Peripheral blood mononuclear cell transcriptome profile in a clinical trial with subcutaneous, grass pollen allergoid immunotherapy

Allergen‐specific immunotherapy (AIT) is the only disease‐modifying treatment in allergic airway diseases. Underlying immunological mechanisms and candidate biomarkers, which may be translated into predictive/surrogate measures of clinical efficacy, remain an active area of research. The aim of this study was to evaluate Pollinex Quattro (PQ) Grass AIT induced immunomodulatory mechanisms, based on transcriptome profiling of peripheral blood mononuclear cells.


| INTRODUC TI ON
Allergic rhinoconjunctivitis (AR) is a chronic inflammatory Type I allergic disease that can be triggered by inhalation of seasonal or perennial allergens. 1,2AR is estimated to affect up to 40% of global population. 3,4Many patients rely on relief pharmacotherapy approaches, which do not alter the natural history of seasonal allergic rhinitis (SAR). 5T, which has been in practice since 1911, 6,7 is the only disease-modifying treatment for SAR and can be administered subcutaneously (SCIT) or sublingually (SLIT). 5Traditional SCIT treatment based on administration of native allergen extracts have a high risk of inducing IgE-mediated anaphylactic reactions.Introduction of depot adjuvants in combination with chemically modified allergens (allergoids) 8,9 have been shown to reduce likelihood of adverse reactions, improve the tolerability profile and maintain immunogenicity. 10,11 Grass formulation is based on chemical modification of 13 grass pollen species, which results in reduction of allergenicity, but maintenance of immunogenic properties. 12Favourable immunogenic characteristics of the allergoid formulation are enhanced by the adjuvant MCT®-MPL system, where MCT® acts as a Th1 depot adjuvant. 8,13,14[16][17] PQ Grass was developed as a pre-seasonal short course form of SCIT for treatment of grass SAR.PQ Grass showed favourable safety and efficacy profiles in the earlier-phase clinical trials 18,19 and in a large phase III field clinical trial. 111][22][23][24] A recent Phase II dose-finding study PQGrass205 25 assessed cumulative doses ranging from 5100 SU to 35,600 SU.The study demonstrated a statistically significant dose response (p < .0001)using the total symptom score (TSS) with the cumulative dose of 27,600 SU being selected for further Phase III development.

Key messages
• Short course subcutaneous AIT with modified grass allergoid inhibited Th2 and induced Th1/Treg immune responses.
• Th17 signalling pathway and associated genes were downregulated following completion of the active treatment regimens.
• Extended treatment regimen demonstrated superior immunomodulatory tolerogenic profile in comparison with conventional treatment regimen.
An exact underlying molecular and cellular immune mechanism of successful AIT is a topic of on-going active research.It is stipulated that both innate and adaptive immune responses contribute to clinical improvements and induction of immune tolerance. 26,27nce 2008, numerous novel immunological markers have become available. 28 Pieter-Jan De Kam et al. 30 It was shown that an optimized cumulative dose of 27,600 SU PQ Grass vaccine was well tolerated and showed statistically significant and clinically relevant field efficacy results for extended and conventional treatment regimens.
However, a more pronounced improvement was observed for the extended regimen: CSMS improvement was 39.5% (p = .011)and 33.1% (p = .033)for the extended and conventional regimen, respectively.Furthermore, significant improvement in the RQLQ-S score was also demonstrated for both regimens: extended −0.72 (p = .022)and conventional −0.44 (p = .171).
The transcriptomic exploratory endpoints of this trial presented hereby were focused on further in-depth prospective investigation of grass SCIT-induced molecular responses comparing different treatment regimens.

| Study objectives and study design
A multi-center, double-blind, placebo-controlled exploratory field clinical trial G309 (Clini caltr ials.gov No.: NCT04687059) with four treatment arms was conducted in the US and Europe.In total, 119 subjects were randomized applying a ratio of 2:2:1:1 to receive PQ Grass conventional regimen (one injection weekly for six weeks), PQ Grass extended regimen (one injection weekly for three weeks followed by three injections monthly), placebo containing MCT® and placebo without MCT®, respectively.Overall, ten pre-seasonal dosing visits were scheduled during the treatment period.Subjects in PQ Grass groups received six injections of PQ Grass and four injections of placebo without MCT® interspersed between the dosing visits to maintain double-blind treatment schedule.Total cumulative dose of PQ Grass per six injections was 27,600 SU.Subjects in placebo groups received either ten injections of placebo without MCT® or four injections of placebo without MCT followed by six injections of placebo with MCT (Figure 1).

