Summary Clonal deletion of autoreactive thymocytes is important for self-tolerance, but the intra-thymic signals that induce clonal deletion have not been clearly identified. We now report that clonal deletion during negative selection requires CD28 costimulation of autoreactive thymocytes at the CD4+CD8lo intermediate stage of differentiation. Autoreactive thymocytes were prevented from undergoing clonal deletion by either absent CD28 costimulation or transgenic over-expression of the anti-apoptotic factors Bcl-2 or Mcl-1, with surviving thymocytes differentiating into anergic T cell receptor αβ+ double negative thymocytes that preferentially migrated to the intestine where they re-expressed CD8α and were sequestered as CD8αα intraepithelial lymphocytes (IELs). This study identifies CD28 costimulation as the intrathymic signal required for clonal deletion and identifies CD8αα IELs as the developmental fate of autoreactive thymocytes that survive negative selection.

Lethal-7 (let-7) microRNAs are the most abundant in the genome but their role in developing thymocytes is unclear. We now report that let-7 miRNAs target Zbtb16 mRNA, which encodes the lineage-specific transcription factor PLZF, to post-transcriptionally regulate PLZF expression and NKT cell effector function. Dynamic up-regulation of let-7 miRNAs during NKT thymocyte development down-regulates PLZF expression and directs terminal differentiation into interferon-γ-producing NKT1 cells. Without let-7 up-regulation, NKT thymocytes maintain high PLZF expression and terminally differentiate into IL-4-producing NKT2 and IL-17-producing NKT17 cells. Let-7 up-regulation in developing NKT thymocytes can be signaled by IL-15, vitamin D and retinoic acid. Such miRNA targeting of a lineage-specific transcription factor constitutes a new level of developmental regulation in the thymus.

SUMMARY Major histocompatibility complex (MHC)-restriction is the cardinal feature of T cell antigen recognition and is thought to be intrinsic to αβ T cell receptor (TCR) structure because of germline-encoded residues which impose MHC specificity. Here, we analyzed TCRs from T cells that had not undergone MHC-specific thymic selection. Instead of recognizing peptide-MHC complexes, the two αβTCRs studied here resembled antibodies in recognizing glycosylation-dependent conformational epitopes on a native self-protein, CD155, and they did so with high affinity independently of MHC molecules. Ligand recognition was via the αβTCR combining site and involved the identical germline-encoded residues that have been thought to uniquely impose MHC specificity, demonstrating that these residues do not only promote MHC binding. Thus, this study demonstrates that, without MHC-specific thymic selection, αβTCRs can resemble antibodies in recognizing conformational epitopes on MHC-independent ligands.