Improving the persistence, distribution and immune killing efficacy of antigen-specific cytotoxic T lymphocytes in tumor by interfering with the tumor microenvironment

Abstract

Objective To observe the role of improving the persistence,distribution and function of cytotoxic T lymphocytes (CTL) in tumor by inhibiting the tumor microenvironment with low-dose cyclophosphamide (CYC) administration.Methods Melanoma antigen-specific CTL were prepared in vitro,labeled with carboxyfluorescin diacetate succinimidyl ester (CFSE) and cultivated.Melanoma-bearing mice models were established and randomly divided into two groups:NSI + CTL group and CYCI + CTL group.The same amount of CYC or normal saline (NS) was intraperitoneally injected in mice,respectively.The levels of regulatory T cells (Tregs),transforming growth factor (TGF)-β and interleukin (IL)-10 were detected at different time points after injection.The cultured CTL were transfused at the 4th day after injection.The fluorescence intensity and cell cycle of CTL were analyzed by flow cytometry (FCM) and the morphological distribution of CTL were observed using laser scanning confocal microscopy.Perforin,granzyme B,IL-2,and interferon (IFN)-γwere assessed by real-time quantitative polymerase chain reaction (Real-time PCR).Tumor volumes in each group were compared.Results In CYCI + CTL group,Tregs (0.05 ±0.00 vs.0.27±0.07,P＜0.05) and IL-10 [(26.89±0.12) ng/L vs.(47.64±1.40) ng/L,P＜ 0.05] were clearly declined on the 4th day after CYC injection,but a reduction in TGF-β was delayed until the 7th day [(4.00 ± 0.12) ng/L vs.(9.80 ± 0.36) ng/L,P ＜ 0.05].The fluorescence intensity of CTL in tumors of CYCI + CTL group was significantly higher and lasted longer than that in NSI + CTL group from the 3rd day (91.90 ± 1.37 vs.39.70 ± 2.10,P ＜0.05).However,no significant difference existed in the cell cycle of lymphocytes (P ＞ 0.05).The amounts of granzyme B (P ＜ 0.05),perforin (P ＜ 0.05),IL-2 (P ＜ 0.05),and IFN-γ (P ＜ 0.05) in tumors of CYCI + CTL group were greater than in NSI + CTL group.The difference in tumor growth between the groups was most evident from the 6th to the 15th day.Conclusion Tumor immunosuppressive microenvironment was successfully inhibited by low-dose CYC administration,with decreases in the levels of Tregs,IL-10,and TGF-β simultaneously.As a result,more recruitment into tumor tissues and more sustainable effective time of the transferred CTL in vivo lead to the strenthened killing efficacy. Key words: Cellular immunotherapy; Cytotoxic T lymphocytes; Immune escape