Immunological alterations in mice irradiated with low doses
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1 Immunological alterations in mice irradiated with low doses "Frédéric Joliot-Curie" National Research Institute for Radiobiology and Radiohygiene, Budapest, Hungary
2 The structure of the immune system INNATE IMMUNITY ADAPTIVE IMMUNITY granulocyte DC B-cell humoral immune response NK cell T-cell CD4 + T-cell cellular immune response macrophage mast cell CD8 + T-cell Hours Days
3 Interrelation between innate and adaptiv immune response Dendritic cells (DC) Professional antigen presenting cells Main roles: antigen uptake and presentation to T cells CD4 + (helper) T-cell (Th) Responsible for cellular immune response (secretion of cytokines and inflammatory factors) CD8 + (citotoxic) T-cells (Tc) Responsible for cellular immune response (removal of virus infected and malignant cells) clonal proliferation of cells CD4 + CD25 + (regulatory) T-cells (Treg) Responsible for the regulation of cellular immune response Main roles: inhibition of the proliferation of CD4 + and CD8 + T-cells, inhibition of DC maturation, establishment of immune tolerance
4 Objectives: - quantitative parameters of main lymphocyte subpopulations in the spleen (changes in relative cell number, apoptosis frequency) - cytokine expression of murine splenocyes - quantitative and functional alterations in regulatory T cells (Treg)
5 1. Quantitative alterations in the lymphocyte subpopulations Experimental outline: - Total body irradiation of mice - Isolation of total splenocytes at different time points after irradiation: 1, 3, 7 days. - Identifying the different splenocyte subpopulations by fluorescently labelled antibodies directed against specific phenotypic markers - Determining the proportion of these cells in the spleen by FACS
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7 2. Apoptotic frequency of lymphocyte subpopulations Experimental outline: - Total body irradiation of mice - Isolation of total splenocytes four hours after irradiation - Immuno-phenotyping of the splenocyte subpopulations - Determining the apoptotic fraction of each splenocyte subpopulation by the TUNEL assay
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9 3. Changes in cytokine expression of irradiated total splenocytes Experimental outline: - Total body irradiation of mice - Isolation of total splenocytes at different time points after irradiation: 4 hours, 1 and 3 days. - RNA isolation - Determination of cytokine expression by realtime RT-PCR
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11 Irradiation induced functional alterations in Treg cells Dendritic cells are potent antigen-presenting cells (APC) with the ability to activate or suppress T cells T reg cells are CD4+CD25+Foxp3+ T cells, suppress activation of the immune system Splenic DC-s are mainly CD11c+ and MHCII+
12 Treg cells the main regulators of the immune system - transplantation tolerance - immune tolerance during pregnancy - suppression of autoimmune reactions BUT - suppression of antitumor immune activation
13 Numerical changes in splenic Treg cells after irradiation Radiation-induced time-dependent alterations in splenocyte cell numbers Days after irradiation Total spleen cells CD4+ splenocytes CD4+Foxp3+CD25+ Treg cells control (±26.06) (±4.91) 1.93 (±0.53) (±3.27) 5.7 (±0.62) 0.66 (±0.12) (±2.57)* 4.85 (±1.04) 0.64 (±0.16) (±9.49)* 7.15 (±1.96)* 0.97 (±0.37)*
14 Radiation induced enrichment the fraction of splenic Tregs in Possible mechanisms: - decreased apoptosis - increased proliferation - increased conversion of peripheric CD4+Foxp3- cells into Foxp3+ - increased thymic de novo production of Tregs
15 Apoptosis in CD4+ cells
16 Proliferation kinetics of CD4+ cells
17 Conversion of CD4+ effector T cells into Foxp3+ Tregs
18 Radiation induced enrichment in the fraction of splenic Tregs Possible mechanisms: - decreased apoptosis - increased proliferation - increased conversion of peripheric CD4+Foxp3- cells into Foxp3+? - increased thymic output of Tregs
19 Influence of radiation on the suppressive capacity of Tregs effector T cell suppression DC suppression reduced suppressive capacity
20 Tregs: - radioresistant lymphocyte subpopulation - functionally compromised by irradiation
21 Collaborators: Enikő Noémi Bogdándi Andrea Balogh Eszter Persa Anett Benedek Tünde Szatmári Géza Sáfrány
22 Thank you for your attention! Thank You for Your Attention!
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