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Background CMV is an important immunotherapeutic oncological target for many tumor types, including pancreatic tumors and glioblastomas. Autologous CMV-specific T cells or CMV-specific CARs therapy could be utilized as treatment options in conjugation with other therapies. However, existing antigen-specific cellular expansion technologies are unable to specifically and rapidly expand a small number of target cells from a mixed population without isolation or enrichment steps and such fail to recapitulate the nuanced signaling and structure of the immune synapse. Here, we have developed a novel Programmable Cellular Interface (sbPCI) to promote selective expansion of antigen specific TCR from a small population by designing immune synaptic interactions without required isolation, enrichment, or depletion protocols.Methods Human CMV -ve RBCs were modified with specialized anchors to display CD3, CD28, CD137, OX40L, and the pp65 epitope at a configuration optimized for CMV-specific T-cell activation. sbPCI bioactivity was demonstrated through 8-day and 16-day expansion assays. Non-isolated T cells from CMV+ and CMV-ve donors (3 donors each condition) were stimulated at an optimized ratio with sbPCI on Days 1 and 8 while exogenous cytokines were administered thrice weekly. Cultures were enumerated and phenotyped whereas gene expression analysis determined mRNA changes.Results sbPCI drove exponential CMV-specific TCR expansion with a mean viability ≥ 92% and > 200-fold expansion over 8 days without prior isolation or depletion steps. At 48 hours, ≥ 58% of PBMCs were identified as activated T cells defined by elevated Granzyme B, Perforin and IFN-y expression. By Day 8 in the CMV + donor cultures, > 85% were CMV-specific CD3+CD8+T cells when stimulated with a CMV-sbPCI. Gene expression was consistent with activation in response to CMV presentation with Zap70 mRNA expression increasing post co-culture with sbPCI. Importantly, sbPCI stimulation did not result in irreversible exhaustion.Conclusions This study shows that sbPCI can be customized to produce antigen specific T cells quickly without additional isolation and enrichment steps. These results indicate that sbPCI can improve CAGT efficiency by narrowing the production window and removing cumbersome extraneous steps. Subsequent experiments will focus on developing CAR T cell expansion as well as other protocols.