Initial experimental systems used to define the nature of the positively selecting peptide repertoire in the thymus aimed to trim the repertoire to either a single peptide or sparse mixtures of peptides presented either on MHC1 or MHC2. Such systems allowed researchers to question whether special peptides were being generated solely for the purpose of carrying out positive selection. To study MHC1 restricted positive selection, β-2 microglobulin (β-2m)18
knockout (KO) mice which effectively abrogated CD8 lineage T cell development were used. In these mice, the deletion of TAP1 or β-2m results in a drastic reduction in surface expression of MHC1. An increase in the level of MHC complexes ensues when exogenous peptides are added to TAP1-deficient cultures. In the case of β-2m deficient mice, the combined addition of recombinant β-2m and exogenous peptide stabilizes surface expression of MHC1 molecules, hence resulting in an increase in MHC1 surface levels.18
Experiments were conducted in which thymocytes expressing particular MHC1 restricted transgenic TCRs were added into TAP1 and β-2m knockout fetal thymic organ culture (FTOC). Agonists or altered peptide ligands (APLs) for the TCR were then systematically added back to cultures in an effort to determine the identity of peptides capable of selecting them. For the lymphocytic choriomeningitis virus (LCMV) peptide specific TCR clone, P14, low and high concentrations of the LCMV peptide agonist added to the TAP1 KO FTOC caused positive and negative selection, respectively.21
When the peptide was added back to the TAP1- sufficient FTOC, however, negative selection was induced even at low peptide concentrations. For the same TCR in the β-2m KO system, a qualitatively similar relationship was found where higher concentrations of peptide induced negative selection, while lower amounts induced positive selection.22
A different strain of mice was bred which displayed Ld
at either 35 or 2% of total MHC1 occupancy. These thymi were tested for their ability to positively select the 2C TCR which is normally negatively selected on Ld
Although negative selection proceeded normally in the 35% Ld
thymus, the 2C TCR was positively selected in the 2% Ld
thymus. Further work in the β-2m KO system showed that an antagonist APL of a TCR specific for OVA peptide presented on Kb
supported positive selection of T cells that expressed normal amounts of CD8 and TCR.24
But when the antagonist was presented in the β-2m (+/-) system, it resulted in negative selection. Work in the F5 TCR model, specific for a peptide derived from the nucleoprotein of Influenza A, demonstrated that an antagonistic APL positively selected two times more thymocytes than the agonist peptide added to TAP1 deficient FTOC at identical concentrations.25
More recently, mice were generated in which MHC1 molecules containing a single peptide were presented to a polyclonal population of developing thymocytes.26
In this system, only 10% of the normal number of CD8 lineage cells developed. Although individual TCRs must have been selected by promiscuous recognition of the same peptide, mature T cells taken from these animals after immunization with particular peptides showed strict peptide specificity, as judged by in vitro lysis of target cells loaded with the peptide used for immunization. Taken together, the body of work indicates that positive selection shows exquisite sensitivity for the strength of signal.
The next step in identification of positively selecting peptides led researchers to begin the difficult task of purifying peptides eluted from MHC1 molecules from wild-type thymi. For the OT-1 transgenic system, Hogquist et al. found seven naturally-occurring peptides which were capable of interacting with the TCR.27
Capability of interaction with the TCR was gauged by culturing thymocytes with APCs presenting the various eluted peptides to mimic negative selection, which was manifested by “dulling” of coreceptor expression. Using a mass-spectrometry analysis, the identity of one of the peptides was determined, and they showed by FTOC add back experiments that it was capable of supporting positive selection. The sequence of the peptide, however, was only related to the agonist peptide at a few positions near the C terminus. Further work from the group using more sensitive detection techniques combined with bioinformatics tools rediscovered this peptide along with another derived from β-catenin.28
The latter peptide acts as an antagonist for mature OT-I T cells, shares significant homology with the agonist ligand and is capable of supporting positive selection.
Consistent with the observation that limited TCR signaling during positive selection directs cells into the CD8 lineage and the demonstrated role for TCR antagonists, recent work has now shown that the antigen-processing machinery in the thymic cortex is poised to generate a repertoire of peptide-MHC complexes that is likely inhibitory toward MHC1 restricted TCR signaling.29–31
A special proteosome, aptly termed the thymoproteosome, has recently been described. It is expressed in 70–80% of cTECs and has not been detected in other tissues. Due to the incorporation of the special subunit, β5t, into the 20s proteolytic site of the traditional proteosome, the thymoproteosome does not cleave polypeptides at hydrophobic amino acid residues. A review of biophysical data of the nearly 1,500 peptides presented by human MHC1 alleles showed that 80% of peptides loaded with high affinity possess hydrophobic amino acids at the C terminus.30
The N terminal anchor shows less stringent specificity. Neither the thymic architecture nor the level of MHC1 expression are altered in mice deficient in the β5t subunit, but the knockout does result in a reduction in the number of CD8 T cells expressing high levels of TCR.31
CD4 lineage T cell development in these animals remains unaffected. Others have suggested that MHC1 molecules loaded with low-binding affinity peptides, like those generated by cTECs, imply that two types of MHC ligands which have not been traditionally regarded as ligands for positive selection are likely to be presented on cTECs.32
These namely include MHC-peptide complexes in which only one side of the peptide is anchored or empty MHC molecules from which peptides have completely disassociated. Antibodies that recognize MHC molecules in a peptide-dependent and independent manner have provided supporting evidence for the presence of such complexes on cortical cells but not within the medulla.33
The presentation of these types of MHC ligands, then, could be a mechanism that precludes high-affinity interactions between TCR and peptide-MHC1 complexes during positive selection. Since knockout of the β5t subunit does not significantly alter MHC1 expression, its role could be envisioned as diluting the pool of available positively selecting interactions such that signaling through MHC1-restricted TCRs happens only very infrequently, allowing CD8 T cell lineage commitment to occur.