Selection in the thymus: pathway divergence and the commitment to life or death

Abstract

Positive and negative selection are processes specific to thymocytes and are crucial to establish a diverse, yet self-tolerant T cell repertoire. Millions of thymocytes each contain a unique T cell receptor (TCR) due to random rearrangement of TCR gene segments, creating immense diversity. However, as part of this randomly created diversity there will be thymocytes without a functioning TCR as well as thymocytes that respond to self-antigen. Both of these groups need to be eliminated. Thymocytes with a non-functional TCR do not transduce any signal and undergo apoptosis. Thymocytes with a high affinity/avidity TCR-self MHC interaction also undergo apoptosis, called negative selection. Negative selection is necessary to avoid the development of auto-reactive T cells and autoimmune disorders. Only thymocytes with a TCR able to correctly recognize MHC on antigen-presenting cells in the thymus are positively selected. Knock-out mice have confirmed the requirement for the majority of proteins involved in proximal TCR signaling and both positive and negative selection. Recently, deletions of Bim and calcineurin have lead to our theory of pathway divergence, since these proteins are necessary for either negative or positive selection (respectively), but not both. The currently accepted model is that thymocyte selection depends on TCR-MHC affinity/avidity or signal ‘strength’. In case of a strong TCR interaction with self-MHC, the resulting signal induces negative selection through a pathway requiring Bim. Only when the TCR-MHC interaction induces the correct signal (of weak/ intermediate intensity), the double positive (DP) thymocytes survive and differentiate to become mature CD4 or CD8 single positive T cells. This process of positive selection requires calcium dependent calcineurin-NFAT signaling. However, knock-out mice for the individual NFATc proteins have revealed unimpaired or only mildly defective T cell development, whereas the defect in the absence of calcineurin is complete. In this thesis, the contribution of NFATc3 to T cell development is addressed by the generation of a conditional NFATc3 knock-out mouse. In the absence of NFATc3, positive selection is only partially blocked. I concluded that NFATc3, together with NFATc1, regulates T cell development at several stages: pre-TCR development at the DN stage and positive selection at the DP stage. Bim is part of the pro-apoptotic Bcl-2 family of proteins and expressed at the highest level in immune cells. In the absence of Bim, negative selection is impaired, resulting in increased numbers of peripheral T cells and autoimmunity. I investigated the pathway leading to Bim induction and negative selection and found that Bim is induced transcriptionally and requires both calcium and one or more isoforms of the protein kinase C (PKC) family. Because calcium is also required for calcineurin activity and positive selection, I propose that the discriminating factor that translates signals of different intensities into either positive or negative selection is the calcium influx in terms of duration or frequency. Calcineurin-NFAT signaling requires a low, but continuous level of calcium influx. If PKC requires a higher level of calcium influx, this could explain how negative selection and bim transcription are induced only after a signal of strong intensity.