In Vitro Primary Responses of Mouse T Lymphocytes to Toxoplasma Gondii Antigens
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Date
2007-06-??
Authors
林大盛
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國立臺灣師範大學生命科學學系
Department of Life Science, NTNU
Department of Life Science, NTNU
Abstract
一般很難在體外以傳統非回憶性抗原，特別是可溶性蛋白質，刺激未免疫過T 細胞增生。此限制已嚴重影響到體外T 細胞反應之研究及T 細胞疫苗之發展。有報告指出在一特殊的培養系統，未致敏T 細胞可和非回憶性抗原反應。據此，本研究乃設計以建立體外小鼠未致敏淋巴球對弓蟲抗原之反應系統。使用兩種培養液 [RPMI-1640 及alpha modification of Eagles medium (MEM)]，並比較其對細胞增生之影響。取自弓蟲免疫過小鼠之淋巴細胞作為陽性對照細胞，而T 細胞mitogen-concanavalin A 當作陽性對照刺激物質。使用3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide 呈色試驗作為細胞反應之偵測。結果顯示，未免疫過小鼠之淋巴細胞只在添加menadione 之MEM 對弓蟲體抗原有反應。免疫過細胞在兩種培養液對弓蟲體抗原反應一樣好。在添加menadione之後，免疫過細胞在MEM 對弓蟲體抗原反應比在RPMI-1640 好。當使用弓蟲排泄/分泌抗原時，亦得到相似的結果。免疫和未免疫過細胞在兩種培養液中對concanavalin A反應一樣好。然而menadione在MEM 對concanavalin A反應的增強效果比在RPMI-1640好。去除CD4⁺ T細胞或附著性巨噬細胞，但非CD8⁺細胞，導致未免疫過細胞對弓蟲體抗原反應喪失，表示本系統有CD4⁺T細胞及抗原呈獻細胞參與。
In general, it has been difficult to stimulate proliferation of non-immunized T lymphocytes in vitro using conventional non-recall antigens, particularly soluble proteins. This limitation has placed severe restrictions on the study of T-cell responses in vitro and the development of T-cell vaccine. A report has indicated that naive T cells do respond to a non-recall antigen in a novel culture system. Accordingly, this study was designed to establish an in vitro system of responsiveness of naive mouse T lymphocytes to Toxoplasma gondii antigens. The effects of two media, RPMI-1640 and alpha modification of Eagles medium (αMEM), on cellular proliferation were compared. Lymphocytes from T. gondii-immunized mice were used as a positive control cells and T-cell mitogen-concanavalin A was used as a positive control stimulant. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide based colorimetric assay was utilized to evaluate cellular response. The results showed that lymphocytes from unimmunized mice responded to T. gondii somatic antigen only in αMEM with the addition of menadione. Immune cells responded to T. gondii somatic antigen equally well in both culture media. In the presence of menadione, immune cells proliferated significantly better to T. gondii somatic antigen in αMEM than in RPMI-1640. Similar results were observed when T. gondii excretory/secretory antigen was used. Both immune and non-immune cells responded equally well to concanavalin A in both culture media. However, menadione had more dramatic additive effect on the responsiveness to concanavalin A in αMEM than in RPMI-1640. The deletion of CD4⁺ T cells or adherent macrophages, but not CD8⁺ cells, abolished the responsiveness of non-immune cells to T. gondii somatic antigen indicating the involvement of CD4⁺ T cells and antigenpresenting cells in this system.
In general, it has been difficult to stimulate proliferation of non-immunized T lymphocytes in vitro using conventional non-recall antigens, particularly soluble proteins. This limitation has placed severe restrictions on the study of T-cell responses in vitro and the development of T-cell vaccine. A report has indicated that naive T cells do respond to a non-recall antigen in a novel culture system. Accordingly, this study was designed to establish an in vitro system of responsiveness of naive mouse T lymphocytes to Toxoplasma gondii antigens. The effects of two media, RPMI-1640 and alpha modification of Eagles medium (αMEM), on cellular proliferation were compared. Lymphocytes from T. gondii-immunized mice were used as a positive control cells and T-cell mitogen-concanavalin A was used as a positive control stimulant. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide based colorimetric assay was utilized to evaluate cellular response. The results showed that lymphocytes from unimmunized mice responded to T. gondii somatic antigen only in αMEM with the addition of menadione. Immune cells responded to T. gondii somatic antigen equally well in both culture media. In the presence of menadione, immune cells proliferated significantly better to T. gondii somatic antigen in αMEM than in RPMI-1640. Similar results were observed when T. gondii excretory/secretory antigen was used. Both immune and non-immune cells responded equally well to concanavalin A in both culture media. However, menadione had more dramatic additive effect on the responsiveness to concanavalin A in αMEM than in RPMI-1640. The deletion of CD4⁺ T cells or adherent macrophages, but not CD8⁺ cells, abolished the responsiveness of non-immune cells to T. gondii somatic antigen indicating the involvement of CD4⁺ T cells and antigenpresenting cells in this system.