BACKGROUND: Allergen-specific immunotherapy (AIT) is a disease-modifying treatment that induces long-term T cell tolerance. OBJECTIVE: To evaluate the role of circulating CXCR5+PD-1+T follicular helper (cTFH) and T follicular regulatory (TFR) cells following grass pollen subcutaneous (SCIT) and sublingual (SLIT) immunotherapy and the accompanying changes in their chromatin landscape. METHODS: Phenotype and function of cTFH cells were initially evaluated in grass pollen-allergics (GPA, n= 28) and non-atopic controls (NAC, n=13) by mathematical algorithms developed to manage high-dimensional data and cell culture, respectively. cTFH and TFR cells were further enumerated in NAC (n=12), GPA (n=14), SCIT (n=10) and SLIT (n=8)-treated groups. Chromatin accessibility in cTFH and TFR cells was assessed by ATAC-seq to investigate epigenetic mechanisms underlying the differences between NAC, GPA, SCIT and SLIT. RESULTS: cTFH cells were shown to be distinct from TH2 and TH2A cell subsets, capable of secreting IL-4 and IL-21. Both cytokines synergistically promoted B cell class switching to IgE and plasma cell differentiation. Grass pollen allergen induced cTFH cell proliferation in GPA but not in NAC (P<.05). cTFH cells were higher in GPA compared to NAC and were lower in SCIT and SLIT (P<.01). Time-dependent induction of IL-4, IL-21 and IL-6 were observed in nasal mucosa following intranasal allergen challenge in GPA but not in SCIT and SLIT groups. TFR and IL-10+ cTFH cells were induced in SCIT and SLIT (all, P<.01). ATAC-seq analyses revealed differentially accessible chromatin regions in all groups. CONCLUSION: For the first time, we showed dysregulation of cTFH cells in GPA compared to NAC, SCIT and SLIT and induction of TFR and IL-10+ cTFH cells following SCIT and SLIT. Changes in the chromatin landscape were observed following AIT in cTFH and TFR cells.