Mice were immunized with 3 μg of each respective mRNA vaccine followed by treatment with 50 μg of α4-1BB or control antibodies on day 4. (A) Summary of LCMV-specific CD8⁺ T cell responses. (B) Representative FACS plots of LCMV-specific CD8⁺ T cells. (C) Pie diagrams showing CD8⁺ T cell subsets (gated on LCMV-specific CD8⁺ T cells). (D) Summary of OC43 spike–specific CD8⁺ T cell responses. (E) Summary of HIV env–specific CD8⁺ T cell responses. (F) Summary of OVA-specific CD8⁺ T cell responses. Data from A–C and F are after tetramer staining; data from D and E are after intracellular cytokine stimulation using overlapping peptide pools (IFN-γ⁺). Data from A–F are from day 14 after vaccination, and are from 2 experiments, one with n = 5 per group/experiment and one with n = 2–5 per group/experiment. (G) Experimental outline for measuring 4-1BB following mRNA vaccination. P14 cells were transferred into C57BL/6 mice. One day after transfer, recipient mice were immunized with 3 μg of an mRNA-LCMV GP vaccine, and 4-1BB was measured on P14 cells at various time points. (H) 4-1BB on P14 cells after mRNA vaccination. Representative histograms showing 4-1BB expression on P14 cells. We utilized this P14 chimera model using a high number of P14 cells to allow us to detect 4-1BB expression on virus-specific CD8⁺ T cells at hyperacute points; endogenous virus-specific CD8⁺ T cells cannot be detected at hyperacute time points due to their low precursor frequency. Mean fluorescence intensity (MFI) is indicated on the x axis to denote “per-cell expression” of 4-1BB. This adoptive transfer experiment was performed 2 times, with n = 3 per group, showing similar results (peak of 4-1BB expression on day 4 after vaccination). All data are shown. Indicated P values in A and D–F were calculated by the Mann-Whitney test.