Enhancing Adoptive Immunotherapy: Targeting Epstein-Barr Virus with Highly Active, Durable and Immunosuppression-resistant Cytotoxic CD4+T Cells

Mithil Soni – Assistant Research Scientist, Medicine, Hematology and Oncology, Colulbia Universty; Vera Kim – Postdoctoral Researcher, Medicine, Hematology and Oncology, Columbia University; Rodica Ciubotariu – Associate Research Scientist, Medicine, Hematology and Oncology, Columbia University; Pawel Muranski – Assitant Professor, Medicine, Hematology and Oncology, Columbia Unviersity

Abstract Text: Solid organ transplantation (SOT) requires prolonged/life-long immunosuppression, often causing Epstein-Barr Virus (EBV) reactivation and post-transplant lymphoproliferative disease (EBV-PTLD). Adoptive transfer of EBV-specific cytotoxic T cells (EBV-CTLs) may rapidly restore immune competency and control EBV-PTLD. However, clinical efficacy is often limited by exhaustion of in vitro expanded CTLs (mostly CD8+) and/or by ongoing immunosuppression. Here, we hypothesized that EBV-specific Th1/17-like CD4+-CTLs programmed to maintain high potency, self-renewal, and resistance to immunosuppression could be a superior therapeutic strategy. Ex vivo generated EBV-specific CD4+-CTLs (Th1/17-CTLs) demonstrated superior recognition and polyfunctional responses, concurrently secreting TNF-α, IFN-γ, IL-2, and inducible Granzyme B against EBV-antigens compared to the standard Th1-CTLs. Th1/17-CTLs displayed less differentiated central memory (TCM) (CCR7hi, CD27hi) and resident memory (TRM) (CD103hi, CD69hi) phenotype, unlike the predominantly effector memory (TEM) (CCR7low, CD103low) Th1-CTLs. Paradoxically, a second stimulation further enhanced Th1/17-CTLs’ polyfunctionality, robust proliferative capacity, superior bioenergetic/mitochondrial stability, and spare respiratory capacity, unlike the Th1-CTLs. Importantly, Th1/17-CTLs overexpressed Multi-Drug Resistance Protein 1 (MDR1) and displayed resistance to tacrolimus-mediated immunosuppression. Functionally, Th1/17-CTLs exhibited superior recognition and cytotoxicity against autologous EBV lymphoblastoid cell lines (EBV-LCLs). Adoptive transfer of Th1/17-CTLs led to the eradication of systemic PTLD-like disease in humanized mice, demonstrating in vivo persistence and superior self-renewal ability without cytokine support. This study introduces a novel, highly functional, non-exhausted oncoprotein-specific T cells with superior cytotoxic capability and resistance to immunosuppression, addressing key limitations of existing T-cell therapies in immunocompromised patients, paving the way for more robust immune effectors targeting other cancer antigens in solid and hematological malignancies.