(A) Cohort overview depicting all features used for unsupervised machine learning analysis: 244 children (24–59 months of age) vaccinated with LAIV; mucosal and blood samples collected on day 0 (prevaccination) and day 21 (postvaccination). Vaccine-induced immune responses calculated as fold-change relative to prevaccination levels. (B) Workflow schematic for automated clustering pipeline applying t-SNE dimensionality reduction, KNN graph construction, and Louvain community detection to identify distinct immunophenotypic clusters. (C and D) Louvain resolution sweep results used to assess cluster stability and select optimal number of clusters. (C) Modularity score plotted against Louvain resolution parameter, colored by number of clusters identified (3–6). High modularity indicates well-separated clusters. Red diamond indicates selected clustering parameters. (D) Number of clusters identified plotted against Louvain resolution parameter, colored by modularity score. Stability of 3-cluster solution (red diamond) is observed across range where modularity is maximal (Q ≈ 0.717). (E) Clustered t-SNE plot of fold-change data (post/pre-LAIV) revealing 3 distinct LAIV response phenotypes: group 1 (green, n = 82), group 2 (orange, n = 88), and group 3 (purple, n = 74). (t-SNE parameters: perplexity: 30; exaggeration factor: 4; max iterations: 10,000; theta: 0; eta: 500; K: 60 for KNN graph; final silhouette score: 0.40). (F and G) Clustering patterns overlaid with demographic factors on t-SNE map. (F) Clustering by sex (female, green; male, orange). (G) Clustering by study year (2017, green; 2018, orange). (H) Heatmap and hierarchical clustering display fold-change data for key immune features across 3 clusters (columns: groups 2, 1, and 3 from left to right). Rows represent immune features, clustered using Euclidean distance and Ward’s D2 method. Heatmap cells are colored based on scaled FC values from –1 (blue, low FC) to 1 (red, high FC). The top color bar indicates responder groups (group 1, green; group 2, orange; group 3, purple). Side color bars indicate qualitative response classifications derived from assays: HAI (purple: high, dark; low, light), IgA (orange: high, dark; low, light), CD4⁺ T cell (blue: high, dark; low, light), and CD8⁺ T cell (green: high, dark; low, light). Column cluster ordering optimized for visual clarity.