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 3

The GC DZ spatial signature in aggressive B cell lymphomas is associated with reduced T cell infiltration.

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The GC DZ spatial signature in aggressive B cell lymphomas is associated...
(A) DZ enrichment scores correlating DZ gene expression and xCell T cell cytotype scores calculated in 8 DLBCL datasets. Positive DZ enrichment values indicate a positive association between the DZ spatial signature and the xCell cytotype scores, while negative values indicate a negative association. Statistical significance is shown with Wilcoxon’s adjusted P values. (B) UMAP projection of 1,078 harmonized DLBCL cases classified based on the DZ/LZ spatial signature; DZ-like cases (red), LZ-like cases (light blue), intermediate cases (green). (C) Kaplan-Meier survival plot showing overall survival (OS) of DZ-like, LZ-like, and intermediate groups from the harmonized dataset (1,078 cases). (D) DSP images of 11 ROIs selected within CD20 (green signal) and CD3E (red signal) infiltrates of a lymph node with DLBCL. Total expression of the DZ signature is shown from low (pink) to high (red). Original magnification, ×50. Scale bar: 250 μm. (E) Pie charts showing SpatialDecon cytotype scores across 11 ROIs, ranked by DZ signature expression. (F) Scatterplot with correlation line of DZ spatial signature expression and SpatialDecon T cell score across 11 ROIs (Kendall’s correlation, P < 0.05). (G) DSP images of lowest DZ signature expression ROI (001) and highest DZ signature expression ROI (004). (H) Schematic of DLBCL tissue microarray (TMA) consisting of 103 patient samples, analyzed using DSP WTA with DAPI, CD20 (red signal​), and CD3E (cyan signal) to observe T cell content and DZ signature expression. (I) Box plot comparing CD3 AOI nuclear count percentages between high and low DZ signature groups. Statistical analysis was performed using a 2-tailed unpaired Mann-Whitney test. Values are shown as mean ± SEM; ****P < 0.0001. (J) Scatterplot with correlation line of DZ spatial signature expression within the CD20+ segment and the percentage of CD3+ cells per ROI across 79 DLBCL samples. Statistical significance was assessed using Spearman’s correlation coefficient (R) and P value.