Cutting edge: germinal centers can be induced in the absence of T cells.

Immunization of mice containing mutations that inactivate the TCR Cbeta and Cdelta genes with the T cell-independent (TI) type 2 Ag (4-hydroxy-3-nitrophenyl)acetyl-Ficoll induces clusters of peanut agglutinin-binding B cells in the spleen. These clusters are histologically indistinguishable from germinal centers (GCs) typical of T cell-dependent immune responses. They are located in follicles, and contain mature follicular dendritic cells, immune complex deposits, and B cells that display the phenotypic qualities of conventional GC B cells. However, the kinetics of this TI GC response differ from T cell-dependent GC responses in being rapidly induced and of short duration. Moreover, the Ab V genes expressed in TI GCs have not undergone somatic hypermutation. Therefore, T cells may be required for B cell differentiation processes associated with the intermediate and latter stages of the GC reaction, but they are dispensable for the induction and initial development of this response.     

Humoral immune responses in Cr2-/- mice: enhanced affinity maturation but impaired antibody persistence

Deficiency in CD21/CD35 by disruption of the Cr2 loci leads to impaired humoral immune responses. In this study, we detail the role of CD21/CD35 on Ab responses to the hapten (4-hydroxy-3-nitrophenyl)acetyl conjugated to chicken gamma-globulin. Surprisingly, Cr2-/- mice generate significant Ab responses and germinal center (GC) reactions to low doses of this Ag in alum, although the magnitude of their responses is much reduced in comparison with those of Cr2+/- and C57BL/6 controls. Increasing Ag dose partially corrected this deficit. In situ study of the somatic genetics of GC B cells demonstrated that VDJ hypermutation does not require CD21/CD35, and Cr2-/- mice exhibited enhanced affinity maturation of serum Ab in the post-GC phase of the primary response. On the other hand, Cr2-/- mice displayed accelerated loss of serum Ab and long-lived Ab-forming cells. These observations suggest that B cell activation/survival signals mediated by CD21 and/or the retention of Ag by CD21/CD35 play important roles in the generation, quality, and maintenance of serum Ab.     

Regulation of memory antibody levels: the role of persisting antigen versus plasma cell life span

Protective Ab levels can be maintained for years upon infection or vaccination. In this study, we studied the duration of Ab responses as a function of the life span of plasma cells and tested the role of persisting Ag in maintaining B cell memory. Our analysis of B cell responses induced in mice immunized with virus-like particles demonstrates the following: 1) Ab titers are long-lived, but decline continuously with a t(1/2) of approximately 80 days, which corresponds to the life span of plasma cells; 2) the germinal center (GC) reaction, which lasts for up to 100 days, is dependent on Ag associated with follicular dendritic cells; and 3) early GCs produce massive numbers of plasma and memory B cell precursors, whereas the late Ag-dependent GCs are dispensable for the maintenance of Ab levels and B cell memory.     

B cell-activating factor belonging to the TNF family acts through separate receptors to support B cell survival and T cell-independent antibody formation

The TNF-related ligand, B cell-activating factor belonging to the TNF family (BAFF), is necessary for normal B cell development and survival, and specifically binds the receptors transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI), B cell maturation Ag (BCMA), and BAFF-R. Similarities between mice completely lacking BAFF and A/WySnJ strain mice that express a naturally occurring mutant form of BAFF-R suggest that BAFF acts primarily through BAFF-R. However, the nearly full-length BAFF-R protein expressed by A/WySnJ mice makes unambiguous interpretation of receptor function in these animals impossible. Using homologous recombination we created mice completely lacking BAFF-R and compared them directly to A/WySnJ mice and to mice lacking BAFF. BAFF-R-null mice exhibit loss of mature B cells similar to that observed in BAFF(-/-) and A/WySnJ mice. Also, mice lacking both TACI and BCMA simultaneously exhibit no B cell loss, thus confirming that BAFF-R is the primary receptor for transmitting the BAFF-dependent B cell survival signal. However, while BAFF-R-null mice cannot carry out T cell-dependent Ab formation, they differ from BAFF-deficient mice in generating normal levels of Ab to at least some T cell-independent Ags. These studies clearly demonstrate that BAFF regulates Ab responses in vivo through receptors in addition to BAFF-R.     

