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Background Epstein-Barr virus (EBV), infects more than 90% of humans during childhood, establishing lifelong latency. Impaired immune surveillance may lead to viral reactivation and uncontrolled B cell lymphoproliferation manifesting as post-transplant lymphoproliferative disease (PTLD) and malignant transformation into B-cell lymphomas. Adoptive transfer of EBV-specific cytotoxic T cells (EBV-CTLs) may rapidly restore immune competency and control EBV-driven lymphomas. However, clinical efficacy is often limited by exhaustion of in vitro expanded CTLs (mostly CD8+) and/or by ongoing immunosuppression. Here, we hypothesized that novel EBV-specific Th1/17-like CD4+-CTLs programmed to maintain high potency, self-renewal, and resistance to immunosuppression could be a superior therapeutic strategy.Methods We developed a method for generating novel CD4 + cytotoxic T cells (CD4+-CTLs) simultaneously targeting EBNA1, LMP1, and LMP2A. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors, and either total T cells or CD4+T cells were purified. These cells were then stimulated with PBMCs pulsed with overlapping peptide libraries spanning LMP1, LMP2A, and EBNA1 antigens, either under neutral (Th1) conditions (IL2 and IL-7) or in the presence of pro-inflammatory cytokines for 12-14 days. The phenotypes, reactivity, and cytotoxic capability of these CD4+-CTLs against autologous EBV-Lymphoblastoid cell lines (LCLs) were assessed in vitro and in vivo upon adoptive transfer into tumor-bearing NSG mice.Results Th1.17-CTLs exhibited significantly higher frequency of antigen specific responses and superior polyfunctionality, concurrently secreting TNF-α, IFN-γ, IL-2, and inducible Granzyme B, as compared to the counterpart Th1-CTLs and standard (unfractionated) EBV-CTLs generated from total PBMC. Th1.17-CTLs displayed less differentiated central memory (T CM) (CCR7hi, CD27hi) and resident memory (TRM) (CD103hi, CD69hi) phenotype, unlike the predominantly effector memory (TEM) (CCR7low, CD103low) Th1-CTLs. Paradoxically, second stimulation further enhanced Th1.17-CTLs’ polyfunctionality, while maintaining robust proliferative capacity, superior bioenergetic/mitochondrial stability, and spare respiratory capacity, unlike the Th1-CTLs that displayed the effector memory (TEM) phenotype, mitochondrial dysfunction and poor proliferative responses. Importantly, Th1.17-CTLs overexpressed Multi-Drug Resistance Protein 1 (MDR1) and displayed resistance to tacrolimus-mediated immunosuppression. Functionally, Th1.17-CTLs exhibited superior cytotoxicity against autologous EBV lymphoblastoid cell lines (EBV-LCLs). Adoptively transferred Th1.17-CTLs eradicated systemic PTLD-like disease in humanized mice, demonstrating in vivo persistence and self-renewal without cytokine support, unlike Th1-CTLs that failed to function and persist.Conclusions This study introduces novel, highly functional, non-exhausted oncoprotein-specific T cells with superior cytotoxic capability, bioenergetic stability and resistance to immunosuppression, addressing key limitations of existing T-cell therapies targeting EBV-associated malignancies and paving the road to more robust immune effectors targeting other hematological and solid cancers.Acknowledgements Study is supported by the Nelson Family Transplant Innovation Award.Ethics Approval Study was performed under the Institutional Animal Care and Use Commitee protocol AC-AABN9557.