Combination Of Fractionated Radiotherapy And Programmed Death-1 Monoclonal Antibody Blockade In The Treatment Of Lymphoma In Mice

Object:The programmed death-1(PD-1)/ PD-ligand 1(PD-L1)pathway is frequently present in the tumor microenvironment(TME)and suppresses tumor immunity by inhibiting the activity of tumor-infiltrating lymphocytes(TILs),particularly CD8+ lymphocytes.PD immunotherapy involves stimulation of the immune response in the region surrounding the tumor,but is insufficient to prevent tumor progression.Emerging evidence demonstrates that fractionated radiotherapy(RT)can not only result in tumor cell death directly but also promote the generation of antitumor immunity.Therefore,in this study,we examined the effects of combined PD immunotherapy with fractionated RT on antitumor immunity and tumor growth in lymphoma.Methods:1.The analysis of PD-L1 expression in irradiated EL4 cells and the lymphoma model was established in mice.EL4 cells were irradiated with different doses of fractionated RT in vitro,and the expression of PD-L1 following fractionated RT was assessed with immunohistochemistry(IHC)and flow cytometric analysis.C57BL/6 mice were inoculated subcutaneously(s.c.)with EL4 cells in the both flank,one of which in the right flank was primary tumor(irradiated)and in the left flank was out-of-field irradiated tumor.Animals were randomly assigned to various groups when tumors volume reached approximately 100-150 mm3.Mice were treated with control Ig G,RT plus control Ig G,αPD-1 monoclonal antibody(m Ab)alone,and RT plus αPD-1 m Ab,respectively.Tumor volume and survival rates was evaluated and recorded.2.The immune cell profiles of the TME were determined.We harvested tumors in the both flank of mice 7 days after different treatment.Immunohistochemical method was adopted to mark CD3,CD4,CD8,FOXP3,PD-1and PD-L1 molecules in the tumor samples of each group.According to the proportion of the positive cells and the color of the positive parts in each visual field,a comprehensive analysis and semi-quantitative statistics were carried out.Results were averaged and used for statistical analysis to assess changes in the immune cell profiles of the TME.3.Systemic immune responses occurred in the combination therapy group.The blood,spleen,and draining lymph node(DLN)were harvested on day 7 after different treatment,which were analyzed by flow cytometry.Antibodies targeting CD45,CD4,CD8,CD3,Gr1,CD11 b were used.Data were analyzed using Flow Jo software.4.Combination therapy stimulated the antigen-specific CD8+ T-cell response.We harvested serum from treated mice and analyzed the levels of IFN-γ and IL-2 in the serum using ELISAs.And the cytotoxic activity of CTLs in the spleen was analyzed by MTT assays.5.To determine the optimal schedule for combining anti-αPD-1 mAbs with fractionated RT.we designed four combination therapy schemes as follows: mice harboring EL4-derived tumors received two fractionated of 8 Gy RT accompanied by application of anti-αPD-1 m Abs beginning on day 1 of the fractionated RT cycle(schedule A),on day 3 of the cycle(i.e.,on the day of the second fractionated RT;schedule B),4 days before starting fractionated RT(schedule C),or 4 days after completion of the fractionated RT cycle(schedule D).Mice were monitored for tumor growth and survival.Results:1.The expression of PD-L1 was elevated after exposure to fractionated RT.Our data demonstrated that fractionated RT plus anti-αPD-1 m Abs more effectively inhibited tumor growth than monotherapy in our mice model of EL4 lymphoma.Morever,combination therapy mediated synergetic abscopal responses in nonirradiated tumor lesions.2.The frequencies of CD3+,CD8+,and CD4+ TILs in the combination therapy group were higher than those in the control Ig G or monotherapy groups for both irradiated and out-of-field tumors.However,the number of FOXP3+ T regulatory cells(Tregs)was reduced in mice receiving combination therapy compared with that in mice receiving monotherapies or control Ig G.In addition,we assessed changes in PD-1 and PD-L1 expression and found increased expression of both targets in fractionated RT-treated mice.3.The synergistic effects of anti-αPD-1 m Abs and fractionated RT on increased CD3+,CD8+,and CD4+ T lymphocytes in the blood,spleen and DLN led to substantial tumor regression.However,the proportion of myeloid-derived suppressor cells(MDSCs(CD11b+Gr1+))decreased in the combination therapy group.4.Serum from mice in the combination therapy group contained significantly higher levels of IFN-γ and IL-2 than mice in the control or monotherapy groups.The cytotoxic activities in response to target cells EL4 in the combination therapy group were significantly higher than those in the corresponding control or monotherapy groups.5.Fractionated RT for 4 days followed by anti-αPD-1 mAb therapy was the superior schedule in mouse EL4 lymphoma.Conclusion: Our data indicated that systemic immune responses occurred following combination therapy with fractionated RT and anti-αPD-1 m Ab immunotherapy in mouse EL4 lymphoma.This combination therapy inhibited the growth of irradiated tumors and out-of-field tumors(abscopal effects).Furthermore,our data revealed that the best combination therapy was fractionated RT for 4 days,followed by anti-αPD-1 m Ab immunotherapy.