Cdc42 is a key regulator of B cell differentiation and is required for antiviral humoral immunity

The small Rho GTPase Cdc42, known to interact with Wiskott–Aldrich syndrome (WAS) protein, is an important regulator of actin remodeling. Here, we show that genetic ablation of Cdc42 exclusively in the B cell lineage is sufficient to render mice unable to mount antibody responses. Indeed Cdc42-deficient mice are incapable of forming germinal centers or generating plasma B cells upon either viral infection or immunization. Such severe immune deficiency is caused by multiple and profound B cell abnormalities, including early blocks during B cell development; impaired antigen-driven BCR signaling and actin remodeling; defective antigen presentation and in vivo interaction with T cells; and a severe B cell–intrinsic block in plasma cell differentiation. Thus, our study presents a new perspective on Cdc42 as key regulator of B cell physiology.B cells provide a critical line of defense from pathogenic infections through the production of highly specific antibodies. The initial stages of B cell development occur in the bone marrow, where hematopoietic stem cells undergo stepwise rearrangements of the genes encoding the B cell receptor (BCR) and changes in the expression of cell surface receptors (Hardy et al., 1991). Immature B cells egress the bone marrow and migrate to the spleen to complete their development, going through transitional stages. Mature follicular B cells then recirculate throughout the body in search for cognate antigen, continually entering secondary lymphoid organs, including the LNs and spleen. Specific recognition of antigen by the BCR provides the first signal required for B cell activation. Typically, a second signal is required for maximal activation and is provided by CD4⁺ helper T cells after the presentation of processed antigen on the B cell surface. These two signals in combination trigger the proliferation and differentiation of B cells, which go on to form antibody-secreting plasma cells and to establish germinal center responses for affinity maturation (Rajewsky, 1996).B cell activation in vivo is predominantly triggered by antigen on the surface of a presenting cell (Batista and Harwood, 2009). The prevalence of this mode of activation has brought about a reevaluation of the importance of the cytoskeleton, given that the recognition of tethered antigen requires considerable alteration in B cell morphology (Fleire et al., 2006). Antigen-induced BCR signaling leads to radical reorganization of the actin cytoskeleton resulting in the modification of the BCR dynamics at the cell surface (Hao and August, 2005; Treanor et al., 2010; Treanor et al., 2011). Moreover the binding of membrane-bound antigen to cognate BCR triggers a cascade of intracellular signaling events that induces actin-dependent spreading of the B cell across the antigen-containing surface (Weber et al., 2008; Sohn et al., 2008; Depoil et al., 2008). However the mediators that link BCR signaling with reorganization of the actin cytoskeleton are currently not well defined.Among actin regulators, the RhoGTPases are a highly conserved family that function as molecular switches by cycling between inactive GDP (guanosine diphosphate) and active GTP (guanosine triphosphate) bound states (Tybulewicz and Henderson, 2009). RhoGTPase activity is modulated by G-nucleotide exchange factors (GEF) that promote the formation of the GTP-bound state and binding to various effectors involved in actin reorganization. Conversely, GTPase-activating proteins (GAP) catalyze the hydrolysis of GTP and thereby switch off RhoGTPase activity. The importance of the RhoGTPases as a whole in the regulation of B cell responses is highlighted by the far-reaching consequences that impaired activity of several GEFs, such as Vav and DOCK8, has on humoral immune responses (Doody et al., 2001; Fujikawa et al., 2003; Randall et al., 2009; Zhang et al., 2009).The importance of Rho GTPases in B cell physiology has been well established. For example, RhoA has been shown to regulate BCR signaling by influencing inositol-3 phosphate synthesis and calcium signaling (Saci and Carpenter, 2005). Moreover, B cell–specific inactivation of both Rac1 and Rac2 leads to virtually complete absence of B cells (Walmsley et al., 2003), and inactivation of Rac1 results in defects in spreading in transitional cells (Brezski and Monroe, 2007). However, although the inactivation of Rac2 leads to defects in B cell adhesion and synapse formation, it is unclear whether these proteins are involved in actin-dependent spreading in mature B cells (Arana et al., 2008).Cdc42 has been little characterized in B cells, in spite of its proven chief role as an essential regulator of cell cycle (Johnson and Pringle, 1990), cell polarity (Etienne-Manneville, 2004), and actin cytoskeleton in other cellular systems. This is likely due, at least in part, to the reported mild phenotype of mice lacking Cdc42 in B cells (Guo et al., 2009) compared with the severe deficiencies observed in animals lacking Rac family members (Walmsley et al., 2003). However, the mild phenotype is somehow surprising given that Cdc42 directly or indirectly associates with Wiskott–Aldrich Syndrome Protein (WASp) and in complex with Arp2/3 regulates cytoskeleton remodeling (Symons et al., 1996; Aspenström et al., 1996; Kolluri et al., 1996). Importantly, mutations in WAS gene lead to a X-linked, recessive disease characterized by recurrent infections, abnormal lymphocyte function, as well as an increased risk for systemic autoimmunity (Derry et al., 1994; Sullivan et al., 1994). WASp deficient B cells play a primary role in driving autoimmunity (Becker-Herman et al., 2011). The Cdc42 effectors WASp and N-WASp have both been implicated the regulation of actin reorganization in response to BCR antigen engagement (Westerberg et al., 2012; Liu et al., 2013). Besides, expression of a dominant negative form of Cdc42 in B cells leads to alterations of the actin cytoskeleton (Westerberg et al., 2001). In addition, Cdc42 has been shown to play a role in the polarization and secretion of lysosomal protein involved in antigen extraction (Yuseff et al., 2011).Here, we used a strategy harnessing the mb1 promoter to generate mice with a selective and very effective deletion of Cdc42 in early B cell progenitors (Hobeika et al., 2006). Using this model, we demonstrated that Cdc42 plays an essential role in many aspects of B cell biology, including the formation of mature B cells and the establishment of antibody responses. We went on to dissect the underlying cause of the severe immunodeficiency of these mice and found that Cdc42-deficient B cells exhibit defects in BCR signaling and presentation of internalized antigen, leading to reduced B–T cell interactions and the absence of germinal center responses in vivo. Moreover, Cdc42-deficient B cells can normally proliferate and class switch when stimulated with CD40 or LPS, but they are completely impaired in their ability to differentiate into plasma cells. Together, these attributes render Cdc42-deficient mice unable to mount antibody responses after immunization with model antigen or viral infection, and highlight a fundamental role for this RhoGTPase in the regulation of B cell responses.