Gene dose-dependent maturation and receptor editing of B cells expressing immunoglobulin (Ig)G1 or IgM/IgG1 tail antigen receptors

Conserved differences between the transmembrane and cytoplasmic domains of membrane immunoglobulin (Ig)M and IgG may alter the function of antigen receptors on naive versus memory B cells. Here, we compare the ability of these domains to signal B cell allelic exclusion and maturation in transgenic mice. A lysozyme-binding antibody was expressed in parallel sets of mice as IgM, IgG1, or a chimeric receptor with IgM extracellular domains and transmembrane/cytoplasmic domains of IgG1. Like IgM, the IgG1 or chimeric IgM/G receptors triggered heavy chain allelic exclusion and supported development of mature CD21(+) B cells. Many of the IgG or IgM/G B cells became CD21(high) and downregulated their IgG and IgM/G receptors spontaneously, resembling memory B cells and B cells with mutations that exaggerate B cell antigen receptor signaling. Unlike IgM-transgenic mice, "edited" B cells that carry non-hen egg lysozyme binding receptors preferentially accumulated in IgG and IgM/G mice. This was most extreme in lines with the highest transgene copy number and diminished in variant offspring with fewer copies. The sensitivity of B cell maturation to transgene copy number conferred by the IgG transmembrane and cytoplasmic domains may explain the diverse phenotypes found in other IgG-transgenic mouse strains and may reflect exaggerated signaling.     

Ig beta tyrosine residues contribute to the control of B cell receptor signaling by regulating receptor internalization

Immunoglobulin (Ig)alpha and Igbeta initiate B cell receptor (BCR) signaling through immune receptor tyrosine activation motifs (ITAMs) that are targets of SH2 domain-containing kinases. To examine the function of Igbeta ITAM tyrosine resides in mature B cells in vivo, we exchanged these residues for alanine by gene targeting (Igbeta(AA)). Mutant mice showed normal development of all B cell subtypes with the exception of B1 cells that were reduced by fivefold. However, primary B cells purified from Igbeta(AA) mice showed significantly decreased steady-state and ligand-mediated BCR internalization and higher levels of cell surface IgM and IgD. BCR cross-linking resulted in decreased Src and Syk activation but paradoxically enhanced and prolonged BCR signaling, as measured by cellular tyrosine phosphorylation, Ca(++) flux, AKT, and ERK activation. In addition, B cells with the ITAM mutant receptor showed an enhanced response to a T-independent antigen. Thus, Igbeta ITAM tyrosines help set BCR signaling threshold by regulating receptor internalization.     

B cell receptor isotypes differentially associate with cell signaling,kinetics, and outcome in chronic lymphocytic leukemia

In chronic lymphocytic leukemia (CLL), the B cell receptor (BCR) plays a critical role in disease development and progression, as indicated by the therapeutic efficacy of drugs blocking BCR signaling. However, the mechanism(s) underlying BCR responsiveness are not completely defined. Selective engagement of membrane IgM or IgD on CLL cells, each coexpressed by more than 90% of cases, leads to distinct signaling events. Since both IgM and IgD carry the same antigen-binding domains, the divergent actions of the receptors are attributed to differences in immunoglobulin (Ig) structure or the outcome of signal transduction. We showed that IgM, not IgD, level and organization associated with CLL-cell birth rate and the type and consequences of BCR signaling in humans and mice. The latter IgM-driven effects were abrogated when BCR signaling was inhibited. Collectively, these studies demonstrated a critical, selective role for IgM in BCR signaling and B cell fate decisions, possibly opening new avenues for CLL therapy.     

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

We describe a mouse strain in which B cell development relies either on the expression of membrane-bound immunoglobulin (Ig) gamma1 or mu heavy chains. Progenitor cells expressing gamma1 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, gamma1-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 Ig alpha cytoplasmic tail compromises their development, it does not affect their maintenance, as it does in WT cells. IgG1-expressing B cells showed an enhanced Ca(2+) response upon B cell receptor cross-linking, which was not due to a lack of inhibition by CD22. The enhanced Ca(2+) 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 gamma1 chain can exert a unique signaling function that can partially replace that of the Ig alpha/beta heterodimer in B cell maintenance and may contribute to memory B cell physiology.     

