Lars Nitschke

Compensation between Vav-1 and Vav-2 in B cell development and antigen receptor signaling

Vav-1 and Vav-2 are closely related Dbl-homology GTP exchange factors (GEFs) for Rho GTPases. Mutation of Vav-1 disrupts T cell development and T cell antigen receptor–induced activation, but has comparatively little effect on B cells. We found that combined deletion of both Vav-1 and Vav-2 in mice resulted in a marked reduction in mature B lymphocyte numbers. Vav-1−/−Vav-2−/− B cells were unresponsive to B cell antigen receptor (BCR)-driven proliferation in vitro and to thymus-indepen-dent antigen in vivo. BCR-stimulated intracellular calcium mobilization was greatly impaired in Vav-1−/−Vav-2−/− B cells. These findings establish a role for Vav-2 in BCR calcium signaling and reveal that the Vav family of GEFs is critical to B cell development and function.

Reduction of marginal zone B cells in CD22-deficient mice.

CD22 is a B cell‐specific member of the immunoglobulin superfamily and binds to sialic acid. CD22 inhibits B cell receptor signaling. Mice deficient for CD22 show a largely normal B cell development. Here, we have performed a detailed analysis of the splenic B cell population and found that the subset of marginal zone (MZ) B cells was selectively reduced in CD22‐deficient mice. CD22‐deficient mice showed a lack of TNP‐ficoll capturing cells in the MZ and a reduced response to TNP‐ficoll, particularly when the antigen was applied intravenously. CD22‐deficient B cells showed both enhanced motility as well as enhanced chemotaxis to certain chemokines. The altered chemokine responsiveness or the higher signaling capacity of CD22‐deficient B cells may lead to the compromised MZ B cell compartment, as both processes have previously been shown to affect MZ composition.

Siglec-G is a B1 cell-inhibitory receptor that controls expansion and calcium signaling of the B1 cell population.

B1 cells are an important cell population for the production of natural antibodies and for antibacterial immunoglobulin responses. Here we identified the mouse protein Siglec-G as a B1 cell inhibitory receptor. Siglec-G was expressed in a B cell–restricted way, with large amounts present in B1 cells. When overexpressed, Siglec-G inhibited B cell receptor–mediated calcium signaling. Siglec-G-deficient mice had massive expansion of the B1a cell population, which began early in development and was B cell intrinsic. Siglec-G-deficient mice had higher titers of natural IgM antibodies but not a higher penetrance of IgG autoantibodies. Siglec-G-deficient B1 cells showed a strongly enhanced calcium signaling. Our results demonstrate that Siglec-G-dependent negative regulation exists in B1 cells, which may explain the naturally muted signaling response of B1 cells.

CD22 and Siglec-G: B-cell inhibitory receptors with distinct functions.

Summary:  Siglecs (sialic acid‐binding immunoglobulin‐like lectins) are sialic acid‐binding proteins, which are expressed on many cell types of the immune system. B cells express two members of the Siglec family, CD22 (Siglec‐2) and Siglec‐G, both of which have been shown to inhibit B‐cell signaling. CD22 recruits the tyrosine phosphatase Src homology 2 domain‐containing phosphatase 1 (SHP‐1) to immunoreceptor tyrosine‐based inhibitory motifs (ITIMs) and inhibits B‐cell receptor (BCR)‐induced Ca2+ signaling on normal B cells. CD22 interacts specifically with ligands carrying α2–6‐linked sialic acids. Interaction with these ligands in cis regulates the association of CD22 with the BCR and thereby modulates the inhibitory function of CD22. Interaction of CD22 to ligands in trans can regulate both B‐cell migration as well as the BCR signaling threshold. Siglec‐G is a recently identified protein with an inhibitory function restricted to a B‐cell subset, the B1 cells. Siglec‐G inhibits Ca2+ signaling specifically in these cells. In addition, it controls the cellular expansion and antibody secretion of B1 cells. Thus, both Siglecs modulate BCR signaling on different B‐cell populations in a mutually exclusive fashion.

IgG1 B cell receptor signaling is inhibited by CD22 and promotes the development of B cells whose survival is less dependent on Igα/β

We describe a mouse strain in which B cell development relies either on the expression of membrane-bound immunoglobulin (Ig) γ1 or μ heavy chains. Progenitor cells expressing γ1 chains from the beginning generate a peripheral B cell compartment of normal size with all subsets, but a partial block is seen at the pro– to pre–B cell transition. Accordingly, γ1-driven B cell development is disfavored in competition with developing B cells expressing a wild-type (WT) IgH locus. However, the mutant B cells display a long half-life and accumulate in the mature B cell compartment, and even though partial truncation of the Igα cytoplasmic tail compromises their development, it does not affect their maintenance, as it does in WT cells. IgG1-expressing B cells showed an enhanced Ca2+ response upon B cell receptor cross-linking, which was not due to a lack of inhibition by CD22. The enhanced Ca2+ response was also observed in mature B cells that had been switched from IgM to IgG1 expression in vivo. Collectively, these results suggest that the γ1 chain can exert a unique signaling function that can partially replace that of the Igα/β heterodimer in B cell maintenance and may contribute to memory B cell physiology.

CD22 x Siglec-G double-deficient mice have massively increased B1 cell numbers and develop systemic autoimmunity.

