcεRI stimulation of cultured human mast cells, PCI-32765 inhibited release of histamine, PGD(2), TNF-α, IL-8 and MCP-1.
PCI-32765 is efficacious in CIA, and in IC models that do not depend upon autoantibody production from B cells. Thus PCI-32765 targets not only B lymphocytes but also monocytes, macrophages and mast cells, which are important Btk-expressing effector cells in arthritis.