The trial was conducted in accordance with Declaration of
Helsinki and the International Conference on Harmonization (ICH) guideline E6: Good Clinical Practice (GCP).The study included four periods: screening, treatment, GPS and follow-up.
During screening visit, the subjects' eligibility for the study was assessed and blood samples were collected for the baseline assessment of gene expression in the subgroup of subjects (n = 30).

| Subject population
Eligible subjects were male or female (aged 18 to 65 years) with a history of moderate to severe symptoms of SAR and/or rhinoconjunctivitis due to grass pollen (Pooideae spp.).Subjects were required to have positive skin prick test (SPT) to grass pollen and histamine at screening, and grass specific IgE class ≥2 (0.70-3.49kU A /L).

| Study materials
PQ Grass active product 1.0 mL injections (900 SU, 2700 SU, 6000 SU) were administered subcutaneously and contained extract of

| Microarray gene expression analysis
Subgroup of 30 eligible subjects, who have signed optional biomarker informed consent form, were prospectively randomly selected at two clinical sites in Europe.Peripheral blood samples were collected at screening (baseline), Visit 12 (~3-5 weeks after the last dose administration) and Visit 15 (~13-17 weeks after the last injection).RT 2 profiler PCR array gene expression analysis was performed using RT 2 SYBR® Green ROX qPCR master mix and custom RT 2 Profiler PCR array platform (Qiagen, Germany).
Expression level for each gene was calculated using delta delta Ct method (ΔΔCt).The relative fold change (FC) for each gene between baseline (control) and treatment time points was calculated as 2 −ΔΔC .Genes with the cut-off fold difference ≥1.2 and p-value <.05 (95% CI) were identified as differentially expressed and underwent further Ingenuity Pathway Analysis (IPA).Activation z score was used to predict direction of pathway regulation.Positive z-score represented predictions of activation for canonical pathway or upstream regulator, while negative z-score predicted inhibition.

| Statistical methods
Student's t-test (2-tail distribution) was used to identify differentially expressed genes between the control (baseline) and treatment time points.Canonical pathway enrichment analysis and upstream regulator analysis (URA) were performed using causal analysis algorithms based on Fisher's exact test in IPA. 31 Adjusted p-value (False Discovery Rate) threshold of 0.05 was used to map significantly enriched pathways and associated upstream regulator molecules.An activation z-score with an absolute value IzI ≥ 2 was considered to be statistically significant (95% CI).

| Demographics and baseline characteristics
Overall, 114 (95.8%) subjects completed the study.Five subjects prematurely discontinued the study with four subjects (9.8%) from PQ Grass conventional and one subject (2.5%) from PQ Grass extended group.From the pool of 114 subjects there were 30 randomly selected subjects, who agreed to participate in exploratory gene expression biomarker study: PQ conventional regimen (n = 10), PQ extended regimen (n = 10), placebo without MCT (n = 5), placebo with MCT (n = 5) (Figure S1).Four treatment groups were comparable with respect to the most demographic variables.All subjects had allergic rhinitis (100%) and/or allergic conjunctivitis (80.7%).At screening baseline all subjects had grass-specific IgE by ImmunoCAP class ≥2 (Table S1).

| Gene expression signature of PQ Grass
Microarray analysis was performed to understand the underlying immunological mechanism associated with PQ Grass immunotherapy.
Before the start of GPS following administration of modified AIT, several pro-allergic cytokines and transcription factors were moderately upregulated in PQ Grass conventional treatment group (IL-17F, IL-4, GATA3) (Table 1).At post-GPS pronounced changes towards F I G U R E 1 Study design.The treatment period was conducted approximately for 14 weeks (3-4 months).Last administration of the PQ Grass or placebo occurred approximately 3 to 7 weeks prior to the long-term predicted start of the GPS.PQ Grass conventional regimen 6 injections updosing and maintenance (900 SU, 2700 SU, 6000 SU, 6000 SU, 6000 SU, 6000 SU) was administered sequentially one injection per week for 6 weeks.PQ Grass extended regimen 3 injections updosing (900 SU, 2700 SU, 6000 SU) was administered sequentially one injection per week for 3 weeks and 3 injections maintenance (6000 SU, 6000 SU, 6000 SU) every 4 weeks.
TA B L E 1 PQ Grass conventional treatment regimen: differentially expressed genes at the start and the end of GPS compared to baseline.tolerogenic immune profile were noticed.Hallmark Th1 cytokines and transcription factor (IFNy, IL-12A, TBX21) were significantly upregulated (p < .05)(Table 1).