CD22 and Siglec-G are inhibitory coreceptors for BCR-mediated signaling. Although CD22-deficient mice show increased calcium signaling in their conventional B2 cells and a quite normal B cell maturation, Siglec-G–deficient mice have increased calcium mobilization just in B1 cells and show a large expansion of the B1 cell population. Neither CD22-deficient, nor Siglec-G–deficient mice on a pure C57BL/6 or BALB/c background, respectively, develop autoimmunity. Using Siglec-G × CD22 double-deficient mice, we addressed whether Siglec-G and CD22 have redundant functions. Siglec-G × CD22 double-deficient mice show elevated calcium responses in both B1 cells and B2 cells, increased serum IgM levels and an enlarged population of B1 cells. The enlargement of B1 cell numbers is even higher than in Siglecg−/− mice. This expansion seems to happen at the expense of B2 cells, which are reduced in absolute cell numbers, but show an activated phenotype. Furthermore, Siglec-G × CD22 double-deficient mice show a diminished immune response to both thymus-dependent and thymus-independent type II Ags. In contrast, B cells from Siglec-G × CD22 double-deficient mice exhibit a hyperproliferative response to stimulation with several TLR ligands. Aged Siglec-G × CD22 double-deficient mice spontaneously develop anti-DNA and antinuclear autoantibodies. These resulted in a moderate form of immune complex glomerulonephritis. These results show that Siglec-G and CD22 have partly compensatory functions and together are crucial in maintaining the B cell tolerance.

Forced usage of positively charged amino acids in immunoglobulin CDR-H3 impairs B cell development and antibody production

Tyrosine and glycine constitute 40% of complementarity determining region 3 of the immunoglobulin heavy chain (CDR-H3), the center of the classic antigen-binding site. To assess the role of DH RF1-encoded tyrosine and glycine in regulating CDR-H3 content and potentially influencing B cell function, we created mice limited to a single DH encoding asparagine, histidine, and arginines in RF1. Tyrosine and glycine content in CDR-H3 was halved. Bone marrow and spleen mature B cell and peritoneal cavity B-1 cell numbers were also halved, whereas marginal zone B cell numbers increased. Serum immunoglobulin G subclass levels and antibody titers to T-dependent and T-independent antigens all declined. Thus, violation of the conserved preference for tyrosine and glycine in DH RF1 alters CDR-H3 content and impairs B cell development and antibody production.

NFATc1 affects mouse splenic B cell function by controlling the calcineurin--NFAT signaling network.

Mouse B cells lacking NFATc1 exhibit defective proliferation, survival, isotype class switching, cytokine production, and T cell help.

Regulation of B Cell Functions by the Sialic Acid-Binding Receptors Siglec-G and CD22

B cell antigen receptor (BCR) engagement can lead to many different physiologic outcomes. To achieve an appropriate response, the BCR signal is interpreted in the context of other stimuli and several additional receptors on the B cell surface participate in the modulation of the signal. Two members of the Siglec (sialic acid-binding immunoglobulin-like lectin) family, CD22 and Siglec-G have been shown to inhibit the BCR signal. Recent findings indicate that the ability of these two receptors to bind sialic acids might be important to induce tolerance to self-antigens. Sialylated glycans are usually absent on microbes but abundant in higher vertebrates and might therefore provide an important tolerogenic signal. Since the expression of the specific ligands for Siglec-G and CD22 is tightly regulated and since Siglecs are not only able to bind their ligands in trans but also on the same cell surface this might provide additional mechanisms to control the BCR signal. Although both Siglec-G and CD22 are expressed on B cells and are able to inhibit BCR mediated signaling, they also show unique biological functions. While CD22 is the dominant regulator of calcium signaling on conventional B2 cells and also seems to play a role on marginal zone B cells, Siglec-G exerts its function mainly on B1 cells and influences their lifespan and antibody production. Both Siglec-G and CD22 have also recently been linked to toll-like receptor signaling and may provide a link in the regulation of the adaptive and innate immune response of B cells.

B Cell–specific Transgenic Expression of Bcl2 Rescues Early B Lymphopoiesis but Not B Cell Responses in BOB.1/OBF.1-deficient Mice

Mice deficient for the transcriptional coactivator BOB.1/OBF.1 show several defects in B cell differentiation. Numbers of immature transitional B cells in the bone marrow are reduced and fewer B cells reach the periphery. Furthermore, germinal center B cells are absent and marginal zone (MZ) B lymphocytes are markedly reduced. Increased levels of B cell apoptosis in these mice prompted us to analyze expression and function of antiapoptotic proteins. Bcl2 expression is strongly reduced in BOB.1/OBF.1-deficient pre–B cells. When BOB.1/OBF.1-deficient mice were crossed with Bcl2-transgenic mice, B cell development in the bone marrow and numbers of B cells in peripheral lymphoid organs were normalized. However, neither germinal center B cells nor MZ B cells were rescued. Additionally, Bcl2 did not rescue the defects in signaling and affinity maturation found in BOB.1/OBF.1-deficient mice. Interestingly, Bcl2-transgenic mice by themselves show an MZ B cell defect. Virtually no functional MZ B cells were detected in these mice. In contrast, mice deficient for Bcl2 show a relative increase in MZ B cell numbers, indicating a previously undetected function of Bcl2 for this B cell compartment.