T-cell profiles from animals with hyperacute GVHD contain an abundance of transcripts associated with proliferation, and exhibit Th1 skewing. (A) First and third principal component projections reveal clustering of transplanted animals by immunoprophylactic strategy. Each dot represents an array sample. The center of inertia ellipses corresponds to the mean projections of the group (P < .05). (B) The first principal component shows a significant correlation with survival in a linear regression model (adjusted [Adj.], R² = 0.3299; P < .004). (C) Heatmap of the top 25 gene sets enriched in the hyperacute cohort using ssGSEA. The constellation map to the right of the heatmap allows for the identification of clusters of these gene sets. Those gene sets plotted closer to the center have a greater degree of similarity to the phenotype of interest (measured by the normalized mutual information [NMI] score) whereas the angular distance corresponds to gene-set similarity with one another. A dark-green edge further indicates gene-set similarity with the thickness of the line proportional to the Jaccard index.^10,11  (D) GSEA of transcripts previously shown to differentiate Th1 from Th17 cells.¹²  These molecules were found to be overrepresented in T cells from animals with hyperacute GVHD compared with healthy controls and the breakthrough acute cohort. *False discovery rate [FDR] < 0.01. (E) Flow cytometric analysis of PBMCs at the time of terminal analysis stimulated with phorbol myristate acetate (PMA)/ionomycin and measured for the production of IFNγ. *P < .05 using an unpaired t test.