Gene conversion and hypermutation during diversification of VH sequences in developing splenic germinal centers of immunized rabbits

The young rabbit appendix and the chicken bursa of Fabricius are primary lymphoid organs where the B cell Ab repertoire develops in germinal centers (GCs) mainly by a gene conversion-like process. In human and mouse, V-gene diversification by somatic hypermutation in GCs of secondary lymphoid organs leads to affinity maturation. We asked whether gene conversion, somatic hypermutation, or both occur in rabbit splenic GCs during responses to the hapten DNP. We determined DNA sequences of rearranged heavy and light chain V region gene segments in single cells from developing DNP-specific GCs after immunization with DNP-bovine gamma-globulin and conclude that the changes at the DNA level that may lead to affinity maturation occur by both gene conversion and hypermutation. Selection was suggested by finding some recurrent amino acid replacements that may contribute increased affinity for antigen in the complementarity-determining region sequences of independently evolved clones, and a narrower range of complementarity-determining region 3 lengths at day 15. Some of the alterations of sequence may also lead to new members of the B cell repertoire in adult rabbits comparable with those produced in gut associated lymphoid tissues of young rabbits.     

Divergent effects of BAFF on human memory B cell differentiation into Ig-secreting cells

B cell-activating factor belonging to the TNF family (BAFF) plays a critical role in B cell maturation, yet its precise role in B cell differentiation into Ig-secreting cells (ISCs) remains unclear. In this study, we find that upon isolation human naive and memory B (MB) cells have prebound BAFF on their surface, whereas germinal center (GC) B cells lack detectable levels of prebound BAFF. We attribute their lack of prebound BAFF to cell activation, because we demonstrate that stimulation of naive and MB cells results in the loss of prebound BAFF. Furthermore, the absence of prebound BAFF on GC B cells is not related to a lack of BAFF-binding receptors or an inability to bind exogenous BAFF. Instead, our data suggest that accessibility to soluble BAFF is limited within GCs, perhaps to prevent skewing of the conventional B cell differentiation program. In support of this concept, whereas BAFF significantly enhances ISC differentiation in response to T cell-dependent activation, we report for the first time the ability of BAFF to considerably attenuate ISC differentiation of MB cells in response to CpG stimulation, a form of T cell-independent activation. Our data suggest that BAFF may be providing regulatory signals during specific T cell-independent events, which protect the balance between MB cells and ISCs outside GCs. Taken together, these data define a complex role for BAFF in humoral immune responses and show for the first time that BAFF can also play an inhibitory role in B cell differentiation.     

Contrasting the in situ behavior of a memory B cell clone during primary and secondary immune responses.

Whether memory B cells possess altered differentiative potentials and respond in a qualitatively distinct fashion to extrinsic signals as compared with their naive precursors is a current subject of debate. We have investigated this issue by examining the participation of a predominant anti-arsonate clonotype in the primary and secondary responses in the spleens of A/J mice. While this clonotype gives rise to few Ab-forming cells (AFC) in the primary response, shortly after secondary immunization its memory cell progeny produce a massive splenic IgG AFC response, largely in the red pulp. Extensive clonal expansion and migration take place during the secondary AFC response but Ab V region somatic hypermutation is not reinduced. The primary and secondary germinal center (GC) responses of this clonotype are both characterized by ongoing V gene hypermutation and phenotypic selection, little or no inter-GC migration, and derivation of multiple, spatially distinct GCs from a single progenitor. However, the kinetics of these responses differ, with V genes containing a high frequency of total as well as affinity-enhancing mutations appearing rapidly in secondary GCs, suggesting either recruitment of memory cells into this response, or accelerated rates of hypermutation and selection. In contrast, the frequency of mutation observed per V gene does not increase monotonically during the primary GC response of this clonotype, suggesting ongoing emigration of B cells that have sustained affinity- and specificity-enhancing mutations.     

