-Author name in bold denotes the presenting author
-Asterisk * with author name denotes a Non-ASH member
Clinically Relevant Abstract denotes an abstract that is clinically relevant.

PhD Trainee denotes that this is a recommended PHD Trainee Session.

Ticketed Session denotes that this is a ticketed session.

4830 The Ig Superfamily Ligand B7H6 Subjects Activated T Cells to NK Cell Surveillance and Limits CAR T Cell Persistence

Program: Oral and Poster Abstracts
Session: 703. Cellular Immunotherapies: Basic and Translational: Poster III
Hematology Disease Topics & Pathways:
Fundamental Science, Lymphoid Leukemias, Biological therapies, Research, Translational Research, Lymphomas, Chimeric Antigen Receptor (CAR)-T Cell Therapies, Checkpoint Inhibitor, Diseases, immune mechanism, Therapies, Immunotherapy, immunology, Lymphoid Malignancies, Biological Processes, Technology and Procedures, gene editing, Study Population, Human, Animal model
Monday, December 11, 2023, 6:00 PM-8:00 PM

Mirco Julian Friedrich, MD, PhD1,2*, Michael Kilian, PhD2*, Kevin Hai-Ning Lu2*, Selina Jansky2*, Julius Michel2*, Anna Keib3*, David Vonhören2*, Nicolaj Hackert3*, Markus Hahn2*, Adelheid Cerwenka4*, Felix Sahm5*, Frank Westermann2*, Peter Dreger, MD3, Marc S. Raab3*, Nadja M. Meindl-Beinker6*, Matthias Ebert6*, Lukas Bunse, MD2*, Carsten Müller-Tidow, MD3*, Michael Schmitt3* and Michael Platten, MD2,7*

1Broad Institute of MIT and Harvard, Cambridge, MA
2German Cancer Research Center (DKFZ), Heidelberg, Germany
3Department of Hematology, Oncology and Rheumatology, Heidelberg University Hospital, Heidelberg, Germany
4Mannheim Institute of Innate Immunoscience (MI3), Medizinische FakultäT M, Mannheim, Germany
5Department of Neuropathology, Heidelberg University Hospital, Heidelberg, Germany
6Department of Medicine II, Medical Faculty Mannheim, Mannheim, Germany
7Department of Neurology, Medical Faculty Mannheim, Mannheim, Germany

Understanding the mechanisms that regulate T cell immunity is critical for the development of effective therapies for diseases associated with T cell dysfunction. Co-inhibitory ‘checkpoint molecules’ are tightly connected to T cell activation and balance excessive or prolonged immune activation by suppressing clonal expansion. Previous studies have suggested that NK cells might also suppress lymphocyte expansion, but the specific ligands enabling T cell recognition by natural cytotoxicity receptors (NCRs) remain unknown.

In this study, we describe the physiological expression of the immunoglobulin superfamily ligand B7H6 on activated T cells and elucidated the functional and clinical implication of its recognition by autologous NK cells.

In a targeted proteomics screen for mediators of NK cell recognition on T cells, we identified the Ig superfamily ligand B7H6 (NCR3LG1) to be highly expressed by T cells upon activation. We show that, unlike other checkpoint molecules, B7H6 mediates NKp30-dependent recognition and subsequent cytolysis of activated T cells by autologous NK cells. Time-resolved deep RNA-sequencing of anti-CD3/CD28-stimulated peripheral blood-derived T cells from n=24 human donors revealed that NCR3LG1 transcript expression changes dynamically upon stimulation and that B7H6 surface protein recognition by NK cells initiates Granzyme B-mediated ‘fratricide’ as early as 8 hours post T cell stimulation. Genomic knockout of NCR3LG1 rendered activated T cells resistant to NK cell-mediated killing without affecting their viability or proliferative capacity.

By analyzing transcriptomic profiles of 313,303 CD8+ and 283.930 CD4+ T cells in datasets of multiple sclerosis (MS, n = 62 patients), inflammatory bowel disease (IBD, n = 4 patients), hepatitis B virus infection (HBV, n = 23 patients) and 9 different cancer entities (n = 47 patients), we found a conserved B7H6+ T cell population that is prevalent in the tissue and blood of most individuals at 1-5% frequency. In line with our experimental data, B7H6 positivity was coupled to a highly activated cell state of T cells.

Considering CAR T cell manufacturing involves robust activation and clonal expansion, we hypothesized the B7H6-NKp30 axis is a yet unknown, but relevant factor in limiting CAR T cell persistence. Clinical-grade CD19.CD28.4-1BBzeta-CAR T cell products as well as circulating CAR T cells in lymphoma patients indeed uniformly expressed NCR3LG1 transcripts and B7H6 surface protein and were lysed by autologous NK cells in vitro. We then co-injected CD19.CAR T cells and patient-autologous NK cells in leukemia-engrafted mice and observed reduced CAR T cell persistence in the peripheral blood of mice with NK cell co-injection. Co-injection of NK cells further increased tumor burden and resulted in significantly decreased survival in leukemia-bearing mice treated with CD19-recognizing CAR-T cells, while these effects could be minimized by NKp30 antibody blockade or prior genetic editing of NCR3LG1 on CAR T cells.

Given the physiological coupling T cell activation and B7H6 expression, any T cell-directed immunotherapy regimen might trigger the B7H6-NKp30-cytolysis mechanism. We therefore analyzed tissue of patients that were treated with nivolumab and ipilimumab (NCT03416244). In contrast to the previous notion of largely tumor-targeting NK cell function, we observed an increased NK cell abundance in patients that failed to respond to treatment. Notably, we found both B7H6+ CD8+ and CD4+ T cells in situ and that their abundance and a high NK/T ratio was associated with reduced progression-free survival (PFS), even in initial clinical responders, and experimentally deducted a NKp30 dependent cytolysis of these T cells.

Together, our data demonstrates a mechanism that couples T cell expansion to eventual clearance by NK cells. This concept differs from the previously known immune checkpoints, as those molecules act through intracellular downstream signaling in T cells. We show that NK cell surveillance via B7H6 counteracts anti-tumor activity of CAR-T cells in vivo and that the genetic or antibody-mediated inhibition of B7H6 enhances T cell persistence. We therefore suggest an alternative immune checkpoint that limits T cell expansion and persistence in physiological and cellular engineering contexts. Targeting the B7H6-NKp30 axis may offer a means to modulate T cell immunity across multiple disease entities.

Disclosures: Friedrich: Pfizer: Speakers Bureau. Dreger: Abbvie: Consultancy, Speakers Bureau; AstraZeneca: Consultancy, Speakers Bureau; Beigene: Consultancy, Honoraria; Gilead: Consultancy, Speakers Bureau; BMS: Consultancy, Honoraria; Novartis: Consultancy, Speakers Bureau; Riemser: Consultancy, Research Funding, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Miltenyi: Consultancy. Raab: Heidelberg University Hospital: Current Employment; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Heidelberg Pharma: Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Schmitt: TolerogenixX: Current Employment; MSD: Membership on an entity's Board of Directors or advisory committees; Kite: Other: travel grant, educational activities and conferences; Novartis: Other: educational activities and conferences, Research Funding; Hexal: Other: travel grant, Research Funding; Apogenix: Research Funding.

*signifies non-member of ASH