Background The majority cutaneous T cell lymphoma (CTCL) run an indolent clinical course. However, advanced stages (>=IIB) or certain CTCL subtypes such as folliculotropic Mycosis Fungoides (FMF) and Sézary Syndrome (SS) are associated with significantly worse prognosis and treatment options are limited. In previous studies, our group has described increased numbers of CTCL tissue infiltrating B cells as an adverse prognostic factor. However, the underlying mechanisms are still poorly understood.This project therefore aims to characterize the role of B cells within the CTCL Tumor Microenvironment with a special focus on spatial distribution and immunometabolic and cellular interaction patterns.

Materials and Methods An independent cohort of clinically annotated, Formalin fixed and Paraffin embedded (FFPE) clinical CTCL probes were analyzed by multiplex Immunohistochemistry (IHC) to characterize the entire tumor and immune cell infiltrate and to determine spatial distribution and interaction patterns. Gene expression within these probes was quantified from consecutive tissue sections by Nanostring^© and spatial transcriptomic analyses via the 10x Genomics^© and compared with multiplex IHC results. In vitro functional and metabolic characterization of CTCL cells (co-culture experiment, migration experiments, extracellular flux analysis) and the consequences of distinct B cell subtype interaction were addressed.

Results In a total of 36 CTCL cases, B cells were enriched in aggressive CTCL subtypes in this independent cohort and formed a complex network, which differed from patterns found in indolent CTCL cases. Panel gene expression analysis confirmed B cell enrichment seen IHC and furthermore showed distinct gene expression patterns in aggressive CTCL cases associated with the B cell infiltrate, showing also a strong correlation of tumor associated and immunosuppressive inhibitory M2 Macrophages (TAMs). Metabolic pathways were also found to be significantly altered depending on the clinical course. Characterization of cellular metabolic programs of CTCL cells compared to activated non-malignant CD4⁺ T cells showed that cell lines HuT78 and HH (aggressive types of CTCL) have distinct glycolytic and mitochondrial capacities compared to CTCL lines representing more indolent subtypes, CD30⁺Anaplastic large T cell Lymphoma (ALCL) and normal CD4⁺ T cells. Notably, B cell conditioned media exhibited higher metabolic capacities in SS cell line SeAx compared to non-conditioned media after 24 hours of cultivation.

Conclusions Our preliminary data further support the observation that tumor infiltrating B cells and TAMs play a functional role in CTCL and are associated with more aggressive clinical courses of the disease. The ongoing experiments aim to provide a detailed characterization of spatio-temporal distribution, cellular interaction patterns, and immunometabolic factors within these networks.

S. Oganesian: None. M. Funk: None. A. Hadzic: None. M. Kirmaier: None. G. Piontek: None. J. Watter: None. M. Heide: None. S. Krebs: None. S. Mages: None. H. Blum: None. C. Posch: None. O. Weigert: None. J. Klughammer: None. M. Rudelius: None. M. Bergwelt-Baildon: None. S. Theurich: None.