Impact of age and time on immune profiles at day 100 of patients that went on to develop cGVHD. (A) Evaluation of the impact of the recipient age on the day-100 immune profile. Volcano plots showing significant markers that meet all 3 criteria of a P ≤ .05 (y-axis), ROC AUC ≥0.60 (circle, ≥ 0.60; cross, <0.6), and effect ratio ≥1.3 or ≤0.75 (x-axis). Cell populations are identified by color, with dark blue representing B cells, orange myeloid populations, yellow NK cells, purple NK_reg cells, green T cells, light blue T_reg cells, and dark red plasma cytokines. We divided each of the subject groups (cGVHD and controls) at their respective median age (∼12.5 years and 9 years) and repeated the above analysis for younger cGVHD vs younger controls. We restricted our analysis to the markers that were significant in the initial analysis (Figure 1A). The younger age group is represented by a solid circle the older age group by an open circle. (B) Evaluation of the impact of the time of onset of cGVHD on the day-100 immune profile. To examine the effect of cGVHD onset time, we divided the cGVHD subjects into 2 onset groups. The early-onset group (solid circles) was less than or equal to the median onset time, and the later onset group (open circles) was greater than the median onset time (166 days after HSCT). We contrasted each onset group against the group that did not develop cGVHD. (C) Group difference visualization with t-SNE. Each patient is represented as a point, where patients with similar marker values are projected onto similar locations in the t-SNE plot. Patients who later developed cGVHD are in blue, those with late aGVHD are in yellow, and non-cGVHD controls are in orange. The ellipses correspond to 1 standard deviation of from the mean of each group.