TLR and B cell receptor signals to B cells differentially program primary and memory Th1 responses to Salmonella enterica.

Protective Th1 responses to Salmonella enterica do not develop in the absence of B cells. Using chimeric mice, we dissect the early (innate) and late (cognate) contributions of B cells to Th programming. B cell-intrinsic MyD88 signaling is required for primary effector Th1 development, whereas Ag-specific BCR-mediated Ag presentation is necessary for the development of memory Th1 populations. Programming of the primary T cell response is BCR/B cell MHC II independent, but requires MyD88-dependent secretion of cytokines by B cells. Chimeras in which B cells lack IFN-gamma or IL-6 genes make impaired Th1 or Th17 responses to Salmonella.

Cutting edge: HLA-E binds a peptide derived from the ATP-binding cassette transporter multidrug resistance-associated protein 7 and inhibits NK cell-mediated lysis.

HLA-E is an MHC class Ib molecule that binds nonamer peptides derived from the leader sequence of MHC class 1a molecules and is the major ligand for CD94/NKG2 receptors found on NK and T cells. Using the MHC class Ia-null cell line 721.221, we find that surface HLA-E increases following heat shock at 42 degrees C and NK cell-mediated lysis is inhibited using heat-stressed 721.221 targets. We have used mass spectrometry to identify and sequence a novel peptide from HLA-E following heat shock, ALALVRMLI, derived from the transmembrane domain of the human ATP-binding cassette protein, multidrug resistance-associated protein, MRP7. Pulsing 721.221 targets with synthetic MRP7 peptide results in strong inhibition of NK cell-mediated lysis that is reversible using anti-CD94 and anti-class I mAbs. This report is the first to identify a non-MHC leader inhibitory peptide bound to HLA-E and provides insight into the immunoregulatory role of HLA-E during cell stress.

Adaptive immune responses are dispensable for isolated lymphoid follicle formation: antigen-naive, lymphotoxin-sufficient B lymphocytes drive the formation of mature isolated lymphoid follicles.

Isolated lymphoid follicles (ILFs) are recently appreciated members of the mucosal immune system. The architecture, composition, and inducible nature of these structures indicates that these structures are tertiary lymphoid structures. The process leading to the formation of tertiary lymphoid structures, lymphoid neogenesis, has been observed in a number of inflammatory and autoimmune conditions. Given this association, there is considerable interest in identifying the factors promoting lymphoid neogenesis, and understanding the steps in this process. Using murine ILF formation as a model, we have examined the roles of different cellular sources of lymphotoxin (LT) and the adaptive immune response in lymphoid neogenesis. In this study, we report that, although other cellular sources of LT may supplant B lymphocytes in the formation of immature ILFs (loosely organized clusters of B lymphocytes), LT-sufficient B lymphocytes are required for the progression of immature ILFs to mature ILFs (organized lymphoid aggregates with a follicle-associated epithelium). ILF formation occurs in the absence of T lymphocytes and Ag-specific B lymphocyte responses, and ILF B lymphocytes express elevated levels of LT in the absence of antigenic stimulation. Consistent with a role for chemokines inducing LT expression in Ag-naive B lymphocytes, and a chemokine-driven positive-feedback loop driving mature ILF formation, mature ILFs express elevated levels of B lymphocyte chemoattractant in the absence of Ag-specific B lymphocyte stimulation. These observations indicate that ILFs contain Ag-naive lymphocytes, and suggest that events occurring within ILFs shape subsequent immune responses mediated by these lymphocytes.

Distinct expression profiles of the peripheral cannabinoid receptor in lymphoid tissues depending on receptor activation status.

Using two distinct anti-CB2 receptor Abs, we investigated the expression patterns of the peripheral cannabinoid receptor CB2 in human secondary lymphoid organs. Immunohistochemical analysis using an N-terminal specific anti-CB2 Ab revealed high protein expression in the germinal centers (GCs) of secondary follicles. A C-terminal specific anti-CB2 Ab, which only recognizes a nonphosphorylated inactive receptor, showed positivity in the mantle zones (MZs) and marginal zones (MGZs) of the secondary follicles where resting cells reside, and in the primary follicles. In contrast, no positivity was observed in GCs using the C-terminal Ab, suggesting that active CB2 receptors are mainly present on cells in the GCs. Dual immunohistochemical analysis revealed that B lymphocytes express the CB2 protein abundantly. In contrast to B cells in the MZ or MGZ, CB2-expressing cells in the GCs coexpress the costimulatory membrane protein CD40, which is mainly expressed in the GCs and at very low levels in the MZs and MGZs and the proliferation marker Ki-67. Using the human Raji B cell line as a model, we demonstrate in a transwell assay that moderate migration occurs upon stimulation of the CB2 receptor with the endocannabinoid 2-arachidonoylglycerol, which is enhanced by CD40 costimulation. Our findings, that GC-related cells express active CB2 and that CB2-dependent migration requires CD40 costimulation, suggest that CB2 is involved in B cell activation.

