1. Mechanisms of sensitization, disease development and desensitization: towards novel approaches for prevention and therapy Ronald van Ree Academic Medical Center, Amsterdam

2. Immunology: B-cells and antibodies Immunotherapy: mechanisms and monitoring Atopic march: eczema asthma rhinitis asthma? Priorities for allergy/asthma research

3. Exposure to allergen results in: no response? protective response (active tolerance)? sensitization? +/- symptoms? Interaction between allergen and IgE is at the basis of allergy and (extrinsic) asthma. How is the process of B-cell activity towards or away from allergic inflammation regulated? B-cells and antibodies

4. Ig? B-cell DC Th1 Th2 naïve Th IgM B-cell Th1 Th2 no allergy IL4/IL13 IFNγ Th1 Th2 allergy IgE B-cell genetic factors exogenous factors (environment incl. allergens infections, diet, lifestyle)

5. Ig? B-cell DC Th1 Th2 naïve Th IgM B-cell Th1 Th2 no allergy IL4/IL13 IFNγ Th1 Th2 allergy IgE B-cell genetic factors exogenous factors (environment incl. allergens infections, diet, lifestyle) Scheme of a simple Th1-Th2 disbalance to distinguish between allergic and non-allergic has actually been dismissed as being too simple.

6. IgG/A? B-cell DC Th1 and/or Treg Th2 naïve Th IgM B-cell Th1Th2 no allergy IL4/IL13 IFNγ Th1 Th2 allergy IgE B-cell genetic factors exogenous factors (environment incl. allergens infections, diet, lifestyle) Treg no antibody response? IgG? IgA? ? healthy non-atopic? IL10 TGFβ

7. IgG/A? B-cell DC Th1 and/or Treg Th2 naïve Th IgM B-cell Th1Th2 no allergy IL4/IL13 IFNγ Th1 Th2 allergy IgE B-cell genetic factors exogenous factors (environment incl. allergens infections, diet, lifestyle) Treg no antibody response? IgG? IgA? ? healthy non-atopic? IL10 TGFβ Questions and Discrepancies healthy immune response against allergens: Treg/IL10/TGFβ or Th1/IFNγ or both? do B-cells of non-atopic individuals ignore allergens or produce IgG/IgA antibodies? Is this different for low-exposure allergens (e.g. pollen and mite) and high- exposure allergens (e.g. cat, bee venom and occupational allergens)?

8. IgG1 B-cell DC Th2 naïve Th IgM B-cell no allergy IL4/IL13 IL10 Th1 Th2 allergy IgE B-cell genetic factors exogenous factors timing/dose/context of allergen exposure modTh2 IgG4 B-cell Th1 modTh2 IgE? B-cell healthy atopic (or atopic treated by immunotherapy) and non-atopic?

9. IgG1 B-cell DC Th2 naïve Th IgM B-cell no allergy IL4/IL13 IL10 Th1 Th2 allergy IgE B-cell genetic factors exogenous factors timing/dose/context of allergen exposure modTh2 IgG4 B-cell Th1 modTh2 IgE? B-cell healthy atopic (or atopic treated by immunotherapy) and non-atopic? Questions and Discrepancies is a modified Th2 response in atopic individuals IgG4 without IgE or with (little/harmless) IgE? is there an early-life window of opportunity for inducing the protective modified Th2? why are IgG4 responses to allergens higher in atopics than in non-atopics? atopic: predisposition to produce IgE and high IgG4? non-atopic: no tendency to develop IgE and only low IgG4? atopic background seems to be less important for IgE/IgG4 production in case of high exposures: cats, bee venom, occupational allergens, parasites.

10. Increased exposure to cat allergen Allergy (sensitization) IgG (exposure / protection) Lancet 2001 ; Tom Platts-Mills et al. High early exposure to cat allergen is protective

11. Why are IgE responses always so low compared to IgG responses? Half-life of IgE is very short but this can not explain the 1000-fold difference in serum titers. A major cause most likely is the poor generation of memory B-cells for IgE caused by inefficient processing of mRNA for membrane IgE. Circulating IgE is derived from long-lived plasma cells hiding in survival niches like the bone-marrow and inflammatory sites. Two situations: low allergen exposure (e.g. pollen/mite) i.e. a weak Th2 response that will not effectively induce a germinal center necessary for induction of memory B-cells high allergen exposure (e.g. cat, bee venom) i.e. a strong Th2 response that will a generate mature germinal center facilitating induction of memory

12. Low exposure situation: no memory only plasma cells plasma cells Atopics respond with all three but IgG is not protective under these conditions Non-atopics only make a little IgG / are hypo-responsive overall compared to atopics. From: Aalberse RC et al. J Allergy Clin Immunol. 2004 May;113(5):983-6.

