A dynamic T cell-limited checkpoint regulates affinity-dependent B cell entry into the germinal center

The germinal center (GC) reaction is essential for the generation of the somatically hypermutated, high-affinity antibodies that mediate adaptive immunity. Entry into the GC is limited to a small number of B cell clones; however, the process by which this limited number of clones is selected is unclear. In this study, we demonstrate that low-affinity B cells intrinsically capable of seeding a GC reaction fail to expand and become activated in the presence of higher-affinity B cells even before GC coalescence. Live multiphoton imaging shows that selection is based on the amount of peptide-major histocompatibility complex (pMHC) presented to cognate T cells within clusters of responding B and T cells at the T-B border. We propose a model in which T cell help is restricted to the B cells with the highest amounts of pMHC, thus allowing for a dynamic affinity threshold to be imposed on antigen-binding B cells.     

High affinity germinal center B cells are actively selected into the plasma cell compartment

A hallmark of T cell-dependent immune responses is the progressive increase in the ability of serum antibodies to bind antigen and provide immune protection. Affinity maturation of the antibody response is thought to be connected with the preferential survival of germinal centre (GC) B cells that have acquired increased affinity for antigen via somatic hypermutation of their immunoglobulin genes. However, the mechanisms that drive affinity maturation remain obscure because of the difficulty in tracking the affinity-based selection of GC B cells and their differentiation into plasma cells. We describe a powerful new model that allows these processes to be followed as they occur in vivo. In contrast to evidence from in vitro systems, responding GC B cells do not undergo plasma cell differentiation stochastically. Rather, only GC B cells that have acquired high affinity for the immunizing antigen form plasma cells. Affinity maturation is therefore driven by a tightly controlled mechanism that ensures only antibodies with the greatest possibility of neutralizing foreign antigen are produced. Because the body can sustain only limited numbers of plasma cells, this "quality control" over plasma cell differentiation is likely critical for establishing effective humoral immunity.     

Increased IL-12 inhibits B cells' differentiation to germinal center cells and promotes differentiation to short-lived plasmablasts

B cells activated by antigen in T cell-dependent immune responses can become short-lived plasma cells, which remain in the spleen, or germinal center-derived memory or plasma cells, which show evidence of affinity maturation and, in the case of plasma cells, migrate to the bone marrow. We show that this cell fate decision can be governed by the cytokine environment engendered by activated dendritic cells (DCs). DCs from mice lacking the Fc receptor gamma chain exhibited an activated phenotype in vitro. They secreted more of the proinflammatory cytokine IL-12, which led to the preferential generation of short-lived splenic plasma cells, with ensuing low affinity antibodies and a diminished recall response. Understanding the factors that regulate antigen-activated B cell differentiation and memory cell formation has implications for both antibody-mediated autoimmune disease and protective antibody responses.     

FLICE-inhibitory protein is a key regulator of germinal center B cell apoptosis

Affinity maturation of the B cell response to antigen (Ag) takes place in the germinal centers (GCs) of secondary follicles. Two sequential molecular mechanisms underpin this process. First, the B cell repertoire is diversified through hypermutation of the immunoglobulin (Ig) variable region genes. Second, mutant B cell clones with improved affinity for Ag are positively selected by Ag and CD40 ligand (L). This selection step is contingent upon "priming" of GC B cells for apoptosis. The molecular means by which B cell apoptosis is initiated and controlled in the GC remains unclear. Here, we show that GC B cell apoptosis is preceded by the rapid activation of caspase-8 at the level of CD95 death-inducing signaling complex (DISC). We found that GC B cells ex vivo display a preformed inactive DISC containing Fas-associated death domain-containing protein (FADD), procaspase-8, and the long isoform of cellular FADD-like IL-1beta-converting enzyme-inhibitory protein (c-FLIP(L)) but not the CD95L. In culture, c-FLIP(L) is rapidly lost from the CD95 DISC unless GC B cells are exposed to the survival signal provided by CD40L. Our results suggest that (a) the death receptor signaling pathway is involved in the affinity maturation of antibodies, and (b) c-FLIP(L) plays an active role in positive selection of B cells in the GC.     

