Primary Study Of CTL Effect Induced By Autologous DC Pulsed With Gp96-associated Petides On Circling Tumor Cells

Objective:To test specific cytotoxic lymphocyte (CTL) effect induced by gp96-peptide complexes isolated from renal clear cell carcinoma (RCC) tissue, against autologous peripheral blood circulating tumor cells.Contents:gp96-peptide complexes were isolated from renal clear cell carcinoma (RCC) tissue using BIO-RAD duoflow protein purification system. The presence of circulating tumor cells were identified in peripheral blood from patients with RCC and cells were isolated for in vitro culture. After pulsing with gp96-peptide complexes, autologous dendritic cells were co-cultured with autologous lymphocytes for 72 hours, then cultured circulating tumor cells were added. Specific CTL effect was evaluated by microscope, detecting secretion of cytokines and calculating killing activity.Methods:1. gp96-peptide complexes were isolated from 30 tumor tissues, excised from patients who were diagnosed with renal clear cell carcinoma, using BIO-RAD duoflow protein purification system. Purified proteins were identified by SDS-PAGE and Western blot and densities were evaluated by BCA.2.10ml peripheral blood was harvested and processed density gradient centrifugation. The presence of circulating tumor cells were determined by though blood smears, or identified by further cell culture.3. Mononuclear cells were isolated from RCC patients identified having circulating tumor cells using COBE spectra TM blood separation system.4. Autologous dendritic cells and T lymphocytes were cultured from isolated mononuclear cells; purified gp96-peptide complexes were added on day 7; 3 days hours later, cultured circulating tumor cells were added.5. The secretion of IFN-γby activated T cells was tested and killing activity to circulating tumor cells was calculated.Results: 1. SDS-PAGE and Western blot identified the purified proteins from RCC tissue were gp96-peptide complexes.2. Through HE stain and microscope detection,20 RCC patients were identified to have circulating tumor cells in the peripheral blood.3 Dendritic cells and lymphocytes grew well in vitro; some circulating tumor cell adhered to flask and processed nuclear division.4. gp96-peptide complexes activated T cells could attack circulating tumor cells to cause cell disintegrating, and released IFN-γ.Conclusions:1. gp96-peptide complexes can be successfully purified from RCC tissue using ammonium sulfate grading salting-out and BIO-RAD duoflow protein purification system.2. Circulating tumor cells could present in peripheral blood from patients with RCC and can be identified by blood smears and in vitro ceil culture.3. Autologous gp96-peptide complexes could activate T cell and induce effective CTL response to kill circulating tumor cells...