Population mean and 95% CI of longitudinal variation (CV) in absolute frequencies are shown for all donors. C: Absolute frequencies of all islet antigen epitope-specific CD8 + T-cells were pooled at each visit for each donor. Percentage of total visits for each donor that were positive for each antigenic epitope is shown. B: At each visit, donors were marked positive if the frequencies were above the threshold (0.01% for all except IGRP, which was 0.005%). Each connected line represents an individual donor. A: Absolute frequencies of insulin B 10–18 epitope-specific CD8 + T-cells detected in each donor, over all available visits, are shown for T1D (left panel) and control (right panel) donors. The frequencies of CD8 + T-cells recognizing the diabetes-associated antigenic epitopes of insulin B 10–18, PPI 15–24, GAD65 114–123, IA-2 797–805, IGRP 265–273, and ppIAPP 5–13 bound to HLA-A2 were determined using flow cytometry in HLA-A2 + T1D (n=18), and controls (n=4). Longitudinal variation in multiple antigenic epitope specific CD8 + T-cells. E: Population mean and 95% CI of longitudinal variation (CV) in frequencies of CD4 +CD25 +Foxp3 + T-cells are shown for each donor cohort. D: CD4 +CD25 +Foxp3 + T-cell frequencies in all donors from each cohort at each visit are shown. C: The frequencies of CD4 +CD25 +Foxp3 + T-cell frequencies detected at each visit in one representative subject from each cohort (T1D n=33, HD n = 10) are shown. B: Frequencies of CD4 +CD25 +Foxp3 + assessed once in the morning and once in the afternoon of the same day, in the same T1D subject, shows similar frequencies. Frequencies of CD4 +CD25 +Foxp3 + cells (shown in FACS plots) were determined as a subset of CD4 + cells. For statistical analysis, non-parametric Mann-Whitney test was used to determine significance.ĬD4 +CD25 +Foxp3 + T-cell frequencies show similar longitudinal variation between HD or T1D donors.Ī: Gating strategy used to identify CD4 +CD25 +Foxp3 + T-cells, from a representative T1D donor is shown. Population mean and 95% CI of longitudinal variation (CV) in net spot numbers are shown for all donors. Net spot numbers per 2 × 10 5 PBMC for all three antigens (GAD-65, IA-2 and PI) were pooled. E: All T1D or control (HD) donors, irrespective of DR3- or DR4-status were included. D: Population mean and 95% confidence intervals of longitudinal variation (CV) in net spot numbers are shown for each antigen, in only DR3 + (GAD-65) or DR4 + (IA-2 and PI) donors. C: Population mean and 95% CI of longitudinal variation in SIs assessed as CV (standard deviation ÷ mean) is shown for each antigen, in DR3 + (GAD-65) or DR4 + (IA-2 and PI) donors. Each line represents an individual donor. B: Longitudinal variations in net spots per 2 × 10 5 PBMC (left panel) and SIs are shown for DR4 + donors and IA-2 antigen-specific responses. Each individual donor is shown on the Y-axis while each independent visit is shown on the X-axis. SI values observed at each visit were plotted as a heat map showing minimum, median, and maximum values calculated based on values for all donors and all visits for each antigen. A: Data were analyzed by restricting to DR3 + T1D donors (GAD-65 specific response) or by restricting to DR4 + donors (PI- and IA-2-specific responses). Each subject's responses to each of the three epitopes at each visit were determined as net spots (spots with antigen – spots in media) or as stimulation index (SI, spots with antigen ÷ spots in media). Longitudinal variation in IFN-γ production in T1D donors.Īt each visit, IFN-γ production against DR4-restricted epitopes of IA-2 (752–775), proinsulin (C19-A3) and a DR3-restricted epitope of GAD65 (335–352), or without stimulation (media only, no antigen) were determined.
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