Dorottya Kövesdi

Phosphoinositide 3-Kinase Activity in T Cells Regulates the Magnitude of the Germinal Center Reaction

The generation of high-affinity Abs is essential for immunity and requires collaboration between B and T cells within germinal centers (GCs). By using novel mouse models with a conditional deletion of the p110δ catalytic subunit of the PI3K pathway, we established that p110δ is required in T cells, but not in B cells, for the GC reaction. We found the formation of T follicular helper (TFH) cells to be critically dependent on p110δ in T cells. Furthermore, by deleting phosphatase and tensin homolog deleted on chromosome 10, which opposes p110δ in activated T cells, we found a positive correlation between increased numbers of TFH cells and GC B cells. These results are consistent with the hypothesis that T cell help is the limiting factor in the GC reaction. P110δ was not required for the expression of B cell lymphoma 6, the downregulation of CCR7, or T cell entry into primary follicles. Instead, p110δ was the critical catalytic subunit for ICOS downstream signaling and the production of key TFH cytokines and effector molecules. Our findings support a model in which the magnitude of the GC reaction is controlled by the activity of the PI3K pathway in TFH cells.

B-cell responses to B-cell activation factor of the TNF family (BAFF) are impaired in the absence of PI3K delta.

B‐cell activating factor of the TNF family (BAFF) is critical for the survival and maturation of B cells. The molecular mechanisms by which BAFF regulates the survival of developing B cells are becoming better understood. Recent evidence has begun to emerge demonstrating a role for the PI3K/Akt signalling pathway in response to BAFF. However, the importance of the PI3K family for BAFF‐signalling and the effects of loss of PI3K function on BAFF responses are still unknown. We therefore investigated the BAFF‐mediated responses of B cells deficient for the PI3K catalytic subunit P110δ. We find that the loss of P110δ impairs the BAFF‐mediated survival of cultured B cells demonstrating a direct role for this member of the PI3K family in regulating the survival of B cells in response to BAFF. P110δ was required for the growth of B cells in response to BAFF and was critical for the upregulation of the receptor for BAFF following BCR crosslinking. Our findings reveal an important role for p110δ in regulating B‐cell responses to BAFF.

BCR mediated signal transduction in immature and mature B cells

Ligation of B cell receptors (BCR) on immature B cells may induce apoptosis, while in mature B cells it stimulates cell activation and growth. The signaling pathway regulating the differential functional response, death or survival of the B cell is not fully characterized. We have tested the intracellular signaling requirement of these processes using B cells isolated from the spleen of irradiated auto-reconstituted (transitional immature B cells) and untreated mice (mature B cells), respectively. We compared the BCR induced intracellular [Ca2+] transient, protein tyrosine phosphorylation and ERK phosphorylation, furthermore, the activation of Elk-1 and CREB transcription factors. The BCR induced rise of intracellular [Ca2+] did not significantly differ in the two populations, only a slight difference in the late phase of the response was observed. Immature B cells responded with a maximum tyrosine phosphorylation to a five times lower dose of anti-IgM compared to the mature population. Most importantly, we have found a significant difference in the tyrosine phosphorylation of the Gab family adaptor proteins, Gab1/2. In contrast to mature B cells, crosslinking of BCR on immature B cells did not induce tyrosine phosphorylation of Gab2, thus the Gab2-organized signal amplification complex could not be produced. Furthermore, we detected a significant difference in the kinetics of BCR induced ERK, Elk-1 and CREB phosphorylation. In immature B cells, ERK was transiently phosphorylated, ceasing after 120 min, while in mature cells, ERK phosphorylation was sustained. Elk-1 and CREB activation was also transient in immature B cells, followed the kinetics of ERK phosphorylation. The lack of sustained Erk1/2 activation suppresses the transcription factors necessary for the proliferation signal. Since ERK is regulated by the phosphorylated Gab1/2, these data demonstrate that BCR triggered phosphorylation and signal amplification of Gab1/2 is a critical step in a life or death decision of B cells.

