Detection And In Vitro Induction Of AFP_158-166-specific T Cells

Objective:To detect AFP158-166-specific T cells in peripheral blood of patients with hepatocellular carcinoma and in vitro induct AFP158.166-specific T cells using lymphocytes from healthy doner. Lay a foundation for liver cancer immunotherapy.Method:1) FITC labelled HLA-A2antibodies and high resolution HLA-A2genotyping kit (PCR-SSP) were used to screen HLA-A0201positive patients. Peripheral blood mononuclear cells (PBMC) were harvested from peripheral blood through density gradient centrifugation separation method, T cell subsets were analyzed using FCM; AFP158-166specific-T cells in peripheral blood of patients with HCC were analyzed through R-PE labelled MHC-AFP158-166peptide Pentamers staining, detecting by flow cytometry.2) PBMC were harvested from the blood of HLA-A0201healthy volunteers, stimulated for AFP-specific T cell according to the following two methods.(1) T2hybridoma cells were cultured and loaded with antigen-epitope peptide AFP158-166. After irradiation with gamma-ray at75Gy, mixed with PBMC at1:1, maintained with IL-2, and stimulated repeatedly,(2) Dendritic cells were cultured with midium containing cytokines rhGM-CSF, rhIL-4and TNF-a, loaded with AFP158-166peptide, then co-cultured with the fresh lymphocytes at1:10in the presence of IL-2. Cells were harvested after apparent proliferation to evaluate cytotoxic activity and stain with pentamers.Results:1) There were18cases with HLA-A0201genotype from28patients with hepatocellular carcinoma. Approximatel×107PBMCs were harvested from2ml peripheral blood, but no MHC-AFP158-166peptide Pentamers+CD8+T cells were detected.2) After incubation with AFP158-166, the expression of HLA-A2molecules at T2cells surface increased. Mean fluorescence intensity and expression rate gradually increased with the increase of peptide concentration, Maximum effectiveness was achieved at the peptide concentration of45ug/ml and60μg/ml, and HLA-A2molecules expression decreased at the concentration of90μg/ml. Three cases of HLA-A0201genotype were found in10cases of healthy volunteers, DC in the number of (1.94±0.79)×106were harvested from20ml peripheral blood. Surface antigen was detected at the7th day. The expression of CD83, CD80, CD86and HLA-DR were (62.6±4.2)%,(90.4±2.4)%,(98.2±0.3)%and (87.9±4.2)%, respectively.3) There were a large number of cells proliferated after co-culture DC loaded with AFP with lymphocyte for14days. Pentamer+CD8+cells ratio was7.5%by FCM. No CD8+Pentamer+cells were detected in the lymphocytes co-cultured with T2after loaded with AFP.4) Cytotoxicity assay showed that the cytotoxicity of T cells stimulated by DC loaded with AFP to HepG2cells and T2cells loaded with AFP were significant higher than that to T2and T2loaded with Her2. At the effector to target ratios of20:1, the cytotoxity are (45.12±10.60)%and (39.06±6.48)%vs (14.83±9.82)%and (2.51±0.14)%, respectively. The cytotoxicity of T cells stimulated by T2loaded with AFP at effector to target ratios20:1were:HepG214.03±11,T2cells loaded with AFP (8.49±7.34)%, T2(3.26±0.32)%and T2loaded with Her2was (3.36±2.45)%. DC stimulated lymphocytes showed higher cytotoxicity than that of T2stimulated cells (p<0.05).Conclusion:No AFP158-166specific T lymphocytes in peripheral blood from patients with AFP+hepatocellular carcinoma were detected by MHC-peptide Pentamer, indicating that the immune system may have tolerance or incompetence to AFP.AFP158-166specific T lymphocytes can be stimulated using DC loaded with antigen peptide AFP158-166to stimulate PBMC from healthy volunteers in vitro, which can lyse AFP+hepatocellular carcinoma cells with specificity. These results supply an experimental basis for adoptive cellular immunotherapy of liver carcinoma.