Mechanotransduction governs CD40 function and underlies X-linked Hyper IgM syndrome.

B cell maturation in germinal centers (GCs) depends on cognate interactions between the T and B cells. Upon interaction with CD40 ligand (CD40L) on T cells, CD40 delivers co-stimulatory signals alongside B cell antigen receptor (BCR) signaling to regulate affinity maturation and antibody class-switch during GC reaction. Mutations in CD40L disrupt interactions with CD40, which lead to abnormal antibody responses in immune deficiencies known as X-linked Hyper IgM syndrome (X-HIgM). Assuming that physical interactions between highly mobile T and B cells generate mechanical forces on CD40-CD40L bonds, we set out to study the B cell mechanobiology mediated by CD40-CD40L interaction. Using a suite of biophysical assays we find that CD40 forms catch bond with CD40L where the bond lasts longer at larger forces, B cells exert tension on CD40-CD40L bonds, and force enhances CD40 signaling and antibody class-switch. Significantly, X-HIgM CD40L mutations impair catch bond formation, suppress endogenous tension, and reduce force-enhanced CD40 signaling, leading to deficiencies in antibody class switch. Our findings highlight the critical role of mechanotransduction in CD40 function and provide insights into the molecular mechanisms underlying X-HIgM syndrome.

Dysregulated PI3K Signaling in B Cells of CVID Patients.

The altered wiring of signaling pathways downstream of antigen receptors of T and B cells contributes to the dysregulation of the adaptive immune system, potentially causing immunodeficiency and autoimmunity. In humans, the investigation of such complex systems benefits from nature's experiments in patients with genetically defined primary immunodeficiencies. Disturbed B-cell receptor (BCR) signaling in a subgroup of common variable immunodeficiency (CVID) patients with immune dysregulation and expanded T-bethighCD21low B cells in peripheral blood has been previously reported. Here, we investigate PI3K signaling and its targets as crucial regulators of survival, proliferation and metabolism by intracellular flow cytometry, imaging flow cytometry and RNAseq. We observed increased basal but disturbed BCR-induced PI3K signaling, especially in T-bethighCD21low B cells from CVID patients, translating into impaired activation of crucial downstream molecules and affecting proliferation, survival and the metabolic profile. In contrast to CVID, increased basal activity of PI3K in patients with a gain-of-function mutation in PIK3CD and activated PI3K delta syndrome (APDS) did not result in impaired BCR-induced AKT-mTOR-S6 phosphorylation, highlighting that signaling defects in B cells in CVID and APDS patients are fundamentally different and that assessing responses to BCR stimulation is an appropriate confirmative diagnostic test for APDS. The active PI3K signaling in vivo may render autoreactive T-bethighCD21low B cells in CVID at the same time to be more sensitive to mTOR or PI3K inhibition.

A fluorescent probe for STED microscopy to study NIP-specific B cells.

We have developed a series of monovalent fluorophore-conjugated affinity probes based on the hapten 3-nitro-4-hydroxy-5-iodophenylacetyl (NIP), which is widely used as a model antigen to study B lymphocytes and the functional principles of B cell antigen receptors (BCRs). We successfully used them in flow-cytometry, confocal and super-resolution microscopy techniques.

Vav family proteins constitute disparate branching points for distinct BCR signaling pathways.

Antigen recognition by B-cell antigen receptors (BCRs) activates distinct intracellular signaling pathways that control the differentiation fate of activated B lymphocytes. BCR-proximal signaling enzymes comprise protein tyrosine kinases, phosphatases, and plasma membrane lipid-modifying enzymes, whose function is furthermore coordinated by catalytically inert adaptor proteins. Here, we show that an additional class of enzymatic activity provided by guanine-nucleotide exchange factors (GEFs) of the Vav family controls BCR-proximal Ca2+ mobilization, cytoskeletal actin reorganization, and activation of the PI3 kinase/Akt pathway. Whereas Vav1 and Vav3 supported all of those signaling processes to different extents in a human B-cell model system, Vav2 facilitated Actin remodeling, and activation of Akt but did not promote Ca2+ signaling. On BCR activation, Vav1 was directly recruited to the phosphorylated BCR and to the central adaptor protein SLP65 via its Src homology 2 domain. Pharmacological inhibition or genetic inactivation of the substrates of Vav GEFs, small G proteins of the Rho/Rac family, impaired BCR-induced Ca2+ mobilization, probably because phospholipase Cγ2 requires activated Rac proteins for optimal activity. Our findings show that Vav family members are key relays of the BCR signalosome that differentially control distinct signaling pathways both in a catalysis-dependent and -independent manner.