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1624 Cd70 Deficiency Impairs CD4+ and CD8+ T-Cell Immune Surveillance in Bcl6-Driven Diffuse Large B-Cell Lymphomas

Program: Oral and Poster Abstracts
Session: 621. Lymphomas: Translational – Molecular and Genetic: Poster I
Hematology Disease Topics & Pathways:
Research, Lymphomas, non-Hodgkin lymphoma, Translational Research, B Cell lymphoma, immune mechanism, Diseases, aggressive lymphoma, immunology, Lymphoid Malignancies, Biological Processes, molecular biology, Study Population, Animal model
Saturday, December 9, 2023, 5:30 PM-7:30 PM

Elisa Mandato, PhD1*, Eleonora Calabretta, MD1*, Gali Bai, MS2,3*, Li Song, PhD2,4*, Tianfang Ma, PhD1*, Vignesh Shanmugam, MD5,6, Julia Paczkowska, PhD1*, Yanbo Sun, PhD1,7*, Il-Kyu Choi, PhD1,8*, Robert A. Redd, MS2*, Donna S. Neuberg, ScD2, Sumbul Jawed Khan, PhD1*, Franziska Michor, PhD2,9,10,11,12*, Scott Rodig, MD, PhD5,13, Baochun Zhang, MD, PhD1* and Margaret A. Shipp, MD1

1Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA
2Department of Data Science, Dana-Farber Cancer Institute, Boston, MA
3Department of Biomolecular Engineering in the Baskin School of Engineering, University of California Santa Cruz, Santa Cruz, CA
4Department of Biomedical Data Science, Dartmouth College, Hanover, NH
5Department of Pathology, Brigham and Women's Hospital, Boston, MA
6Cancer Program, Broad Institute of MIT and Harvard, Cambridge, MA
7Moderna, Cambridge, MA
8Daegu Gyeongbuk Institute of Science and Technology, Daegu, Korea, Republic of (South)
9Department of Biostatistics, Harvard T.H. Chan School of Public Health, Boston, MA
10Department of Stem Cell and Regenerative Biology, Harvard University, Boston, MA
11Broad Institute of MIT and Harvard, Cambridge, MA
12Ludwig Center at Harvard, Boston, MA
13Center for Immuno-Oncology, Dana-Farber Cancer Institute, Boston, MA

Anti-tumor immune responses to lymphoid malignancies are modulated by co-stimulatory and co-inhibitory pathways. Our group previously characterized the recurrent genetic alterations in primary diffuse large B-cell lymphomas (DLBCL) and identified frequent inactivating mutations or copy loss of the CD70 co-stimulatory ligand in these tumors. CD70 co-stimulation of CD27+ T cells induces antigen-dependent T-cell expansion and immune surveillance of normal and malignant B cells. Given the frequent combination of CD70 and BCL6 alterations in our human DLBCL series, we investigated the consequences of CD70 deficiency (Cd70-/-) in a murine model of BCL6-driven lymphomagenesis (Bcl6tg/+).

Cd70-/-; Bcl6tg/+ animals developed predominantly splenic DLBCLs with earlier onset and increased penetrance when compared to Bcl6tg/+ mice (p<.01). Hence, to elucidate potential differences in T-cell mediated anti-tumor responses in Cd70-/-; Bcl6tg/+ versus Bcl6tg/+ mice (and wild-type (WT) and Cd70-/- animals) and to characterize the concurrent processes of B-cell transformation and immune evasion over time, we performed single-cell RNA sequencing of splenic cell suspensions from animals euthanized for symptoms and additional asymptomatic mice at pre-determined timepoints (6, 14 and 18 months). We applied unsupervised clustering and defined the major B- and T-cell types based on canonical lineage markers. Additionally, we reconstructed the splenic T-cell receptor (TCR) and B-cell receptor (BCR) sequences with the TRUST4 algorithm to assess their diversity and clonal expansion.

TRUST4 analyses revealed clonally expanded B cells that were primarily found in specific clusters. These clusters also had an increased number of aberrant cells identified with an orthogonal approach that detects copy number alterations, InferCNV. Certain of these clusters were more abundant in aged asymptomatic Bcl6tg/+ and Cd70-/-; Bcl6tg/+ animals than in symptomatic mice. Aberrant cells in these clusters had a memory-like IgM+ phenotype (Nt5e, Zeb2, Cd38, Cd44, Itgb1) and included two discrete populations: 1) previously described age-associated/autoimmune-like cells (Tbx21, Cd11c, Cd11b) and 2) newly appreciated atypical memory cells. Additional clusters that were predominantly found in symptomatic animals included aberrant cells with increased expression of cycling genes and downregulation of lineage-defining markers and MHC II (p<.001). We interpret the aberrant memory-like B-cells in asymptomatic Cd70-/-; Bcl6tg/+ and Bcl6tg/+ animals to be precursors of the ones detected in symptomatic mice. Importantly, malignant clonal expansion and splenomegaly occurred at earlier timepoints and to a greater extent in Cd70-/-; Bcl6tg/+ than Bcl6tg/+ mice (p<.01).

To further characterize potential anti-tumor immune responses, we examined splenic CD8+ and CD4+ T-cell subsets in tumor-bearing cohorts (Bcl6tg/+ and Cd70-/-; Bcl6tg/+) and control animals (WT and Cd70-/-). In the tumor-bearing cohorts, we detected selective expansion of CD8+ cytotoxic T cells (CTLs) with a spectrum of activation and exhaustion markers. CD8+ CTL expansion was significantly greater in Bcl6tg/+ mice than Cd70-/-; Bcl6tg/+ animals.

We also identified selective expansion of a major CD4+ CTL compartment (GzmB, Prf1, Eomes) in the tumor-bearing cohorts that persisted significantly longer in Bcl6tg/+ than Cd70-/-; Bcl6tg/+ mice. In the tumor-bearing cohorts at 18 months, there was significantly greater clonal expansion of CD4+ CTLs, in comparison to CD8+ CTLs, highlighting the likely importance of this understudied cytotoxic CD4+ T-cell subset. In this regard, the downregulation of MHC II on the malignant B cells of symptomatic mice likely favored CD4+ CTL immune escape and accelerated the development of overt lymphoma.

Taken together, our data indicate that genetic perturbation of CD70/CD27 co-stimulation limits the development of an effective T-cell mediated immune response to Bcl6tg/+-driven DLBCL and highlights the unanticipated role of CD4+ CTLs and MHC II down-regulation in the attempted development and subsequent loss of an anti-tumor immune response. Additionally, we identified previously unappreciated precursor populations of murine DLBCL that warrant further investigation in human tumors.

Disclosures: Sun: Moderna: Current Employment; Repertoire Immune Medicines: Ended employment in the past 24 months. Neuberg: Madrigal Pharmaceuticals: Current equity holder in private company. Khan: New England Biolabs: Current Employment. Michor: Harbinger Health: Consultancy, Current equity holder in private company; Zephyr AI: Consultancy, Current equity holder in private company; Exscientia Plc: Membership on an entity's Board of Directors or advisory committees. Rodig: Bristol Myers Squibb: Research Funding; Immunitas Therapeutics: Membership on an entity's Board of Directors or advisory committees; KITE/Gilead: Research Funding. Shipp: Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Research Funding; Abbvie: Research Funding.

*signifies non-member of ASH