1. Research On The Anti-tumor Immunological Mechanism Of Plasmodium Infection In Mouse Three-negative Breast Cancer Animal Model 2. Establishment Of The Detection Method Of Fine Immune Cell Subsets And Its Application In AIDS Monitoring

This paper contains two parts,in the first part we aim to investigate the effects of Plasmodium infection on the tumor inhibition and antitumor T cell responses in a murine triple negative breast cancer(TNBC)model.Tumor antigen-specific immune response is dominated by T cell-mediated cellular immune response.After encountering tumor antigens presented by dendritic cells in the draining lymph nodes,CD8+ T cells differentiate into effector and memory subsets,then travel to tumor tissue and other tissues through the blood for killing tumor cells and providing long term protective immunity to prevent relapse.CD4+ T cells can also help the activation and differentiation of CD8+ T cells and help optimizes cytotoxic,migratory and invasive capacities of the CTL.Here,we reported that Plasmodium infection significantly inhibited the tumor growth,and increased the survival rate and prolonged the survival of the tumor-bearing mice.Multi-color flow cytometry analysis revealed that both effector and memory T cells were increased in peripheral blood and tumor-draing lymph nodes(DLN)in the Plasmodium-infected animals.The co-stimulatory(CD40L,GITR and OX-40)and co-inhibitory(PD-1,CTLA-4,TIM-3,LAG3)immune checkpoints were up-regulated on CD8+ T cells in infected mice.Similarly,the co-stimulatory(CD40L,GITR)and co-inhibitory(PD-1,CTLA-4,TIM-3,LAG3)immune checkpoints were also up-regulated on CD4+ T cells.Importantly,although the co-inhibitory immune checkpoints were up-regelated,the expression of granzyme B and perforin were significantly increased in CD8+ effector and memory T cells either in peripheral blood or in DLN of the infected mice.In summary,the results suggested that the effects of Plasmodium infection on murine 4T1 breast cancer might relate to the induction of CD8+ T cell-mediated antitumor immune responses.This finding may provide a novel strategy for the treatment of triple negative breast cancer.In the second part,we aim to establish a flow cytometry technology based precise immune cell subsets detection method,evaluate its methodological performance and investigate its application in AIDS monitor.According to the classification of the human immune cells subsets and their immunophenotyping characteristics,design a immune cell subsets detection panel.Detect different fluorescent dyes labeled monoclonal antibodies panel by flow cytometer,classify different immune cell subsets according to the antigen expression profiles,and analyze different immune subsets' proportion and absolute count results which are calculated according to the CBC results.The repeatability of the detection method is evaluated by intra-assay and inter-assay precision analysis.Ten healthy samples are analyzed by this panel,evaluate the distribution of the proportion and absolute count result of the immune cell subsets and calculate the median,minimum and maximum results of the immune cell subsets.Three AIDS patients samples are selected for the immunophenotypic testing,to analyze the changes of each immune cell subsets and t-test is used to compare the statistical differences between the results of AIDS group and the healthy control group.The results shown that in this method,the immune response relative innate immune cells such as granulocytes,monocytes / macrophages,natural killer cells and adaptive immune cells such as T lymphocytes and B lymphocytes are preliminarily classified and quantified by detecting different monoclonal antibodies panel.According to different antigens expression pattern,the functional subsets and activation states of various types of immune cells were further subdivided.Sixty-seven immune cell subgroups could be identified and the functional state of each cell subgroup could be evaluated more accurately and comprehensively.The results of intra-assay precision experiment show that most of the CV value of the proportion and the absolute count results of sixty-seven cell subsets are less than 20%,median values are 3.87% and 5.70%.The CV values of proportion results are greater than 20% in nine cell subsets,and the results of which are all less than 5%.The CV value of absolute count results was greater than 20% in 9 cell subsets,and the results of which are all less than 20 /ul.The results of inter-assay precision experiment show that most of the CV value of the proportion and the absolute count results of sixty-seven cell subsets are less than 20%,median values are 5.13% and6.25%.The CV values of proportion results are greater than 20% in six cell subsets,and the results of which are all less than 5%.The CV value of absolute count results was greater than 20% in six cell subsets,and the results of which are all less than 20 /ul.This method preliminarily summarized the median,minimum and maximum values of the proportion and the absolute count results of each immune cell subgroup in Chinese population,which can be used as a reference for evaluating the immune status of the body.This method is used to test the samples from AIDS patients,and the proportion and absolute count of sixty-seven cell subsets in the AIDS group and the healthy control group are analyzed.The results show that nineteen cell subsets in the AIDS group are increased compared with the healthy control group,which include T lymphocytes,Ts lymphocytes,immature natural killer cells,naive B lymphocytes,B2 cells,CD4+ Effector T cells,CD4+ Effector memory T cells,CD4+CD27-Differentiated/senescent T cells and CD8+ Effector T cells,CD8+ Effector memory T cells,CD8+CD27-Differentiated/senescent T cells,CD8+CD28-Differentiated/senescent T cells,CD8+CD57+ Differentiated/senescent T cells,early stage of activated monocyte,Later stage activated T cells,Later stage activated Th cells,CD16 positive myeloid dendritic cells,memory regulatory T cells,and natural regulatory T cells.The proportion and/or the absolute count results are statistically significant(P<0.05).Compared with the healthy control group,fourteen cell subsets in the AIDS group are decreased,which include Th cells,natural killer cells,mature natural killer cells,Intermediate monocytes,Marginal zone B cells,Memory B cells,B1 cells,Naive CD4+ T cells,Central memory CD4+Tcells,Naive CD8+ T cells,Central memory CD8+Tcells,Plasmoid dendritic cells,middle stage activated Th cells,naive regulatory T cells.The proportion and/or the absolute count results are statistically significant(P<0.05).There is no statistically significant(P>0.05)on the results of other subsets.So we conclude that detect different fluorescent dyes labeled monoclonal antibodies panel by flow cytometer can precisely classify the functional subsets of the innate immune cells which involve granulocytes,monocytes/macrophages,natural killer cells,dendritic cells and the adaptive immune cells which involve T lymphocytes and B lymphocytes.The amount of these cells can be calculated which can help to evaluate the state of the immune function comprehensively.This method has a good repeatability,and the minimum and maximum range of each immune cell subgroup in Chinese population can be used as a reference for clinical use to identify the abnormal changes of cell subsets.This method has a wide range of clinical application.This study takes AIDS as an example to conduct a preliminary analysis on the changes of each immune cell subset.Some of the results verify the existing research conclusions and also have some new findings which provide more ideas for the follow-up research.