Development of self-reactive germinal center B cells and plasma cells in autoimmune Fc gammaRIIB-deficient mice

Abnormalities in expression levels of the IgG inhibitory Fc gamma receptor IIB (FcγRIIB) are associated with the development of immunoglobulin (Ig) G serum autoantibodies and systemic autoimmunity in mice and humans. We used Ig gene cloning from single isolated B cells to examine the checkpoints that regulate development of autoreactive germinal center (GC) B cells and plasma cells in FcγRIIB-deficient mice. We found that loss of FcγRIIB was associated with an increase in poly- and autoreactive IgG(+) GC B cells, including hallmark anti-nuclear antibody-expressing cells that possess characteristic Ig gene features and cells producing kidney-reactive autoantibodies. In the absence of FcγRIIB, autoreactive B cells actively participated in GC reactions and somatic mutations contributed to the generation of highly autoreactive IgG antibodies. In contrast, the frequency of autoreactive IgG(+) B cells was much lower in spleen and bone marrow plasma cells, suggesting the existence of an FcγRIIB-independent checkpoint for autoreactivity between the GC and the plasma cell compartment.     

Identification of a human follicular dendritic cell molecule that stimulates germinal center B cell growth

The initial interaction between B cells and follicular dendritic cells (FDCs) appears to be essential for germinal center (GC) formation. To identify molecules regulating this interaction, we generated FDC-staining monoclonal antibodies (mAbs) and screened them for their ability to block FDC-mediated costimulation of growth and differentiation of CD40-stimulated B cells. Using one of the inhibitory mAbs, 8D6, we expression cloned the cDNA encoding the 8D6 antigen (Ag) from a human FDC line, HK. The 8D6 Ag is a novel protein of 282 amino acids that is expressed abundantly on FDCs. Monolayers of COS cells transiently transfected with the 8D6 Ag cDNA stimulate B cell growth. The mAb 8D6 blocks the costimulatory function completely. The inhibitory activity of the mAb 8D6 was demonstrated to be due to an inhibition of cell cycle progression of CD40 ligand-stimulated GC B cells. In addition, the mAb 8D6 inhibits the growth of a lymphoma of GC origin, L3055, which depends on FDCs or HK cells for its growth. These findings suggest that the primary function of FDCs in the GC is to stimulate B cell growth. An FDC signal molecule, 8D6 Ag, may be an important molecule to mediate this function.     

Cyclin D3 drives inertial cell cycling in dark zone germinal center B cells

During affinity maturation, germinal center (GC) B cells alternate between proliferation and somatic hypermutation in the dark zone (DZ) and affinity-dependent selection in the light zone (LZ). This anatomical segregation imposes that the vigorous proliferation that allows clonal expansion of positively selected GC B cells takes place ostensibly in the absence of the signals that triggered selection in the LZ, as if by “inertia.” We find that such inertial cycles specifically require the cell cycle regulator cyclin D3. Cyclin D3 dose-dependently controls the extent to which B cells proliferate in the DZ and is essential for effective clonal expansion of GC B cells in response to strong T follicular helper (Tfh) cell help. Introduction into the Ccnd3 gene of a Burkitt lymphoma–associated gain-of-function mutation (T283A) leads to larger GCs with increased DZ proliferation and, in older mice, clonal B cell lymphoproliferation, suggesting that the DZ inertial cell cycle program can be coopted by B cells undergoing malignant transformation.     

