Purified recombinant Bet v 1 and Phl p 5 were purchased from Biomay AG (Vienna, Austria). Seven Bet v 1-derived long peptides (L1-L7) with a length of 31 to 42 amino acids spanning the entire sequence of Bet v 1 ( Table 1 ) and seventeen Bet v 1-derived short peptides (S1-S17) of 12 amino acids in length spanning amino acids 85 to 160 of Bet v 1 ( Table 1 ) were produced by chemical synthesis using Fmoc amino acid protection and HBTU coupling on a peptide synthesizer (Liberty Blue, CEM Corporation, USA). The short peptides were overlapping for at least 5 amino acids. The seven long Bet v 1 peptides (L1-L7) are similar to a set of peptides reported to lack IgE reactivity and allergenic activity in birch pollen allergic patients but were optimized to span the complete Bet v 1 sequence (32). The 14mer peptide BV139 (MGETLLRAVESYLL) which has been previously described as major T cell epitope in Bet v 1-sensitized BALB/c mice (31) is part of the long peptides L6 and L7, whereas peptide L5 contains only the first six amino acids of BV139. Likewise, the short peptides do not contain complete BV139. S14 and S15 contain ten amino acids of BV139 and S13 as well as S16 only 6 amino acids thereof. Peptides were purified by HPLC (Dionex UltiMate 3000; Thermo Fisher Scientific) to at least 90% purity and their molecular weights confirmed by MALDI-TOF mass spectrometry (Microflex, Bruker, Billerica, MA) as described (32). Lyophilised peptides were stored at -20°C and reconstituted in endotoxin-free water for use. The Bet v 1-derived synthetic peptide sequences and their biochemical characteristics are summarized in Table 1 . The ability of the six long Bet v 1 peptides P1-P6 to stimulate CD4⁺ T cell responses in blood samples obtained before, during and after the birch pollen season in a population of highly birch pollen-exposed subjects (birch pollen allergic patients: n=6; patients with allergy without birch sensitization: n=4; non-allergic subjects: n=9) from the Moscow region in Russia was studied by carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution assay using two different concentrations (1.2nM, 12nM) as described (24). Together the six peptides P1-P6 comprised the Bet v 1-specific human CD4⁺ T cell epitopes as tested in the Moscow population ( Supplementary Figure 1 ).

Female BALB/c mice of different age groups (3-4 weeks for tolerance induction experiments, 6-8 weeks for immunization experiments) were purchased from Charles River (Sulzfeld, Germany) and were maintained according to the guidelines for animal welfare. All experimental procedures were approved by the Animal Ethics Committee of the Medical University of Vienna and the Austrian Federal Ministry of Science, Research and Economy BMWFW (66.009/0431-V/3b/2019). Experiments were performed in accordance with national and international guidelines of laboratory animal care.

Groups of mice, 6 to 8 weeks of age (n = 5), were sensitized by a single subcutaneous (s.c.) injection with 5µg, 10µg, 20µg or 40µg of recombinant Bet v 1 adsorbed to 75μl of aluminum hydroxide (Al(OH)₃) (Alu-Gel-S, SERVA, Heidelberg, Germany) and made up to a final injection volume of 150μl per mouse with PBS or 75μl of PBS and 75μl of Al(OH)₃ alone (negative control). Blood samples were taken from the tail veins before sensitization and at days 14, 28 and 42 and serum was prepared by centrifugation. Specific IgE sensitization was determined by the measurement of allergen-specific IgE antibodies (33, 34).

For mapping of epitopes recognized by CD4⁺ T cell epitopes, groups of mice, 6 to 8 weeks of age (n = 6), received three s.c. injections of 10µg or rBet v 1 adsorbed to Al(OH)₃ every two weeks (days 0, 14 and 28). Blood samples were taken from the tail veins before sensitization as well as on days 20 and 34 to measure Bet v 1-specific antibody responses. Mice were sacrificed 7 days after the last sensitization (day 35) and spleen cells were isolated from each mouse and pooled for cellular analyses (33). More pronounced T cell responses were obtained when mice received 2 sensitizations with Bet v 1 10 weeks apart and were sacrificed at week 25 (12 weeks after last sensitization).

The mapping of CD4⁺ T cell epitopes was performed in three independent experiments obtaining identical results.

