Establishment Of A Humanized Mouse Model With Human Immune System Reconstitution And Allogeneic Human Melanoma Growth

Objective:To construct a humanized mouse model with both human immune system and human tumor growth,and to provide basis for human cancer immunological research and verify the efficacy of cancer immunotherapeutic regimens in vivo.Methods:Humanized mice with functional human immune system were established by intravenous injection of human CD34 fetal liver cells combined with implantation of human fetal thymic tissues under the renal capsule of NOD/SCID IL2rg^-/-after sub-lethal total body irradiation.Immune cell reconstitutions were monitored every 3 weeks from the 3th week post humanization,including in CD45,CD3,CD3CD4,CD3CD8,CD20,CD33 and CD56 cells.Human A375 melanoma cells were subcutaneously inoculated in the15th week after humanization.Six weeks after tumor inoculation,the mice were sacrificed and human immune combinations as well as their phenotype in blood,bone marrow,spleen,lymph node and tumor were measured by flow cytometry and immunohistochemistry.Results:At the 15th week after humanization,the percentage of human T cells in the peripheral blood of the humanized mice was 33.53%±8.57%and the percentage of human B cells was 43.33%±10.05%.The human tumors grew slowly during the first 3 weeks,thereafter the human tumor sizes expanded rapidly and reached 1564±334.3 mm³ at 6th week after tumor inoculation when the animals were sacrificed for detailed analysis.The immunohistochemical and flow cytometric analysis demonstrate that a large number of human T cells,which mainly composed by CD8 T subsets,infiltrated in human tumor.Additionally,the CD8 T/CD4 T ratios within human tumor infiltrated T cells were found significantly higher than those in peripheral blood?t=7.544,P<0.0001?,bone marrows?t=7.549,P<0.0001?,lymph nodes?t=6.544,P<0.0001?,and spleens?t=6.923,P<0.0001?.Further analysis reveals that the levels of PD-1 molecules expressed in tumor infiltrated human CD4 T and CD8T cells were higher than those in peripheral blood and lymphoid organs.Conclusion:In this study,high levels of human CD45 white blood cells,including human T cells,were detected in the peripheral blood,like lymph nodes immune organs and human melanoma in the mouse models,which indicating the successful establishment of a humanized mouse models with human immune system reconstitution and allogeneic human melanoma growth.This study found that the ratio of CD8 T/CD4 T cells in melanoma was much higher than that in peripheral blood and immune organs.In addition,these results showed that a large number of CD8 T cells with potential anti-tumor ability were infiltrated in the melanoma.This study found that the surface of these tumors infiltrated CD4 T and CD8 T cells with high levels of PD-1 molecular expression.This explains why the infiltration of a large number of human T cells into the tumor can still escape the immune attack and grow rapidly,and also indicates that the mouse model established in this study can be used for the functional evaluation of PD-1 and other immune checkpoint inhibitors.