Understanding T-cell receptor (TCR) specificity is not only essential for fundamental research, but could open up novel avenues for diagnostics, cancer immunotherapy, and the targeted treatment of autoimmune diseases. The immune system responds to challenges through groups of T-cells with similar TCR sequences. In recent years, searching for TCRs with an enrichment of similar sequences - neighbors - in a TCR repertoire has become a standard procedure for antigen-specific TCR identification. This study provides a systematic comparison of computational algorithms-ALICE, TCRNET, GLIPH2, and tcrdist3-that leverage neighborhood enrichment for antigen-specific TCR identification. Using published murine datasets from Lymphocytic choriomeningitis virus (LCMV) infection and novel datasets from Sputnik V vaccination and Mycobacterium tuberculosis (Mtb) infection, we evaluated the performance of these algorithms. To facilitate reproducible analysis, we developed TCRgrapher, an R library that integrates these pipelines into a user-friendly framework. TCRgrapher enables efficient identification of antigen-specific TCRs from single repertoire snapshots and supports flexible parameter customization. Our comparative analysis revealed that ALICE and TCRNET consistently outperformed GLIPH2 and tcrdist3 across most datasets, achieving higher area under precision-recall curve. While murine datasets provide valuable insights into algorithm performance, caution is advised when extrapolating these results to other species or different experimental conditions. TCRgrapher is freely available on GitHub (https://github.com/KseniaMIPT/tcrgrapher), offering researchers a robust tool for investigating TCR specificity and advancing immunological studies.

Abu Dhabi Stem Cells Center 25 Mahdar Qutouf Street Rowdhat Abu Dhabi United Arab Emirates

Center for Molecular and Cellular Biology Skolkovo Institute of Science and Technology The Territory of the Skolkovo Innovation Center Bolshoy Boulevard 30 bld 1 121205 Moscow Russian Federation

Center for Precision Genome Editing and Genetic Technologies for Biomedicine Pirogov Russian National Research Medical University 1 6 Ostrovitianova St 117513 Moscow Russian Federation

Central European Institute of Technology Žerotínovo nám 617 9 601 77 Brno Czech Republic

Department of Genomics of Adaptive Immunity Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry 16 10 Miklukho Maklaya St 117997 Moscow Russian Federation

NRC Institute of Immunology Federal Medico Biological Agency 24 Kashirskoe Highway 115522 Moscow Russian Federation

Pirogov Russian National Research Medical University 1 6 Ostrovitianova St 117513 Moscow Russian Federation

Saint Petersburg Scientific Research Institute of Vaccines and Serums of the Federal Medical Biological Agency of Russia g Krasnoe Selo ul Svobody d 52 198320 St Petersburg Russian Federation

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