Negative selection of human germinal center B cells by prolonged BCR cross-linking

The antigen receptors on T and B lymphocytes can transduce both agonist and antagonist signals leading either to activation/survival or anergy/death. The outcome of B lymphocyte antigen receptor (BCR) triggering depends upon multiple parameters which include (a) antigen concentration and valency, (b) duration of BCR occupancy, (c) receptor affinity, and (d) B cell differentiation stages. Herein, using anti- immunoglobulin kappa and lambda light chain antibodies, we analyzed the response of human naive, germinal center (GC) or memory B cells to BCR cross-linking regardless of heavy chain Ig isotype or intrinsic BCR specificity. We show that after CD40-activation, anti-BCR (kappa + gamma) can elicit an intracellular calcium flux on both GC and non-GC cells. However, prolonged BCR cross-linking induces death of CD40- activated GC B cells but enhances proliferation of naive or memory cells. Anti-kappa antibody only kills kappa + GC B cells without affecting surrounding gamma + GC B cells, thus demonstrating that BCR- mediated killing of GC B lymphocytes is a direct effect that does not involve a paracrine mechanism. BCR-mediated killing of CD40-activated GC B cells could be partially antagonized by the addition of IL-4. Moreover, in the presence of IL-4, prestimulation through CD40 could prevent subsequent anti-Ig-mediated cell death, suggesting a specific role of this combination in selection of GC B cells. This report provides evidence that in human, susceptibility to BCR killing is regulated along peripheral B cell differentiation pathway.     

CD5 maintains tolerance in anergic B cells

Clonal anergy of autoreactive B cells is a key mechanism regulating tolerance. Here, we show that anergic B cells express significant surface levels of CD5, a molecule normally found on T cells and a subset of B-1 cells. Breeding of the hen egg lysozyme (HEL) transgenic model for B cell anergy onto the CD5 null background resulted in a spontaneous loss of B cell tolerance in vivo. Evidence for this included elevated levels of anti-HEL immunoglobulin M (IgM) antibodies in the serum of CD5(-/-) mice transgenic for both an HEL-specific B cell receptor (BCR) and soluble lysozyme. "Anergic" B cells lacking CD5 also showed enhanced proliferative responses in vitro and elevated intracellular Ca(2+) levels at rest and after IgM cross-linking. These data support the hypothesis that CD5 negatively regulates Ig receptor signaling in anergic B cells and functions to inhibit autoimmune B cell responses.     

Attenuation of HIV-associated human B cell exhaustion by siRNA downregulation of inhibitory receptors

Chronic immune activation in HIV-infected individuals leads to accumulation of exhausted tissue-like memory B cells. Exhausted lymphocytes display increased expression of multiple inhibitory receptors, which may contribute to the inefficiency of HIV-specific antibody responses. Here, we show that downregulation of B cell inhibitory receptors in primary human B cells led to increased tissue-like memory B cell proliferation and responsiveness against HIV. In human B cells, siRNA knockdown of 9 known and putative B cell inhibitory receptors led to enhanced B cell receptor-mediated (BCR-mediated) proliferation of tissue-like memory but not other B cell subpopulations. The strongest effects were observed with the putative inhibitory receptors Fc receptor-like-4 (FCRL4) and sialic acid-binding Ig-like lectin 6 (Siglec-6). Inhibitory receptor downregulation also led to increased levels of HIV-specific antibody-secreting cells and B cell-associated chemokines and cytokines. The absence of known ligands for FCRL4 and Siglec-6 suggests these receptors may regulate BCR signaling through their own constitutive or tonic signaling. Furthermore, the extent of FCLR4 knockdown effects on BCR-mediated proliferation varied depending on the costimulatory ligand, suggesting that inhibitory receptors may engage specific pathways in inhibiting B cell proliferation. These findings on HIV-associated B cell exhaustion define potential targets for reversing the deleterious effect of inhibitory receptors on immune responses against persistent viral infections.     

Signal transduction by immunoglobulin is mediated through Ig alpha and Ig beta

Immunoglobulin (Ig) antigen receptors are composed of a noncovalently- associated complex of Ig and two other proteins, Ig alpha and Ig beta. The cytoplasmic domain of both of these Ig associated proteins contains a consensus sequence that is shared with the signaling proteins of the T cell and Fc receptor. To test the idea that Ig alpha-Ig beta heterodimers are the signaling components of the Ig receptor, we have studied Ig mutations that interfere with signal transduction. We find that specific mutations in the transmembrane domain of Ig that inactivate Ca2+ and phosphorylation responses also uncouple IgM from Ig alpha-Ig beta. These results define amino acid residues that are essential for the assembly of the Ig receptor. Further, receptor activity can be fully reconstituted in Ca2+ flux and phosphorylation assays by fusing the cytoplasmic domain of Ig alpha with the mutant Igs. In contrast, fusion of the cytoplasmic domain of Ig beta to the inactive Ig reconstitutes only Ca2+ responses. Thus, Ig alpha and Ig beta are both necessary and sufficient to mediate signal transduction by the Ig receptor in B cells. In addition, our results suggest that Ig alpha and Ig beta can activate different signaling pathways.     

