Harnessing the immune adjuvant properties of natural killer T (NKT) cells is an effective strategy to generate anticancer immunity. The objective of this study was to increase the potency and durability of vaccine-induced immunity against B cell lymphoma by combining α-galactosylceramide (α-GalCer)-loaded tumor cell vaccination with an agonistic antibody targeting the immune checkpoint molecule 4–1BB (CD137). We observed potent synergy when combining vaccination and anti-4–1BB antibody treatment resulting in significantly enhanced survival of mice harboring Eμ-myc tumors, including complete eradication of lymphoma in over 50% of mice. Tumor-free survival required interferon γ (IFNγ)-dependent expansion of CD8+ T cells and was associated with 4–1BB-mediated differentiation of KLRG1+ effector CD8+ T cells. 'Cured' mice were also resistant to lymphoma re-challenge 80 days later indicating successful generation of immunological memory. Overall, our results demonstrate that therapeutic anticancer vaccination against B cell lymphoma using an NKT cell ligand can be boosted by subsequent co-stimulation through 4–1BB leading to a sustainable immune response that may enhance outcomes to conventional treatment.

Combinations of mAbs that target various components of T-cell activation/inhibition may work synergistically to improve antitumor immunity against cancer. In this study, we investigated the therapeutic potential of combining an anticancer vaccination strategy with antibodies targeting an immune stimulatory (4-1BB) and immune inhibitory (PD-1) receptor, in a preclinical model of spontaneously arising c-Myc-driven B-cell lymphoma. In Eμ-myc transgenic mice, we reveal that 4-1BB agonistic mAb treatment alone was sufficient to drive antitumor immunity and prevent disease progression in 70% of mice. When combined with an α-GalCer-loaded, irradiated tumor cell vaccine, 4-1BB mAb treatment led to increased expansion of effector CD8 T-cell populations and protection of long-term surviving mice against tumor rechallenge. Unexpectedly, PD-1 blockade did not provide therapeutic benefit. The T-cell-promoting effects and antitumor activity of 4-1BB mAb were diminished when used simultaneously with a PD-1-blocking mAb. This was associated with a rapid and dramatic reduction in effector CD8 T-cell subsets in the presence of PD-1 blockade. These findings reveal that supporting T-cell activation therapeutically is effective for controlling B-cell lymphomas; however, caution is required when combining antibody-mediated modulation of both costimulatory and coinhibitory T-cell receptors. .

Monocytosis is considered a poor prognostic factor for many cancers, including B cell lymphomas. The mechanisms by which different monocyte subsets support the growth of lymphoma is poorly understood. Using a pre-clinical mouse model of B cell non-Hodgkin's lymphoma (B-NHL), we investigated the impact of tumor progression on circulating monocyte levels, subset distribution and their activity, with a focus on immune suppression. B-NHL development corresponded with significant expansion initially of classical (Ly6Chi) and non-classical (Ly6Clo) monocytes, with accumulation and eventual predominance of Ly6Clo cells. The lymphoma environment promoted the conversion, preferential survival and immune suppressive activity of Ly6Clo monocytes. Ly6Clo monocytes expressed higher levels of immunosuppressive genes including PD-L1/2, Arg1, IDO1 and CD163, compared to Ly6Chi monocytes. Both monocyte subsets suppressed CD8 T cell proliferation and IFN-γ production , but via different mechanisms. Ly6Chi monocyte suppression was contact dependent, while Ly6Clo monocytes suppressed via soluble mediators, including IDO and arginase. Ly6Clo monocytes could be selectively depleted in tumor-bearing hosts by liposomal doxorubicin treatment, further enhanced by co-administration of anti-4-1BB monoclonal antibody. This treatment led to a reduction in tumor growth, but failed to improve overall survival. Analogous immunosuppressive monocytes were observed in peripheral blood of diffuse large B cell lymphoma patients and actively suppressed human CD8 T cell proliferation. This study highlights a potential immune evasion strategy deployed by B cell lymphoma involving accumulation of circulating non-classical monocytes with immunosuppressive activity.