Gene
PQ Grass extended regimen showed higher degree of changes at gene expression level towards Th1/Treg immunological profile in comparison with conventional regimen (Table 2).Major Th1 signature cytokines (IFNy, IL-12A) were significantly upregulated throughout GPS.There was significant stable induction of Treg cell activation marker-LRRC32 (GARP) and significant downregulation of the Th2 cytokines IL-4 and IL-13 (p < .05)(Table 2).

| Canonical pathway functional enrichment analysis
Canonical pathway enrichment analysis was performed to de-  S2).In both placebo groups, main pro-allergic inflammatory pathways were downregulated before the start of grass season (Figure 2A) and opposite trends towards upregulation was observed at the end of the GPS (Figure 2B).
The most pronounced immunomodulatory effect of PQ Grass treatment was observed at the end of grass season with several pro-allergic pathways significantly inhibited (Table S2).
Furthermore, PQ extended treatment regimen demonstrated significant inhibition not only for classical pro-allergic pathways but also inhibition of IL-17 signalling, Th17, IL-23 signalling and HMGB1.
Of note, upregulation of Th1 pathways and Th17 was shown for the PQ Grass conventional treatment group post-GPS.On the other hand, in PQ Grass extended treatment group, the Th1 and Th17 pathways were predicted to be activated at pre-GPS and inhibited at the end of season.EBI3 (IL-35B), were predicted to be upregulated (Figure 3B).
URA was also conducted for grass-specific IgG.PQ Grass extended regimen demonstrated stable upregulation of IgG throughout GPS.Upregulation of IgG was also seen in conventional treatment group at post-GPS (Figure S3).

| DISCUSS ION
An adjuvant based allergoid immunotherapy, PQ Grass, is classed as a novel short-course AIT approach for the treatment of grass induced SAR. 11,18,25For the first time in this clinical trial, two treatment regimens of PQ Grass were evaluated in parallel. 30It was reported earlier this year that PQ Grass extended regimen had enhanced efficacy profile for primary (CSMS) and secondary (RQLQ-S) endpoints in comparison with conventional treatment (Figure S4). 30 Furthermore, results reported for grass-specific serum IgG 4 confirmed consistent superior efficacy profile of PQ Grass extended regimen.
The concept of prolonged antigen exposure with administration of the maintenance dose on a monthly rather than weekly basis leads to enhanced immunological responses. 32Early phase immunological responses following AIT are accompanied by a transient short-lived increase of allergen-specific IgE, which has no functional or clinical relevance, and induction of allergen-specific blocking IgG antibody. 26,33Control over the vaccine dose and frequency of administration by prolonging antigen exposure and thus, regulating B-cells expansion, plasma cell formation and IgG production in germinal centers may induce potent long-term humoral immune responses. 34,35Therefore, an extended treatment regimen using a For the first time, in this study, we have seen dynamic changes through the grass pollen season in the expression level of IL-17 cytokine family.The major role of Th17 cells in several inflammatory diseases is well documented. 36However, there are limited studies that report on the role of Th17 cells in allergic diseases. 37,38nonical pathway analysis demonstrated IL-17 signalling and Th17 open design model.However, in this study, reduction only in Th2 cell responses was reported, but not in Th17 cell responses. 35rthermore, immunomodulatory tolerogenic mode of action for PQ Grass AIT was strengthened by the fact that grass-specific IgE was significantly inhibited as an upstream regulator at post-GPS.
Induction of immune tolerance is also driven by the allergen-specific blocking IgG antibody. 26,33Analysis of the upstream regulators demonstrated stable upregulation of IgG throughout GPS for extended regimen and at the end of GPS for conventional regimen.an important role in bridging innate and adaptive immune mechanisms.[45] Even though we could not show stable induction of DCreg cell subset, we were able demonstrate stable significant downregula- There are some limitations to this study such as relatively small sample size of the sub-groups and selected limited gene panel.
Also, the underlying immunological mechanism was explored using PBMCs, not specifically isolated cell subsets.PBMCs is a heterogeneous cell population and potentially could lead to "masking" molecular signature from the rare cell subsets.Confirmation of these findings at cellular and protein level would be needed based on larger sample population size.