Protective T cell-independent antiviral antibody responses are dependent on complement.

Complement is part of the innate immune system and one of the first lines of host defense against infections. Its importance was evaluated in this study in virus infections in mice deficient either in soluble complement factors (C3(-/-), C4(-/-)) or in the complement signaling complex (complement receptor [CR]2(-/-), CD19(-/-)). The induction of the initial T cell-independent neutralizing immunoglobulin (Ig)M antibody response to vesicular stomatitis virus (VSV), poliomyelitis virus, and recombinant vaccinia virus depended on efficient antigen trapping by CR3 and -4-expressing macrophages of the splenic marginal zone. Neutralizing IgM and IgG antibody responses were largely independent of CR2-mediated stimulation of B cells when mice were infected with live virus. In contrast, immunizations with nonreplicating antigens revealed an important role of B cell stimulation via CR2 in the switch to IgG. The complement cascade was activated after infection with VSV via the classical pathway, and active complement cleavage products augmented the effector function of neutralizing IgM and IgG antibodies to VSV by a factor of 10-100. Absence of the early neutralizing antibody responses, together with the reduced efficiency of neutralizing IgM in C3(-/-) mice, led to a drastically enhanced susceptibility to disease after infection with VSV.     

B cell intrinsic TLR signals amplify but are not required for humoral immunity

Although innate signals driven by Toll-like receptors (TLRs) play a crucial role in T-dependent immune responses and serological memory, the precise cellular and time-dependent requirements for such signals remain poorly defined. To directly address the role for B cell–intrinsic TLR signals in these events, we compared the TLR response profile of germinal center (GC) versus naive mature B cell subsets. TLR responsiveness was markedly up-regulated during the GC reaction, and this change correlated with altered expression of the key adaptors MyD88, Mal, and IRAK-M. To assess the role for B cell–intrinsic signals in vivo, we transferred MyD88 wild-type or knockout B cells into B cell–deficient μMT mice and immunized recipient animals with 4-hydroxy-3-nitrophenylacetyl (NP) chicken gamma globulin. All recipients exhibited similar increases in NP-specific antibody titers during primary, secondary, and long-term memory responses. The addition of lipopolysaccharide to the immunogen enhanced B cell-intrinsic, MyD88-dependent NP-specific immunoglobulin (Ig)M production, whereas NP-specific IgG increased independently of TLR signaling in B cells. Our data demonstrate that B cell–intrinsic TLR responses are up-regulated during the GC reaction, and that this change significantly promotes antigen-specific IgM production in association with TLR ligands. However, B cell–intrinsic TLR signals are not required for antibody production or maintenance.     

Functional reconstitution of an immunoglobulin antigen receptor in T cells

Humoral immune responses are initiated by binding of antigen to the immunoglobulins (Igs) on the plasma membrane of B lymphocytes. On the cell surface, Ig forms a complex with several other proteins, two of which, MB-1 and B29, have been implicated in receptor assembly. We have reconstituted Ig receptor function in T lymphocytes by transfection of cloned receptor components. We found that efficient transport of IgM to the surface of T cells required coexpression of B29. Furthermore, IgM and B29 alone were sufficient to reconstitute antigen-specific signal transduction by Ig in the transfected T cells. Crosslinking of IgM with either antireceptor antibodies or antigen induced a calcium flux, phosphoinositol turnover, and interleukin secretion in T cells. These experiments establish a requirement for B29 in Ig receptor function, and suggest that the signaling apparatus of T and B cells is structurally homologous.     

Ia-mediated signal transduction leads to proliferation of primed B lymphocytes

One of the most controversial questions in immunology is the molecular basis by which Th lymphocytes deliver activating signals to quiescent B lymphocytes during T cell-dependent immune responses. Recent studies suggest that T cell-dependent activation of quiescent B lymphocytes may involve signaling mediated by direct T helper cell-B cell contact. Since B cell membrane-associated MHC-encoded class II molecules (Ia) must be recognized by Th lymphocytes for generation of T cell-dependent humoral immune responses, they are obvious candidates for receptors of this signal. Here we report that stimulation of quiescent murine B cells with IL-4 and antibodies against the B cell antigen receptor for 12-16 h primes cells to proliferate in response to immobilized mIa binding ligands. In the presence of additional lymphokines, these B cells differentiate to secrete Ig of IgM and IgG classes. These results suggest that Ia molecules are receptors for direct, T helper cell-B cell contact mediated signaling that results in B cell proliferation.     

