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761 Polyfunctional Effector PD-1+ CD8+ T Cells and M1 Macrophages Promote Immune Checkpoint Blockade Response in Richter Syndrome Mouse Models

Program: Oral and Poster Abstracts
Type: Oral
Session: 641. Chronic Lymphocytic Leukemia: Basic and Translational: CLL and Richter Transformation: Functional Genomics and Molecular Mechanisms
Hematology Disease Topics & Pathways:
Research, Translational Research, Immune mechanism, Immunology, Biological Processes
Monday, December 9, 2024: 11:30 AM

Elisa Ten Hacken, PhD1,2, Johan Gustafsson, PhD3,4,5*, Chiara Tomasoni, PhD1*, Gabriela Brunsting Hoffmann6*, Kayla Cruz6*, Wesley S Lu, BS7*, Shuqiang Li8*, Nicholas Haradhvala, PhD3*, Connor Johnson, BA3*, Stephanie Deng2*, Xia Bu, PhD2*, Donna S. Neuberg, ScD9, Othman Al-Sawaf, MD10, Barbara F. Eichhorst, MD10, Clare Sun, MD11, Erin M. Parry, MD, PhD2,3,12,13, Gordon J. Freeman, PhD2,13*, Gad Getz, PhD3,14,15* and Catherine J. Wu, MD2,3,12,13*

1Division of Hematology and Medical Oncology, Weill Cornell Medical College, Weill Cornell Medicine, New York City, NY
2Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA
3Broad Institute of MIT and Harvard, Cambridge, MA
4TIMM Laboratory at Sahlgrenska Center for Cancer Research, University of Gothenburg, Gothenburg, Sweden
5Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
6Department of Medical Oncology, Dana Farber Cancer Institute, Boston, MA
7Translational Immunogenomics Lab, Dana-Farber Cancer Institute, Boston, MA
8Dana Farber Cancer Institute, Translational Immunogenomics Laboratory, Boston, MA
9Department of Data Science, Dana-Farber Cancer Institute, Boston, MA
10Faculty of Medicine and University Hospital Cologne, Department I of Internal Medicine, Center for Integrated Oncology Aachen Bonn Cologne Duesseldorf; German CLL Study Group, University of Cologne, Cologne, Germany, Cologne, Germany
11National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD
12Brigham and Women's Hospital, Boston, MA
13Harvard Medical School, Boston, MA
14Department of Pathology, Massachusetts General Hospital, Boston, MA
15Krantz Family Center for Cancer Research and Dept. of Pathology, Massachusetts General Hospital, Boston, MA

Richter syndrome (RS) displays dismal prognosis and remains largely refractory to most existing therapies. Immune checkpoint blockade (ICB) and bi-specific antibodies have shown promising activity, yet determinants of response and resistance to these immunotherapies remain incompletely understood. We leveraged our previously developed mouse models of RS, achieved via CRISPR-Cas9 gene editing of combinatorial loss-of-function drivers in immune competent mice, to interrogate microenvironmental determinants of disease transformation and response to therapy.

We profiled immune cells from the spleen of 18 primary murine tumors displaying either CLL (n=4), CLL/RS (n=3) or RS (n=11) histology by sc-RNAseq. Analysis of a total of 104,731 T/NK cells revealed pronounced changes in the CD4+ and CD8+ T cell compartment, with CD8+ effector/exhausted T cells and CD4+ cytotoxic cells as the predominant subsets in RS compared to CLL (pAdj<0.038). Flow cytometric analysis of an independent set of 17 cases (5 CLL, 3 CLL/RS, 9 RS) confirmed increased CD8/CD4 T cell ratio as typical of RS (p=0.008), together with abundant effector/memory CD8+ T cell subsets expressing high PD-1 and TOX levels (p=0.001). Enrichment in cytotoxic/exhausted CD8+ T cells in RS was validated in bulk external RNA-seq data from 34 human RS lymph node (LN) samples and 34 CLL-LNs using a gene score based on human orthologs of 13 genes derived from the mouse data (p=0.0023). Importantly, ex vivo stimulation of murine splenocytes (5 CLL, 6 RS) with PMA/ionomycin showed marked secretion of IFNg, TNFa and Granzyme B in PD-1+ CD8+ T cells from RS cases (p<0.02 for all combinations). In contrast, CLL CD8+ PD-1+ T cells only showed induction of TNFa. TCR-seq revealed pronounced clonal expansion in RS compared to CLL (Shannon index p=0.008), indicative of chronic antigenic stimulation in RS.

