Distinct CD28 signaling requirements determine the alternative fates of thymic agonist selection

Abstract During thymic T cell development, T cells strongly reactive to self-Ag are eliminated from the conventional T cell repertoire by clonal deletion. Alternatively, however, a number of T cell subsets are positively selected by strong recognition of self-Ag/MHC by TCR in thymus, termed agonist selection. Those T cell subsets include 1) Foxp3+ Treg cells, 2) invariant NKT (iNKT) cells, and 3) αβTCR+ PD-1+ CD4− CD8− precursors of CD8αα IELs (pIEL). In addition to a strong TCR signal, B7-CD28 co-stimulation has a role in these agonist selections. This role is context dependent. For instance, B7-CD28 has a positive role for thymic Treg and iNKT cell generation, while it has a negative role for thymic pIEL generation. These differences may indicate involvement of distinct CD28 signaling in these events. To test this hypothesis, we analyzed CD28 cytoplasmic tail mutant mice in which tyrosine 170 residue was substituted to phenylalanine (Y170F), proline residues in PYAP motif were substituted to alanine (AYAA), or both mutation were present (Y170F/AYAA). Generation of self-Ag-specific Treg, defined by pMHCII tetramer binding, was attenuated in AYAA mice but not in Y170F mice, consistent with the reported role of the PYAP motif for total Treg generation. Meanwhile, interestingly, the negative role of CD28 for self-Ag-specific pIEL generation was not affected by Y170F and/or AYAA substitution, indicating that this CD28 effect was mediated by motif(s) other than Y170 and PYAP. Analysis of thymic iNKT cell (NKT1, 2 and 17) development in CD28 mutant mice is currently in progress. These results suggest that the differing roles of B7-CD28 costimulation for agonist selection fates are mediated via qualitatively distinct CD28 signaling.