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A system to innocuously visualize T cell lineage commitment is described. Using a "knock-in" approach, we have generated mice expressing a β-galactosidase reporter in place of CD4; expression of β-galactosidase in these animals appears to be an accurate and early indicator of CD4 gene transcription. We have exploited this knock-in line to trace CD4/CD8 lineage commitment in the thymus, avoiding important pitfalls of past experimental approaches. Our results argue in favor of a selective model of thymocyte commitment, demonstrating a fundamentally symmetrical process: engagement of either class of major histocompatibility complex (MHC) molecule by a differentiating CD4+CD8+ cell can give rise to T cell antigen receptor (TCR)hi thymocytes of either lineage. Key findings include (a) direct demonstration of a substantial number of CD4-committed, receptor/coreceptor-mismatched cells in MHC class II– deficient mice, a critical prediction of the selective model; (b) highly efficient rescue of such "mismatched" intermediates by forced expression of CD8 in a TCR transgenic line, and an explanation of why previous experiments of this nature were less successful—a major past criticism of the selective model; (c) direct demonstration of an analogous, though smaller, population of CD8-committed mismatched intermediates in class I–deficient animals. Finally, we found no evidence of a CD4 default pathway.
Key Words: homologous recombination • transgenesis • positive selection • CD4 • β-galactosidase
Abbreviations used: β2m, β2-microglobulin; B6, C57Bl/6; βgal, β-galactosidase; DN, double-negative; DP, double-positive; ES, embryonic stem; FDG, fluorescein digalactopyranoside; I0, MHC class I-negative; II0, MHC class II–negative; I0II0, MHC double-deficient; PGK, phosphoglycerate kinase; RT, reverse transcriptase; SP, single-positive; tg, transgenic.
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