Abstract
The thymic medulla plays a key role in negative selection (self-tolerance induction) and contains differentiated T cells en route to the extrathymic environment. However, being relatively mature, medullary T cells are thought to be beyond the stage of tolerance induction. This paradox is resolved by the finding that medullary T cells (CD4+8- thymocytes) comprise two distinct subsets. Medullary thymocytes expressing a fully mature (HSAlo) phenotype are strongly resistant to tolerance induction, whereas cells with a semimature (HSAhi) phenotype are tolerance susceptible. These findings suggest that the differentiated T cells reaching the medulla from the cortex remain sensitive to tolerance induction for a brief period before acquiring a fully mature tolerance-resistant phenotype. The semimature subset of medullarsy T cells displays unique requirements for tolerance induction; depending upon the conditions used, tolerizing these cells can involve either a Fas (CD95)-dependent or a Fas-independent pathway.
Highlights
The results suggest that immature CD4ϩ8ϩ and semimature HSAhi CD4ϩ8Ϫ thymocytes are both susceptible to negative selection
Acute negative selection of thymocytes can be induced by injecting mice with anti-TCR mAb i.p. [30]; in this model, costimulation is presumably provided by bystander APC [31, 32]
Similar findings applied when anti-TCR mAb was injected into neonatal (1-wk-old) mice. In this situation, the elimination of thymocytes was prominent for semimature HSAhi CD4ϩ8Ϫ cells but not for CD4ϩ8ϩ cells, suggesting that the destruction of CD4ϩ8ϩ cells in adult mice (Fig. 1 A) was probably largely mediated by cytokines and/or increased steroid levels released via anti-TCR stimulation of mature postthymic T cells
Summary
Adult C57BL/6 (B6) and B6 lpr/lpr mice aged 8–12 wk were obtained from The Scripps Research Institute breeding facility. Antibodies specific for the following markers were previously described [26]: CD3 (C363.29B, rat IgG), CD4 (RL172, rat IgM), CD8 (3.163.8, rat IgM), CD25 (7D4, rat IgM), HSA (J11D, rat IgM), and class II (M5/114, rat IgG). Purification of TCRlo CD4ϩ8ϩ thymocytes was performed as described previously [29]. For purification of HSAhi CD4ϩ8Ϫ cells, thymocytes were treated with mAbs specific for CD8 (3.168.8) and CD25 (7D4) plus guinea pig complement (C) for 45 min at 37ЊC, positively panned with anti-CD4 (RL172) mAb, positively panned with anti-HSA (J11d) mAb; panning was performed at 4ЊC. CD4ϩ LN T cells were purified by treating pooled LN cells with mAbs specific for HSA, CD8, and class II plus guinea pig C, followed by positive panning with anti-CD4 mAb
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