Abstract 1551: Phenotypic characteristics of T cells co-expressing SOX2, OCT3/4, and NANOG
Authors: Cerniglia Michael , Escuin-Ordinas Helena , Porter Caroline , Aleshin Maria Alexandrovna , Cheung-Lau Gardenia , Galic Zoran , Abraham-Davidi Inbal , Shin Daniel Sanghoon , Rosenblatt-Rosen Orit , Ribas Antoni , Comin-Anduix Begoña 
Published: July 2021

Abstract
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.