The Immunosuppressive Function And Its Mechanism Of Intratumoral CD11b~+CD15~+Granulocytes In Human Gastric Cancer Microenvironment

Objective:To explore the immunosuppressive function and its mechanism of IntratumoralCD11b~+CD15~+granulocytes in human gastric cancer microenvironmentMethods:1. According to the current research progress, CD11b and CD15were used as surfacemarkers. We separated the Intratumoral CD11b~+CD15~+granulocytes in gastric cancerand peripheral tissues and detected the proportion and phenotypic changes by usingflow cytometry.2. Activated CD15~+granulocytes infiltrating in gastric cancer or peripheral tissues wereisolated by inmunomagnetic beads and cultured together with T cells from healthyhuman5days for exploring the immunosuppressive function.3. Purified CD15~+granulocytes from healthy donors were incubated with mediacontaining IL-10or PBS for3days, and then cells were co-cultured with T cells foranother3days as described before to test whether IL-10could induce CD15~+granulocytes more suppressive.4. Arginase I levels were determined by ELISA in the co-culture system to determine itspossible mechanism.Results:1. Flow cytometry results show that: High levels of CD11b~+CD15~+granulocytes were detected in the tumor tissues from gastric cancer patients compared with pairedadjacent tissues (30±5%vs4±2%, P<0.05). Granulocytes in gastric cancer tissueshad a significantly decreased HLA-DR expression compared with adjacent tissues(8±3%vs40±5%, P<0.05).2. T-cells co-cultured with CD11b~+CD15~+granulocytes from gastric cancer tissuesexhibited markedly diminished expression of granzyme B（7±2%vs45±4%, P<0.05).The production of IL-2and IFN-γ in those T-cells was also significantly reduced（10±2%vs50±5%, P<0.05）IFN-γ（8±3%vs48±9%, P<0.05).3. Granulocytes cultured with IL-10had an increased suppressive activity. The productionof IL-2and IFN-γ in those T-cells was significantly reduced. IL-2（control group48±5%vs anti-ARG-1group40±3%vs IL10introduction group15±2%, P<0.05）IFN-γ （control group40±5%vs anti-ARG-1group28±4%vs IL10introduction group12±2%, P<0.05).4. ELISA was confirmed that arginase I concentration was higher in supernatants whichwere then collected from CD15~+granulocytes from gastric cancer tissues co-culturedwith T cells (0.68±0.10ng/ml) than CD15~+granulocytes from healthy donors(0.10±0.02ng/ml).（P<0.05).Conclusion:1. Gastric cancer patients have elevated numbers of CD11b~+CD15~+granulocytes.2. T-cells function is highly suppressed by CD11b~+CD15~+granulocytes isolated from gastric cancertissues.3. IL-10could induce CD15~+granulocytes more suppressive. Granulocytes inhibit T-cellproliferation via Arginase I.