For mapping of epitopes recognized by IgE and IgG antibodies, groups of mice, 6 to 8 weeks of age (n =10), received three s.c. injections of 10µg or rBet v 1 adsorbed to Al(OH)₃ every two weeks (days 0, 14 and 28). Blood samples were taken from the tail veins before sensitization as well as on days 20, 34, 42 and 56 for ELISA analysis. The mapping of antibody epitopes was performed in two independent experiments and gave identical results.

Three- to four-week-old female Balb/c mice were used in the tolerance induction experiments.

In one set of experiments mice (n= 6 to 7) received an intraperitoneal (i.p.) injection of a 100µg mix containing 5 long Bet v 1-derived peptides (peptides L1, L2, L3, L4 and L6) and 17 short Bet v 1-derived peptides (peptides S1 to S17) ( Table 1 ) on days 2, 4 and 6 ( Figure 1 , Groups 1 and 2). The 100µg peptide mixes were prepared by adding 4.5µg of each peptide from 1mg/ml stock concentrations of reconstituted lyophilized peptides in endotoxin free water and final injection volumes were made up to 150µl per mouse with 50µl of PBS. Group 3 mice received only PBS but not peptides. Group 1 and 3 mice were sensitized with Bet v 1 and all three groups were sensitized to Phl p 5 ( Figure 1 ). A second sensitization with Bet v 1 and Phl p 5 was given to group 1 and group 3 mice at week 13 but not to group 2. At week 18, group 1 mice received a second administration of Bet v 1 peptides by the subcutaneous route ( Figure 1 ). Serum samples were collected at week 11, 15 and 20. Mice were sacrificed and spleens excised at week 25 for the analysis of CD4⁺ T cell responses. The design of the first tolerance induction experiment allowed to investigate if the administration of peptides would induce allergic sensitization or Bet v 1-specific CD4⁺ T cell or IgG antibody responses because group 2 was not sensitized to Bet v 1. Furthermore, the co-sensitization with Bet v 1 and Phl p 5 of groups 1 and 3 permitted to study the effects of the administration of the Bet v 1 peptides on antibody and T cell responses to the corresponding allergen and to an unrelated allergen.

The design of the second set of tolerance experiments was similar to the first experiments with the exception that instead of 3 peptide administrations, 5 were given, a second sensitization and later peptide administration was avoided but a lung challenge was performed ( Figure 2 ). Furthermore, the numbers of mice in each group were higher than in the first experiment (i.e., n=10). Respiratory function of mice was measured at days 56, 57 and 58 (week 9) by challenging mice with either PBS or with 0.625mg birch pollen extract dissolved in 125µl of PBS and aerosolized per mouse using Whole Body Plethysmography (Buxco FinePointe, Data Sciences International, USA). Mice were sacrificed and spleens excised at day 59. Serum samples were collected at weeks 6, 8 and 9.

Mouse spleens were removed under aseptic conditions and single cell suspensions were prepared by physical disruption of spleens through 70µM nylon cell strainers (FalconTM, BD Biosciences, New Jersey, USA). Thereafter, isolated splenocytes were labelled with CFSE (Life Technologies, Bleiswijk, Netherlands). Cells were diluted to a final concentration of 2 x 10⁵ cells/well and seeded into 96-well round bottom cell culture plates in triplicates (200µl/well) in the presence or absence of antigens in supplemented RPMI 1640 medium (Gibco, Bleiswijk, The Netherlands), using 5µg/well or 10µg/well of Bet v 1 or Phl p 5, 2.5µg/well or 5µg/well of Bet v 1-derived long peptides L1 to L7, respectively ( Table 1 ). Cells were cultured for 5 days and stained with fixable viability dye, eFluorTM 780 (Invitrogen, California, USA), PE/Cy7 anti-mouse CD3 antibody (clone: 17A2, Biolegend) and PE Rat anti-mouse CD4 antibody (Clone: RM4-5, BD Biosciences). Flow cytometry was performed with a FACS Canto flow cytometer (Becton Dickinson) and analysis with the FlowJo software (TreeStar, Ashland, Oregon) (33). The measurement of allergen-specific cytokine production in splenocyte cultures is described in the supplement.

