The calculations were performed from every cell in one time point to every cell in the next time point within an individual. many individual immune markers such as IL-13, CD25, IL-17A, IL-4, and ITG47 between comparison groups. Interestingly, during IT some markers (CD28, CD27) seemed to normalize to healthy control levels but others, such as CCR7, CD25, and forkhead box P3 (FOXP3), remained significantly different ( 0.00057, Table 1). Open in a separate window Fig. 2. CD4+ T cells form clusters with distinct gene expression, with allergen-specific cells preferentially occupying IL4+/IL13+/CD69+ cluster 4 and FOXP3+/IL10+/CD25+ cluster 5. ( 0.0073, ns = not significant (2 tests with Bonferroni-corrected values for tests of pairwise comparisons of individual gene expression of CD4+ cells for healthy vs. pretreatment cells, healthy vs. IT cells, pretreatment vs. IT cells, and dextramer+ vs. dextramer? cells. Bonferonni-corrected 0.00057. Pretreatment cells include all cells from all pretreatment time points, and IT cells include all cells from all IT time points (IT-1, IT-2, IT-3, IT-4). Pretreatment cells and IT cells are from the same individuals. *Bonferroni-corrected = 7), pretreatment (= 5), IT-1 (= 5), IT-2 (= 5), IT-3 (= 2), and IT-4 (= 2) participants. * 0.01, ns = not significant (tests comparing each time point to healthy controls with Bonferroni-corrected = 5), IT-2 (= 5), IT-3 (= 2), and IT-4 participants (= 2), pretreatment (= 5), and healthy controls (= 7). ( 0.001, ns = not significant (one-way ANOVAs). The calculations were performed from every cell in one time point to every cell in the next time point within an individual. The total number of cellCcell comparisons are summarized in Table S2. Results CD4+ T-cell Transcriptional Profiling. We performed transcriptional profiling of individual dextramer+ and dextramer? CD4+ T lymphocytes throughout the course of IT in vivo, using a regimen of peanut oral IT to test our hypothesis. IT was given to peanut-allergic participants, who had no other known allergies, under a published protocol (7), and peripheral blood was collected from these participants at different time points before treatment (pretreatment time points) and during IT at 3 Dansylamide mo (IT-1), 6C7 mo (IT-2), 9C10 mo (IT-3), and Dansylamide 11C18 mo (IT-4) (Fig. 1). One IT-3 blood draw was performed at 9 mo and the other was performed at 10 mo, whereas one IT-4 blood draw was performed at 11 mo and the other at 18 mo. Dansylamide Participants from whom blood was drawn pretreatment are the same individuals from whom blood was drawn during IT. CD4+ lymphocytes from each participant were labeled with dextramers specific for the peanut-derived antigen Ara h 2 23 (Fig. 1), the most widely recognized peanut antigen among allergic individuals (23) and dextramer+ and dextramer? CD4+ T cells were sorted separately into single-cell wells, followed by profiling of genes expressed in T cells like CD69, Ki67, CD28, CD38, CD27, CD127, IL-4, IL-13, IFN-, ITG47, FOXP3, and IL-10 and others (Table S1) to generate heat maps and determine immunophenotyping of CD4+ T-cell subtypes (Fig. S1) (24). Table S1. Biomarker panel markers show clustering of markers based on similarity of Dansylamide expression profile using the complete linkage clustering. Table S2. RMSD cell comparisons tests of individual gene expression for dextramer+ CD4+ T cells between healthy controls vs. pretreatment (all pretreatment time points), healthy controls vs. IT treatment (all IT time points), pretreatment vs. IT treatment, and dextramer+ vs. dextramer? CD4+ T cells, identified several shared significant markers ( 0.00057) across two or more comparisons, particularly CD28, IL-10, FOXP3, IL-17a, ITG47, IL-13, CCR7, CCR8, and CD25 (Table 1). The most frequent statistically RGS17 significant changes ( 0.00057) were detected in the pretreatment vs. IT treatment comparison. In addition, there were several markers that were statistically different between dextramer+ and dextramer? CD4+ T cells (Table 1). Notably, the elbow method for gap statistics performed on all data (including all healthy, pretreatment, and IT cells) identified seven clusters of CD4+ T cells with distinct gene-expression patterns (Fig. 2and tests showed statistically significant ( 0.01) different proportions of antigen-specific CD4+ T Dansylamide cells in each cluster, except cluster 7 (Fig. 2and and and 0.01) (Fig. 4 0.05) fluctuations in clusters were observed (Fig. S3 and = 3) and at IT-2 (= 3) from all individuals from whom negatively sorted cells were acquired, (= 7) and 6 mo later without IT (= 7), and (= 5) and 6 mo later without IT (= 5). Importantly, we juxtaposed the aggregated clinical symptoms of the participants undergoing IT.