Fig. 3: Distinct cell subclusters drive LC signatures in NK cells, CD8⁺ T cells and CD14⁺ monocytes.

a, UMAP of CD8⁺ T cells (left) and violin plots of AUC scores of TNF and TLR1–TLR2 pathways (right) within the identified subclusters CD226⁺ CD8⁺ T cells (C0), S100A4⁺ CD8⁺ T cells (C1), CD69⁺GZMK⁺ CD8⁺ T cells (C2), CD69^hiGZMK⁺ CD8⁺ T cells (C3) and KLRC2⁺KLRD1⁺ CD8⁺ T cells (C4) from all donors and all time points in cohort 1, as in Fig. 1a. Data were analyzed by two-sided Wilcoxon rank-sum test; ****P < 0.00001. b, UMAP of NK cells (left) and violin plots of AUC scores of TNF and TLR1–TLR2 pathways (right) in identified subclusters PRF1⁺GZMB⁺ NK cells (C0), GZMB⁺KLRF1⁺ NK cells (C1), GZMK⁺TGFB1⁺ NK cells (C2), IFNG⁺ NK cells (C3) and CALR⁺S100A9⁺ NK cells (C4) from all donors at all time points as in Fig. 1a. Data were analyzed by two-sided Wilcoxon rank-sum test; ****P < 0.00001. c, UMAP of CD14⁺ monocytes from all cohort 1 donors at all time points showing subclusters IL1B⁺ (MC1), S100A4⁺ (MC2), FCN1⁺ (MC3) and TGFB1⁺ (MC4) cells. d, Top significantly upregulated markers in MC1–MC4 CD14⁺ monocyte subclusters as in c. Plotted genes were significant with a Benjamini–Hochberg method-adjusted P value of <0.05 (two-sided Wilcoxon test). e, Differential enrichment of neighborhoods representing transcriptional states in LC^AM compared to AI^M (top) and LC^AM compared to R^LC (bottom) at months 1.7–2.9, 3–5.9, 6–8.9 and 9–11. Each dot represents a neighborhood of ~150–400 cells. Transcriptional states show significant enrichment with a spatial false discovery rate (FDR) of <0.1 (F-test statistic from the quasilikelihood F-test, graph-weighted FDR). f, AUC scores of TNF, TGFβ and WNT–β-catenin signaling pathways in MC1–MC4 CD14⁺ monocyte subclusters as in c. Data were analyzed by two-sided Wilcoxon rank-sum test; ****P < 0.00001. Horizontal dashed lines in a, b and f serve as visual reference for comparison of relative shifts in pathway AUC scores across clusters. g, Correlation of the percentage of MC1, MC2, MC3 or MC4 CD14⁺ monocyte subclusters with FAS score and pO₂ (LC^AM, n = 38; LC^AS, n = 13; R^LC, n = 9). Data were analyzed by Spearman correlation. P values were determined using the exact/permutation-based test. The gray shaded area indicates the 95% confidence interval. h, Box plot showing FAS score in MC4^hi (>10% of CD14⁺ monocytes found within MC4, n = 13), MC4^lo (<10% of CD14⁺ monocytes within MC4, n = 26) and R^LC (n = 7) samples from individuals with LC^AM, LC^AS and R^LC. Data were analyzed by two-sided Wilcoxon rank-sum test; **P < 0.01. The box plot shows the median (center), first and third quartiles (bounds) and 1.5 times the interquartile range (whiskers).