| CON CLUS ION
In conclusion, we hypothesized that differences in molecular mecha-

K
E Y W O R D S AIT, biomarker, efficacy, PQ Grass, transcriptome G R A P H I C A L A B S T R A C T Short course subcutaneous AIT with modified grass allergoid inhibited Th2, Th17 signalling pathways and induced Th1, Treg protolerogenic immune responses.Extended treatment regimen exhibited superior mechanistic efficacy profile in comparison with PQ conventional regimen.
An aspirational goal of biomarker development is to determine the clinical relevance of biomarkers as surrogate or predictive markers of efficacy.As development of personalized immunotherapy modalities could be used to guide clinical development of the treatment regimens, we aimed to further unravel dominant molecular immune mechanisms, which could potentially serve as early predictors of outcome for AIT.Therefore, the exploratory transcriptomic biomarkers from a previous Phase I PQ Grass SCIT study 29 have been further optimized and evaluated in the current multicentre field study.Primary and secondary clinical efficacy results based on combined symptom medication score (CSMS) and standardized rhinoconjunctivitis quality of life questionnaire (RQLQ-S) for this study were recently reported by 13 cross-reactive grass pollens (Pooideae spp) chemically modified and adsorbed onto 2% L-Tyrosine [w/v] + 0.5% phenol [w/v] 50 μg/1.0mL MPL.Placebo vehicle with MCT® contained buffered saline solution with 2% L-tyrosine [w/v] and 0.5% phenol.Placebo without MCT® contained buffered saline solution with 0.5% phenol [w/v].

31 IL- 4 Interleukin 4
molecule expressed on the differentiated Th1 cells regulates B cells activation and differentiation into plasma cells 1.53 IL-12A Interleukin 12A Th1 cytokine, required for T-cell-independent induction of IFNγ and regulated differentiation of Th1 cells 1.58* (p=.041)TBX21 T-box transcription factor 21 Th1 cell-specific transcription factor controls the expression of Th1 cytokine-IFNγ 1.25 IL-17F Interleukin 17F Th17 cytokine, member of IL-17 family, induces allergen-specific Th2 cell activation, eosinophil and neutrophil accumulation 1.38 GATA3 GATA binding protein 3 DNA binding Th2 cell-specific transcription factor 1.Th2 cytokine regulates activation and proliferation of pro-allergic cell responses, IgE synthesis 1.32 Downregulated IL-17A Interleukin 17A Th17 cytokine, member of IL-17 family, induces allergen-specific Th2 cell activation, eosinophil and neutrophil accumulation −1.48 RIPK4 Receptor-interacting serine/threonineprotein kinase 4 Pro-allergic dendritic cells type 2 (DC2) activation marker, DC2 cells induce differentiation of Th2 cells −1.37 IL-17F Interleukin 17F Th17 cytokine, member of IL-17 family, induces allergen-specific Th2 cell activation, eosinophil and neutrophil accumulation −1.44 IL-25 Interleukin 25 Th2 cytokine, member of IL-17 family, regulates Th2 type of immune responses −cytokine, candidate gene for asthma treatment −1.49GATA3 GATA binding protein 3 DNA binding Th2 cell-specific transcription transcription factor, DC2 marker −1.21 IL-13 Interleukin 13 Th2 cytokine regulates activation and proliferation of pro-allergic cell responses, IgE synthesis −1.38 IL-4 Interleukin 4 Th2 cytokine regulates activation and proliferation of pro-allergic cell responses, IgE synthesis −Data are quantified as changes from baseline (Visit 1) and presented as an average (per treatment group) normalized fold-regulaton (≥1.2) of an individual gene expression.Student's t-test (2-tail distribution), Bold and *p < .05denotes statistical significance at pre-GPS (Visit 12) and post-GPS (Visit 15) compared to baseline.Abbreviations: CD, cluster of differentiation; DC, dendritic cells; GPS, grass pollen season; IFN, interferon; IL-, interleukin; Th1, T helper 1 cells; Th17, T helper 17 cells; Th2, T helper 2 cells.TA B L E 2 PQ Grass extended treatment regimen: differentially expressed genes at the start and the end of GPS compared to baseline.required for T-cell-independent induction of IFNγ and regulated differentiation of member of IL-12 family, induction of IFNy production, regulation of Th1 cell differentiation and proliferation, polarization of DC towards toleragenic DCreg type activation and proliferation of pro-allergic cell responses, IgE synthesis member of IL-17 family, induces allergen-specific Th2 cell activation, eosinophil and neutrophil accumulation −Data are quantified as changes from baseline (Visit 1) and presented as an average (per treatment group) normalized fold-regulation (≥1.2) of an individual gene expression.Student's t-test (2-tail distribution), Bold and *p < .05,**p < .01denotes statistical significance at pre-GPS (Visit 12) and post-GPS (Visit 15) compared to baseline.Abbreviations: CD, cluster of differentiation; DC, dendritic cells; GPS, grass pollen season; IFN, interferon; IL-, interleukin; Th1, T helper 1 cells; Th17, T helper 17 cells; Th2, T helper 2 cells.

