Autoimmune diseases are diverse disorders arising from abnormal cellular and humoral responses to self-antigens in the immune system. However, the underlying cause of autoimmunity remains unclear. In this study, ready-made autoreactive CD4⁺ T cell hybridomas (containing autoreactive CD4⁺ lymphocytes from scurfy TCR^mini mice with BWα⁻β⁻ thymoma line) were cloned and their antigen receptors (TCRs) were characterized to decipher the specificity of autoreactive TCRs. This approach was used to understand the mechanisms of autoreactivity in the immune system and assist in redirecting abnormal CD4⁺ T cells present in the peripheral repertoire of healthy individuals for therapeutic advantage. Some hybridoma clones expressed CD4⁺ and Vα2⁺ TCRs with unique amino acid sequences and they upregulated Nur77^GFP expression upon autologous DCs stimulation and a stronger expression of Nur77^GFP in the presence of aCD3 stimulation. Our data suggests that these activated hybridoma clones TCRs have an elevated binding affinity for self-peptides/MHCII complex and are autoreactive.