Fig. 1: Erythrocytes conjugated with MHC-I molecules efficiently activate T cells in vitro.

a Schematic representation of the MHC-I‒Ery preparation procedure. G-Mal: GAASK-6-maleimide. Mal-LPET*G: the asterisk denotes the 2-hydroxyacetic acid between the threonine and glycine residues. b Diagram illustrating the mouse MHC-I‒E6‒IgG1 Fc construct. c SDS-PAGE analysis of the purified mouse MHC-I‒E6‒IgG1 Fc fusion proteins. d Flow cytometry plots of MHC-I‒E6‒mEry stained for human IgG. e Pharmacokinetic study of MHC-I‒E6‒mEry and mEry in C57BL/6 mice. Far-Red-labeled MHC-I‒E6‒mEry or mEry were injected into C57BL/6 mice intraveneously (i.v.) Blood samples were collected at the specified times, and the percentage of Far-Red-labeled erythrocytes in circulation were determined by flow cytometry (n = 5 mice per group). f IFN-γ secreting T cells were assessed using an ELISpot assay. Splenocytes harvested from MC38-HPV16 tumor-bearing mice were co-incubated with mEry, MHC-I‒E6‒mEry, HPV16 E6 peptide (KCLKFYSKI) or recombinant protein of MHC-I‒E6 for 48 h. Low (0.5 μg/mL) or high (25 μg/mL) represent recombinant MHC-I‒E6 protein dosages used in this experiment. Left: representative images of ELISpot wells. Right: the quantification of IFN-γ spots (n = 8 per group, except for n = 3 for PMA group). g In vitro killing assay. Splenocytes from MC38-HPV16 tumor-bearing mice were cultured with mEry or MHC-I‒E6‒mEry for 48 h. Then, CD8⁺ T cells were isolated and further co-cultured with Calcein AM-labeled MC38-HPV16 tumor cells for additional 72 h. The survival of the MC38-HPV16 tumor cells were subsequently calculated by Calcein AM fluorescence. Left: representative images of tumor cells after the killing assay. Right: the relative cytotoxicity of CD8⁺ T cells against tumor cells upon different treatments (n = 8 per group). Data are presented as means ± SEM. Significance was determined by one-way analysis of variance (ANOVA) with Dunnett’s multiple-comparison test (f) or unpaired t-test (g). The significance levels are indicated as follows: ns, not significant; ****P < 0.0001.