Figure 3

Expamers generate distinct but consistent T cell phenotypic and genetic profiles. (A,B) T cell phenotypic profiling was conducted by measuring surface expression of indicated surface markers by flow cytometry. Bar graph and Heat map present cumulative data from four donors. Error bars in (A) represent mean ± SD. (C) Analysis by PCA indicated that the Expamers titration did result in titration-dependent surface markers expression profiles for selected donors with the Dynabeads treatments distinctly different. Low Expamers and Dynabeads conditions resulted in more comparable T cell profiles. Greater difference was observed between donors (Component 2) than among conditions (Component 1). PCA analysis was based of T cell surface marker expression of 12 donors detected by flow cytometry (left panel, each donor indicated by different color and symbol shape). T cells were left unstimulated (grey shapes on right panel) or were stimulated for 24 h before data collection using either varying concentrations of Expamers (900%, 300%, 100%, 30%, 10% of standard concentration per 1 × 10⁶ T cells; blue gradient on right panel, darker color equals to higher Expamer dose) or different bead-to-cell ratios (3:1, 1:1, 1:3, 1:9, 1:18; orange-to-brown gradient on right panel, darker color equals higher Dynabeads content). (D) Depicted are volcano plots of the -log₁₀ adjusted P value vs. log₂ fold change with differentially expressed genes highlighted in color for one out of four representative Expamers condition. Differentially expressed genes were selected by imposing a log₂FC cutoff of 1 and Benjamini–Hochberg adjusted FDR cutoff of 0.1. Similar results were achieved for other three Expamers formulations (not shown). (E) The Venn diagram was generated to show that the different individual Expamers conditions (as in D) yielded similar differentially expressed genes compared to Dynabeads stimulation independent of titration conditions for both upregulated (top) and downregulated (bottom) genes. Differentially expressed genes were selected by imposing a log₂FC cutoff of 1 and Benjamini–Hochberg adjusted FDR cutoff of 0.1.