TNF family member B cell-activating factor (BAFF) receptor-dependent and -independent roles for BAFF in B cell physiology

The cytokine TNF family member B cell-activating factor (BAFF; also termed BLyS) is essential for B cell generation and maintenance. Three receptors have been identified that bind to BAFF: transmembrane activator, calcium modulator, and cyclophilin ligand interactor (TACI); B cell maturation Ag (BCMA); and BAFF-R. Recently, it was shown that A/WySnJ mice, which contain a dramatically reduced peripheral B cell compartment due to decreased B cell life span, express a mutant BAFF-R. This finding, together with normal or enhanced B cell generation in mice deficient for BCMA or TACI, respectively, suggested that the interaction of BAFF with BAFF-R triggers signals essential for the generation and maintenance of mature B cells. However, B cells in mice deficient for BAFF differ phenotypically and functionally from A/WySnJ B cells. Residual signaling through the mutant BAFF-R could account for these differences. Alternatively, dominant-negative interference by the mutant receptor could lead to an overestimation of the importance of BAFF-R. To resolve this issue, we generated BAFF-R-null mice. Baff-r(-/-) mice display strongly reduced late transitional and follicular B cell numbers and are essentially devoid of marginal zone B cells. Overexpression of Bcl-2 rescues mature B cell development in Baff-r(-/-) mice, suggesting that BAFF-R mediates a survival signal. CD21 and CD23 surface expression are reduced on mature Baff-r(-/-) B cells, but not to the same extent as on mature B cells in BAFF-deficient mice. In addition, we found that Baff-r(-/-) mice mount significant, but reduced, Ag-specific Ab responses and are able to form spontaneous germinal centers in mesenteric lymph nodes. The reduction in Ab titers correlates with the reduced B cell numbers in the mutant mice.     

Identification of memory B cells using a novel transgenic mouse model

Memory B cells help to protect the host from invading pathogens by maintaining persistent levels of Ag-specific serum Ab and generating rapid Ab responses upon re-exposure to Ag. Unambiguous identification of memory B cells has been a major obstacle to furthering our knowledge concerning both the development of B cell memory and secondary Ab responses due to an absence of specific cell surface markers. Germinal centers (GCs) are thought to be the major site of Ig hypermutation and Ag-driven selection of memory B cells. To develop a model that would identify GC-derived memory B cells, we generated transgenic mice that expressed cre recombinase in a GC-specific fashion. Interbreeding these mice with the cre-reporter strain, ROSA26R, produced progeny in which beta-galactosidase (beta-gal) was permanently expressed in B cells of the GC-memory pathway. Analysis following immunization with (4-hydroxy-3-nitrophenyl)acetyl coupled to chicken gamma globulin showed that long-lived beta-gal+ B cells exclusively contained somatically mutated lambda1 V regions and were capable of producing Ag-specific Ab-forming cell (AFC) responses that were >100-fold higher than those afforded by beta-gal- B cells following adoptive transfer to naive hosts. Secondary challenge of immune mice showed that only approximately 20% of secondary AFCs expressed beta-gal. Interestingly, we found that somatic hypermutation of rearranged lambda1 V regions within secondary AFCs showed a strong correlation with beta-gal expression, suggesting that nonmutated B cells contribute significantly to secondary Ab responses. This model should provide useful insights into memory B cell development, maintenance, and differentiation following immunization or pathogenic infection.     

