Extended Data Fig. 3: Flow cytometric analysis of metabolism in the proliferative-exhausted subpopulation (cluster 4) of CD8⁺ T cells.

(a) Heatmap summary of proliferation and exhaustion markers across metabolically defined CD8⁺ T cell clusters. Metabolic cluster 4 is the proliferative-exhausted subpopulation. (b) Representative flow cytometry plot of the gating strategy for proliferative-exhausted cells. Gating was performed on all CD8⁺ T cells. Since all CD8⁺ Ki-67⁺ cells were also PD-1⁺ in scRNA-seq analysis and because of flow cytometer color limitations, we gated on this population with Ki-67. (c) Expression of HK2 mRNA from COVID-19 patient scRNA-seq data in the proliferative-exhausted CD8⁺ T cell subpopulation (that is, metabolic cluster 4) vs. other CD8⁺ T cells (non-metabolic cluster 4: n = 24,958 cells; metabolic cluster 4: n = 1,008). (d) Protein expression level of HK2 from flow cytometric measurements of COVID-19 patient samples in the gated, proliferative-exhausted CD8⁺ T cell subpopulation vs. other CD8⁺ T cells (other: n = 13 patient samples; proliferative-exhausted: n = 14). For (c) and (d), boxes indicate 25^th, 50^th, and 75^th percentiles. Whiskers indicate 1.5 interquartile ranges below and above the 25^th and 75^th percentiles, respectively. Statistical significances were calculated by two-sided Mann-Whitney U test. ***: p < 0.001. (e) Two-sided Pearson correlation of HK2 mRNA expression from scRNA-seq data and HK2 protein level from flow cytometry. (f) Two-sided Pearson correlation of % proliferative-exhausted cells among CD8⁺ T cells as determined from scRNA-seq data vs. flow cytometry. Shaded bands in (e) and (f) each indicates 95% confidence interval of linear regression. (g) Integrated metabolic activity of CD8⁺ T cells per disease severity category: mild/healthy (WHO 0–2), moderate (WHO 3–4), and severe (WHO 5–7). The contributions from CD8⁺ T cell subpopulations are shown with cluster-specific color. Metabolic activity is defined as expression levels of metabolic pathway-related genes from each metabolically defined CD8⁺ T cell subpopulation, multiplied by the fraction of CD8⁺ T cells that are in each subpopulation.