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Immune checkpoint blockade (ICB) has revolutionized the treatment of many cancers but has been ineffective for the treatment of microsatellite stable (MSS) PDAC. The lack of efficacy of immunotherapies in PDAC is due to: 1) a desmoplastic tumor microenvironment (TME); 2) the presence of suppressive cells, including myeloid derived suppressor cells and regulatory T cells; and 3) the lack of antigen-presenting dendritic cells (DCs) that are important in priming an effective immune response to generate functionally effective tumor antigen-specific T cells. We recently completed a pilot study of dual ICB (Ipilumamab and Nivolumab) with radiation therapy (SBRT 8Gy for 3 fractions) in a cohort of 25 metastatic PDAC patients that had progressed on conventional chemotherapy; this combination conferred an impressive 18% ORR and 29% disease control rate measured on non-irradiated lesions (historical 0% ORR with ICB in PDAC). This led to a phase 2 study in 30 metastatic PDAC patients using this dual modality treatment paradigm. To understand the role of radiation and ICB in altering the PDAC tumor microenvironment we performed single-cell RNA-sequencing and TCR-sequencing (>180k cells), and single-nucleus RNA-sequencing (>300k cells) on 36 tumor biopsies (23 pre-treatment, 13 paired on-treatment between day 10 and 21) from patients undergoing treatment in our phase 2 study. Tumor tissue was taken from distinct tissue sites, including primary tumors in the pancreas, and liver and abdominal wall metastases. We identified distinct tumor cell state distributions within different tissues, and a redistribution of cells from basal/mesenchymal states to classical states after radiation. We identified several state-specific interferon stimulated gene programs thus cataloging distinct responses of epithelial cells with different transcriptional states. Importantly, we found a redistribution of T cells states towards proliferating and exhausted T cells with unique clonality after radiation. Additionally, the myeloid compartment after radiation was enriched for C1QC+ and MHCII+ macrophage subsets, as well as infiltrating CD16/CD16 monocytes and CD14 monocytes, each showing induction of unique sets of interferon stimulated genes (ISGs). We next sought to better understand immunotherapy resistance mechanisms within these PDAC patients despite finding strong ISG induction in several subsets. We analyzed covarying gene programs and identified multicellular communities of cells before and after radiation that underlie interaction networks associated with radiation. Together our data provides the most comprehensive single-cell atlas of paired biopsies to study tumor and immune cell states in the context of radiation and ICB response. Citation Format: Arnav Mehta, Aparna Parikh, Milan Parikh, Ryan Park, Moshe Sade-Feldman, Lynn Bi, Nicole Carzo, Tarin M. Grillo, Islam Baiev, Olanike Asupoto, Irena Gushterova, Tom LaSalle, Anna Gonye, Emily Blaum, Sebastien Vigneau, Ronan Chaligne, Ana Lako, Thomas Lila, David Nelson, Caroline Porter, Orr Ashenberg, Karthik Jagadesh, William L. Hwang, Christopher Smillie, David P. Ryan, David T. Ting, Theodore Hong, Dana Pe'er, Nir Hacohen. Dissecting the reorganization of pancreatic tumor microenvironments after radiation and immunotherapy reveals insights into immunotherapy resistance [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C012.

Introduction: A small subset of T cells from patients treated with MART-1 engineered adoptive T cell transfer (ACT) were found to co-express Yamanaka transcription factors SOX2, OCT3/4, and NANOG (TSON). Identifying these T cells may have potential use in future T cell based immunotherapy. Methods: Flow (FC) and mass cytometry (MC) were used to evaluate T cells with induced pluripotent stem cell (iPS)-like markers. Peripheral mononuclear cells (PBMC) were collected from 3 melanoma patients (MP) treated with ACT and 1 healthy donor (HD0). Bone marrow (BM) samples were collected from patients with Non-Hodgkin's lymphoma (NHL) and multiple myeloma (MM). T cell populations were then screened for stem cell markers: SOX2 OCT3/4 NANOG TRA-1-81 SSEA4 TRA-1-60 CD3+ CD34-. Naïve T cells (CD45RO- CD62L+) from HD0 were exposed to IL-7/IL-15/IL-21 with or without pan-AKT inhibitor (AKTi). These cells were analyzed after 7 days for TSON expression and the following surface markers: CD27 CD28 CD45RO CD45RA CD57 CD62L CD95 CD122 CD127 CCR4 CCR6 CCR7. PBMC collections from 5 healthy donors (HD) were expanded with or without IL-7/IL-15/IL-21 exposure in G-Rex bioreactors for 7 days. TSON expression was analyzed using MC and characterized for the already mentioned markers as well as CXCR3 and Ki67. Lastly, expanded CD95+ cells were single-cell sorted for TSON and their RNA was extracted using the FRISCR method for single-cell RNA sequencing (scRNA-seq). The iPS cell line H1 was included as a positive control. Results: T cells across all 3 MP had a small percentage of TSON expression (0.0002%, 0.0001%, 0.0001%, respectively). This small cohort of T cells co-expressed TRA-1-81, SSEA4, and TRA-1-60 (0.05%, 0.05%, 0.8%, respectively). TSON expression for HD0 was 0.0000013%, while co-expression of TRA-1-81, SSEA4, and TRA-1-60 was undetectable. BM samples NHL and MM also showed TSON expression (0.18% vs 0.06%). Naïve T cells treated with IL-7/IL-15/IL-21 had a significantly higher percentage of TSON after 7 days of exposure compared to T cells treated with IL-7/IL-15/IL-21/AKTi (0.008% vs 0.001%, respectively). Furthermore TSON cell subsets in both treatment populations displayed a phenotype similar to T memory stem cells (TMSC) and central memory T cells (TCM) (CD45RA+ CD27+ CD28+ CD62L+ CCR7+ CD95+ CD57- CD45RO+). Concerning PBMCs from the 5 HD, the proliferation marker Ki67 was displayed on 3 non-stimulated HD and all stimulated HD with a range of median intensities [10, 67]. The presence of CD62L CD27 CD95 CCR7 CD28 CD45RO in both non-stimulated and stimulated cell populations was similar to TCM phenotype. Validation of scRNA-seq is still in process. Early results show that OCT3, NANOG, and cMYC are detected in both TSON and H1 while SOX2 could only be detected in H1. Conclusion: Our data show evidence of T cells with natural TSON expression. However, additional studies are needed to further understand the role of TSON in immunotherapy. Citation Format: Michael Cerniglia, Helena Escuin-Ordinas, Caroline Porter, Maria Alexandrovna Aleshin, Gardenia Cheung-Lau, Zoran Galic, Inbal Abraham-Davidi, Daniel Sanghoon Shin, Orit Rosenblatt-Rosen, Antoni Ribas, Begoña Comin-Anduix. Phenotypic characteristics of T cells co-expressing SOX2, OCT3/4, and NANOG [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1551.