Abstract LT02: Aggressive B-cell lymphomas retain ATR-dependent determinants of T-cell exclusion from the Germinal Center Dark Zone

Abstract Diffuse large B-cell lymphomas (DLBCLs) with a dark zone (DZ)-like transcriptional profile correlate with poor outcomes to rituximab-based chemoimmunotherapy, but the mechanisms underlying this resistance remain unclear. We hypothesize that DZ-like DLBCLs retain immune-evasion properties of the physiological germinal center (GC) DZ, contributing to treatment resistance. We investigated the molecular basis of spatial T-cell exclusion in GCs and its relevance to immune resistance in DZ-like lymphomas. Digital Spatial Profiling (DSP) and spatial transcriptomics were performed on DZ and light zone (LZ) regions of 10 tonsil GCs to define DZ gene signatures. DZ transcriptional programs in normal DZ and malignant B cells were enriched for cell cycle checkpoints, DNA damage response, ATR activation, and chromatin compaction pathways, correlating with reduced T-cell infiltration. Given that activation-induced cytidine deaminase (AID) is a known driver of the DZ program, we evaluated its role in T-cell exclusion. We observed persistent DZ signatures in AID-deficient and WT murine GCs, with no associated T-cell infiltration. While AID-high B cells enriched for DZ features, AID-low cells overlapped with ATR activation within the DZ program, suggesting ATR-dependent T-cell exclusion independent of AID mutagenesis. ATR inhibition in DZ-like DLBCL cell lines reversed the DZ spatial signature and increased T-cell attraction in microfluidic co-culture systems. In vivo, ATR inhibition in immunized mice significantly increased overall GC T-cell infiltration, particularly CD8+ T cells within the DZ. Finally, in PDX models, ATR inhibition significantly improved CAR-19-mediated cytotoxicity, with the most pronounced effect in DZ-like DLBCL clones, which otherwise exhibited resistance to CAR-T cell killing. These findings support ATR inhibitors as potential adjuncts to chemoimmunotherapy, immune checkpoint blockade, or CAR-T cell therapy in lymphomas, particularly in lymphomas characterized by immune exclusion. This abstract is included in the 18-ICML Abstract Book, https://doi.org/10.1002/hon.70094_172 Citation Format: Valeria Cancila, Giorgio Bertolazzi, Allison S. Y. Chan, Giovanni Medico, Giulia Bastianello, Gaia Morello, Daniel Paysan, Clemence Lai, Hong Liang, Girija Shenoy, Patrick W. Jaynes, Giovanna Schiavoni, Fabrizio Mattei, Silvia Piconese, Maria V. Revuelta, Francesco Noto, Luca Businaro, Adele De Ninno, Ilenia Cammarata, Fabio Pagni, Saradha Venkatachalapathy, Sabina Sangaletti, Arianna Di Napoli, Giada Cicio, Davide Vacca, Silvia Lonardi, Luisa Lorenzi, Andrés J. M. Ferreri, Beatrice Belmonte, Min Liu, Manikandan Lakshmanan, Michelle S. N. Ong, Zhang Biyan, Tingyi See, Kong-Peng Lam, Gabriele Varano, Mario P. Colombo, Silvio Bicciato, Giorgio Inghirami, Leandro Cerchietti, Maurilio Ponzoni, Roberta Zappasodi, Evelyn Metzger, Joe Beechem, Fabio Facchetti, Marco Foiani, Stefano Casola, Anand D. Jeyasekharan, Claudio Tripodo. Aggressive B-cell lymphomas retain ATR-dependent determinants of T-cell exclusion from the Germinal Center Dark Zone [abstract]. In: Proceedings of Frontiers in Cancer Science 2025; 2025 Nov 5-7; Singapore. Philadelphia (PA): AACR; Cancer Res 2026;86(13_Suppl):Abstract nr LT02.