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Background Cytotoxic CD4 T cells have emerged as promising effectors in antitumor immunity, capable of both direct tumor killing and immune modulation. Despite clinical successes with CD4-based adoptive cell transfer (ACT) therapy, scalable methods to selectively expand rare antigen-specific, cytotoxic CD4 T cells remain limited. Existing platforms primarily focus on CD8 T cells and often lack the precision or yield needed for clinical translation of CD4-directed therapies.Methods Our lab utilizes artificial antigen presenting cells (aAPCs) made of iron dextran particles conjugated with MHC class II proteins and the co-stimulatory molecule anti-CD28 to activate T cells ex vivo. We isolate human CD4 T cells from peripheral blood mononuclear cells (PBMC) of healthy donors who are HLA-DP4 +. We then stimulate CD4 T cells with HLA II aAPCs in media with Th1 skewing cytokines and assess the antigen specificity, effector function, and memory profiles through tetramer staining, intracellular cytokine staining, and peptide-pulsed lymphoblastoid cell line (LCL) killing assays. Clonal expansion and repertoire architecture were analyzed by bulk TCRa and TCRβ sequencing.Results We used aAPCs to stimulate human CD4 T cells and achieved robust expansion of antigen-specific populations. We expanded CD4 T cells specific to tetanus toxoid (p30) and herpes simplex virus (HSV). After 14 days of culture, the percentage of antigen-specific population increased significantly from approximately 0.5% to over 70% in p30-aAPC-stimulated groups and to over 10% in HSV-aAPC-stimulated groups.The expanded antigen-specific CD4 T cells exhibited a polyfunctional effector phenotype, characterized by high expression of TNF-α, IFN-γ, IL-2, perforin, and granzyme B. They also showed increased levels of activation (CD69, CD40L), adhesion (LFA-1, NCAM), and degranulation (CD107a) markers compared to tetramer- counterparts. In functional assays, these cells mediated antigen-specific cytotoxicity, achieving up to 60% target cell killing at an E:T of 10. The majority of tetramer+ cells are effector memory T cells, with a small percentage of Tscm, supporting their relevance for lasting ACT applications.TCR sequencing revealed key distinctions in repertoire dynamics. p30-expanded cells were enriched for hyperexpanded clonotypes with reduced diversity, whereas HSV-expanded cells maintained higher diversity. All dominant clonotypes were private, highlighting donor-specific solutions to shared antigens and emphasizing the need for individualized ACT approaches.Conclusions Our findings demonstrate that aAPCs can drive the expansion of functional, antigen-specific cytotoxic CD4 T cells with distinct clonal profiles. This platform offers a scalable and tunable approach for developing next-generation ACT strategies, highlighting the potential of CD4 T cells in personalized immunotherapy.Ethics Approval All uses of human material in this study have been approved by the ethical committee of Johns Hopkins University; approval number NA-00027947.Consent All recruited volunteers provided written informed consent. A copy of the written consent is available for review by the Editor of this journal.