Allergen-specific IgE, IgG₁ and IgG_2a antibody levels were measured by ELISA. ELISA plates (Greiner bio-one, Frickenhausen, Germany) were coated in duplicates with 1µg/ml of antigen dissolved in PBS (Bet v 1, Phl p 5, Mal d 1, Cor a 1, Dau c 1, Api g 1, Aln g 1, Bet v 1-derived long peptides L1 to L7) (50µL/well) for five hours at room temperature. Plates were washed twice with PBS 0.05% Tween 20 (200µL/well) and then blocked with 2%BSA PBS 0.05% Tween-20 (100µL/well) at 4°C overnight. Thereafter, sera were added (50µL/well) in duplicates per mouse for incubation at 4°C overnight with the following dilutions: 1:100 for the detection of IgG₁, 1:10 for the detection of IgE and 1:50 for the detection of IgG_2a. Plates were then washed five times with PBS 0.05% Tween-20 followed by incubation with a 1:1000 dilution (50µL/well) of purified rat anti-mouse IgE (Clone: R35-72), purified rat anti-mouse IgG₁ (Clone: A85-1) or purified rat anti-mouse IgG_2a (Clone R19-15) (BD Pharmingen, San Diego, CA, USA) at 4°C overnight. Plates were again washed five times as above and then incubated with a 1:1000 dilution (50µL/well) of ECL™ anti-rat IgG, horseradish peroxidase linked whole antibody from goat (Sigma-Aldrich, UK) at 37°C for one hour. Plates were washed five times and colorimetric detection was done with 2,2′-azino-bis 3-ethylbenzothiazoline-6-sulphonic acid (ABTS) (Sigma-Aldrich, St. Louis, Mo, USA) solution in citric acid buffer (50µL/well). Optical densities (OD) were measured using a Tecan infinite F50 instrument (OD at 405nm and reference OD at 492nm). Results are expressed as means of duplicates per mouse with a deviation of less than 10%.

Rat basophil leukaemia cells (RBL-2H3) grown in supplemented RPMI 1640 medium (Gibco, Bleiswijk, The Netherlands) were seeded at 4 x 10⁵ cells/well into 96-well flat bottom cell culture plate (Corning incorporated, Kennebank, OH) and cultured overnight at 37°C in 5% CO₂. Cells were then incubated with 1:10 dilutions of a serum pool generated from each mouse-group in triplicates at 37°C and 5% CO₂ for 2 hours. Thereafter, supernatants were removed and cells were washed twice with Tyrode’s buffer/0.1% BSA (Sigma-Aldrich, UK). IgE-loaded cells were then stimulated with different concentrations of antigens at 37°C for 30 minutes and mediator release was detected in the cell supernatants with the addition of 4-methylumbelliferyl β-D-galactopyranoside (4-MUG, Sigma Aldrich). For determination of 100% mediator release, cells were lysed with 10% v/v Triton X-100 (Merck Millipore, Darmstadt, Germany). The fluorescence of β-hexosaminidase release was measured between wavelengths of 360nm to 465nm using an Infinite 200 PRO microplate reader (Tecan, Maennedorf, Switzerland). The results are calculated as the percentages of total β-hexosaminidase released with the addition of 10% triton.

Data were analysed using Stata 13.1 (StataCorp, College Station, TX). Peptides were analysed by a general linear model with a log link, comparisons of peptide-induced antibody reactivities and CD4⁺ T cell proliferations against medium control were done by Dunnett’s tests. For prophylactic experiments, data were analysed by a general linear model with a log link, comparing group 1 and 3 by linear contrast. P values of less than 0.05 were considered statistically significant (* P < 0.05; ** P < 0.01; *** P < 0.001).

In order to establish a model for Bet v 1-specific sensitization, a single injection was selected to determine the effect of one dose on allergen-specific IgE and IgG production. Bet v 1-specific antibody responses could be induced with a single subcutaneous injection of 5μg of Bet v 1. The antibody levels were higher in the group receiving 10μg than in the 5μg group and decreased again when higher Bet v 1-doses were applied ( Figures 3A, B ). Moreover, 10µg dose had consistently (both for IgG₁ and IgE) the highest average OD values; for IgE this was significantly higher against 20µg (p=0.045) and 40µg (p=0.033). No Bet v 1-specific antibodies were detected in mice immunized only with adjuvant and PBS alone.