FcRn Overexpression in Transgenic Mice Results in Augmented APC Activity and Robust Immune Response with Increased Diversity of Induced Antibodies

Our previous studies have shown that overexpression of bovine FcRn (bFcRn) in transgenic (Tg) mice leads to an increase in the humoral immune response, characterized by larger numbers of Ag-specific B cells and other immune cells in secondary lymphoid organs and higher levels of circulating Ag-specific antibodies (Abs). To gain additional insights into the mechanisms underlying this increase in humoral immune response, we further characterized the bFcRn Tg mice. Our Western blot analysis showed strong expression of the bFcRn transgene in peritoneal macrophages and bone marrow derived dendritic cells; and a quantitative PCR analysis demonstrated that the expression ratios of the bFcRn to mFcRn were 2.6- and 10-fold in these cells, respectively. We also found that overexpression of bFcRn enhances the phagocytosis of Ag-IgG immune complexes (ICs) by both macrophages and dendritic cells and significantly improves Ag presentation by dendritic cells. Finally, we determined that immunized bFcRn mice produce a much greater diversity of Ag-specific IgM, whereas only the levels, but not the diversity, of IgG is increased by overexpression of bFcRn. We suggest that the increase in diversity of IgG in Tg mice is prevented by a selective bias towards immunodominant epitopes of ovalbumin, which was used in this study as a model antigen. These results are also in line with our previous reports describing a substantial increase in the levels of Ag-specific IgG in FcRn Tg mice immunized with Ags that are weakly immunogenic and, therefore, not affected by immunodominance.

Grb2 associated binder 2 couples B-cell receptor to cell survival

B-cell fate during maturation and the germinal center reaction is regulated through the strength and the duration of the B-cell receptor signal. Signaling pathways discriminating between apoptosis and survival in B cells are keys in understanding adaptive immunity. Gab2 is a member of the Gab/Dos adaptor protein family. It has been shown in several model systems that Gab/Dos family members may regulate both the anti-apoptotic PI3-K/Akt and the mitogenic Ras/MAPK pathways, still their role in B-cells have not been investigated in detail. Here we studied the role of Gab2 in B-cell receptor mediated signaling. We have shown that BCR crosslinking induces the marked phosphorylation of Gab2 through both Lyn and Syk kinases. Subsequently Gab2 recruits p85 regulatory subunit of PI3-K, and SHP-2. Our results revealed that Ig-alpha/Ig-beta, signal transducing unit of the B-cell receptor, may function as scaffold recruiting Gab2 to the signalosome. Overexpression of Gab2 in A20 cells demonstrated that Gab2 is a regulator of the PI3-K/Akt but not that of the Ras/MAPK pathway in B cells. Accordingly to the elevated Akt phosphorylation, overexpression of wild-type Gab2 in A20 cells suppressed Fas-mediated apoptosis, and enhanced BCR-mediated rescue from Fas-induced cell death. Although PH-domain has only a stabilizing effect on membrane recruitment of Gab2, it is indispensable in mediating its anti-apoptotic effect.

Coadministration of antigen-conjugated and free CpG: effects of in vitro and in vivo interactions in a murine model.

CpG oligodeoxynucleotides (CpG) are widely studied as promising adjuvants in vaccines against a range of diseases including infection, cancer or allergy. Conjugating antigen to CpG has been shown to potentiate the adjuvant effect via enhancing antigen uptake and danger signaling by the very same cell. In the present study, using biotinylated CpG and streptavidin as a model system, we demonstrate that CpG motif containing free and antigen-conjugated oligonucleotides do not compete in terms of cell activation via TLR9, but do compete for cellular uptake. Antigen-conjugated CpG enhances cellular association and uptake of the antigen by antigen-presenting cells (APC) and T cells. Free CpG efficiently competes with antigen-CpG conjugates in BMDC and T cells, but shows weak or no competition in B cells that have higher TLR9 expression. Vaccination with antigen-conjugated CpG or with a mixture of antigen and CpG elevates the level of antigen-specific antibodies but co-administration of CpG-antigen conjugates and free CpG adversely effects immunogenicity. These observations may help optimize CpG-based vaccine formulation.

