Highly regulated activation of B cells is required for the production of specific antibodies necessary to provide protection from pathogen infection. This process is initiated by specific recognition of antigen through the B cell receptor (BCR), leading to early intracellular signaling followed by the late recruitment of T cell help. In this study we demonstrate that specific BCR uptake of CD1d-restricted antigens represents an effective means of enhancing invariant natural killer T (iNKT)-dependent B cell responses in vivo. This mechanism is effective over a wide range of antigen affinities but depends on exceeding a tightly regulated avidity threshold necessary for BCR-mediated internalization and CD1d-dependent presentation of particulate antigenic lipid. Subsequently, iNKT cells provide the help required for stimulating B cell proliferation and differentiation. iNKT-stimulated B cells develop within extrafollicular foci and mediate the production of high titers of specific IgM and early class-switched antibodies. Thus, we have demonstrated that in response to particulate antigenic lipids iNKT cells are recruited for the assistance of B cell activation, resulting in the enhancement of specific antibody responses. We propose that such a mechanism may operate to potentiate adaptive immune responses against pathogens in vivo.innate ͉ B cell activation ͉ antigen affinity

IntroductionB lymphocytes comprise a critical component of the adaptive immune system, producing antibodies necessary for both immediate and long-lived protection from pathogenic infection. B cells encounter antigen (Ag) within specialized tissues, such as the spleen or lymph nodes, known as secondary lymphoid organs. After Ag-induced activation, B cells can follow 2 distinct pathways of differentiation. In extrafollicular (EF) regions of the secondary lymphoid organs, including the medullary cords of lymph nodes or the red pulp of the spleen, activated B cells give rise to short-lived plasma cells (PCs) responsible for the rapid production of lowaffinity antibodies. 1 Alternatively, activated B cells can form germinal centers (GCs) where they undergo affinity maturation, resulting in differentiation into either long-lived PCs, which maintain the immune response by secreting high-affinity antibodies, or into long-lasting memory cells. [2][3][4] Though at this stage the precise combination of factors responsible for determining the outcome of B-cell differentiation remains unclear, it has been proposed that Ag binding strength can influence the fate of activated B cells. As such, high-affinity Ag favors differentiation into EF PCs, while lower affinity Ag induces the preferential entry of B cells into GCs. 5 B cells recognize and respond to Ag through surface B-cell receptors (BCRs) that possess a remarkably wide range of potential affinities. [6][7][8][9] The BCR is a heterotrimeric complex containing a membrane immunoglobulin (Ig) responsible for extracellular Ag binding, together with an Ig␣/␤ sheath required for mediating intracellular signaling through its immunoreceptor tyrosine-based activation motifs (ITAMs). 10 Engagement of the BCR by specific Ag induces signaling that results in a variety of cellular processes, 11 including the internalization of accumulated Ag. The subsequent presentation of processed antigenic peptide-loaded major histocompatibility complex-II (MHC-II) molecules on the surface of the B-cell membrane stimulates the recruitment of specific helper CD4 ϩ T cells, required for the full activation of B cells. 12,13 As the process of BCR-mediated internalization provides a mechanism whereby Ag can access intracellular compartments, it would seem likely that several other receptors expressed within the B cell could potentially participate in shaping the outcome of B-cell activation in a similar manner.In addition to the BCR, B cells express germline encoded Toll-like receptors (TLRs) more usually associated with innate immune cell function. 14 The specificity of TLRs for pathogenassociated ligands is dependent on either the restricted expression of the ligand within bacteria or viruses, or the atypical cellular location of more universally expressed ligands. 15 The expression of TLR9 in humans is restricted to plasmacytoid dendritic cells (pDCs) and B cells. 16,17 While the majority of TLR9 appears to be concentrated in the endoplasmic reticulum, 18,19 TLR9 has also been observed within smal...

BackgroundGrass pollen is one of the most important sources of respiratory allergies worldwide.ObjectiveThis study describes the development of a grass pollen allergy vaccine based on recombinant hypoallergenic derivatives of the major timothy grass pollen allergens Phl p 1, Phl p 2, Phl p 5, and Phl p 6 by using a peptide-carrier approach.MethodsFusion proteins consisting of nonallergenic peptides from the 4 major timothy grass pollen allergens and the PreS protein from hepatitis B virus as a carrier were expressed in Escherichia coli and purified by means of chromatography. Recombinant PreS fusion proteins were tested for allergenic activity and T-cell activation by means of IgE serology, basophil activation testing, T-cell proliferation assays, and xMAP Luminex technology in patients with grass pollen allergy. Rabbits were immunized with PreS fusion proteins to characterize their immunogenicity.ResultsTen hypoallergenic PreS fusion proteins were constructed, expressed, and purified. According to immunogenicity and induction of allergen-specific blocking IgG antibodies, 4 hypoallergenic fusion proteins (BM321, BM322, BM325, and BM326) representing Phl p 1, Phl p 2, Phl p 5, and Phl p 6 were included as components in the vaccine termed BM32. BM321, BM322, BM325, and BM326 showed almost completely abolished allergenic activity and induced significantly reduced T-cell proliferation and release of proinflammatory cytokines in patients' PBMCs compared with grass pollen allergens. On immunization, they induced allergen-specific IgG antibodies, which inhibited patients' IgE binding to all 4 major allergens of grass pollen, as well as allergen-induced basophil activation.ConclusionA recombinant hypoallergenic grass pollen allergy vaccine (BM32) consisting of 4 recombinant PreS-fused grass pollen allergen peptides was developed for safe immunotherapy of grass pollen allergy.

Injections of BM32 induced allergen-specific IgG, improved clinical symptoms of seasonal grass pollen allergy, and were well tolerated.

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Background-Increasing evidence suggests that the low-affinity receptor for IgE, CD23, plays an important role in controlling the activity of allergen-specific T cells through IgE-facilitated allergen presentation.

Background-Recent data obtained in mouse models have initiated a controversy whether basophils are the key antigen-presenting cells (APCs) in allergy. Here, we investigate whether basophils are of importance for the presentation of allergen and the induction of T cell proliferation in allergic patients.

Key Points• Cell autonomous BCR interactions and interactions with low-affinity autoantigens drive leukemia development in an in vivo model of CLL.• BCR signals induced by binding to external antigen can increase the aggressiveness of CLL.Chronic lymphocytic leukemia (CLL) is a common B-cell malignancy characterized by a highly variable course and outcome. The disease is believed to be driven by B-cell receptor (BCR) signals generated by external antigens and/or cell-autonomous BCR interactions, but direct in vivo evidence for this is still lacking. To further define the role of the BCR pathway in the development and progression of CLL, we evaluated the capacity of different types of antigen/BCR interactions to induce leukemia in the Em-TCL1 transgenic mouse model. We show that cell autonomous signaling capacity is a uniform characteristic of the leukemia-derived BCRs and represents a prerequisite for CLL development. Low-affinity BCR interactions with autoantigens generated during apoptosis are also positively selected, suggesting that they contribute to the pathogenesis of the disease. In contrast, high-affinity BCR interactions are not selected, regardless of antigen form or presentation. We also show that the capacity of the leukemic cells to respond to cognate antigen correlates inversely with time to leukemia development, suggesting that signals induced by external antigen increase the aggressiveness of the disease. Collectively, these findings provide in vivo evidence that the BCR pathway drives the development and can influence the clinical course of CLL. (Blood. 2015;125(10):1578-1588