Fig. 3.

Inflammation levels at onset of type 1 diabetes correlate with duration of the post-onset partial remission. (a) Among 19 TN-09 participants in the placebo arm (ages 8–35 years), Weibull (solid line, grey shaded area = 1 SE) and Cox regression (dashed line) analyses identified an inverse relationship between I.I.₃₅₉ and median remission duration (p = 0.012). The partial remission period was defined as the length of time from diagnosis in which the 2 h stimulated C-peptide AUC was >0.2 nmol/l. (b) Weibull (solid line, grey shaded area = 1 SE) and Cox regression (dotted line) analyses identified a significant relationship between I.I.₃₅₉ and median remission duration among 26 local individuals with diabetes (p = 6.8 × 10⁻¹⁰). These participants were aged 4–16 years and possessed high-risk HLA haplotypes; samples were collected 2–7 months after diagnosis (see ESM Table 1 for additional characteristics). As dynamic testing was not performed on the local cohort, the IDAA1c was determined from HbA_1c and total daily insulin doses at post-onset clinic visits as described in Mortensen et al [27]. IDAA1c ≤9 is reflective of a stimulated C-peptide >0.3 nmol/l; thus, the remission duration was defined as the last quarterly clinic visit when IDAA1c was ≤9. Among TN-09 participants in the placebo arm, remission lengths determined using the IDAA1c were highly correlated with those determined through dynamic testing (r = 0.79); however, as anticipated [49], on average, the use of the IDAA1c underestimated partial remission durations relative to dynamic testing (1.2 ± 1.0 years vs 2.2 ± 1.5 years, respectively). In (a, b) participant age range is indicated by colour: green, 0–6 years; black, >6–12 years; red, >12–18 years; blue, >18 years; boxes indicate participants in remission at the last visit. (c, d) Kaplan–Meier analysis: the proportion of TN-09 placebo-arm participants (n = 19) (c) and local individuals with diabetes (n = 26) (d) in remission was compared for those with I.I.₃₅₉ above the median (dashed line) and those with I.I.₃₅₉ below the median (solid line). A logrank test found participants with I.I.₃₅₉ above the median were significantly different in both populations (TN-09: p = 0.016; local individuals: p = 0.005). (e) Representative flow cytometry profiles showing the gating strategy for resting and activated Treg populations in cryopreserved PBMCs from local individuals with diabetes. Resting and activated CD4⁺ Tregs were respectively defined as CD45RA⁺/FOXP3^low and CD45RA⁻/FOXP3^high. Since CD45RA and CD45RO are different CD45 isoforms that are respectively expressed on naive and activated/memory T cells, the expression of CD45RO as well as CD25 confirmed the phenotype of resting (CD45RO⁻/CD25⁺) and activated (CD45RO⁺/CD25^high) Tregs. Each analysis included fluorescence minus one controls to ensure correct gating. (f) Per cent activated Tregs among total (active + resting) Tregs in cryopreserved PBMCs collected from 23 local individuals with diabetes during the immediate post-onset period. Eight of these participants were among the 26 analysed by plasma-induced transcription. The data were classified as having shorter or longer partial remissions using the Jenks natural breaks method. The resulting groups had ages of 8.4 ± 3.3 and 11.3 ± 3.8 years, respectively (not significant, p > 0.05). The per cent activated Treg was higher in those with longer vs shorter partial remissions (p = 0.016). The data are similar if plotted as percentage of activated Treg among total CD4⁺ T cells