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Updated: Aug 8, 2026
Examination of Thymic Positive and Negative Selection by Flow Cytometry
Published on: October 8, 2012
Distinct requirements of positive and negative selection for selecting cell type and CD8 interaction
G Schönrich1, G Strauss, K P Müller
1Department of Somatic Genetics, Deutsches Krebsforschungszentrum DKFZ, Heidelberg, Germany.
Insights
Positive selection in the thymus requires CD8 interaction and specific cell types, unlike negative selection. Thymic epithelial cells, not thymocytes, are crucial for positive selection, even with increased MHC class I expression.
Area of Science:
- Immunology
- T cell development
- Thymus biology
Background:
- Positive and negative selection in the thymus are critical for T cell repertoire development.
- Thymic epithelial cells are known to mediate positive selection, but the role of thymocytes is unclear.
- The precise requirements for CD8 interaction and selecting cell type in thymic selection remain to be fully elucidated.
Purpose of the Study:
- To investigate the necessity of CD8 interaction for positive selection.
- To determine the role of cell type (thymic epithelial cells vs. thymocytes) in mediating positive selection.
- To differentiate the requirements for positive and negative T cell selection in the thymus.
Main Methods:
- Generation of CD2.Kb transgenic mice with high MHC class I expression on thymocytes.
- Cross-mating transgenic mice with T cell receptor (TCR) transgenic mice (Des.TCR and 2C.TCR).
- Creation of hybrid MHC class I genes (Kk/A2, Kb/A2) to impair CD8 binding for functional assessment.
Main Results:
- Despite high MHC class I expression, thymocytes failed to induce positive selection in CD2.Kb transgenic mice.
- Impaired CD8 interaction using hybrid MHC class I genes abolished positive selection.
- Negative selection remained functional in thymocytes even when CD8 interaction was impaired.
Conclusions:
- Positive selection is highly dependent on CD8 interaction and specific cell types, unlike negative selection.
- Thymic epithelial cells possess unique properties essential for positive selection that thymocytes lack.
- These findings highlight distinct cellular and molecular requirements for positive and negative T cell selection in the thymus.
Abstract:
We investigated the requirements of positive and negative selection in the thymus for CD8 interaction and the selecting cell type. Thymic epithelial cells are known to mediate positive selection, whereas thymocytes fail to do so. The reason for this failure could be either the low amount of MHC class I molecules on thymocytes or the lack of other properties required for positive selection. To address this question a CD2.Kb transgenic mouse was prepared in which the expression of the Kb gene is under control of the CD2 promoter. In these mice the thymocytes exhibited very high levels of Kb. The mice were crossed with two TCR transgenic mice expressing either the Des.TCR (anti-Kb), which is positively selected on Kk and negatively on Kb, and the 2C.TCR (anti-Ld) with positive selection on Kb and negative on Ld. Despite the high Kb expression on thymocytes in CD2.Kb x 2C.TCR mice no positive selection was observed, whereas efficient negative selection was found in CD2.Kb x Des.TCR F1 mice. Thus, although thymocytes can negatively select, even a strong increase of MHC class I expression cannot convert them into positively selecting cells. This is consistent with the notion that thymic epithelium is specialized for positive selection, but the respective difference between thymocytes and thymic epithelium is not clear. The influence of CD8 was investigated using transgenic mice expressing a Kk/A2 or a Kb/A2 hybrid gene in which the promoter, the alpha 1, alpha 2 domains were of mouse origin and the alpha 3 domain of human origin. Because the murine CD8 molecule does not efficiently bind to human HLA class I, in these mice the CD8 interaction was impaired. In Kb/A2 x 2C.TCR and Kk/A2 x Des.TCR mice no positive selection of the respective TCR was found, whereas in Kb/A2 x Des.TCR mice negative selection was still functional. Altogether, the results indicate that positive selection depends more strictly on CD8 interaction and cell type than negative selection.
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