Supplementary Material for: T Cell Epitopes of the Timothy Grass Pollen Allergen Phl p 5 of Mice and Men and the Detection of Allergen-Specific T Cells Using Class II Ultimers

<i>Background:</i> Knowledge of allergen-specific T cell epitopes is a prerequisite not only for therapeutic approaches but also for elucidating immunological mechanisms of type I allergy. Ex vivo detection of allergen-specific T cells using class II tetramer technology has become an important tool for investigating immune responses in atopic and healthy individuals. <i>Methods:</i> Using ³H-thymidine incorporation assays, T cell epitopes specific for the major timothy grass pollen allergen Phl p 5.0101 were mapped in 11 allergic donors and two different mouse strains. Different protocols for expansion/restimulation of T cells from the blood of allergic donors and detection of allergen-specific T cells by Class II Ultimer staining were evaluated. <i>Results:</i> We identified several new Phl p 5.0101 class II T cell epitopes in allergic patients and confirmed previously published ones. Additionally, we discovered the major T cell epitopes in BALB/c and C57BL/6 mice. Using a novel Class II Ultimer, we detected epitope-specific T cells expanded from the blood of an allergic donor. <i>Conclusions:</i> Epitope mapping of Phl p 5.0101 revealed an immunodominant epitope in BALB/c and C57BL/6 mice and an immunodominant region in humans (amino acids 259–282), which was recognized by 8 out of 11 allergic donors. Detection of Phl p 5-specific T cells was demonstrated using a Class II Ultimer specific for epitope 196–210. Successful detection of ultimer-positive T cells was strongly dependent on a resting phase after in vitro expansion

Supplementary Material for: Inflammatory Properties and Adjuvant Potential of Synthetic Glycolipids Homologous to Mycolate Esters of the Cell Wall of Mycobacterium tuberculosis

<p>The cell wall of mycobacteria is characterised by glycolipids composed of different classes of mycolic acids (MAs; alpha-, keto-, and methoxy-) and sugars (trehalose, glucose, and arabinose). Studies using mutant <i>Mtb</i> strains have shown that the structure of MAs influences the inflammatory potential of these glycolipids. As mutant <i>Mtb </i>strains possess a complex mixture of glycolipids, we analysed the inflammatory potential of single classes of mycolate esters of the <i>Mtb</i> cell wall using 38 different synthetic analogues. Our results show that synthetic trehalose dimycolate (TDM) and trehalose, glucose, and arabinose monomycolates (TMM, GMM, and AraMM) activate bone marrow-derived dendritic cells in terms of the production of pro-inflammatory cytokines (IL-6 and TNF-α) and reactive oxygen species, upregulation of costimulatory molecules, and activation of NLRP3 inflammasome by a mechanism dependent on Mincle. These findings demonstrate that Mincle receptor can also recognise pentose esters and seem to contradict the hypothesis that production of GMM is an escape mechanism used by pathogenic mycobacteria to avoid recognition by the innate immune system. Finally, our experiments indicate that TMM and GMM, as well as TDM, can promote Th1 and Th17 responses in mice in an OVA immunisation model, and that further analysis of their potential as novel adjuvants for subunit vaccines is warranted.</p