Diacylglycerol kinase ζ promotes actin cytoskeleton remodeling and mechanical forces at the B cell immune synapse.

Diacylglycerol kinases (DGKs) limit antigen receptor signaling in immune cells by consuming the second messenger diacylglycerol (DAG) to generate phosphatidic acid (PA). Here, we showed that DGKζ promotes lymphocyte function-associated antigen 1 (LFA-1)-mediated adhesion and F-actin generation at the immune synapse of B cells with antigen-presenting cells (APCs), mostly in a PA-dependent manner. Measurement of single-cell mechanical force generation indicated that DGKζ-deficient B cells exerted lower forces at the immune synapse than did wild-type B cells. Nonmuscle myosin activation and translocation of the microtubule-organizing center (MTOC) to the immune synapse were also impaired in DGKζ-deficient B cells. These functional defects correlated with the decreased ability of B cells to present antigen and activate T cells in vitro. The in vivo germinal center response of DGKζ-deficient B cells was also reduced compared with that of wild-type B cells, indicating that loss of DGKζ in B cells impaired T cell help. Together, our data suggest that DGKζ shapes B cell responses by regulating actin remodeling, force generation, and antigen uptake-related events at the immune synapse. Hence, an appropriate balance in the amounts of DAG and PA is required for optimal B cell function.

Distinct Roles for Bruton's Tyrosine Kinase in B Cell Immune Synapse Formation.

Bruton's tyrosine kinase (Btk) has a key role in the signaling pathways of receptors essential for the B lymphocyte response. Given its implication in B cell-related immunodeficiencies, leukemias/lymphomas and autoimmunity, Btk is studied intensely and is a target for therapy. Here, using primary B cells from distinct mouse models and the pharmacological inhibitors ibrutinib and acalabrutinib, we report distinct roles for Btk in antigen-triggered immune synapse (IS) formation. Btk recruitment to the plasma membrane regulates the B cell ability to trigger IS formation as well as its appropriate molecular assembly; Btk shuttling/scaffold activities seem more relevant than the kinase function on that. Btk-kinase activity controls antigen accumulation at the IS through the PLCγ2/Ca2+ axis. Impaired Btk membrane-recruitment or kinase function likewise alters antigen-triggered microtubule-organizing center (MTOC) polarization to the IS, B cell activation and proliferation. Data also show that, for B cell function, IS architecture is as important as the quantity of antigen that accumulates at the synapse.

Diacylglycerol kinase ζ limits the polarized recruitment of diacylglycerol-enriched organelles to the immune synapse in T cells.

The antigen-induced formation of an immune synapse (IS) between T cells and antigen-presenting cells results in the rapid generation of the lipid second messenger diacylglycerol (DAG) in T cells. Diacylglycerol kinase ζ (DGKζ) converts DAG into phosphatidic acid (PA). Cytotoxic T lymphocytes (CTLs) from mice deficient in DGKζ have enhanced antiviral and antitumor activities, indicating that the amount of DAG controls the effectiveness of the T cell response. We characterized the second C1 domain of protein kinase Cθ (PKCθ), a DAG-binding protein that is specifically recruited to the IS, as a biological sensor to observe the generation of a DAG gradient during IS formation. In experiments with transgenic mouse CTLs expressing the OT-I T cell receptor (TCR), we showed that both strong and weak interactions between antigen and the TCR led to the rapid generation of DAG, whereas only strong interactions induced the movement of DAG-enriched organelles toward the IS. In DGKζ-deficient CTLs, antigen stimulation led to the enhanced accumulation of DAG-containing organelles at the IS; however, impaired activation of the PA effector PKCζ resulted in lack of reorientation of the microtubule-organizing center toward the IS, a process needed for effective T cell activation. Together, these data suggest that the activation of DGKζ downstream of antigen recognition provides a mechanism that ensures the activation of PA-dependent signaling as a direct result of the strength of TCR-dependent DAG mobilization.

Homeobox NKX2-3 promotes marginal-zone lymphomagenesis by activating B-cell receptor signalling and shaping lymphocyte dynamics.

NKX2 homeobox family proteins have a role in cancer development. Here we show that NKX2-3 is overexpressed in tumour cells from a subset of patients with marginal-zone lymphomas, but not with other B-cell malignancies. While Nkx2-3-deficient mice exhibit the absence of marginal-zone B cells, transgenic mice with expression of NKX2-3 in B cells show marginal-zone expansion that leads to the development of tumours, faithfully recapitulating the principal clinical and biological features of human marginal-zone lymphomas. NKX2-3 induces B-cell receptor signalling by phosphorylating Lyn/Syk kinases, which in turn activate multiple integrins (LFA-1, VLA-4), adhesion molecules (ICAM-1, MadCAM-1) and the chemokine receptor CXCR4. These molecules enhance migration, polarization and homing of B cells to splenic and extranodal tissues, eventually driving malignant transformation through triggering NF-κB and PI3K-AKT pathways. This study implicates oncogenic NKX2-3 in lymphomagenesis, and provides a valid experimental mouse model for studying the biology and therapy of human marginal-zone B-cell lymphomas.