Biodistribution analyses

mRBC-CTRL or mRBC-OVA-4-1BBL-IL-12 cells were incubated with 10 μM CellTrace Far Red dye at room temperature for 6 min followed by quenching with FBS and extensive washing before dosing. Pep Boy (CD45.1 B6) mice were dosed with 1 × 10⁹ CellTrace Far-Red dye-labeled mRBC-CTRL or mRBC-OVA-4-1BBL-IL-12. At 1 h or 17 h post mRBC-aAPC dosing, mice were euthanized and perfused with PBS. One quarter of the spleen, the whole mandibular lymph node (LN), one of the mesenteric LNs, the bottom half of the left lung lobe, a 5-mm³ portion of liver, the bottom half of the heart, a 5-mm³ portion of brain, a 5-mm³ portion of large intestine, a whole kidney, an ovary, and the testes were frozen in Tissue-Tek^® Cryomold^® plastic molds (VWR International, LLC) containing Tissue-Tek O.C.T. Compound (VWR International, LLC) in a liquid nitrogen bath. Frozen tissues in O.C.T. Compound were stored at −80 °C before preparation of 7 μM sections that were placed onto slides. Slides were fixed with 4% formalin (Sigma-Aldrich), washed, and incubated with Hoechst dye (Thermo Fisher Scientific). After washing, coverslips were mounted on slides with ProLong^™ Gold Antifade Mountant (Thermo Fisher Scientific) and scanned on a 3DHISTECH PANNORAMIC™ scanner v2.1.1.10094 RTM (Thermo Fisher Scientific). Images were analyzed for mRBC density per tissue area using HALO image analysis software V2.2.1870.44 with High Plex FL module V2.0. Samples that were not sectioned or stained properly were excluded from analysis and noted in the source data.