In a first set of experiments, we analyzed T cell proliferations on day 35 after three s.c. injections of 10µg of Bet v 1 every two weeks (days 0, 14 and 28) and found that Bet v 1 and three peptides derived from the Bet v 1 C-terminal portion, peptides L4 (aa 91-126), L6 (aa 119-160) and L7 (aa 130-160) were able to induce specific CD4+ T cell responses above the medium background (data not shown). Bet v 1-specific T cell responses were most pronounced when mice received 2 sensitizations with Bet v 1 10 weeks apart and were sacrificed at week 25 (12 weeks after last sensitization) showing that Bet v 1-specific T cell epitopes in mice include L4, L6 and L7 but are scattered along the sequence with proliferation that were significantly higher than the subtracted medium background ( Figure 4 ).

Next, sera from Bet v 1-sensitized mice were used to identify peptides that react with IgE, IgG₁ and IgG_2a antibodies. In this context it is of note that Bet v 1-allergic patients lack IgE antibodies specific for sequential Bet v 1 epitopes but only recognize the complete folded Bet v 1 molecule (32, 35). We found that IgE antibodies from Bet v 1-sensitized mice recognized not only the complete folded Bet v 1 allergen but also sequential epitopes represented by peptides L2, L6 and L7 (strength of peptide IgE reactivity: peptide L6 = peptide L7 > peptide L2) and also showed some low reactivity with peptides L3, L4 and L5 ( Figure 5A ). Peptides L6 and L7 were significantly higher compared to all peptides except L2. Peptides L6 and L7 form a homogenous subgroup significantly (p<0.05) different from all other peptides for IgG_2a. For IgE and IgG₁ L2 joins L6 and L7 as a homogenous subgroup as per Tukey’s HSD test. The same sequential epitopes were recognized with comparable intensity by IgG ₁ and to a lesser extent by IgG_2a antibodies ( Figures 5B, C ).

We also investigated if the peptides can induce specific basophil activation by cross-linking Bet v 1-specific IgE on basophils ( Figure 6 ). Bet v 1 induced dose-dependent basophil activation starting already at a concentration of 0.001µg/ml. Specific basophil activation was also obtained with peptides L6 and L7 but an approximately 10,000-fold higher concentration in terms of microgram and more than 40,000-fold higher in terms of molarity was needed to induce comparable basophil activation. Approximately 40% ß-hexosaminidase release was obtained with 0.001µg/ml rBet v 1 whereas 10µg/ml of peptides L6 and L7 were required to achieve a comparable release ( Figure 6 ).

Bet v 1-allergic patients show IgE cross-reactivity to Bet v 1-related pathogenesis-related proteins (PR10 allergens) in pollen and plant foods (28). IgE antibodies from Bet v 1-sensitized mice showed comparable reactivity to the related pollen allergens from hazel, Cor a 1 and alder, Aln g 1 ( Supplementary Figure 2A ). Lower IgE reactivity was observed with the major apple allergen, Mal d 1 forming an intermediate subset whereas almost no reactivity with Dau c 1 and Api g 1 from carrot and celery, respectively, was found ( Supplementary Figure 2A ). Regarding IgG₁ strong cross-reactivity with Mal d 1, Cor a 1 and Aln g 1 was found whereas IgG₁ reactivity to Dau c 1 and Api g 1 was low and they form a homogenous subset significantly different from all other allergens ( Supplementary Figure 2B ). Similar results were obtained for IgG_2a. Cross-reactivity was most pronounced for Mal d 1, Cor a 1 and Aln g 1 and almost no reactivity to Dau c 1 and Api g 1 was found ( Supplementary Figure 2C ). Thus, for IgG_2a reactivity there are three homogenous subsets: Dau c 1 and Api g 1, Mal d 1 and Cor a 1, Aln g 1 and Bet v 1. Subsets significantly differ from each other at p<0.05 as per Tukey’s HSD test.

Aln g 1 induced also strong basophil activation when basophils had been loaded with IgE from Bet v 1-sensitized mice, but Bet v 1 was approximately tenfold more potent in inducing basophil activation ( Supplementary Figure 3 ). We noted also basophil activation with the other Bet v 1-related PR10 allergens but only at the highest concentrations tested (i.e., 10µg/ml) ( Supplementary Figure 3 ).

