Deciphering the specificity of T-cell receptor (TCR) repertoires is crucial for monitoring adaptive immune responses and developing targeted immunotherapies and vaccines. To elucidate the specificity of previously unseen TCRs, many methods employ the BLOSUM62 matrix to find TCRs with similar amino acid (AA) sequences. However, while BLOSUM62 reflects the AA substitutions within conserved regions of proteins with similar functions, the remarkable diversity of TCRs means that both TCRs with similar and dissimilar sequences can bind the same epitope. Therefore, reliance on BLOSUM62 may bias detection towards epitope-specific TCRs with similar biochemical properties, overlooking those with more diverse AA compositions. In this study, we introduce tcrBLOSUMa and tcrBLOSUMb, specialized AA substitution matrices for CDR3 alpha and CDR3 beta TCR chains, respectively. The matrices reflect AA frequencies and variations occurring within TCRs that bind the same epitope, revealing that both CDR3 alpha and CDR3 beta display tolerance to a wide range of AA substitutions and differ noticeably from the standard BLOSUM62. By accurately aligning distant TCRs employing tcrBLOSUMb, we were able to improve clustering performance and capture a large number of epitope-specific TCRs with diverse AA compositions and physicochemical profiles overlooked by BLOSUM62. Utilizing both the general BLOSUM62 and specialized tcrBLOSUM matrices in existing computational tools will broaden the range of TCRs that can be associated with their cognate epitopes, thereby enhancing TCR repertoire analysis.