3. 2 . 3 |
Upstream regulator analysisURA was conducted to identify the molecules upstream of the differentially expressed genes that were molecular drivers of the observed gene expression.At the start of the GPS, a URA heatmap showed noticeable differences between the top upstream regulators for PQ Grass conventional and extended regimens.Transcriptome profile changes in PQ Grass extended group were driven predominantly by anti-allergic Th1 type of cytokine molecules IL-12 (FDR = 1.07E × 10 −17 ; z-score[2.00])and IFNy (FDR = 2.09E × 10 −13 ; zscore[1.81]).In addition, immunomodulatory profile of PQ Grass extended regimen was enhanced by activation of transcription factor STAT1 and inhibition of STAT6 (Figure3A).URA at pre-GPS for PQ Grass conventional regimen did not demonstrated predicted activation of Th1 upstream regulators.However, positive signs of the early immune modulation were supported by predicted activation of IL-10 and inhibition of IL-23 upstream regulators (Figure3A).URA predicted enhanced tolerogenic dynamics shift towards Th1/Treg systemic immune responses at the end of season.Several top pro-allergic upstream regulators were significantly inhibited in both PQ Grass treatment groups: IgE, IL-25 (IL-17E), IL-17A/F, IL-4, STAT3 (Figure 3B).One of the top predicted upstream regulators at post-GPS for both treatment regimens was IgE.IgE molecule showed significantly high inhibition z-score in conventional (FDR = 4.43E × 10 −9 ; z-score[−2.94]) and extended (FDR = 1.88E × 10 −15 ; z-score[−2.83])treatment groups, which resulted in downregulation of main pro-inflammatory molecules and dampening of allergic responses (Figure 4).IL-25 (IL-17E) was another top upstream regulator, which was predicted to be significantly inhibited.All three members of IL-17 cytokine family (IL-25, IL-17A, IL-17F) appeared to be the dominant inhibited upstream regulators at the end of GPS.At the same time, several members of the IL-12 family, IL-12A (IL-12p35), IL-27 and