Salmonella induces a switched antibody response without germinal centers that impedes the extracellular spread of infection

T-dependent Ab responses are characterized by parallel extrafollicular plasmablast growth and germinal center (GC) formation. This study identifies that, in mice, the Ab response against Salmonella is novel in its kinetics and its regulation. It demonstrates that viable, attenuated Salmonella induce a massive early T-dependent extrafollicular response, whereas GC formation is delayed until 1 mo after infection. The extrafollicular Ab response with switching to IgG2c, the IgG2a equivalent in C57BL/6 mice, is well established by day 3 and persists through 5 wk. Switching is strongly T dependent, and the outer membrane proteins are shown to be major targets of the early switched IgG2c response, whereas flagellin and LPS are not. GC responses are associated with affinity maturation of IgG2c, and their induction is associated with bacterial burden because GC could be induced earlier by treating with antibiotics. Clearance of these bacteria is not a consequence of high-affinity Ab production, for clearance occurs equally in CD154-deficient mice, which do not develop GC, and wild-type mice. Nevertheless, transferred low- and high-affinity IgG2c and less efficiently IgM were shown to impede Salmonella colonization of splenic macrophages. Furthermore, Ab induced during the infection markedly reduces bacteremia. Thus, although Ab does not prevent the progress of established splenic infection, it can prevent primary infection and impedes secondary hemogenous spread of the disease. These results may explain why attenuated Salmonella-induced B cell responses are protective in secondary, but not primary infections.     

Impaired apoptotic cell clearance in the germinal center by Mer-deficient tingible body macrophages leads to enhanced antibody-forming cell and germinal center responses

Germinal centers (GCs) are specialized microenvironments that generate high-affinity Ab-forming cells (AFCs) and memory B cells. Many B cells undergo apoptosis during B cell clonal selection in GCs. Although the factors that regulate the AFC and GC responses are not precisely understood, it is widely believed that dysregulated AFCs and GCs contribute to autoimmunity. The Mer receptor tyrosine kinase (Mer) facilitates macrophage clearance of apoptotic cells. The Tyro-3, Axl, and Mer receptors, including Mer, suppress TLRs and cytokine-mediated inflammatory responses. We report in this study that tingible body macrophages (TBMφs) in GCs express Mer. Compared to C57BL/6 (B6) controls, Mer-deficient (Mer(-/-)) mice had significantly higher AFC, GC, and Th1-skewed IgG2 Ab (especially IgG2c) responses against the T cell-dependent Ag (4-hydroxy-3-nitrophenyl) acetyl-chicken γ globulin. Mer(-/-) mice had a significantly higher percentage of GC B cells on days 9, 14, and 21 postimmunization compared with B6 controls. Significantly increased numbers of apoptotic cells accumulated in Mer(-/-) GCs than in B6 GCs, whereas the number of TBMφs remained similar in both strains. Our data are the first, to our knowledge, to demonstrate a critical role for Mer in GC apoptotic cell clearance by TBMφs and have interesting implications for Mer in the regulation of B cell tolerance operative in the AFC and GC pathways.     

Cutting edge: critical role of inducible costimulator in germinal center reactions

Inducible costimulator (ICOS) is a new member of the CD28/CTLA-4 family that is expressed on activated and germinal center (GC) T cells. Recently, we reported that ICOS-deficient mice exhibited profound defects in T cell activation and effector function. Ab responses in a T-dependent primary reaction and in a murine asthma model were also diminished. In the current study, we investigate the mechanism by which ICOS regulates humoral immunity and examine B cell GC reactions in the absence of ICOS. We found that ICOS(-/-) mice, when immunized with SRBC, had smaller GCs. Furthermore, IgG1 class switching in the GCs was impaired. Remarkably, GC formation in response to a secondary recall challenge was completely absent in ICOS knockout mice. These data establish a critical role of ICOS in regulation of humoral immunity.     

