Fig. 2: MHC-I‒E7‒hEry specifically activates cytotoxic CD8⁺ T cells from HPV16⁺ cervical cancer patients.

a Left: flow cytometry plots of MHC-I‒E7‒hEry stained for human IgG. Right: quantification of MHC-I‒E7 proteins on erythrocytes analyzed by ELISA (n = 5 per group). b IFN-γ-secreting T cells were assessed using an ELISpot assay. PBMCs isolated from HPV16⁺ CESC patients or healthy donors (HD) were co-incubated with hEry, MHC-I‒E7‒hEry, or HPV16 E7 peptide (YMLDLQPET) for 48 h. Left: representative images of ELISpot wells. Right: the quantification of IFN-γ spots (CESC patients, n = 8; HD, n = 4). c Left: representative flow cytometry plots of HPV16-specific (tetramer⁺) CD8⁺ T cells after hEry or MHC-I‒E7‒hEry treatment. Right: HPV16-specific (tetramer⁺) CD8⁺ T cells from CESC patients after different treatments (n = 4 per group). d In vitro killing assay. PBMCs isolated from HPV16⁺ CESC patients or healthy donors were cultured with hEry or MHC-I‒E7‒hEry for 48 h. Following stimulation, CD8⁺ T cells were isolated and further co-cultured with Calcein AM-labeled Ca-ski tumor cells for additional 72 h. Left: representative images of tumor cells after the killing assay. Right: the relative cytotoxicity of CD8⁺ T cells against tumor cells under different treatments (CESC patients, n = 8; HD, n = 4). e Left: visualization of PBMCs from the CESC patient or the healthy donor after treatment with hEry or MHC-I‒E7‒hEry analyzed by mass cytometry using t-distributed stochastic neighbor embedding (t-SNE) plot. Right: proportion of effector CD8⁺ T cells in total T cells was estimated by R_O/E values. Data are presented as means ± SEM. Significance was determined by one-way ANOVA with Dunnett’s multiple-comparison test (b), unpaired t-test (c, d) or chi-square test (e). The significance levels are indicated as follows: ns, not significant; *P < 0.05; **P < 0.01; ****P < 0.0001.