Essential role of membrane cholesterol in accelerated BCR internalization and uncoupling from NF-kappa B in B cell clonal anergy

Divergent hypotheses exist to explain how signaling by the B cell receptor (BCR) is initiated after antigen binding and how it is qualitatively altered in anergic B cells to selectively uncouple from nuclear factor kappaB and c-Jun N-terminal kinase pathways while continuing to activate extracellular signal-regulated kinase and calcium-nuclear factor of activated T cell pathways. Here we find that BCRs on anergic cells are endocytosed at a very enhanced rate upon binding antigen, resulting in a large steady-state pool of intracellularly sequestered receptors that appear to be continuously cycling between surface and intracellular compartments. This endocytic mechanism is exquisitely sensitive to the lowering of plasma membrane cholesterol by methyl-beta-cyclodextrin, and, when blocked in this way, the sequestered BCRs return to the cell surface and RelA nuclear accumulation is stimulated. In contrast, when plasma membrane cholesterol is lowered and GM1 sphingolipid markers of membrane rafts are depleted in naive B cells, this does not diminish BCR signaling to calcium or RelA. These results provide a possible explanation for the signaling changes in clonal anergy and indicate that a chief function of membrane cholesterol in B cells is not to initiate BCR signaling, but instead to terminate a subset of signals by rapid receptor internalization.     

PLC-γ2 is essential for formation and maintenance of memory B cells

Resting antigen-experienced memory B cells are thought to be responsible for the more rapid and robust antibody responses after antigen reencounter, which are the hallmark of memory humoral responses. The molecular basis for the development and survival of memory B cells remains largely unknown. We report that phospholipase C (PLC) γ2 is required for efficient formation of germinal center (GC) and memory B cells. Moreover, memory B cell homeostasis is severely hampered by inducible loss of PLC-γ2. Accordingly, mice with a conditional deletion of PLC-γ2 in post-GC B cells had an almost complete abrogation of the secondary antibody response. Collectively, our data suggest that PLC-γ2 conveys a survival signal to GC and memory B cells and that this signal is required for a productive secondary immune response.Humoral memory is characterized by recall immune responses, which are more rapid than the primary response, and by production of higher serum titers of antigen-specific antibodies, mostly of the IgG isotype. The prevailing view is that antigen-specific B cells are maintained as a pool of memory B cells after clonal expansion during the primary immune response (1–4). Most memory B cells have been thought to originate from the germinal center (GC) reaction. In the GC, the combined processes of somatic hypermutation and selection based on the affinity of the B cell receptor (BCR) for the antigen are responsible for the generation of high-affinity antibody variants that ultimately differentiate into long-lived plasma cells or long-lived memory B cells (5, 6). The GC is also a preferential site of antibody class switching. In the GC reaction, de novo–generated antigen-specific memory B cells are thought to acquire intrinsically different traits from their naive predecessors, accounting for faster and heightened secondary responses. Thus, understanding the mechanism by which memory B cells are generated and maintained, as well as the intrinsic functional differences between naive and memory B cells, is of fundamental interest to reveal the basis of immunological memory.The analysis of gene-targeted mice lacking the cytoplasmic tail of the IgG1 or IgE BCR has revealed its essential function in secondary responses (7, 8). In response to T cell–dependent antigens, mice harboring the tailless IgG1 had ∼25-fold fewer IgG1-expressing B cells, presumably reflecting a reduced number of GC and memory B cells and raising the possibility that the IgG1 cytoplasmic tail is involved in the generation and/or maintenance of memory B cells or their direct precursors. Two non–mutually exclusive models have been proposed to explain the function of the IgG1 tail (9). First, it may be required for efficient BCR-mediated internalization and, hence, presentation of antigen to T cells (10). As T cells facilitate productive IgG1 memory responses, inefficient antigen presentation by mutant B cells could lead to defective proliferation of GC B cells and, consequently, diminished generation of memory B cells. Second, the IgG1 tail may contribute to memory responses by modifying the BCR signal, for example by transmitting survival signals to memory B cells and/or their direct precursors (11, 12).To define the signaling molecules required for the establishment and maintenance of memory B cells, we focused on the function of phospholipase C (PLC) γ2 because this enzyme is well recognized as an important component of the BCR signaling pathway (13, 14). Indeed, PLC-γ2–deficient mice show a differentiation block between the immature and mature B cell stages owing to defective BCR signaling (15, 16). However, given the expression of PLC-γ2 in several immune cell types (17, 18) and the premature block in B cell development in conventional PLC-γ2 KO mice, these mice are not ideal for analyzing the role of PLC-γ2 in a B cell–intrinsic manner during T cell–dependent antibody responses. Thus, we used conditional mice in which PLC-γ2 function was specifically inactivated in GC B cells and in an inducible manner. We show in this paper that PLC-γ2 is required for the efficient generation and maintenance of memory B cells, probably through the delivery of a prosurvival signal.     