CD27 is acquired by primed B cells at the centroblast stage and promotes germinal center formation

Studies on human B cells have featured CD27 as a marker and mediator of the B cell response. We have studied CD27 expression and function on B cells in the mouse. We find that B cells acquire CD27 at the centroblast stage and lose it progressively upon further differentiation. It is not a marker for somatically mutated B cells and is present at very low frequency on memory B cells. Enrichment of CD27 among centroblasts and the presence of its ligand CD70 on occasional T and B cells in or near germinal centers (GCs) suggested a role for CD27/CD70 interactions in clonal B cell expansion. Accordingly, GC formation in response to influenza virus infection was delayed in CD27 knockout mice. CD27 deficiency did not affect somatic hypermutation or serum levels of virus-specific IgM, IgG, and IgA attained in primary and recall responses. Adoptive transfer of T and B cells into CD27/CD28(-/-) mice revealed that CD27 promotes GC formation and consequent IgG production by two distinct mechanisms. Stimulation of CD27 on B cells by CD28(+) Th cells accelerates GC formation, most likely by promoting centroblast expansion. In addition, CD27 on T cells can partially substitute for CD28 in supporting GC formation.     

Axon growth and guidance genes identify T-dependent germinal centre B cells

Selection of B cells subjected to hypermutation in germinal centres (GC) during T cell-dependent (TD) antibody responses yields memory cells and long-lived plasma cells that produce high affinity antibodies biased to foreign antigens rather than self-antigens. GC also form in T-independent (TI) responses to polysaccharide antigens but failed selection results in GC involution and memory cells are not generated. To date there are no markers that allow phenotypic distinction of T-dependent and TI germinal centre B cells. We compared the global gene expression of GC B cells purified from mice immunized with either TD or TI antigens and identified eighty genes that are differentially expressed in TD GC. Significantly, the largest cluster comprises genes involved in growth and guidance of neuron axons such as Plexin B2, Basp1, Nelf, Shh, Sc4mol and Sult4alpha. This is consistent with formation of long neurite (axon and dendrite)-like structures by mouse and human GC B cells, which may facilitate T:B cell interactions within GC, affinity maturation and B cell memory formation. Expression of BASP1 and PLEXIN B2 protein is very low or undetectable in resting and TI GC B cells, but markedly upregulated in GC B cells induced in the presence of T cell help. Finally we show some of the axon growth genes upregulated in TD-GC B cells including Basp1, Shh, Sult4alpha, Sc4mol are also preferentially expressed in post-GC B cell neoplasms.     

Germinal center-independent affinity maturation in tumor necrosis factor receptor 1-deficient mice

Germinal centers (GCs) have been identified as site at which the somatic mutation of immunoglobulins occurs.However, somatic mutations in immunoglobulins have also been observed in animals that normally do not harbor germinal centers. This clearly indicates that somatic mutations can occur in the absence of germinal centers.We therefore attempted to determine whether or not GCs exist in TNFR1-deficient mice, and are essential for the somatic mutation of immunoglobulins, using (4-hydroxy-3-nitropheny)acetyl-ovalbumin (NP-OVA). Both wild-type and TNFR1-deficient mice were immunized with NPOVA, and then examined with regard to the existence of GCs. No typical B-cell follicles were detected in the TNFR1-deficient mice. Cell proliferation was detected throughout all splenic tissue types, and no in vivo immunecomplex retention was observed in the TNFR1-deficient mice. All of these data strongly suggest that no GCs were formed in the TNFR1-deficient mice. Although TNFR1-deficient mice are unable to form GCs, serological analyses indicated that affinity maturation had been achieved in both the wild-type and TNFR1-deficient mice. We therefore isolated and sequenced several DNA clones from wild-type and the TNFR1-deficient mice. Eight out of 12 wild-type clones, and 11 out of 14 clones of the TNFR-1-deficient mice contained mutations at the CDR1 site. Thus, the wild-type and TNFR1-deficient mice were not extremely different with regard to types and rates of somatic mutation. Also, high-affinity antibodies were detected in both types of mice. Collectively, our data appear to show that affinity maturation may occur in TNFR1-deficient mice, which completely lack GCs.     

