Thymic selection is responsible for generating the immune system's repertoire of mature T cells, a pool of individual antigen reactivities whose effectiveness relies on maintaining a balance between two opposed requirements. In order to combat combat infection, the T cell repertoire must contain at least one T cell specific for each invading pathogen it might encounter: thus the repertoire must be as broad as possible. But if the repertoire includes T cells specific for the body's own tissues, the immune system has the potential to itself become pathogenic: to avoid this, the repertoire must be constrained to exclude such autoreactive cells. Two opposed processes guarantee adherence to these requirements. Positive selection ensures the breadth of the T cell repertoire, while negative selection eliminates potentially autoimmune T cells.
Positive selection begins in the thymic cortex, where thymocytes having recently rearranged their T cell receptor (TCR) genes can experience peptide-MHC for the first time. At this CD4+CD8+ (DP) stage, young thymocytes are exquisitely sensitive to TCR signals, even moreso than their more mature T cell brethren, both in terms of the magnitude of the signal that can be recognized and responded to, and in terms of the response itself. Because unlike mature T cells, DP thymocytes are programmed with the capacity for self-annihilation—negative selection—should the signal be too strong.
In this work we will define precisely how sensitive DP thymocytes are, in terms of the number of ligands that evoke negative selection, and we will go on to show how thymocytes find such small numbers of self-determinants. We will also demonstrate a molecular mechanism that gives rise to this heightened sensitivity to ligands at the DP stage. Finally we will identify novel self-peptides that promote positive selection of a class II-restricted TCR specificity, and will show how these peptides can influence mature T cells during a response toward antigen.
We know from prior work that immature DP thymocytes respond to negatively selecting peptide-MHC ligands by forming an immune synapse that sustains contact with the presenting APC. We show here, using fluorescently labeled peptides, that as few as two agonist ligands can promote APC contact and subsequent apoptosis in reactive thymocytes. Furthermore we show that productive signaling for positive selection—as gauged by nuclear translocation of a GFP-labeled NFATc construct—does not involve formation of a synapse between thymocytes and selecting epithelial cells in reaggregate thymus cultures. Antibody blockade of endogenous positively selecting ligands prevents NFAT nuclear accumulation in reaggregate thymus cultures, and reverses that accumulation in previously stimulated thymocytes. Together these data suggest a 'gauntlet' model in which thymocytes mature by continually acquiring and reacquiring positively selecting signals without sustained contact with epithelial cells, which allows them to sample many cell surfaces for potentially negatively selecting ligands.
Whereas DP thymocytes can mount an apoptotic response to only two agonist ligands, mature T cells require 8-10 agonist ligands to fully respond. This higher sensitivity to ligand at the DP stage is well-documented, albeit not at the level of individual peptide-MHC: but the genetic or epigenetic programs that underlie that sensitivity were not previously known. Here we show that disruption of a microRNA—miR-181a, which is normally highly expressed in DP thymocytes but not mature T cells—dampens this sensitivity and renders DP thymocytes unable to respond to positively or negatively selecting ligands.
Immature thymocytes are driven to mature by self-peptide-MHC ligands that are too weak to provoke a response from their peripheral T cell counterparts. The identity of some of the self-peptides responsible for positively selecting a particular TCR are known only in two cases, the OT-1 and 2C TCRs, both of which are class I-restricted. Here we show that several endogenously derived peptides, in complex with the I-Ek class II MHC molecule, can drive positive selection thymocytes bearing the 5c.c7 TCR into the CD4+ T cell lineage.
More recently, similarly weak peptides have also been shown to boost mature T cell responses, but a relationship between the two has yet to be shown. Here we show by antibody blockade that naturally occurring positively selecting peptides can contribute to mature T cells' response to agonist ligands. Furthermore, the most potent among our newly identified positively selecting peptides enhances mature T cell stimulation in a peptide- and TCR-specific and CD4-dependent manner These results establish a connection between the peptides that induce positive selection and those that enhance peripheral T cell activation, suggesting that TCRs may be selected in the thymus in part for their ability to utilize endogenous ligands during an antigen response.
