IL-10 Inhibiting BCG-induced Memory Th1 Cell Development

Objective:Bacillus Calmette-Guerin(BCG)is the only human vaccine that is currently approved for prevention and treatment in Tuberculosis,but it has lost the immunological efficacy on adults.In this study,we investigated the effects of IL-10 on the immune memory formation of CD4~+ T cells in mice immunized with BCG.CD62L is used for identification and segregation of central memory T(TCM)and effector memory T(TEM)cells.We evaluated the effects of experimental conditions on surface CD62L expression of primary murine CD3~+CD4~+ T cells.Next,we assessed the effects of IL-10 on the generation of memory CD4 + T cells in mice immunized with BCG.Methods:1)The effects of collagenase digestion,culture time and preservation conditions on CD62L expression were evaluated by flow cytometry.2)To optimize the immunological memory animal model induced by BCG immunization,different doses of BCG were used to immune mice.The murine CD4~+Tcm and TEM cells were determined by flow cytometry.3)The IL-10 in serum of BCG-immunized mice was detected by ELISA and the cell types of IL-10-producing cells was detected by flow cytometry.4)The levels of CD4~+ TCM and TEM cells from BCG-immunized mice(WT vs.IL-10-/-)were determined by flow cytometry.After immunization with BCG,the production of IL-4,IFN-y and IL-17A by the CD4~+ T cells upon restimulation with BCG lysis in vitro were also determined by flow cytometry.5)The expression of metalloproteinase 17(ADAM 17)in CD4~+ T cells were determined by Western blotting.Results:1)The number of CD4~+ T cells in mouse bronchial alveolar lavage fluid(BALF)was very few but it was highly enriched in mouse lung tissue.The surface molecule CD62L on CD4 + T cell was intolerant of ?,?,? collagenase digestion,but D-type collagenase digestion had a minor effect on CD62L expression.2)The higher levels of CD4~+ Tcm and TEM developed when mice was immunized subcutaneously with 1 x 108 CFU/mouse,while immunization with the lower doses of BCG did not efficiently induce an immune memory response.3)The expression of IL-10 in serum was increased 15 days after BCG immunization.The cells secreting IL-10 in the BCG-immunized mice were mainly DC and macrophages.4)BCG immunization can induce higher levels of memory CD4~+ T cells in IL-10-/-mice compared with WT mice.IFN-? secreted by CD4~+T cells from BCG-immunized IL-10-/-mice was significantly increased,5)The expression of AD AM 17 in CD4~+ T cells from IL-10-/-mice was significantly decreased compared with WT control group.Conclusion:1)We found that CD62L expression were more resistant to collagenase D treatment.Prolonged period of culture(at 37 ?)and preservation(at 4 ?)resulted in modulation of surface CD62L expression in vitro.These results could provide valuable information for the development and optimization of a reliable method for the detection of CD62L expression in vitro,especially when detect memory T cells by flow cytometry.2)Our data suggest that the absence of IL-10 is beneficial to the increase in the proportion of memory CD4~+ T cells in BCG-immunized mice.