The T cell-dependent B cell immune response and germinal center reaction are intact in A-myb-deficient mice

Expression of the protooncogene A-myb is restricted to the developing CNS, adult testes, breasts in late pregnancy, and germinal centers of secondary B cell follicles. The functional relevance of A-myb expression at three of these sites has been demonstrated previously via the generation and analysis of A-myb-deficient mice, which display behavioral abnormalities, male sterility, and perturbed breast development during pregnancy. In contrast, here we show that the germinal center response driven by T cell-dependent Ag immunization and the associated processes of Ab V gene somatic hypermutation, affinity maturation, and heavy chain class switching are overtly normal in A-myb-deficient mice. Nonetheless, these mice display mild splenic white pulp hypoplasia and blunted primary serum Ab responses, suggesting that although A-myb is not directly involved in the regulation of the memory B cell response, it may play a role in enhancing peripheral B cell survival or proliferative capacity.     

A spatial model of germinal center reactions: cellular adhesion based sorting of B cells results in efficient affinity maturation

Affinity maturation of humoral responses to T-cell-dependent antigens occurs in germinal centers (GC). In GCs antigen-specific B cells undergo rounds of somatic mutations that alter their affinity. High-affinity mutants take over GCs very soon after they appear; the replacement rate is as high as 4 per day (Radmacher et al., Immunol. Cell Biol. 76 (1998) 373). To gain more insight into this selection process, we present a spatial model of GC reactions, where B cells compete for survival signals from follicular dendritic cells (FDC). Assuming that high-affinity B cells have increased cellular adhesion to FDCs, we obtain an affinity-based sorting of B cells on the FDC. This sorting imposes a very strong selection and therefore results in a winner-takes-all behavior. By comparing our sorting model with "affinity-proportional selection models", we show that this winner-takes-all selection is in fact required to account for the fast rates at which high affinity mutants take over GCs. Another important feature of in vivo GC reactions is that they are non-mixed, i.e. GCs contain either no high-affinity cells at all or they are dominated by high-affinity cells. We here show that this all-or-none behavior can be obtained if B cells are sorted based on their affinity on the FDC surface. Affinity-proportional selection models, in contrast, always produce mixed GCs.     

Bcl-2 rescues the germinal center response but does not alter the V gene somatic hypermutation spectrum in MSH2-deficient mice

Ab V genes in mice deficient for the postreplication mismatch repair factor MutS homolog (MSH2) have been reported to display an abnormal bias for hypermutations at G and C nucleotides and hotspots. We previously showed that the germinal center (GC) response is severely attenuated in MSH2-deficient mice. This suggested that premature death of GC B cells might preclude multiple rounds of hypermutation necessary to generate a normal spectrum of base changes. To test this hypothesis, we created MSH2-deficient mice in which Bcl-2 expression was driven in B cells from a transgene. In such mice, the elevated levels of intra-GC apoptosis and untimely GC dissolution characteristic of MSH2-deficient mice are suppressed. However, the spectrum of hypermutation is unchanged. These data indicate that the effects of MSH2 deficiency on GC B cell viability and the hypermutation process are distinct.     

Plexin-D1 is a novel regulator of germinal centers and humoral immune responses

Long-lived humoral immune responses depend upon the generation of memory B cells and long-lived plasma cells during the germinal center (GC) reaction. These memory compartments, characterized by class-switched IgG and high-affinity Abs, are the basis for successful vaccination. We report that a new member of the plexin family of molecules, plexin-D1, controls the GC reaction and is required for secondary humoral immune responses. Plexin-D1 was not required for B cell maturation, marginal zone precursor development, dark and light zone formation, Igλ(+) and Igκ(+) B cell skewing, B1/B2 development, and the initial extrafollicular response. Plexin-D1 expression was increased following B cell activation, and PlxnD1(-/-) mice exhibited defective GC reactions during T-dependent immune activation. PlxnD1(-/-) B cells showed a defect in migration toward the GC chemokines, CXCL12, CXCL13, and CCL19. Accordingly, PlxnD1(-/-) mice exhibited defective production of IgG1 and IgG2b, but not IgG3 serum Ab, accompanied by reductions in long-lived bone marrow plasmacytes and recall humoral memory responses. These data show a new role for immune plexins in the GC reaction and generation of immunologic memory.