B Cell Receptor Inactivation Boosts NK Cell Killing of MYC-Driven Lymphomas
Authors: Varano Gabriele , Arima Hiroshi , Parr Nicara C , Sala Eleonora , Mangione Marta , Mladenic Karlo , Morello Gaia , Mainoldi Federica , Brambillasca Silvia , Andronache Adrian , Imsirovic Vanna , Mercurio Ciro , Wensveen Felix , Polic Bojan , Kuka Mirela , Tripodo Claudio , Casola Stefano 
Published: December 2024

Survival of normal and transformed B-lymphocytes hinges on signals emanating from the B cell receptor (BCR), rendering the BCR a preferred target of therapy. In the λ-MYC murine B cell lymphoma model, we previously showed that inducible extinction of BCR signals leads to reduced competitive fitness of the malignant B cells, which could be corrected inhibiting glycogen synthase kinase 3 catalytic function. In addition to tumor cell-intrinsic mechanisms, we hypothesized that extinction of BCR signals influenced lymphoma immune surveillance. To address this, we performed longitudinal studies in mice reconstituted with BCR-proficient and -deficient λ-MYC lymphoma cells, combining estimation of absolute number and phenotype of malignant B cells, with immune cell characterization in tumor-infiltrated primary (bone marrow, BM) and secondary (spleen, SPL and lymph node) lymphoid organs. Intravenous inoculation of BCR-expressing λ-MYC lymphomas favored efficient engraftment and rapid proliferation of tumor cells in the bone marrow and spleen of immuno-competent syngeneic hosts. Conversely, λ-MYC lymphomas transplanted after inducible genetic BCR extinction, suffered from a significant delay in in vivo expansion, as revealed by flow cytometric and histological analyses. Transplantation of λ-MYC lymphomas into immunodeficient NOS/SCID/IL2R common-gamma chain deficient mice lacking B/T and Natural killer (NK) cells, showed a significant acceleration in in vivo tumor growth irrespective of BCR status. These results implicate effective adaptive immune cell clearance of λ-MYC lymphomas, in particular upon extinction of BCR signals. Immune cell profiling of tumor-infiltrated bone marrow of immunocompetent mice revealed an enrichment for CD8+ cytotoxic T cells and NK1.1+ NK cells at sites of BCR-less lymphoma growth, when compared to their BCR+ counterparts. CD8+ T cells infiltrating isles of BCR-less MYC lymphomas in BM and SPL expressed the proliferation marker Ki-67 and interferon gamma indicative of local activation. Antibody-mediated depletion of CD8+ T cells significantly improved recovery of BCR- lymphoma cells in lymphoid organs of transplanted mice, yet never normalizing those of BCR+ counterparts. In contrast, depletion of NK1.1+ NK cells showed a dramatic improvement of λ-MYC lymphoma growth in vivo, in particular in tumor inoculated after BCR extinction. Mechanistically, BCR inactivation causes significant reduction in MHC-class-I levels via downregulation of the transcription factor NLRC5, matched to increased surface expression of ligands for the NK cell activating receptor NKG2D. In vitro NK cytotoxicity assays showed preferential killing of BCR-less MYC lymphoma cells by NK cells, inhibited by NKG2D blocking antibodies. Serial transplantation of BCR-less lymphomas into immunocompetent hosts selected variants with improved immune cell evasion. Comparison of Ig-less tumor cells collected from early and late passages pointed to increased MHC-I surface levels, enhanced PI3K activity and BCR restoration via Rag1/2-dependent secondary IgH variable gene rearrangements as recurrent mechanisms to evade NK cell killing. Altogether, our results assign a central role to the BCR in protecting MYC-driven lymphomas from host immune cell clearance. In particular, we reveal the essential contribution of NK cells to counteract in vivo growth of BCR-less lymphomas, via engagement of the NKG2D receptor in a context of reduced tumor MHC-I expression. Our findings highlight the potential to boost lymphoma/leukemia therapies based on inhibitors of BCR signaling combining them with NK cell-engaging immunotherapies. The latter treatments may also become useful for the treatment of emerging mature aggressive B cell lymphomas lacking BCR expression (doi: https://doi.org/10.1101/2024.07.13.603066).