Objective To establish a stable in vitro model of CD8+T cell exhaustion.Methods CD8+T cells were isolated and purified from the spleens of ovalbumin-specific CD8+T cell receptor(OT-I)transgenic mice and subjected to chronic antigen stimulation to induce exhaustion in vitro.Flow cytometry was employed to evaluate the expressions of exhaustion markers,secretion of effector cytokines,and transcription factor profiles in CD8+T cells.Exhausted and effector(non-exhausted)CD8+T cells were co-cultured with tumor cells before tumor cell viability was measured to assess the cytotoxic potential of CD8+T cells.Additionally,N-acetyl-L-cysteine(N-AC)was used as a positive control during exhaustion induction to validate the model.Results Chronic stimulation resulted in a significant upregulation of inhibitory receptors,including programmed cell death protein 1(PD-1),T cell immunoglobulin and mucin domain-containing protein 3(TIM-3),T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motifdomain(TIGIT),and lymphocyte activation gene 3(LAG-3).Concurrently,the secretion of key effector cytokines such as tumor necrosis factor-α(TNF-α)and interferon-γ(IFN-γ)was markedly reduced.Exhausted CD8+T cells exhibited diminished cytotoxicity against tumor cells compared to effector CD8+T cells.Notably,treatment with N-AC effectively restored the function of exhausted CD8+T cells and enhanced their anti-tumor activity.Conclusion This study has established an effective in vitro model for CD8+T cell exhaustion.The use of N-AC demonstrates its potential to restore functionality in exhausted CD8+T cells,underscoring the reliability and utility of this model for investigating the anti-tumor potential of exhausted T cells.

BACKGROUND:Calcium metaphosphate has excel ent biocompatibility, degradability, and cel affinity. Human bone marrow mesenchymal stem cel s can grow and proliferate in the pores of the porous calcium metaphosphate, but less is known about calcium metaphosphate nanoparticles.
OBJECTIVE:To prepare calcium metaphosphate nanoparticles, and to analyze the effect of calcium metaphosphate nanoparticles at different concentrations on apoptosis of human bone marrow mesenchymal stem cel s by flow cytometry.
METHODS:The calcium metaphosphate nanoparticles were prepared by wet bal mil ing. Scanning electron microscopy and transmission electron microscopy were used to observe the morphology of the calcium metaphosphate nanoparticles, and the crystal structure of nanoparticles was analyzed by X-ray diffraction. Calcium metaphosphate nanoparticles were mixed in the CYAGON Oricel TM basal medium, and the concentrations of calcium metaphosphate nanoparticles in the medium were 10, 1, 0.1 mg/L. Human bone marrow mesenchymal stem cel s were cultured for 7 days in the above-mentioned media, and apoptosis of human bone marrow mesenchymal stem cel s was analyzed by flow cytometry.
RESULTS AND CONCLUSION:Calcium metaphosphate nanoparticles were successful y prepared by wet bal mil ing, irregular in shape, and the mean diameter was 10-30 nm. X-ray diffraction results showed the crystal structure of nonaparticles was mainlyβ-Ca(PO3)2. The cel ratio of G0/G1 phase and G2/M phase in 10 mg/L group was obviously higher than that in 1, 0.1 mg/L groups (P<0.01). The cel apoptosis rates during the early, middle, late stages in 10 mg/L group were obviously higher than those in 1, 0.1 mg/L groups (P<0.01), and the total cel apoptosis was also significantly increased in 10 mg/L group (P<0.01). These findings indicate that human bone marrow mesenchymal stem cel s proliferation can be inhibited by calcium metaphosphate nanoparticles, and apoptosis rate is increased significantly when the concentration of calcium metaphosphate nanoparticles increases from 1 mg/L to 10 mg/L.