13. Poor expression of membrane form of IgE favors apoptosis resulting in poor memory High exposure situation: memory generation for IgG but not for IgE plasma cells This also reflects the situation during allergen-specific immunotherapy Same specificity From: Aalberse RC et al. J Allergy Clin Immunol. 2004 May;113(5):983-6.

14. For primary prevention it is of the greatest importance to investigate the dose-response relation between allergen and (the quality) of the immune response. The window of opportunity is of great importance. A high-dose protective effect as observed for cat has so far not been found for house dust mite. For food allergens this is even more debated. The outcome has very significant public health impact. Promote cats and peanut butter sandwiches early on or not?

15. What is needed to study the dose-response relation between allergen exposure and (the quality of) the immune response? Analysis of (existing) birth cohort samples for IgG and IgA responses. In most cases only IgE has been measured. Multiple time points in first year of life are required. Multiplex systems now allow in detailed analysis with limited sample. Analysis of (existing) birth cohort samples for mRNA profiling In vitro cellular analyses to study the process of immune skewing, preferably in an allergen specific fashion. Mouse models that go beyond the ovalbumin model by using real allergens.

16. plasma B-cell memory B-cell Breg immature B-cell exogenous factors (environment incl. allergens infections, diet, lifestyle) genetic factors Regulatory B-cells, a missing link? CD1d hi CD5+ major source of IL10 promote Treg development In several mouse models of auto-immune diseases an anti-inflammatory (protective) role has been established for regulatory B-cells. Do they play a protective role in allergy/asthma. If so, how can they be promoted?

17. The quality of IgE antibodies Do they always translate into clinical allergy? No There are several examples of IgE responses that are without clinical relevance, i.e. the ones observed during parasite infections, the ones directed to plant glycans and the ones observed during successful immunotherapy. Tregs or Th1 cells can not explain the lack of biological activity. The explanation can also not (always) simply be found in a protective effect of IgG4 (blocking antibodies). How is the biological activity of IgE assessed?

18. lactic acid treatment: removal of IgE incubation with serum: sensitization with IgE basophils from non-atopic donor stripped basophils from non-atopic donor basophils re-sensitized with IgE under study Stripped basophil protocol

19. Allergen-specific IgE from parasite-infected children lacks activity. This is not explained by high allergen-specific IgG4 titers What explains the poor biological activity?

20. IgE antibodies against plant N-glycans have the same poor biological activity An explanation needs to be found why some IgE antibodies are poor inducers of mediator release, i.e. without clinical relevance.

21. What is different about these type of IgE antibodies? Lower affinity? Something else? Is this also observed during immunotherapy where IgE antibodies are persistent but skin reactivity clearly decreases. Are these IgE antibodies the same as before the start of therapy or are they (partly) newly formed IgE antibodies.

22. B-cells and antibodies have not received the attention they deserve compared to T-cells. They do however produce the antibodies that cause the disease!! Reasons to study IgE/IgG/IgA antibody responses: They are a good read-out of the nature of the immune response It needs to be established when/why they are clinically relevant, i.e. either disease-inducing (IgE) or protective (IgG/IgA). Is induction of IgG/IgA before IgE a protection against future sensitization? What is the difference between IgE from direct (μ to ε) and from indirect (μ to ε via γ4)? Reasons for studying B-cells: What determines the quality of IgE antibodies? What is the role of Bregs in allergy/asthma? How could we target persistent plasma B-cells for IgE?

23. Immunotherapy: mechanism and monitoring Many of the same questions need to be answered. Blocking IgG4 antibodies? What is the difference between IgE before and after therapy? Th1 skewing? Tregs? Or perhaps Bregs? Expression profiling / proteomics for biomarker search Investigator-initiated trials needed. Companies are too small to set up trials that include in depth immunological analyses. Better insight in mechanism is a prerequisite for improvement of therapy. Primary outcome (natural exposure) is an ill-controlled disaster! Towards pollen chamber monitoring in season.