Aggressive B cell lymphomas retain ATR-dependent determinants of T cell exclusion from the germinal center dark zone
Valeria Cancila, et al.
Valeria Cancila, et al.
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Research Article Cell biology Immunology Oncology

Aggressive B cell lymphomas retain ATR-dependent determinants of T cell exclusion from the germinal center dark zone

Abstract

The germinal center (GC) dark zone (DZ) and light zone represent distinct anatomical regions in lymphoid tissue where B cell proliferation, immunoglobulin diversification, and selection are coordinated. Diffuse large B cell lymphomas (DLBCLs) with DZ-like gene expression profiles exhibit poor outcomes, though the reasons are unclear and are not directly related to proliferation. Physiological DZs exhibit an exclusion of T cells, prompting exploration of whether T cell paucity contributes to DZ-like DLBCL. We used spatial transcriptomic approaches to achieve higher resolution of T cell spatial heterogeneity in the GC and to derive potential pathways that underlie T cell exclusion. We showed that T cell exclusion from the DZ was linked to DNA damage response (DDR) and chromatin compaction molecular features characterizing the spatial DZ signature, and that these programs were independent of activation-induced cytidine deaminase (AID) activity. As ATR is a key regulator of DDR, we tested its role in the T cell inhibitory DZ transcriptional imprint. ATR inhibition reversed not only the DZ transcriptional signature, but also DZ T cell exclusion in DZ-like DLBCL in vitro microfluidic models and in in vivo samples of murine lymphoid tissue. These findings highlight that ATR activity underpins a physiological scenario of immune silencing. ATR inhibition may reverse the immune-silent state and enhance T cell–based immunotherapy in aggressive lymphomas with GC DZ–like characteristics.

Authors

Valeria Cancila, Giorgio Bertolazzi, Allison S.Y. Chan, Giovanni Medico, Giulia Bastianello, Gaia Morello, Daniel Paysan, Clemence Lai, Liang Hong, 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, Biyan Zhang, Tingyi See, Kong-Peng Lam, Gabriele Varano, Mario P. Colombo, Silvio Bicciato, Giorgio Inghirami, Leandro Cerchietti, Maurilio Ponzoni, Roberta Zappasodi, Evelyn Metzger, Joseph Beechem, Fabio Facchetti, Marco Foiani, Stefano Casola, Anand D. Jeyasekharan, Claudio Tripodo

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Figure 4

The spatial signature of DZ cells is independent of AICDA-related mutational processes.

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The spatial signature of DZ cells is independent of AICDA-related mutati...
(A) Representative photomicrographs of H&E and IHC for Cre+ and Ki-67+ cells on mesenteric lymph nodes from WT and Aicda–/– mice. Original magnification, ×200. Scale bars: 100 μm. (B and C) Quantitative analyses of Cre+ (B) and Ki-67+ (C) cells in WT and Aicda–/– GCs (n = 20). Statistical analysis was assessed using a 2-tailed unpaired Mann-Whitney test. Values are shown as mean ± SEM; ***P < 0.001, ****P < 0.0001. (D) Representative photomicrographs of H&E-stained sections from WT and Aicda–/– mesenteric lymph nodes involved in the Visium spatial transcriptome experiment profiling. Original magnification, ×50. Scale bars: 250 μm. (E) Spatial visualization of WT and Aicda–/– follicle/GC clusters. (F) Volcano plot showing differentially expressed genes between WT cluster 4 and Aicda–/– clusters 1 and 3 (Wilcoxon’s rank sum test adjusted P values < 0.05, absolute log fold change > 0.025). (G and H) Spatial projection of DZ spatial signature (G) and T cell signature (H) total expression in WT and Aicda–/– samples. (I–L) Representative photomicrographs of triple IHC staining for DZ Ki-67+ (cyan signal), LZ CD21+ (pink signal), and CD4+ (I) or CD8+ cells (K) (brown signal) and quantitative analyses of the percentage of CD4+ (J) or CD8+ (L) T cells in WT and Aicda–/– GCs (n = 10 WT GCs; n = 10 Aicda–/– GCs). Original magnification, ×400. Scale bars: 50 μm. Statistical analysis: 2-tailed unpaired Mann-Whitney test. Mean ± SEM is shown. (M) Gene set enrichment analysis (GSEA) of DZ spatial signature in AICDA-high and AICDA-low DZ B cells. (N) Pathway enrichment of 257 AICDA-high signature genes using Reactome Pathway library. (O) Pathway enrichment of 127 AICDA-low signature genes using Reactome Pathway library.