Significance of haploidentical allogeneic lymphocytes infusion in induction of graft versus host disease and graft versus tumor in mice / 中华消化外科杂志

ABSTRACT

Objective To establish the mice model of immunological tolerance,and investigate the significance of haploidentical allogeneic lymphocytes infusion in induction of graft versus host disease and graft versus tumor in mice.Methods Sixty-four BALB/C female mice were randomly divided into 4 groups with 16 mice in each group.Control groupno special treatment was given after inoculation of tumor cells at the 4th day (CT26 colorectal cancer cell lines with mixture of 1 × 107/mL tumor cells suspension was inoculated to the right subcutaneous axillary of mice) ; Chemotherapy groupchemotherapy was applied at the 7th day after inoculation of tumor cells at the 4th day; DLI grouptumor cells were inoculated at the 4th day,and then haploid donor cells were infused at the 13th,15th and 17th day; Chemotherapy + DLI grouptumor cells were inoculated at the 4th day,chemotherapy was applied at the 7th day,and haploid donor cells were infused at the 13th,15th and 17th day.The pretreatment scheme included haploidentical allogeneic lymphocyte + ring ling amide + haploidentical allogeneic lymphocyte,and the chemotherapy regimen included peritoneal infusion of cyclophosphamide at the 3rd day after inoculation of tumor cells in mice.The time from the first day after vaccination to the day of death of mice and the mass of the tumors were detected to calculate the tumor inhibition rate.The clinical indexes of GVHD were observed,and clinical evaluation was made.The numbers of T lymphocytes in peripheral blood were detected by flow cytometry.Three mice were sacrificed in each group at the 15th day to make the tissue specimens,and they were observed under light microscope after HE staining.All data were analyzed using the analysis of variance or LSD-t test.Results The symptoms of GVHD of mice in the chemotherapy + DLI group were milder than those in other groups.The GVHD scores of the control group,chemotherapy group and the chemotherapy + DLI group were 2.3 ±0.6,1.5 ± 1.1,6.7 ±0.9 and 3.4 ±0.5,respectively,with significant difference between the 4 groups (F =148.68,P ＜ 0.05).The tumor masses of the control group,chemotherapy group,DLI group and the chemotherapy + DLI group were (3.40 ± 0.20) g,(0.80 ± 0.10) g,(2.20 ± 0.20) g and (0.50 ± 0.30) g,respectively,with significant difference between the 4 groups (F =149.17,P ＜ 0.05).The tumor inhibition rates of the control group,chemotherapy group,DLI group and the chemotherapy + DLI group were 0,77％ ± 9％,35％ ± 3％,85％ ± 44％.The levels of CD3 + of the control group,chemotherapy group,DLI group and the chemotherapy + DLI group were 52.3％ ± 2.9％,44.8％ ± 3.1％,62.9％ ± 3.5％,65.9％ ± 3.3％,respectively,with significant difference between the 4 groups (F =28.04,P ＜ 0.05).The levels of CD3 + CD4 + of the control group,chemotherapy group,DLI group and the chemotherapy + DLI group were 32.1％ ± 2.6％,27.1％ ± 1.1％,42.6％ ± 1.8％ and 41.7％ ± 2.4％,respectively,with significant difference between the 4 groups (F =40.29,P ＜ 0.05).The levels of CD3 + CD8 + of the control group,chemotherapy group,DLI group and the chemotherapy + DLI group were 22.7％ ± 2.2％,20.7％ ± 1.8％,26.7％ ± 0.8 ％ and 26.1％ ± 0.7％,respectively,with significant difference between the 4 groups (F =10.74,P ＜ 0.05).The levels of CD3 + CD4 + CD25 + of the control group,chemotherapy group,DLI group and the chemotherapy + DLI group were 8.7％ ±0.6％,6.6％ ±0.6％,11.2％ ±0.4％ and 13.3％ ± 0.7％,respectively,with significant difference between the 4 groups (F =82.88,P ＜ 0.05).Necrosis and bleeding of the tumor tissues were observed in all the 4 groups.Necrosis,shrinking of the tumor cells,inflammatory infiltration were observed in the DLI group and the chemotherapy + DLI group.Proliferation of lymphoid follicles was observed in the chemotherapy + DLI group.The survival time of mice in the control group,chemotherapy group,DLI group,chemotherapy + DLI group were (16.8 ± 2.5) days,(26.3 ± 2.9) days,(23.4 ± 2.5) days and (33.7 ± 4.6) days,respectively,with significant difference between the 4 groups (F =46.45,P ＜ 0.05).Conclusions (1) Pretreatment can induce specific immune tolerance in mice.(2) Haploidentical allogeneic lymphocyte infusion and chemotherapy have synergistic effects,joint application of haploidentical allogeneic lymphocyte infusion and chemotherapy can inhibit the proliferation of tumor cells and prolong the survival time of mice.(3) Chemotherapy can reduce the GVHD of haploidentical allogeneic lymphocyte infusion and enhance the GVT.(4) CD3 + CD4 + CD25 + T lymphocytes play important roles in decreasing GVHD.