Mutant HVEM in lymphoma disrupts BTLA and CD160 regulation of anti-tumor signaling.

INTRODUCTION: Somatic mutations can rewire tumor-immune interactions to favor cancer progression. Here, we define the functional impact of missense mutations in Herpesvirus entry mediator (HVEM, TNFRSF14), a receptor frequently altered in lymphoma and melanoma. METHODS: We first assessed binding interactions between cell-expressed HVEM proteins containing mutations identified in human B cell lymphoma and its ligands LIGHT (TNFSF14), B and T lymphocyte attenuator (BTLA), and CD160. We next assessed the capacity for these ligands in a soluble or membrane expressed format to engage these mutant HVEM receptors and activate NF-κB signaling using luciferase assays. We additionally used NK92 cells in cytotoxicity assays with K562 and JVM13 target cells expressing these HVEM mutants to compare wild-type and mutant HVEM costimulation of NK cell lysis. We used proteomic analysis to identify BTLA and CD160 interacting proteins in NK92 and Jurkat cells, and phospho-proteomic analysis to identify signaling pathways activated by these receptors. We used a mouse model of B cell lymhoma expressing ovalbumin (TBL-OVA) and a mouse model of melanoma (B16) to assess how HVEM expression in these tumors regulated their expansion in vivo. Additionally, we used OT1 transgenic CD8+ T cells that recognize TBL-OVA cells and were derived from wild-type, Btla-deficient, or Cd160-deficient animals to assess the role of BTLA and CD160 in regulating anti-tumor responses. RESULTS: We found that HVEM mutations identified in human B cell lymphoma cluster in the extracellular domain. These mutants selectively disrupt BTLA and CD160 binding, and showed weaker activation of NF-κB in response to ligand binding compared to wild-type HVEM. These mutants also showed reduced costimulation of NK92 lytic function compared to wild-type HVEM. Proteomics revealed that BTLA engagement suppresses interferon signaling, whereas CD160 regulates MYC pathway activity. Functionally, HVEM expression and BTLA activation restrained growth of TBL-OVA tumors in vivo, while CD160 was required for optimal natural killer (NK) and tumor-specific CD8⁺ T cell responses. DISCUSSION: Our data illustrate an immuno-evasion strategy used by lymphoma to interrupt HVEM-activated inhibitory signaling in cis, and CD160 costimulatory signaling in trans, demonstrating the pressure to acquire HVEM mutations. The HVEM network thus plays a critical role in regulation of anti-tumor immunity, making it an attractive target for clinical intervention in cancer.