Developmental differences in B cell receptor-induced signal transduction

We have compared early signaling events at various stages of B cell differentiation using established mouse cell lines. Clustering of pre-B cell antigen receptor (BCR) or BCR induced the tyrosine phosphorylation of various proteins in all cells, although the phosphorylation pattern differed. In spite of the pre-BCR-induced tyrosine phosphorylation, we could not detect an intracellular Ca(2+) signal in pre-B cells. However, co-clustering of the pre-BCR with CD19 did induce Ca(2+) mobilization. In contrast to the immature and mature B cells, where the B cell linker protein (BLNK) went through inducible tyrosine phosphorylation upon BCR clustering, we observed a constitutive tyrosine phosphorylation of BLNK in pre-B cell lines. Both BLNK and phospholipase C (PLC)gamma were raft associated in unstimulated pre-B cells, and this could not be enhanced by pre-BCR engagement, suggesting a ligand-independent PLC gamma-mediated signaling. Further results indicate that the cell lines representing the immature stage are more sensitive to BCR-, CD19- and type II receptors binding the Fc part of IgG (Fc gamma RIIb)-mediated signals than mature B cells.

Differential expression of FCRLA in naïve and activated mouse B cells

FCRLA is an intracellular B cell protein that belongs to the FcR-like family. Using newly generated FCRLA-specific antibodies, we studied the constitutive expression pattern of mouse FCRLA and monitored changes during an immune response and following in vitro B cell activation. All B cell subpopulations examined expressed FCRLA. However, the level of FCRLA expression is determined by the stage of B cell differentiation. Low expression of FCRLA is characteristic of naïve follicular and marginal zone B cells. High expression was detected in a small fraction of activated B cells scattered along migratory pathways in the lymphoid tissues. FCRLA-bright cells could be subdivided into two subpopulations, with high and low/undetectable level of intracellular immunoglobulins, which phenotypically resemble either plasma or memory B cells. High expression of FCRLA in subset(s) of terminally differentiated B-cells suggests that, being an ER protein, FCRLA may participate in the regulation of immunoglobulin assembly and secretion.

Overexpression of bovine FcRn in mice enhances T-dependent immune responses by amplifying T helper cell frequency and germinal center enlargement in the spleen

The neonatal Fc receptor (FcRn) plays key roles in IgG and albumin homeostasis, maternal IgG transport, and antigen presentation of IgG-opsonized antigens. Previously, we reported that transgenic (Tg) mice that overexpress the bovine FcRn (bFcRn) have augmented T-dependent humoral immune response with increased IgG protection, higher level of antigen-specific antibodies, greater number of antigen-specific B cells, and effective immune response even against weakly immunogenic epitopes. In the current study, we analyzed the localization of the bFcRn in secondary lymphoid organs, and focused to demonstrate the in vivo impact of its overexpression in the spleen on the course of antibody production. bFcRn was highly expressed by red pulp macrophages and marginal zone macrophages in the spleen and by subcapsular sinus macrophages and macrophage-like cells in the interfollicular areas in the lymph node cortex. We also demonstrated that splenic dendritic cells of Tg mice express bFcRn and intraperitoneal immunization of these mice with T-dependent antigens led to more than threefold increase in the number of antigen-specific activated T helper cells with increased size and numbers of germinal centers compared to wild-type controls. bFcRn expression in splenic B cells was also detected and that may also contribute to the enhanced B cell activation. Finally, we demonstrated that these Tg mice developed efficient immune response against very low dose of antigen, reflecting another important practical benefit of these Tg mice.

T-bet is a new synergistic meeting point for the BCR and TLR9 signaling cascades

The importance of the BCR and TLR9 in autoimmunity and in the production of auto‐antibodies is well established but the underlying molecular mechanism still needs to be determined. Here, we aim to characterize the BCR‐TLR9 cross‐talk by its effect on T‐bet, as T‐bet is activated and regulated by both receptors and has an important role in class‐switching to pathological IgG2a in mice. Using primary mouse B cells, we demonstrate that T‐bet expression is synergistically elevated by the cross‐talk between the BCR and TLR9. To test the effect of this synergy on IgG2a‐switching, the levels of switched B cells were checked by functional tests. We found that BCR costimulation had no additional effect on TLR9‐induced IgG2a expression, however the expression of Rad51 was synergistically increased. To check the biological significance of the synergy, we compared T‐bet expression in B cells from healthy and collagen‐induced arthritis mice but no differences were found. Taken together, we demonstrate here that signaling cascades driven by the BCR and TLR9 have a newly identified meeting point at T‐bet. The two cascades act synergistically on T‐bet; however additional signals may be needed to induce prolonged functional responses such as class‐switch recombination.