The Small Rho GTPases Rac1 and Rac2 Are Important for T-Cell Independent Antigen Responses and for Suppressing Switching to IgG2b in Mice

Abstract

The Rho GTPases Cdc42, Rac1, and Rac2 coordinate receptor signaling to cell adhesion, migration, and proliferation. Deletion of Rac1 and Rac2 early during B cell development leads to failure in B cell entry into the splenic white pulp. Here, we sought to understand the role of Rac1 and Rac2 in B cell functionality and during the humoral antibody response. To circumvent the migratory deficiency of B cells lacking both Rac1 and Rac2, we took the approach to inducibly delete Rac1 in Rac2−/− B cells in the spleen (Rac1BRac2−/− B cells). Rac1BRac2−/− mice had normal differentiation of splenic B cell populations, except for a reduction in marginal zone B cells. Rac1BRac2−/− B cells showed normal spreading response on antibody-coated layers, while both Rac2−/− and Rac1BRac2−/− B cells had reduced homotypic adhesion and decreased proliferative response when compared to wild-type B cells. Upon challenge with the T-cell-independent antigen TNP-conjugated lipopolysaccharide, Rac1BRac2−/− mice showed reduced antibody response. In contrast, in response to the T-cell-dependent antigen sheep red blood cells, Rac1BRac2−/− mice had increased serum titers of IgG1 and IgG2b. During in vitro Ig class switching, Rac1BRac2−/− B cells had elevated germline γ2b transcripts leading to increased Ig class switching to IgG2b. Our data suggest that Rac1 and Rac2 serve an important role in regulation of the B cell humoral immune response and in suppressing Ig class switching to IgG2b.

The Rac Activator DOCK2 Mediates Plasma Cell Differentiation and IgG Antibody Production

“…Finally, we have shown that CD19-Cre +/− Dock2 lox/lox conditional KO mice fail to mount antigen-specific IgG antibody upon immunization of OVA. This result is in marked contrast to a recent study showing that after treatment with tamoxifen to delete Rac1 in Rac2 –/– B cells, Mb1-Cre-ERT2 Rac1 lox/lox Rac2 –/– mice exhibited increased IgG1 and IgG2b antibody to a TD antigen ( 51 ). As DOCK2 was deleted early during B cell development in CD19-Cre +/− Dock2 lox/lox mice, B cell trafficking is also impaired in this model.…”

Congenital Defects in Actin Dynamics of Germinal Center B Cells

“…Defective entry of mature B cells into the white pulp makes it difficult to study the role of Rac1 and Rac2 in antigen-activated B cells. To circumvent this issue, Rac proteins were inducibly deleted by Tamoxifen in the mature B cell population (Rac1 B−ERT2 Rac2 −/− ) ( 66 ). The TI response to TNP-LPS of Rac1 B−ERT2 Rac2 −/− B cells is greatly compromised, with reduced level of antigen specific IgM and IgG3, whereas the TD response to TNP-SRBC in these mice seems comparable to wildtype mice, with a normal GC response and plasma cell output.…”

Arhgap25 Deficiency Leads to Decreased Numbers of Peripheral Blood B Cells and Defective Germinal Center Reactions

“…Prior to Ab staining, cells were blocked with 5 ng of rat IgG (14131; Sigma-Aldrich, St. Louis, MO). Cell surface Ags were stained with combinations of the following Abs: CD93-FITC (AA4.1; BioLegend, San Diego, CA), CD23-PE (B3B4; eBioscience), IgM-PerCP-Cy5.5 (R6-60.2; BD, Franklin Lakes, NJ), CD19-allophycocyanin (1D3; eBioscience), CD1d-Superbright 645 (1B1; eBioscience), CD3e-allophycocyanin (145-2C11; eBioscience), CD19-BV605 (6D5; BioLegend), IgD-FITC (11-26c [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26]; eBioscience), CD45R/B220-BV787 (RA3-6B2; BD), CD38-FITC (90; BioLegend), CD95-allophycocyanin-R700 (Jo2; BD), IgG1-PE-Cy7 (RMG1-1; BioLegend), IgM-BUV395 (II/ 41; BD), CD267-PE (eBio8F10-3; eBioscience), NP-PE (N-5070-1; Biosearch Technologies, Novato, CA), CD138-BV650 (281-2; BD), CD86-PE (GL-1; BioLegend), and CXCR4-allophycocyanin (L276F12; BioLegend). Cells were stained with the following viability dyes: SYTOX Blue Dead Cell Stain (S34857; Invitrogen, Carlsbad, CA), Zombie Red (423102; BioLegend), and Zombie Aqua (423109; BioLegend).…”

“…Rho family GTPases are critical for B cell development and activation (6)(7)(8)(9). Indeed, deletion of Rac1, Rac2, RhoA, or Cdc42 all result in significant B cell developmental defects (6,(10)(11)(12)(13)(14)(15)(16). In vivo, the activity of these GTPases is moderated by a multitude of GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs).…”