A chimeric multi-human epidermal growth factor receptor-2 B cell epitope peptide vaccine mediates superior antitumor responses.

Immunotherapeutic approaches to cancer should focus on novel undertakings that modulate immune responses by synergistic enhancement of antitumor immunological parameters. Cancer vaccines should preferably be composed of multiple defined tumor Ag-specific B and T cell epitopes. To develop a multiepitope vaccine, 12 high ranking B cell epitopes were identified from the extracellular domain of the human epidermal growth factor receptor-2 (HER-2) oncoprotein by computer-aided analysis. Four novel HER-2 B cell epitopes were synthesized as chimeras with a promiscuous T cell epitope (aa 288-302) from the measles virus fusion protein (MVF). Two chimeric peptide vaccines, MVF HER-2(316-339) and MVF HER-2(485-503) induced high levels of Abs in outbred rabbits, which inhibited tumor cell growth. In addition, Abs induced by a combination of two vaccines, MVF HER-2(316-339) and MVF HER-2(628-647) down-modulated receptor expression and activated IFN-gamma release better than the individual vaccines. Furthermore, this multiepitope vaccine in combination with IL-12 caused a significant reduction (p = 0.004) in the number of pulmonary metastases induced by challenge with syngeneic tumor cells overexpressing HER-2. Peptide Abs targeting specific sites in the extracellular domain may be used for exploring the oncoprotein's functions. The multiepitope vaccine may have potential application in the treatment of HER-2-associated cancers.

B cell positive selection by soluble self-antigen.

It is well established that autoreactive B cells undergo negative selection. This stands in paradox with the high frequency of so-called natural autoreactive B cells producing low affinity polyreactive autoantibodies with recurrent specificities, suggesting that these B cells are selected on the basis of their autoreactivity. We previously described two transgenic mouse lines (with and without IgD) producing a human natural autoantibody (nAAb) that binds ssDNA and human Fcgamma. In the absence of human IgG, nAAb-transgenic B cells develop normally. By crossing these mice with animals expressing knockin chimeric IgG with the human Fcgamma, we now show that the constitutive expression of chimeric IgG promotes the increase of nAAb-expressing B cells. This positive selection is critically dependent on the presence of IgD, occurs in the spleen, and concerns all mature B cell subsets, with a relative preferential enrichment of marginal zone B cells. These data support the view that soluble self-Ags can result in positive clonal selection.

Antibody to the HNK-1 glycoepitope affects fasciculation and axonal pathfinding in the developing posterior lateral line nerve of embryonic zebrafish.

The HNK-1 glycoepitope, carried by many cell recognition molecules, is present in the developing posterior lateral line nerve and on other primary axons of zebrafish. To elucidate the function of HNK-1 in vivo, the antibody 412 to HNK-1 was injected into zebrafish embryos at 16 h post fertilization (hpf). The injected antibody bound specifically to axons carrying HNK-1. This treatment selectively affected the growth of either one or both posterior lateral line nerves in 39% of the experimental cases (13 of 33 animals), which was significantly more (P<0.0002) than in uninjected, vehicle injected, and non-immune IgG injected controls (1.2% of the animals; one of 85 animals), as assessed at 27 or 33 hpf. Other HNK-1 immunoreactive nerves, such as the ventral motor nerves were unaffected, indicating that antibody binding per se did not interfere with axon growth. The primordium of the posterior lateral line was not affected in its caudal migration and in depositing differentiating neuromasts along the trunk, showing that injections did not retard development and that initial formation of lateral line organs is probably independent of contact with nerve fibers. We suggest that the HNK-1 glycoepitope is an important modulator of embryonic nerve growth.

Engagement of the B-cell antigen receptor by antigen negatively regulates IgE production by antigen-specific B cells.

