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Cytomegalovirus (CMV) continues to be a major problem for organ transplant recipients. T cells specific to the CMV antigens immediate-early (IE-1) and pp65 CMV govern the adaptive immune response to CMV. Molecular studies indicate that CMV-specific responses usually comprise clonally expanded viral-specific T cell receptors (TCR) that can be conserved among subjects with a similar HLA haplotype. However, our understanding of the CMV-specific T cell receptor (TCR) repertoire is incomplete and the characteristics of TCR repertoire conferring protection to CMV are not known.The aim of this study is to identify specificity groups in the IE-1-specific TCR repertoire of CMV seropositive A02:01 subjects.Methods: PBMC from three CMV seropositive, IE-1-ELISPOT+ A02:01 healthy subjects were stimulated with IE-1 peptides. Antigen-reactive T cells were FACS-sorted based on the expression of CD137 for CD8+ T cells and CD154 for CD4+ T cells, or the loss of CFSE expression in T cells that had been prelabeled with CFSE prior to stimulation. We sequenced the IE-1 specific TCRβ chains following TCRb CDR3 segment amplification using a PCR template switch methodology that minimizes bias through the use of unique molecular barcodes. IE-1 TCRb sequences were analyzed using the recently published Grouping of Lymphocyte Interactions Paratope Hotspots (GLIPH) algorithm.We obtained a total of 2003 TCRβ sequences from IE-1-specific CD8+ T cells from three CMV seropositive subjects. The IE-1-specific TCR repertoire analysis revealed clonally expanded populations for two of the three subjects, with ~50% of the repertoire comprised of 1-10 dominant clones. In contrast, the corresponding non-IE-1 specific T cells had highly diverse TCR repertoires and a minimal number of expanded clones (<1%). Analysis of all CD8+ IE-1-specific TCRβ sequences by GLIPH identified multiple possible clusters. The best scoring identity cluster grouped 95 TCR sequences across two subjects within either the CD137+, or the CFSE-populations. To note, cluster 1 motiff was found in a published IE-1 TCR sequence in the literature, binding to an HLA A2:01 restricted epitope (VLEETSVML).Cluster 2 grouped 43 TCR sequences across two subjects within either the CD137+, or the CFSE-, populations. Importantly, 58 TCR sequences were found to cluster across the IE-1-specific CD8+ T cell populations in all three donors. Here we demonstrate that is feasible to characterize the CMV-specific TCR repertoire in an antigen-specific manner. This methodology has the potential to cluster TCR sequences in transplant patients controlling CMV replication.