Hsp70

Exosomes were extracted from the cell culture supernatant of bMSCs. The bMSCs were first washed twice with phosphate-buffered saline (Servicebio, Wuhan, China). The culture medium was then replaced with exosome depleted fetal bovine serum (System Biosciences, Palo Alto, CA, USA) for another 48 hours. The resultant supernatant was collected and first centrifuged for 10 minutes at 300 × g. It was then transferred to an ultracentrifuge (Hitachi Limited, Tokyo, Japan, Himac cp80wx) and centrifuged for 30 minutes at 10,000 × g. Further centrifugation of the supernatant was performed at 100,000 × g for 70 minutes to form an exosome pellet. The supernatant was discarded, and the pellet was resuspended in 500 μL phosphate-buffered saline for use in subsequent experiments. The exosomes were then identified using a transmission electron microscope (FEI, Hillsboro, OR, USA). Nanoparticle tracking analysis was performed using ZetaVIEW 8.04 (Particle Metrix, Meerbusch, Germany) to measure the diameter and particle number of the exosomes. Two protein samples from exosomes with different concentrations were prepared. The antibodies of exosome marker tumor susceptibility gene 101 (TSG101; Abcam, Cambridge, UK) and heat shock protein 70 (HSP70; Biolegend, San Diego, CA, USA) were detected using western blot assay.

ASTX660 was acquired from Astex Pharmaceuticals through a cooperative research and development agreement (CRADA) with the National Institute on Deafness and Other Communication Disorders (NIDCD). Pharmaceutical grade mitoxantrone (MTX) was obtained from the National Institutes of Health veterinary pharmacy. Recombinant human IFN-γ, and human and mouse TNFα were obtained from BioLegend. Fluorescent-conjugated antibodies for mouse tumor flow cytometry were obtained from eBioscience (CD3) and BioLegend (CD45.2, CD8a, H-2Kb/H-2Db, CD11b, CD11c, CD80, CD86, I-A/I-E). Viability dyes were obtained from BD Biosciences (7AAD) and Biolegend (Zombie Yellow, Zombie NIR). Unconjugated antibodies for flow cytometry in human cell lines were obtained from LSBio (TAP1) and Abcam (TAP2, LMP2) with corresponding secondary antibodies obtained from BioLegend. Fluorescent-conjugated antibodies for flow cytometry in human cell lines were obtained from Abcam (CRT, HSP70, HMGB1, and ERp57) and Biolegend (HLA-A,B,C). The in vivo anti-mouse MHC class I antibody used for ex vivo T cell impedance assays was from BioXCell (clone M1/42.3.9.8). Antibodies and concentrations used for ICD and APM flow panels are detailed in Supplemental Methods.