384 An investigation into the role of CD4+ tumor-infiltrating lymphocytes (TIL) in metastatic melanoma patients with a complete response to adoptive cell therapy
Authors: MacLean Hall, Holly Branthoover, Patrick Innamarato, Amy Hall, Alex Alfaro, Allison Richards, Jeani Rich, Jonathan Hensel, Jim Bender, Jake Ceccarelli, TJ Langer, Matthew Beatty, John Mullinax, Jamie Teer, Amod Sarnaik, Shari Pilon-Thomas
Journal: Journal for ImmunoTherapy of Cancer (2021)

Background Immunotherapy for cancer has long been focused on the generation of CD8+ cytotoxic T lymphocyte responses, independent of their dynamic CD4+ T cell counterpart. One promising approach, adoptive cell transfer (ACT) of tumor-infiltrating lymphocytes (TIL), has yielded response rates ranging from 28–55%. 1–2 Investigation into the role of CD4+ TIL in this setting remains critically underexplored as an opportunity to improve upon these successes. Methods Two metastatic melanoma patients (PT1 and PT2) were treated with TIL on a completed clinical trial at Moffitt Cancer Center ( NCT01005745 ). Tumor recognition by TIL was assessed via co-culture with tumor. Whole exome (WES) and RNA Sequencing were performed on cryopreserved tumor sections and mutant peptide-MHC binding was predicted. TIL were stimulated with antigen presenting cells (APCs) loaded with neoantigen-derived 25mer peptides and sorted based on 41BB/OX40 upregulation, followed by functional immunologic assays. TCR sequencing was conducted on patient peripheral blood as well as isolated neoantigen-specific TIL clones to determine persistence in vivo and cognate peptide-MHC targets were determined empirically. Results PT1, infused with predominantly CD4+ TIL (88%), achieved a complete response (CR) despite lack of IFNγ detection with conventional in vitro tumor co-culture methods. Infusion product TIL were sorted by upregulation of OX40 and 41BB upon stimulation with APCs loaded with the mutant peptide pool. Neoantigen reactivity arose from a single peptide sequence, which conferred recognition by a CD4+ TIL clone, which comprised 17% of the infusion product and enriched to greater than 80% after sorting via FACS. These CD4+ TIL produced IFNγ, TNFα, and granzyme B in response to peptide-loaded APCs in an HLA-DR dependent manner. TCRβ overlap revealed this CD4+ clone peaked at two weeks post-infusion (40%) and persisted after infusion for at least six weeks. PT2 was infused with highly reactive, primarily CD8+ (88%) TIL and also achieved a CR. Isolated CD4+ TIL were also responsive to tumor antigens in the context of MHC Class II in vitro. Tumor-reactive CD4+ TIL were enriched by IFNγ capture and delayed xenograft growth in vivo (p&lt;0.01). Neoantigen peptides stimulated predominantly CD4+ TIL to upregulate OX40/41BB and produce IFNγ, TNFα, and granzyme B. Conclusions Investigation of these case studies demonstrated evidence of CD4+ TIL involvement in complete clinical responses after ACT. Ongoing studies will define the precise role of tumor-reactive CD4+ T cells in the anti-tumor immune response and provide the framework for future investigation into their function and therapeutic efficacy. Trial Registration NCT01005745 References Bailey SR, et al . Human CD26high T cells elicit tumor immunity against multiple malignancies via enhanced migration and persistence. Nat Commun 2017; 8 (1):1961. Tran E, et al . Cancer immunotherapy based on mutation-specific CD4+ T cells in a patient with epithelial cancer. Science 2014; 344 (6184):641–5. Ethics Approval Approved by USF IRB approval number Ame5_107905. All participants gave informed consent before taking part.