BACKGROUND: Functional differentiation of B lymphocytes can be regulated at different levels. Such B-cell responses, to a large extent, are under the control of T cells and the release of soluble factors from these cells. Crosslinking of B-cell antigen receptors also can direct B-cell differentiation, although the mechanisms are not well defined and the effects on specific antibody production have not been examined. OBJECTIVE: This study was conducted to determine whether crosslinking of the B-cell antigen receptor by antigen or antibody to the B-cell receptor could modulate specific antibody production, especially IgE production. METHODS: Antigen-specific human B cells were isolated and incubated with antigen or antibody to the B-cell antigen receptor. Specific antibody production was examined, as were surface isotype expression and levels of Ig heavy chain mRNA in T cell-independent cultures. RESULTS: The addition of antigen or anti-IgM antibody enhanced IgM and IgG antibody production in the presence of anti-CD40 and IL-4 but downregulated IgE synthesis. These changes in isotope production paralleled changes in the frequencies of surface Ig+ B cells and Ig mRNA levels. Exogenous IL-6 abolished the antigen-mediated downregulation of IgE production. CONCLUSIONS: These studies indicate that signals delivered by means of the B-cell antigen receptor regulate antibody production in a distinct manner, resulting in enhanced IgM and IgG responses but leading to the inhibition of IgE production. Endogenous IL-6 production by B cells may counter the negative regulatory effects of antigen on IgE synthesis. We suggest that although antigen-mediated T/B-cell interactions play a major role in IgM- and IgG-specific antibody production, antigen-independent interactions may be more effective for T-cell dependent, IL-4-induced IgE production by antigen-specific B cells. Furthermore, these findings provide a basis for further understanding of allergic responses and modulation by immunotherapy, where repeated exposure to large concentrations of allergen, administered by means of injection, enhance IgG responses and downregulate IgE synthesis.

B cell responses to a peptide epitope. VII. Antigen-dependent modulation of the germinal center reaction.

Germinal center responses to two analogous peptides, PS1CT3 and G32CT3, that differ in sequence only at one position within the B cell epitopic region were examined. In comparison with peptide PS1CT3, peptide G32CT3 elicited a poor germinal center response. By demonstrating equal facility of immune complexes with IgM and IgG Ab isotypes to seed germinal centers, we excluded differences in isotype profiles of early primary anti-PS1CT3 and anti-G32CT3 Ig as the probable cause. Quantitative differences in germinal center responses to the two peptides were also not due to either qualitative/quantitative differences in T cell priming or variation in the frequency of the early Ag-activated B cells induced. Rather, they resulted from qualitative differences in the nature of B cells primed. Analysis of early primary anti-PS1CT3 and anti-G32CT3 IgMs revealed that the latter population was of a distinctly lower affinity, implying the existence of an Ag affinity threshold that restricts germinal center recruitment of G32CT3-specific B cells. The impediment in anti-G32CT3 germinal center initiation could be overcome by making available an excess of Ag-activated Th cells at the time of immunization. This resulted in the appearance of a higher affinity population of G32CT3-specific B cells that, presumably, are now capable of seeding germinal centers. These data suggest that the strength of a germinal center reaction generated is Ag dependent. At least one regulatory parameter represents the quality of B cells that are initially primed.

Characterization of CD40 signaling determinants regulating nuclear factor-kappa B activation in B lymphocytes.

CD40 signaling to B cells is important for generating an effective humoral immune response. CD40 ligation leads to B cell activation events such as proliferation, Ig secretion, isotype switching, and up-regulation of cell surface molecules, as well as the generation of memory B cells. Many of these events are dependent upon the ability of CD40 to activate the transcription factor NF-kappa B (NF-kappa B). To define the CD40 signaling components upstream of NF-kappa B activation and the functional consequences downstream of NF-kappa B activation, we examined mouse B cell transfectants expressing wild-type or mutant human CD40. Analysis of CD40 cytoplasmic domain truncation and point mutants defined a 10-amino acid CD40 cytoplasmic signaling determinant required for NF-kappa B activation. A threonine residue at position 234, previously shown to be important for CD40 association with TNF receptor-associated factor 2 (TRAF2), TRAF3, and TRAF5, was not required for NF-kappa B activation. This suggests that in B cells, CD40-induced NF-kappa B activation can occur independently of TRAF2 and TRAF5 association. NF-kappa B activation was independent of the transmembrane domain of CD40, suggesting that it is independent of p23, a molecule that associates with CD40 in a region other than the cytoplasmic domain. Proteasome-dependent inhibitory kappa B alpha (I kappa B alpha) and I kappa B beta degradation occurred downstream of CD40 ligation and preceded CD40-mediated NF-kappa B nuclear translocation. CD40- or pervanadate-mediated I kappa B tyrosine phosphorylation was not detected. NF-kappa B activation correlated with the ability of CD40 to induce Ab secretion and the up-regulation of ICAM-1 and LFA-1. However, NF-kappa B activation was insufficient for CD40-mediated up-regulation of B7-1, Fas, and CD23.