Nasal IL-13 production identifies patients with late phase allergic responses

BACKGROUND: There is limited knowledge on how local cytokine secretion patterns after nasal allergen challenge correlate with clinical symptoms especially with regards to the "late allergic response" (LAR) which occurs in approximately 40-50% of allergic patients. OBJECTIVE: In this study we aimed to characterise the immunological and clinical nasal responses to birch pollen allergen challenge with a special focus on the LAR. METHODS: In this randomised double-blinded placebo-control trial, birch pollen allergic participants were challenged with pollen extract (n=20) or placebo (n=10) on three consecutive days. On days one and three nasal secretions were collected at selected time points over a 24h time course for the measurement of 33 inflammatory mediators. Clinical responses were determined through subjective symptom scores and objective nasal airflow measurements. RESULTS: Provoked participants had significantly greater clinical responses and showed significant increases in tryptase and sST2 within minutes compared to placebo. Eight out of 20 provoked participants displayed high IL-13 levels 2-8 hours after allergen provocation. This group also showed significant changes in clinical parameters, with a secondary drop in nasal airflow measured by peak nasal inspiratory flow and increased symptoms of nasal obstruction which significantly differed from IL-13 non responders at 6 hours. CONCLUSION: IL-13 response status correlates with cytokine and clinical responses in the late phase after allergen provocation

BCR-signalling synergizes with TLR-signalling for induction of AID and immunoglobulin class-switching through the non-canonical NF-κB pathway

By diversifying antibody biological effector functions, class switch DNA recombination has a central role in the maturation of the antibody response. Here we show that BCR-signalling synergizes with Toll-like receptor (TLR) signalling to induce class switch DNA recombination. BCR-signalling activates the non-canonical NF-κB pathway and enhances the TLR-dependent canonical NF-κB pathway, thereby inducing activation-induced cytidine deaminase (AID), which is critical for class switch DNA recombination. Escherichia coli lipopolysaccharide (LPS) triggers dual TLR4/BCR-signalling and induces hallmarks of BCR-signalling, including CD79a phosphorylation and Ca2+ mobilization, and activates both the NF-κB pathways to induce AID and class switch DNA recombination in a PI(3)K p85α-dependent fashion. CD40-signalling activates the two NF-κB pathways to induce AID and class switch DNA recombination independent of BCR-signalling. Finally, dual BCR/TLR-engaging NP–lipopolysaccharide effectively elicits class-switched NP-specific IgG3 and IgG2b in mice. Thus, by integrating signals of the non-canonical and canonical NF-κB pathways, BCR and TLRs synergize to induce AID and T-cell-independent class switch DNA recombination

Type I IFN enhances follicular B cell contribution to the T cell–independent antibody response

Humoral immunity to viruses and encapsulated bacteria is comprised of T cell–independent type 2 (TI-2) antibody responses that are characterized by rapid antibody production by marginal zone and B1 B cells. We demonstrate that toll-like receptor (TLR) ligands influence the TI-2 antibody response not only by enhancing the overall magnitude but also by skewing this response to one that is dominated by IgG isotypes. Importantly, TLR ligands facilitate this response by inducing type I interferon (IFN), which in turn elicits rapid and significant amounts of antigen-specific IgG2c predominantly from FO (follicular) B cells. Furthermore, we show that although the IgG2c antibody response requires B cell–autonomous IFN-α receptor signaling, it is independent of B cell–intrinsic TLR signaling. Thus, innate signals have the capacity to enhance TI-2 antibody responses by promoting participation of FO B cells, which then elaborate effective IgG anti-pathogen antibodies

How B cell receptors and Toll-like receptors collaborate in shaping B cell responses

Antigen recognition by B cells results in their activation followed by specific antibody production. These events are initiated by antigen binding to their surface B cell receptors (BCR) which triggers both signalling and internalization of the receptor bound antigen to the endosome. However B cells also express features of the innate immune system such as Toll like receptors (TLRs), that can be located either on the surface of the cell or intracellularly where they recognize bacterial and viral nucleic acids. Engagement of these receptors within B cells is associated with enhancement of humoral responses. The aim of my PhD project was to investigate how endosomal TLR ligands in a particulate form could gain access to their intracellular receptors in the B cell and which impact the subsequent TLR engagement had on B cell fate. To achieve this, I directly linked both antigen and TLR9 ligand to particulates. Immunisation of mice with those particulates resulted in enhanced specific antibody titers compared to stimulation with particulate antigen alone. To dissect the underlying mechanism, I employed transgenic B cells bearing BCR specificity for the same antigen and stimulated them with particulate antigen-TLR9 ligand conjugates. Particulate TLR9 ligand could not gain access to its receptor within B cells via unspecific macropinocytosis and instead depended on BCRmediated internalization. Subsequent engagement of intracellular TLR9 by its ligand present in the conjugates resulted in B cell activation and proliferation, followed by differentiation into plasma cells and antigen specific antibody secretion. The uptake of the antigen-TLR9 ligand particulates both in vitro and in vivo depended on the affinity of the antigen once a defined threshold required for internalization was surpassed. The extent of plasma cell differentiation however could be modulated by the amount of TLR9 ligand present on the particulates. Thus I observed that direct linking of antigen and TLR ligand resulted in PC differentiation through antigen specific BCR mediated internalization and subsequent TLR engagement. This reveals a mechanism that may operate during the initiation of a primary immune response

Poor association of allergenspecific antibody, T and Bcell responses revealed with recombinant allergens and a CFSE dilutionbased assay

Background The adaptive immunity underlying allergy comprises two components, the allergenspecific antibody (i.e. IgE, IgG) and the Tcell response. These two components are responsible for different disease manifestations and can be targeted by different therapeutic approaches. Here, we investigated the association of allergenspecific antibody and T as well as Bcell responses in pollenallergic patients using recombinant (r) major birch pollen allergen rBet v 1 and major timothy grass pollen allergen rPhl p 5 as defined antigens. Methods Allergenspecific IgE and IgG antibody responses were determined by ELISA, and allergenspecific T and Bcell responses were measured in peripheral blood mononuclear cells using a carboxyfluoresceindiacetatesuccinimidylester (CFSE) dilution assay. Results CFSE staining in combination with Tcell and Bcellspecific gating allowed discriminating between allergenspecific Tcell and Bcell responses. Interestingly, we identified patients where mainly T cells and others where mainly B cells proliferated in response to allergen stimulation. No association between the level of allergenspecific Ig responses and B or Tcell proliferation was observed. Conclusion Purified recombinant allergens in conjunction with CFSE staining allow the dissection of allergenspecific B and Tcell responses. The dissociation of allergenspecific antibody, and B and Tcell responses may explain the occurrence of selective IgE and Tcellmediated manifestations of allergic inflammation and may be important for the development of diagnostic and therapeutic strategies selectively targeting B cells and T cells.(VLID)484590

B cell receptor-mediated uptake of CD1d-restricted antigen augments antibody responses by recruiting invariant NKT cell help in vivo.

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