DIVERSITY OF INTRATUMORAL REGULATORY T CELLS IN B‐CELL NON‐HODGKIN LYMPHOMA

Introduction: Tumor-infiltrating regulatory T cells (Tregs) contribute to an immunosuppressive tumor microenvironment. Despite extensive studies, the prognostic impact of infiltrating Tregs in non-Hodgkin lymphomas (NHL) remains unclear. Emerging studies suggest substantial heterogeneity phenotypes and suppressive capacity Tregs, emphasizing importance understanding Treg diversity need for markers identify highly Tregs. Methods: Live CD4+ were obtained by FACS sorting from NHL malignant lymph nodes (diffuse large B cell lymphoma (DLBCL, n = 3), follicular (FL, 3)), healthy donor tonsils (n 3) subjected single-cell RNA sequencing (scRNAseq), CITE-seq scTCR-seq 10X Genomics platform. The computational framework CIBERSORTx was used generate a unique signature matrix subsets identified scRNAseq, facilitate validation separate scRNAseq cohorts (King, Sci Immunol 2021; Roider, Nat Cell Biol 2020; Steen, Cancer 2021), impute frequencies correlate with survival bulk RNAseq data (Steen, Haematologica 2019; Pastore, Lancet Oncol 2015). High-dimensional cytometry functional immunosuppression assays applied characterize suspensions DLBCL, FLand donors peripheral blood tonsils. Spatial neighborhood analysis performed Imaging Mass Cytometry on FL tissue. Results: We three distinct transcriptional states Tregs; resting, activated LAG3+FOXP3- Activated enriched tumors had higher expression checkpoint receptors (TNFRSF4, TNFRSF18, TIGIT), NF-κB pathway (NFKBIA, NFKBIZ, REL), chemokine (CXCR4) transcription factors (JUN, JUNB, BATF) compared resting Phenotypical characterization showed that co-expressed several (PD-1, TIGIT, CTLA4, ICOS, OX40) stronger activity In microenvironment, found closer proximity CD8+ than other types. Using gene each subset, we confirmed major subset FL, high abundance associated adverse outcome two independent cohorts. research funded by: foundation KG Jebsen, Centre B-cell malignancies (Centre no.19 E.B.S. J.H.M), Norwegian Society (162948 K.H., 182694 E.B.S.), Research Council Norway (FRIMEDBIO 230817/F20, E.B.S.) American Association (19-40-12-STEE C.B.S). Keywords: aggressive lymphoma, indolent microenvironment No conflicts interests pertinent abstract.