The cross-reactivity of IgE and IgG antibodies in Bet v 1-sensitized mice with Bet v 1-related PR10 allergens reflected the extent of sequence identity. Supplementary Figure 4 shows that the sequences of Aln g 1 (81% sequence identity), Cor a 1 (73%) and Mal d 1 (56%) are most closely related to Bet v 1 whereas the sequence identity of Bet v 1 with Api g 1 (42%) and Dau c 1 (38%) is low.

Next we were interested to investigate if it was possible to reduce allergic sensitization as well as adaptive immunity to Bet v 1 in terms of IgG and CD4⁺ T cell responses by preventive administration of hypoallergenic T cell-epitope-containing Bet v 1 peptides. Major questions to be addressed in these experiments were i.) if specific tolerance can be induced with a relatively low peptide dose; ii.) if tolerance can be achieved with peptides longer than 30 amino acids without inducing allergic sensitization with the peptides; iii.) if tolerance induction can be achieved with a peptide mix comprising human CD4⁺ T cell epitopes and iv.) if peptide tolerance is specific for the corresponding allergen or if there can be bystander effects.

Testing of the seven long Bet v 1 peptides for their ability to stimulate CD4⁺ T cells revealed that in addition to peptides L6 and L7 containing a previously reported T cell epitope recognized by Bet v 1-sensitized BALB/c mice, peptide L4 contains an additional not yet described CD4⁺ T cell epitope for BALB/c mice ( Figure 4 ). In addition, we added a mix of 17 12mer peptides spanning the Bet v 1 C-terminal portion ranging from amino acid positions 85 to 160. This was done to ensure that the newly discovered CD4⁺ T cell-stimulating peptide in peptide L4 and eventual other T cell epitopes of the C-terminus are covered. On the other hand, the mix of short peptides did not contain the previously reported intact BV139 sequence which was part of the long peptides L6 and L7. The peptide mix contained only 4.5µg of each of the peptides and thus the dose of the individual peptides was at least five-fold lower than in earlier tolerance induction experiments carried out with Bet v 1 peptides (31) and more than 50-fold lower than in earlier reports of peptide-induced tolerance (36, 37).

The seven long Bet v 1 peptides ( Table 1 : L1-L7) even covered more completely the Bet v 1 sequence than 6 similar Bet v 1 peptides ( Table 1 : P1-P6) which comprised human Bet v 1-specific CD4⁺ T cell epitopes (32). The latter six peptides P1-P6 were shown to stimulate CD4⁺ T cell responses in blood samples obtained before, during and after the birch pollen season in a population of highly birch pollen-exposed subjects (birch pollen allergic patients: n=6; patients with allergy without birch sensitization: n=4; non-allergic subjects: n=9) from the Moscow region in Russia ( Supplementary Figure 1 ). None of the latter mentioned birch pollen allergic patients showed IgE reactivity to the peptides (data not shown).

In a first set of experiments ( Figure 1 ), Bet v 1-specific IgE antibody levels eight weeks after the sensitization (i.e., at week 11) were significantly lower in group 1 which had received preventive administration of Bet v 1 peptides as compared to group 3 which had received only PBS ( Figure 7A ). No Bet v 1-specific IgE was detected in group 2. This group had been sensitized only to Phl p 5 but not to Bet v 1 but had received preventive administration of Bet v 1 peptides indicating that the peptides were non-allergenic since they also failed to prime mice for IgE production against Bet v 1 ( Figure 7A ). An approximately tenfold lower Bet v 1-specific allergic sensitization was found for group 1 mice as compared to group 3 mice in the basophil release experiments at the same time point ( Figure 7B ). Group 2 mice showed no Bet v 1-specific basophil degranulation ( Figure 7B ). Even after a second Bet v 1-specific sensitization at week 13, Bet v 1-specific IgE levels remained significantly lower in group 1 mice as compared to group 3 mice as determined at week 15 ( Figure 7C ). Also, at week 15 no Bet v 1-specific IgE was detected in group 2 mice, again suggesting that peptides were non-allergenic ( Figure 7C ). The specificity of the reduction of Bet v 1-specific allergic sensitization by preventive administration of Bet v 1 peptides was demonstrated by the fact that Phl p 5-specific IgE levels were comparable among the three groups of mice at both, week 11 and at week 15 ( Figures 7D, E ).

