Abstract The composition and activation phenotype of immune cells within the tumor microenvironment (TME) can significantly influence therapeutic response to PD-1 based immunotherapy. Using the Farcast TruTumor histoculture platform, we investigated potential factors contributing to the unexpected anti-PD-1 responsiveness in an immune cold TME. Freshly resected Head and Neck Squamous Cell Carcinoma (HNSCC) samples (n=5) and matched blood were collected from consented patients. Explants were generated from the tumors and treated in culture with anti PD-1 (Nivolumab132 µg/ml) for 72 h. The response was evaluated using histopathology and flow cytometry readouts. Among the five samples analysed, one responder (S2) exhibited elevated cleaved caspase-3 expression (2.5-fold increase) in the tumor relative to the control. Interestingly, this sample (immune content 2%), exhibited a cold tumor immune microenvironment compared with the four non-responder (NR) samples (mean immune content = 4.5%). To investigate factors underlying the observed response, we evaluated the baseline immune profile. Sample S2 exhibited a more heterogenous Lo-SSC compartment compared to NR samples. This was characterized by a high proportion of CD23+ follicular B cells (3.9-fold increase), CD11c+ dendritic cells (1.5-fold increase), and DC-LAMP+ expressing mature dendritic cells (2.9-fold increase) compared to NR samples. These sub-populations are indicative of immune cell subsets typically present in or associated with TLS. Although the responder sample contained a relatively low overall proportion of CD8+T cells, it exhibited 15 fold lower proportions of both early (CD8+PD-1+) and late (CD8+CD39+) exhausted T-cells, while displaying a 12-fold increase in stem-like (CD8+TCF1+) T cells. Upon ex vivo culture of tumor explants on TruTumor platform, the responder sample showed a distinct increase of 1.8-fold in pro-inflammatory M1 macrophages in the treated relative to the untreated control, whereas no appreciable change was observed in the non-responder samples. Interestingly, anti PD-1 treatment led to the emergence of an NKT-cell population (CD3+CD56+) and an increase in NK cells expressing Granzyme B (CD3-CD56+Granzyme B+). In summary, our data reveals that response to anti-PD-1 is not solely dependent on the abundance of CD8+ T cells but also involves substantial contributions from TLS representative immune sub-populations within the TME. Specifically, the presence of follicular B cells, stem-like CD8+ T cells, CD11c+ cells, and DC-LAMP+ dendritic cells may contribute to treatment responsiveness, even in the context of an otherwise immune-poor phenotype. The TruTumor histoculture platform provides a translational model to study tumor response to immunotherapy with insights into how tumor responsiveness, transcends beyond mere presence of CD8+ T cells in the TME. Citation Format: Satish Sankaran, Priyanka Chevour, Kowshik Jaganathan, Biswajit Das, Moumita Nath, Abdul Haseeb, Jobin K Paul, Vasanthakumar A, Oliyarasi M, Rajashekar M, Jayaprakash C, Venkatesh T, MS Ganesh, Amritha Prabha, Prakash BV, Upendra K, Méhul Kapur, Ritu Malhotra, Govindaraj K, Pavithira., Mohit Malhotra. Tertiary lymphoid structure (TLS) representative cells elicit response to anti-PD-1+ therapy in a cold tumor abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7761.
Sankaran et al. (Fri,) studied this question.