Heterogeneity and Homogeneity in Treg responses to Interleukin-2 [RNAseq2: Differential activation of Immune cell types by IL2-Immunocytokines]

IL2 is an immune-regulatory cytokine that activates both effector and T regulatory cells—a dual function leveraged in immunotherapy. However, pure IL2 induces both activation and suppression, and requires toxic doses due to its short half-life. To address this, the Spangler Lab at Johns Hopkins developed IL2 immunocytokines (IL2-ICs)—antibody-conjugated IL2 fusion proteins targeting IL2Ra (Tregs) or IL2Rb (effector cells), with extended serum half-life. We tested IL2-ICs in Foxp3IRES-GFP mice, harvesting cells at 4, 24, and 64 hours post-injection for bulk RNA-seq. Transcriptomic analysis showed IL2-ICs induced sustained IL2 responses with partial cell-type bias. Overall design: Five treatments were tested: 1: F5111 IL2-IC, which targets IL-2 activity to Treg cells. 2: F10 IL2-IC, which targets IL-2 activity to NK cells. 3 and 4: Two FITC-IL2 controls, in which IL-2 is bound to a biologically irrelevant antibody that does not direct activity to any specific cell type. To match the N297A mutation present in F10 IL2-IC, the corresponding FITC control also included this mutation. 5: PBS, as a negative control. Each treatment was administered to 20 mice: 10 were injected 24 hours before tissue harvest and 10 were injected 4 hours before. Thus, each treatment group included both time points. Spleens were harvested at 4 and 24 hours post-injection. Treg cells, B cells, and NK cells were sorted for low-input bulk RNA sequencing. B cells, which do not respond to IL2-ICs, served as a reference to distinguish differential responses in Treg and NK cells.