In the thymus, a T-cell repertoire able to confer protection against infectious 
and noninfectious agents in a peptide-dependent, self-MHC-restricted manner is 
selected. Direct detection of Ag-specific thymocytes, and analysis of the impact 
of the expression of the MHC-restricting allele on their frequency or function 
has never been studied in humans because of the extremely low precursor 
frequency. Here, we used a tetramer-based enrichment protocol to analyze the ex 
vivo frequency and activation-phenotype of human thymocytes specific for self, 
viral and tumor-antigens presented by HLA-A*0201 (A2) in individuals expressing 
or not this allele. Ag-specific thymocytes were quantified within both CD4CD8 
double or single-positive compartments in every donor. Our data indicate that 
the maturation efficiency of Ag-specific thymocytes is poorly affected by HLA-A2 
expression, in terms of frequencies. Nevertheless, A2-restricted T-cell lines 
from A2(+) donors reacted to A2(+) cell lines in a highly peptide-specific 
fashion, whereas their alloreactive counterparts showed off-target activity. 
This first ex vivo analysis of human antigen-specific thymocytes at different 
stages of human T-cell development should open new perspectives in the 
understanding of the human thymic selection process.