gp39-CD40 interactions are essential for germinal center formation and the development of B cell memory

gp39, the ligand for CD40 expressed on activated CD4+ T helper cells, is required for the generation of antibody responses to T-dependent (TD) antigens. Treatment of mice with anti-gp39 in vivo inhibits both primary and secondary antibody formation to TD, but not T-independent antigens. However, the role of this receptor-ligand pair in the development of germinal centers and the generation of B cell memory is as yet undefined. Using an antibody to gp39, this study examines the in vivo requirement for gp39-CD40 interactions in the induction of germinal center formation, as well as in the generation of B cell memory. Animals were immunized, treated in vivo with anti-gp39, and evaluated using immunohistochemical staining for the presence of splenic germinal centers 9-11 d after immunization. The results demonstrate that the formation of germinal centers was completely inhibited as a result of treatment with anti-gp39. Moreover, adoptive transfer experiments demonstrate that the generation of antigen- specific memory B cells is also inhibited as a consequence of blocking gp39-CD40 interactions. Taken together, the data demonstrate that gp39- CD40 interactions are critical not only for the generation of antibody responses, but also in the development of B cell memory.     

Distinct germinal center selection at local sites shapes memory B cell response to viral escape

Respiratory influenza virus infection induces cross-reactive memory B cells targeting invariant regions of viral escape mutants. However, cellular events dictating the cross-reactive memory B cell responses remain to be fully defined. Here, we demonstrated that lung-resident memory compartments at the site of infection, rather than those in secondary lymphoid organs, harbor elevated frequencies of cross-reactive B cells that mediate neutralizing antibody responses to viral escape. The elevated cross-reactivity in the lung memory compartments was correlated with high numbers of V_H mutations and was dependent on a developmental pathway involving persistent germinal center (GC) responses. The persistent GC responses were focused in the infected lungs in association with prolonged persistence of the viral antigens. Moreover, the persistent lung GCs supported the exaggerated B cell proliferation and clonal selection for cross-reactive repertoires, which served as the predominant sites for the generation of cross-reactive memory progenitors. Thus, we identified the distinct GC selection at local sites as a key cellular event for cross-reactive memory B cell response to viral escape, a finding with important implications for developing broadly protective influenza vaccines.Protective memory responses provided by parental influenza vaccines primarily depend on neutralizing IgG antibodies (Abs) directed against hemagglutinin (HA), a major glycoprotein on the virus surface (Gerhard, 2001; Plotkin, 2013). The membrane distal region of the HA globular head is highly immunogenic and is the primary target of anti-HA Abs elicited by vaccination (Skehel and Wiley, 2000). However, the HA globular head undergoes continual antigenic evolution (Wiley et al., 1981), making vaccine-induced Abs less effective against drifted viruses. Moreover, new subtypes can emerge rapidly and unexpectedly, as experienced in the 2009 A/H1N1 pandemic virus and sporadic human infection with avian viruses such as H5N1 and H7N9. Thus, the evolving threats of influenza virus underscore the need for influenza vaccines that are more broadly protective.HA conserved regions can be targeted by broadly cross-reactive Abs that exhibit potent virus-neutralizing activity in vitro and in vivo (Okuno et al., 1993; Throsby et al., 2008; Sui et al., 2009; Yoshida et al., 2009; Corti et al., 2010; Krause et al., 2011; Wrammert et al., 2011). Such cross-reactive Abs were observed in IgG and IgA fractions after respiratory exposure of viruses (Tamura et al., 1992; Tumpey et al., 2001; Margine et al., 2013). Of note, cross-reactive IgG Abs were higher in humans infected with influenza virus than in humans parentally boosted with vaccines (Moody et al., 2011; Wrammert et al., 2011; Li et al., 2012; Pica et al., 2012; Margine et al., 2013), suggesting that the cellular pathways for cross-reactive Ab responses are more active after respiratory virus infection.Pulmonary-infected influenza virus initially primes virus-binding B cells in the lung-draining mediastinal LNs (MLNs; Coro et al., 2006). The infected lungs, albeit at delayed kinetics, also participate in the primary immune response, concordant with the ectopic formation of induced bronchus-associated lymphoid tissue (iBALT; Moyron-Quiroz et al., 2004; Halle et al., 2009). iBALTs are able to support germinal center (GC) formation (Moyron-Quiroz et al., 2004), suggesting intraorgan development of long-lived plasma cells and memory B cells, which are crucial cellular components for humoral memory responses (Joo et al., 2008; Onodera et al., 2012; Tarlinton and Good-Jacobson, 2013). Although immediate protection against homologous reinfection is mediated by preexisting neutralizing Abs from long-lived plasma cells, memory B cells serve as a reservoir of cross-reactive Ab repertoires in West Nile virus infection (Purtha et al., 2011). Therefore, it is now postulated that memory B cells are important for the broad protection against escape mutants, against which strain-specific Abs are no longer effective (Baumgarth, 2013). However, the memory B cell subset reserving cross-reactive repertoires and its developmental pathway has not been fully characterized.Here, using two types of fluorochrome-labeled HA probes, we identified the cross-reactive memory B cell subset and dissected its developmental pathway after pulmonary influenza virus infection. Our data revealed a striking heterogeneity in the tissue localization, persistence, and selection for cross-reactivity among virus-specific GC responses. Among such heterogeneous GC responses, persistent GCs in the infected lungs profoundly selected and supplied cross-reactive memory repertoires into local sites, thereby potentiating the cross-protection at the site of infection.     