4 -F I G U R E 2
weekly maintenance dose should be considered as the most optimal choice for the pivotal Phase III study.It is thought that the long-term clinical efficacy is driven by deviation from Th2/ThA cellular immune responses towards Th1 response, reduction in T follicular helper type 2 (Tfh2) cell responses and induction of T and B regulatory cells.[26][27][28]However, it is still a subject of on-going active research if changes induced by AIT at cellular level correlate with molecular changes.An earlier study of PQ Grass induced molecular mechanisms showed that changes in both components of immune response-innate and adaptive were taking place.29 Polarization and Hierarchical clustering heat map of the enriched canonical pathways in PQ Grass treatment groups versus placebo.(A) Canonical pathways enrichment heat map before the start of grass pollen season (pre-GPS) at Visit 12; (B) Canonical pathways enrichment heat map at the end of grass pollen season (post-GPS) at Visit 15.Heatmap is displayed based on the value of zactivation score.Orange z-activation score-predicted activation of the canonical pathway, blue z-activation score-predicted inhibition of the canonical pathway, white z-activation score-close to 0 or there are fewer than four analysis-ready molecules in the dataset associated with the pathway.Placebo without MCT® n = 5; placebo with MCT® n = 5; PQ Grass conventional regimen n = 10, PQ Grass extended regimen n = 10.(FDR < 0.05).differentiation of Th cell subsets was a central molecular mechanism predicted to be driven by polarization of the cytokine milieu and orchestrated by the innate dendritic cells.In the current study, molecular mechanisms induced by PQ Grass AIT were explored further.Transcriptome analysis demonstrated stable significant upregulation of the main Th1 signature cytokines (IFNγ, IL-12A, IL-27) for both regimens.For PQ Grass conventional treatment group, it was observed initial transient inhibition of Th1 pathway with subsequent activation at post-GPS.In PQ Grass extended treatment group Th1 pathway was strongly activated at pre-GPS and showed trend towards inhibition at post-GPS.However, an absolute z-score for Th1 pathways in PQ Grass extended group was <2, which would suggest minor impact on the overall changes in the landscape of Th cells differentiation.These noticeable differences in the time-cause dependant activation of Th1 pathway between treatment regimens could be the result of unique mode of action for each of the treatment regimens.There was a trend of transient upregulation in Th2 signature cytokines (IL-4, IL-13) and transcription factors (GATA3, STAT4, COX2) at the start of grass season following PQ Grass administration and significant downregulation at the end of GPS.Canonical pathway analysis showed initial activation of Th2 pathway at pre-GPS and subsequent significant inhibition at the end of season for PQ Grass conventional treatment group.In PQ Grass extended treatment group, Th2 pathway was stably inhibited over entire GPS.
activation pathways as one of the most significantly enriched pathways.PQ Grass conventional regimen showed a predominantly inhibited pattern of IL-17 and Th17 pathways at the start of GPS and partially activated at the end of GPS.At the same time, PQ Grass extended regimen demonstrated conversion from an early-phase adaptive immune response with predicted activation of IL-17 and Th17 at pre-GPS into initiation of the late-phase with inhibition of the corresponding immune responses, which could indicate a positive trend towards stable long-lasting immune tolerance induction.Upstream regulator analysis at pre-GPS did not show upstream molecules, which were directly involved in induction of IL-17 and Th17 signalling pathways.However, at post-GPS, upstream regulators IL-17A, IL-17F and IL-25 (IL-17E) were predicted to be inhibited for both treatment regimens.Furthermore, classical pro-allergic upstream regulators IgE, IL-4 and Th2/Th17 transcription factor STAT3 were also inhibited at post-GPS.Therefore, it is plausible to suggest that Th17 cells alongside Th2 cells contribute to pro-allergic inflammatory mechanisms.Of note, for the first time in the double-blind placebo-controlled study, we were able to demonstrate inhibitory immunomodulatory mechanisms of PQ Grass on Th17 cell subset, which could potentially become a novel biomarker candidate of clinical efficacy.The potential role of Th17 in allergic immune responses was also reported in an exploratory observational study using an F I G U R E 3 Upstream regulator analysis.(A) Top upstream regulators in PQ Grass conventional and extended treatment regimens at the start of GPS; (B) top upstream regulators in conventional and extended treatment regimens at the end of GPS.Orange z-activation score-predicted activation of the upstream regulator, blue z-activation score-predicted inhibition of the upstream regulator.PQ Grass conventional regimen n = 10, PQ Grass extended regimen n = 10 (IzI-score ≥ 2, FDR < 0.05).
tion of DC2 molecular marker RIPK4 in PQ Grass extended group and trend towards decreased expression of RIPK4 in conventional group.It could be stipulated that downregulation of DC2 was driven by activated upstream regulators IL-35, IL-12, IL-27 and inhibited upstream regulator IL-4.Furthermore, based on polarization milieu for DC type 1 and tolerogenic DCreg, it is plausible to suggest that DC1 and DCreg cell subsets were present in peripheral blood of subjects.These findings are supported by a previous study, where it was found possible induction of tolerogenic DCreg type based on increased expression level of IL-27. 45 nisms, degree of changes in gene expression level and induction of certain canonical pathways highlights the potential differential mechanisms underlying immune tolerance induction by the two applied treatment regimens of PQ Grass.Findings from transcriptome analysis confirmed the more pronounced clinical efficacy profile of PQ Grass extended treatment regimen.In this study, it was demonstrated that the PQ Grass extended regimen showed higher degree of changes in upregulation of key molecular drivers of Th1/Treg immunological profile (IL-12, IFNy, IL-27) and dampening Th2/Th2A molecular drivers (IL-4, IL-13) compared to the conventional regimen.Inhibition of Th17 cell activation, IL-17 signalling and potential link with induction of IL-35 iTregs was an important finding for both PQ Grass treatment regimens.These findings can help to open new insights in underlying mechanism of immune tolerance induction by PQ Grass SCIT AIT and identify potential molecular biomarkers that can be translated into clinical efficacy or become novel treatment targets.