Development of a murine tumor-infiltrating lymphocyte therapy model for cholangiocarcinoma.

Tumor-infiltrating lymphocyte (TIL) therapy is a promising approach, earning U.S. Food and Drug Administration approval in patients with anti-PD-1-resistant melanoma. Extending TIL therapy to patients with cholangiocarcinoma (CCA), an aggressive and largely immune-refractory cancer, is an emerging area of interest. However, cost and manufacturing complexity constrain clinical scalability of TIL therapy for CCA, underscoring the need for a murine model to optimize efficacy. Here, we established a novel orthotopic model of TIL therapy for CCA and tested a new ex vivo expansion strategy. We first characterized the immune landscape of orthotopic CCA and then compared 2 TIL expansion methods: (1) a conventional protocol using CD3 agonist stimulation (CD3 TILs) and (2) a tumor antigen-based protocol using irradiated autologous CCA cells to enrich for tumor-reactive TILs (Tumor Ag TILs). Tumor Ag TILs displayed superior tumor lysis in vitro compared to CD3 TILs. While both TIL products engrafted in vivo, Tumor Ag TILs showed enhanced persistence. Despite this, monotherapy with either TIL product alone had only a modest impact on tumor growth rate, and infused cells had upregulation of inhibitory checkpoint receptors, including PD-1. Further investigations demonstrated that the in vivo antitumor efficacy of both Tumor Ag TILs and CD3 TILs was enhanced when combined with PD-L1 inhibitor therapy. Altogether, our study establishes a preclinical platform for modeling CCA TIL therapy, identifies a rational combination strategy that potentiates TIL efficacy, and provides the field with a foundation to advance adoptive T-cell transfer development for CCA and related solid tumors.