A B cell receptor with two Igalpha cytoplasmic domains supports development of mature but anergic B cells

B cell receptor (BCR) signaling is mediated through immunoglobulin (Ig)alpha and Igbeta a membrane-bound heterodimer. Igalpha and Igbeta are redundant in their ability to support early B cell development, but their roles in mature B cells have not been defined. To examine the function of Igalpha-Igbeta in mature B cells in vivo we exchanged the cytoplasmic domain of Igalpha for the cytoplasmic domain of Igbeta by gene targeting (Igbetac-->alphac mice). Igbetac-->alphac B cells had lower levels of surface IgM and higher levels of BCR internalization than wild-type B cells. The mutant B cells were able to complete all stages of development and were long lived, but failed to differentiate into B1a cells. In addition, Igbetac-->alphac B cells showed decreased proliferative and Ca2+ responses to BCR stimulation in vitro, and were anergic to T-independent and -dependent antigens in vivo.     

Identification of the tyrosine phosphatase PTP1C as a B cell antigen receptor-associated protein involved in the regulation of B cell signaling

Recent data implicating loss of PTP1C tyrosine phosphatase activity in the genesis of the multiple hemopoietic cell defects found in systemic autoimmune/immunodeficient motheaten (me) and viable motheaten (mev) mice suggest that PTP1C plays an important role in modulating intracellular signaling events regulating cell activation and differentiation. To begin elucidating the role for this cytosolic phosphatase in lymphoid cell signal transduction, we have examined early signaling events and mitogenic responses induced by B cell antigen receptor (BCR) ligation in me and mev splenic B cells and in CD5+ CH12 lymphoma cells, which represent the lymphoid population amplified in motheaten mice. Despite their lack of functional PTP1C, me and mev B cells proliferated normally in response to LPS. However, compared with wild-type B cells, cells from the mutant mice were hyperresponsive to normally submitogenic concentrations of F(ab')2 anti- Ig antibody, and they exhibited reduced susceptibility to the inhibitory effects of Fc gamma IIRB cross-linking on BCR-induced proliferation. Additional studies of unstimulated CH12 and wild-type splenic B cells revealed the constitutive association of PTP1C with the resting BCR complex, as evidenced by coprecipitation of PTP1C protein and phosphatase activity with BCR components and the depletion of BCR- associated tyrosine phosphatase activity by anti-PTP1C antibodies. These results suggest a role for PTP1C in regulating the tyrosine phosphorylation state of the resting BCR complex components, a hypothesis supported by the observation that PTP1C specifically induces dephosphorylation of a 35-kD BCR-associated protein likely representing Ig-alpha. In contrast, whereas membrane Ig cross-linking was associated with an increase in the tyrosine phosphorylation of PTP1C and an approximately 140-kD coprecipitated protein, PTP1C was no longer detected in the BCR complex after receptor engagement, suggesting that PTP1C dissociates from the activated receptor complex. Together these results suggest a critical role for PTP1C in modulating BCR signaling capacity, and they indicate that the PTP1C influence on B cell signaling is likely to be realized in both resting and activated cells.     

Limiting dilution analysis of Epstein-Barr virus-induced immunoglobulin production by human B cells

The Epstein-Barr virus (EBV) is a herpes virus that has the capacity to infect human B cells and to induce them to secrete immunoglobulin (Ig). In the current experiments, Poisson analysis of limiting dilution cultures has been used to study the activation of human peripheral B cells by the B95-8 strain of EBV. Under the culture conditions used, 0.2-1% of peripheral blood B cells were activated by EBV to secrete IgM or IgG. In addition, when multiple replicate cultures containing limited numbers of B cells were tested for IgM and for IgG production, the precursors for IgM and IgG segregated independently; thus, individual B cell precursors matured into cells secreting IgM or IgG but not both classes of Ig. Additional experiments using limiting dilutions of EBV were undertaken to study the viral requirements for B cell activation. These studies indicated that B cell activation by EBV to produce Ig was consistent with a "one-hit" model and inconsistent with a "two-hit" model. Taken together, these results indicate that infection by one EBV virion is sufficient to induce a precursor peripheral blood B cell to secrete Ig and that only one isotype of Ig is then secreted.