Here we describe a practical approach to increase the detectability and the yield of these rare cells by flow cytometry through the removal of CD25+ Treg, which opens new possibilities for the deeper classification and enrichment of autoantigen-specific CD4+ T cells in a large variety of settings and autoimmune diseases including the identification of immunodominant peptide sequences for the development of suitable MHCII-peptide multimers. This evidence concerns the gene CD4 and autoimmune disease.