CD40 and B cell antigen receptor dual triggering of resting B lymphocytes turns on a partial germinal center phenotype

Phenotypic alterations occur when resting human B lymphocytes become germinal center (GC) cells. These include the induction of surface CD38, CD95 (FAS/APO-1), and carboxy-peptidase-M (CPM), a recently described GC marker. However, the factors that govern the in vivo induction of these surface molecules on B cells remain unknown. Here, we purified resting (CD38-) human B lymphocytes from tonsils in an attempt to establish culture conditions resulting in the induction of these three GC markers. We show that interferon (IFN) alpha or IFN- gamma, as well as antibodies against the B cell antigen receptor (BCR), could induce CD38 on resting B lymphocytes, a phenomenon further enhanced by CD40 stimulation. Concomitantly, CD95 was upregulated by CD40 ligation and, to a lesser extent, by IFN-gamma. By contrast, CPM expression could be upregulated only through BCR triggering. This CPM induction was specifically enhanced by CD19 or CD40 ligation. CD40 + BCR stimulation of resting B cells with CD40 ligand-transfected fibroblastic cells in the presence of cross-linked anti-BCR monoclonal antibodies resulted in the coexpression of CD38, CD95, and CPM. As GC cells, these cells also expressed CD71, CD80 (B7.1), and CD86 (B7.2), but not CD24. However, CD10+ or CD44- B cells could not be detected in these culture conditions, suggesting that yet other signals are required for the induction of these GC markers. Consistent with a GC phenotype, CD40 + BCR-stimulated cells exhibited reduced viability when cultured for 20 h in the absence of stimulus. These results first demonstrate that cotriggering of resting B cells through BCR and CD40 induces both phenotypic and functional GC features. They also show that IFN and CD19 triggering of resting B cells specifically modulate the expression of GC markers.     

Antigen recognition. V. Requirement for histocompatibility between antigen-presenting cell and B cell in the response to a thymus- dependent antigen, and lack of allogeneic restriction between T and B cells

The restrictions imposed by the major histocompatibility complex on T-B- antigen-presenting cell (APC) interactions were studied with an in vivo adoptive transfer system, using mutually tolerant T and B cells taken from one-way fetal liver chimeras. It was found that the B cells and adoptive recipient (which provides APC function) have to share determinants encoded by the left-hand end of the H-2 complex for cooperation, whereas there is apparently no such requirement for T-B cell syngeneicity. Suppression arising from allogeneic effects between the host and the transferred T or B cells was excluded by the use of tolerant as well as normal adoptive recipients; both were functionally equivalent. We conclude that under experimental conditions, unrestricted helper T cell function and concurrent APC-B cell genetic restriction can be demonstrated in vivo.