Presentation of allergen derived naturally processed HLA-DR associated peptides by DCs from ten different donors (B01–B10).

<p>Allergen derived peptides were detected in samples generated from unmodified Bet v 1 loaded DCs (light grey bars) and in samples generated from Bet v 1 nitro loaded DCs (dark grey bars). In the sequence, tyrosine residues are highlighted grey.</p

Allergen derived peptide clusters and peptide length variants identified in donors B01–B10.

<p>Allergen derived peptide clusters and peptide length variants identified in donors B01–B10.</p

Nitration of Bet v 1 increases the copy number of identified allergen-derived peptides with identical amino acid sequence.

<p>The numbers of detected copies of allergen derived peptides with identical peptide sequence, derived from DCs loaded with unmodified Bet v 1 (grey bars) and allergen derived peptides derived from DCs loaded with Bet v 1 nitro (black bars) are shown for each identical peptide and corresponding donors. Due to the limited amount of available cells for peptide isolation, each sample was measured only once.</p

Structural analysis of the tyrosine residues in Bet v 1.0101.

<p>(A) Cartoon representation with alpha-helices in magenta and beta-strands in yellow. The tyrosine residues are shown as sticks. (B) Electrostatic potential mapped to the protein surface. Positive charges are colored blue and negative charges are colored red. The tyrosine residues are show as spheres, the CH groups are show in green color. (C) The front half of Bet v 1 is cut off providing a view to the large central cavity. Tyrosines Y81 and Y83 are exposed to the cavity. The figure was prepared with UCSF Chimera <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031483#pone.0031483-Pettersen1" target="_blank">[34]</a>.</p

HLA-DRB-alleles of the donors B01 to B10.

#<p>For donor B 07 only the DRB1 alleles were determined.</p

Correlation of HLA-DR associated peptides and peptide clusters measured by MAPPs to the amount of protein present in subvisible particles.

<p>Linear regression analyses of the increase of the HLA-DR associated peptides and clusters as functions of the calculated amount of protein present in the subvisible particles. Left up: HLA-DR associated peptides of mAb1 vs protein amount in subvisible particles (r² = 0.994), left down: HLA-DR associated peptide clusters of mAb1 vs protein amount in subvisible particles (r² = 0.993), right up: HLA-DR associated peptides of mAb2 vs protein amount in subvisible particles (r² = 0.86), right down: HLA-DR associated peptide clusters of mAb2 vs protein amount in subvisible particles (r² = 0.943). HS: aggregates generated by heat and shake stress; FT: aggregates generated by freeze and thaw stress, S: aggregates generated by shear stress mAb1: monoclonal antibody 1, mAb2: monoclonal antibody 2, 1: stress level 1, 2: stress level 2. For further details, please, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086322#s4" target="_blank">Materials and Methods</a>.</p

MAPPs heat map of identified HLA-DR associated peptides in the HS study.

<p>Heat map visualization of mAb-derived HLA-DR associated peptides for both model antibodies in the HS study. Each sequence position is colored according to the presence and number of different mAb-derived peptides identified. In black colored sequence regions, no peptides were identified, in colored regions, peptides were identified, with the color coding for the number of different peptides identified per position. HS: aggregates generated by heat and shake stress; mAb1: monoclonal antibody 1, mAb2: monoclonal antibody 2, un: unstressed, sl1: stress level 1, sl2: stress level 2.</p

Physicochemical characterization of stressed mAb materials.

<p>Representative MFI screenshots after freeze/thaw (FT) stress of mAb1 (A) unstressed, un; (B) stress level 1, sl1; (C) stress level 2, sl2 and mAb2 un, (E) sl1 and (F) sl2. (G) Particle Size distribution obtained by MFI of mAb1 (left) and mAb2 (right). For visualization the size binning 2–2.5, 2.5–5, 5–10 10–25, 25–50 and 50–400 µm was used. Representative images of individual particles formed by (H) heat/shake, HS; (I) freeze/thaw, FT and (J) shear stress, S. Polydispersity in % (PD%) revealed by DLS for (K) mAb1 and (L) mAb2.</p

Illustration of MAPPs assay procedure and data output example.

<p>A: Illustration of MAPPs assay procedure. Monocytes are isolated from human buffy coats and differentiated to immature DCs in the presence of cytokines. Immature DCs are loaded with the model mAb and induced to maturation with lipopolysaccharide. After 24 hours, mature DCs are frozen. After lysis of mature DCs, HLA-DR:peptide complexes are isolated via immunoprecipitation using anti-HLA-DR coated beads. After several wash steps, peptides are eluted from HLA-DR complexes by pH shift and analysed by nano LC-MS with subsequent sequence identification via SEQUEST database search. B: MAPPs assay data output example. HLA-DR associated peptides can originate from different sequence regions of a protein and can occur in multiple length variants. Peptides in a sample with unique sequence are termed “different peptides”, highlighted with blue box. Nested sets of peptide length variants occurring within a sequence region sharing the same HLA-DR binding core are termed “clusters”, highlighted with green boxes. The number of different peptides per amino acid position can be summarized in a heatmap in which the cell colors are reflecting the number of different peptides, highlighted with red box. Different donors can differ in the pattern of presented peptides depending on binding propensities of their 2 HLA-DR alleles.</p

Results from T-cell activation assay.

<p>T-cell responses determined by IFN-γ ELISpot from donors treated with mAb1, un, sl1 and sl2 material from (A) FT condition (8 donors) and from (B) HS condition (13 donors). The respective heat maps specify to which extent each single donor responded to each treatment. The horizontal line shows the geometric mean of the populations. ***: statistically significant (p<0.001); ns: statistically not significant. HS: aggregates generated by heat and shake stress, FT: aggregates generated by freeze and thaw stress, mAb1: monoclonal antibody 1, SI: Stimulation index, un: unstressed, sl1: stress level 1, sl2: stress level 2.</p