The analysis of Bet v 1-specific IgG₁ levels showed that there were no differences between group 1 and group 3 at week 11 whereas at weeks 15 and 20 Bet v 1-specific IgG₁ levels were lower in group 1 compared to group 3, albeit not reaching statistical significance ( Figures 8A–C ). No Bet v 1-specific IgG₁ levels were found in group 2 mice at weeks 11, 15 and 20 indicating that the Bet v 1 peptides were non-immunogenic ( Figures 8A–C ).

There was no significant difference between Phl p 5-specific IgG₁ levels between the three mouse groups at week 11 ( Figure 8D ). At weeks 15 and 20 Phl p 5-specific IgG₁ levels were even significantly lower in group 3 than in group 1 mice ( Figures 8D–F ).

The reduction of Bet v 1-specific antibody production achieved by preventive administration of Bet v 1 peptides was also reflected at the level of CD4⁺ T cell responses. Figure 9A shows that the proliferation of Bet v 1-specific CD4⁺ T cells was significantly lower in group 1 mice as compared to group 3 mice regardless if 5 or 10 µg/ml of Bet v 1 were used for stimulation. However, no proliferation of Bet v 1-specific CD4⁺ T cells was observed in group 2 mice confirming that Bet v 1 peptides despite repeated administrations (three at days 2, 4 and 6) were not immunogenic and did not induce Bet v 1-specific CD4⁺ T cell responses at both concentrations of Bet v 1 used for stimulation of T cells ( Figure 9A ).

There were no significant differences regarding Phl p 5 specific CD4⁺ T cell proliferation between groups 1 and 3 at two different antigen concentrations used for stimulation ( Figure 9B ). The Phl p 5-specific CD4⁺ T cell proliferation in group 2, which had received only one injection with Phl p 5 on day 16, was much lower than that in groups 1 and 3 which had received two injections of Phl p 5, one on day 16 and one at week 13 ( Figures 1 , 9B ).

Furthermore, the analysis of allergen-specific cytokine production in splenocyte cultures showed a significant reduction of Bet v 1-specific IL-4 (5 or 10µg/ml of Bet v 1) and IFN-γ levels (10µg/ml of Bet v 1) in Bet v 1-peptide-tolerized group 1 mice compared to group 3 mice but not for the control allergen Phl p 5 ( Supplementary Figure 5 ). There were no significant differences regarding Bet v 1-induced IL-5, IL-13, IL-10 and TGF-β levels in splenocyte cultures stimulated with 5 or 10µg/ml of Bet v 1 between group 1 and group 3 mice ( Supplementary Figure 5 ). Similar to the absence of Bet v 1-specific CD4⁺ T cell proliferation observed in group 2 mice, there was no relevant induction of Bet v 1 specific IL-4, IL-5 and IL-13 in group 2 mice ( Supplementary Figure 5 ). With few exceptions (i.e., IL-5 and IL-10) cytokine responses to both concentration of the control allergen Phl p 5 were similar in all three mouse groups ( Supplementary Figure 5 ).

The results obtained in the second set of experiments ( Figure 2 ) were almost identical to the ones obtained in the first set of experiments. Preventive administration of Bet v 1 peptides yielded significantly lower Bet v 1-specific IgE levels in group 1 as compared to group 3 ( Figure 10A ). Again, no induction of Bet v 1-specific IgE and basophil sensitivity was noted in group 2 which had been treated only with peptides ( Figure 10A ). Group 1 mice were approximately fivefold less sensitive to Bet v 1 in basophil degranulation experiments ( Figure 10B ). Bet v 1-specific IgG₁ antibody levels were significantly lower in group 1 mice compared to group 3 at weeks 8 and 9 ( Figures 11A, B ) whereas Phl p 5-specific IgG₁ levels were higher in group 1 than in group 3 at week 8 ( Figure 11C ) and lower at week 9 ( Figure 11D ). No Bet v 1-specific CD4⁺ T cells were detected in group 1 mice whereas they were clearly detectable in group 3 mice ( Figure 12A ). No significant difference regarding Phl p 5-specific CD4⁺ T cells were noted between groups 1, group 2 and group 3 ( Figure 12B ).

The single sensitization to Bet v 1 did not induce any relevant airway symptoms in mice when exposed to inhalation of birch pollen extract as compared to inhalation with saline alone ( Supplementary Figure 6 ). Therefore, it was not possible to observe any effects of peptide treatment on lung function.