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 6

Functional impact of ATRi treatment on GC response.

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Functional impact of ATRi treatment on GC response. (A) Schematic repres...
(A) Schematic representation of experimental protocol. In one experiment on ten C57BL/6J mice, immunization with NP-OVA in alum on day 0, followed by treatment with vehicle (n = 5) or AZD6738 (50 mg/kg, n = 5), was performed from day 2 to day 6. mLNs were harvested on day 7 for analysis. (B) Box plots showing quantitative analysis of CD3+, CD4+, and CD8+ T cell infiltration in indicated numbers of total GCs and DZ and LZ compartments in vehicle-treated (CTRL) versus ATRi-treated mice. (C) Representative photomicrographs of double-marker IHC for Ki-67+ (brown) and CD3+ (pink) cells in mLN GCs from vehicle-treated (CTRL) and ATRi-treated mice. Original magnification, ×400. Scale bars: 50 μm. (D) Combined IHC/IF staining for CD3+ (brown), CD21+ (pink), and Ki-67+ (cyan) cells in mLN GCs from vehicle-treated (CTRL) and ATRi-treated mice, showing spatial distribution of T cells in DZ and LZ compartments. Original magnification, ×400. Scale bars: 50 μm. (E) Combined IHC/IF staining for CD4+ (pink), CD8+ (brown), and Ki-67+ (cyan) cells in mLN GCs from vehicle-treated (CTRL) and ATRi-treated mice, illustrating phenotype of infiltrating T cells. Original magnification, ×400. Scale bars: 50 μm. (F) Box plots showing quantitative analysis of Ifnγ+CD8+ T cells, Ifnb1+CD20+ B cells, and MHC-I expression in indicated numbers of total GCs and DZ and LZ compartments in vehicle-treated (CTRL) versus ATRi-treated mice. (G) Representative images of combined mRNA ISH for Ifnγ (brown) and double-marker IHC for CD8 (pink) and Ki-67 (cyan) in mLN GCs from vehicle-treated (CTRL) and ATRi-treated mice, showing localization of activated CD8+ T cells. Original magnification, ×400 and ×630 (insets). Scale bars: 50 μm and 25 μm. (H) Representative images of combined mRNA ISH for Ifnb1 (brown) and double-marker IHC for CD20 (pink) and Ki-67 (cyan) in vehicle-treated (CTRL) and ATRi-treated mice, highlighting induction of type I interferon response in the DZ. Original magnification, ×400 and ×630 (insets). Scale bars: 50 μm and 25 μm. (I) Representative IHC staining for MHC-I (brown) or MHC-I (brown) and Ki-67 (violet) in vehicle-treated (CTRL) and ATRi-treated mice, demonstrating increased MHC-I expression in the DZ in response to ATR inhibition. Original magnification, ×400 and ×630 (insets). Scale bars: 30 μm. Box plot statistical analysis: 2-tailed unpaired Mann-Whitney test. Mean ± SEM is shown; *P < 0.05, **P < 0.01, ***P < 0.001.