The myeloid/monocytic compartment (41,956 cells) displayed more stability at transformation, aside from new enrichment of CXCL9+/CXCL10+ macrophages in RS compared to CLL (pAdj=0.093). This population displayed features of inflammatory M1 cells, including high expression of H2-Ab1 (class II), Cd274 (PD-L1), Cd40, Stat1, and Cd86. Flow cytometric analysis confirmed increased abundance of CXCL9+ macrophages in splenic preparations from RS compared to CLL mice (p= 0.007), and signatures extracted from CXCL9+ macrophages were validated as enriched in bulk RNA-seq from human RS cohorts (p<0.0001). Importantly, proportional levels of CXCL9+ macrophages correlated with those of CD8+ exhausted T cells (Pearson r= 0.69, p<0.0001). Numerous related ligand-receptor pairs, including PD-1/PD-L1, were significantly enriched in RS compared to CLL per the Liana algorithm. Physical proximity of IFNG, GZMB, and CD3D expressing cells, representing cytotoxic T cells, to CXCL9 and CD68 expressing cells, representing CXCL9/10+ macrophages (Moran’s R > 0.1), was evident in one RS-LN analyzed by 10X Visium.

To interrogate determinants of response/resistance to ICB therapy, we treated 4 different RS tumor models (achieved by transplantation of RS primary tumors into syngeneic immune competent recipients) and one CLL for 3 weeks with an anti-PD-1 monoclonal antibody (or isotype control) and analyzed changes in immune microenvironmental features at the end of treatment by combined scRNA-seq and flow cytometry. While CLL transplants remained insensitive to therapy, we observed a range of responses in the RS models, from non-response (1 line) to partial response (1 line) to marked response to treatment (2 lines). Importantly, responding cases showed lower tumor burden in spleen at euthanasia, higher PD-1 or PD-L1 tumor expression before treatment (p<0.001), and increased baseline (or post-treatment) polyfunctionality of CD8+ PD-1+ T cells. Complete tumor regression in one of the RS lines was accompanied by loss of CXCL9+ macrophages (p=0.001) and CD8+ PD-1+ T cells (p=0.0002), suggesting that tumor-intrinsic features favor retention of these subpopulations prior to therapy.

Overall, these data suggest key changes in immune microenvironmental features of RS compared to CLL, which associate to response to ICB in mice. Analysis of spatial datasets from LN of RS patients treated with ICB is ongoing to evaluate whether these features associate to response in humans, together with further investigation of their relevance to outcome through in vivo depletion experiments.

Disclosures: Haradhvala: MorphoSys: Consultancy. Neuberg: Madrigal Pharmaceutical: Current equity holder in publicly-traded company. Al-Sawaf: Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees; Ascentage: Membership on an entity's Board of Directors or advisory committees; BeiGene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Eli Lilly: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Eichhorst: Roche: Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Research Funding, Speakers Bureau; Janssen: Consultancy, Research Funding, Speakers Bureau; BeiGene: Consultancy, Research Funding, Speakers Bureau; Kite: Consultancy, Speakers Bureau; Lilly: Consultancy; MSD: Consultancy, Speakers Bureau; Miltenyi: Consultancy; Hoffmann-La Roche: Research Funding, Speakers Bureau; AbbVie: Consultancy, Research Funding, Speakers Bureau. Sun: Gemab: Research Funding. Freeman: Roche: Patents & Royalties: PD-L1/PD-1 pathway; Merck KGA: Patents & Royalties: PD-L1/PD-1 pathway; AstraZeneca: Patents & Royalties: PD-L1/PD-1 pathway; Bristol-Myers Squibb: Patents & Royalties: PD-L1/PD-1 pathway; Merck MSD: Patents & Royalties: PD-L1/PD-1 pathway; Boehringer-Ingelheim: Patents & Royalties: PD-L1/PD-1 pathway; Dako: Patents & Royalties: PD-L1/PD-1 pathway; Leica: Patents & Royalties: PD-L1/PD-1 pathway; Eli Lilly: Patents & Royalties: PD-L1/PD-1 pathway; Novartis: Patents & Royalties: PD-L1/PD-1 pathway; iTEOS: Consultancy, Current equity holder in publicly-traded company; Nextpoint: Consultancy, Current equity holder in private company, Current holder of stock options in a privately-held company; IgM: Consultancy, Current equity holder in publicly-traded company; GV20: Consultancy, Current equity holder in private company, Current holder of stock options in a privately-held company; IOME: Consultancy; Bioentre: Consultancy, Current equity holder in private company; Santa Ana Bio: Consultancy; Simcere of America: Consultancy; Geode: Consultancy, Current equity holder in private company, Current holder of stock options in a privately-held company; Invaria: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. Getz: Broad Institute: Patents & Royalties: MSMuTect, MSMutSig, POLYSOLVER, SignatureAnalyzer-GPU, MSEye, and MinimuMM-seq; Scorpion Therapeutics: Consultancy, Current equity holder in private company, Other: Founder; PreDICTA Biosciences: Consultancy, Current equity holder in private company, Other: Founder; IBM, Pharmacyclics/Abbvie, Bayer, Genentech, Calico, and Ultima Genomics: Research Funding. Wu: Aethon Therapeutics: Membership on an entity's Board of Directors or advisory committees; Adventris: Membership on an entity's Board of Directors or advisory committees; BioNtech, Inc: Current equity holder in publicly-traded company; Pharmacyclics: Research Funding; Repertoire: Membership on an entity's Board of Directors or advisory committees.

*signifies non-member of ASH