Our study demonstrates that presentation of HLA-DR associated peptides was altered upon nitration of Bet v 1. Nitration resulted in a 2.9-fold increased number of identified peptide clusters, a 7.2-fold increase in the overall number of peptide length variants and a 12.2-fold increase in the copy number of identified peptides derived from major birch pollen allergen. An increase in allergen-derived peptide presentation was observed not only for sequence stretches containing tyrosine residues but also in regions devoid of tyrosine. This indicates a general change in uptake and/or processing of the Bet v 1 nitro protein.
There could be several explanations for the increase in the presentation of Bet v 1 nitro derived peptides; 1) an enhanced uptake of the allergen by the DCs, which could be due to multimerization of the allergen. Consistently, for Bet v 1 nitro an elevated tendency to form dimers and trimers was observed using high performance size exclusion chromatography and Western blot analysis (data not shown). 2) there are membrane bound receptors recognizing nitrotyrosine; this would facilitate the uptake of the nitrated allergen, and/or alter the processing of the allergen within endolysosomal compartments of DCs. 3) nitration of tyrosine residues has the potential to alter the sterical properties and interaction with neighboring amino acids, resulting in changes of the protein conformation and ultimately altered antigen processing. 4) nitrotyrosine residues could render the allergen either more or less susceptible to protease activity of degrading enzymes without affecting the processing and presentation of unmodified proteins 
. A decrease in the stability of nitrated allergens was confirmed in a recent study on food allergy. In this study, the nitrated allergens administered orally in a mouse model had a reduced allergenicity and were more easily digested. In contrast, intravascular injection of nitrated food proteins did increase their allergenicity 
. Our data show that the average length of allergen derived peptides differed only marginally between samples from DCs loaded with unmodified allergen (22.34 amino acids) or samples from DCs loaded with nitrated allergen (22.98 amino acids). However, nitration could still affect the kinetics of nitrated Bet v 1 degradation, consequently leading to a change in the presentation kinetics of Bet v 1-derived peptides and eventually the peptide profile which is presented to the T-cells.
Which of the delineated processes finally contributes to the increase of Bet v 1 nitro derived peptide presentation remains unknown and the interplay seems to be complex. Noteworthy, the nitration of human serum albumin did not lead to an enhanced peptide presentation (data not shown); suggesting that nitration per se does not result in enhanced peptide presentation of the nitrated protein. Thus, the impact of nitration on antigen presentation also seems to depend on the properties of the protein itself. However, formally we cannot rule out that the grade of nitration impacts on antigen presentation as well.
The identification of Bet v 1 derived HLA-DR associated peptides led to the question, whether these peptides would be recognized by T cell receptors of T lymphocytes and if this would result in activation and proliferation of the T lymphocytes. For this purpose PBMCs loaded with Bet v 1 nitro were assessed for their capacity to stimulate Bet v 1 specific T cell lines. In response to Bet v 1 nitro, proliferation was significantly higher as compared to unmodified Bet v 1 using a concentration of 1.25 µg/ml protein. Increasing the protein concentration did not in result in higher proliferation and the impact of nitration was lower. Altogether, our data showed that nitration not only enhanced presentation of Bet v 1 derived HLA-DR associated peptides on DCs, but also had an impact on T cell activation.
It has been hypothesized that nitration of autologous proteins may contribute to autoimmunity 
; additionally, the fact that nitration occurs in inflamed tissue should be taken into account. Nitration of tyrosine residues may have evolved as a strategy to intensify immune responses against foreign proteins derived from viruses or bacteria, while a possible contribution to autoimmunity had to be accepted as an unfortunate side effect. Improved presentation of pathogen derived nitrated peptides may in contrast be beneficial to the host. Tyrosine nitration could also be seen as a danger signal – a type of stimulus which is thought to play an important role in the regulation of immune responses. Airborne allergens bearing nitro-tyrosine mimic nitrated foreign proteins present in inflamed tissue, which may explain our findings that nitration of allergens intensifies the presentation of allergen derived HLA-DR associated peptides. Previous studies have shown increased immunogenicity of Bet v 1 nitro compared to Bet v 1 
: Sera from patients with birch pollen allergy contain higher titers for IgE against Bet v 1 nitro compared to Bet v1; the reactivity against Bet v 1 nitro cannot be fully removed by absorption with normal Bet v 1, indicating a specific recognition of the nitrated allergen. The same study showed that nitrated Bet v 1 and nitrated Ovalbumin were more potent allergens compared to their unmodified forms when tested in mouse models 
Regarding the issue of HLA haplotypes and the predisposition to allergies published studies show diverging results. Several studies have shown associations between IgE reactivity and the presence of distinct HLA-DRB1 alleles; most notably in patients allergic to ragweed Amb a 5, Alternaria Alt a 1, Parietaria Par o 1, birch Bet v 1, cat Fel d 1, as well as cockroach and house dust mite allergens. In these cases HLA-DRB1 haplotypes could favor susceptibility to allergy. However, Jahn-Schmid et al. have recently shown that the dominant T cell epitopes of the major ragweed allergen Amb a 1 were presented by different HLA- DR, DP and DQ molecules 
. These findings suggest that, alternatively, a broad HLA class II restriction profile might contribute to the high allergenic properties of Amb a 1.
Several questions remain to be addressed, e.g. if and/or how nitrated proteins may interfere with uptake and/or processing pathways of DCs or if potential alternative uptake mechanisms for nitro-proteins e.g. via specialized receptors expressed on DCs might exist. Furthermore, the questions whether chemical nitration of the protein compared to nitration by NO2 and ozone in polluted air have different characteristics (e.g. act on different tyrosine residues) and whether they contribute to nitration to a similar extent could not be investigated in the scope of the present study. Environmental pollutants might nitrate tyrosine residues less eagerly and more selectively than the chemical agent used here. These aspects will have to be addressed in follow-up investigations.
In summary, our data show that nitration has an enhancing effect on processing and presentation of Bet v 1 derived HLA-DR associated peptides, by enhancing both the quality and the quantity of the Bet v 1 specific peptide repertoire.