Therapy of lymphoma by immune checkpoint inhibitors: the role of T cells, NK cells and cytokine-induced tumor senescence

Abstract

Background: Although antibodies blocking immune checkpoints have already been approved for clinical cancer treatment, the mechanisms involved are not yet completely elucidated. Here we used a λ-MYC transgenic model of endogenously growing B-cell lymphoma to analyze the requirements for effective therapy with immune checkpoint inhibitors.

Methods: Growth of spontaneous lymphoma was monitored in mice that received antibodies targeting programmed cell death protein 1 and cytotoxic T lymphocyte-associated protein-4, and the role of different immune cell compartments and cytokines was studied by in vivo depletion experiments. Activation of T and natural killer cells and the induction of tumor senescence were analyzed by flow cytometry.

Results: On immune checkpoint blockade, visible lymphomas developed at later time points than in untreated controls, indicating an enhanced tumor control. Importantly, 20% to 30% of mice were even long-term protected and did never develop clinical signs of tumor growth. The therapeutic effect was dependent on cytokine-induced senescence in malignant B cells. The proinflammatory cytokines interferon-γ (IFN-γ) and tumor necrosis factor (TNF) were necessary for the survival benefit as well as for senescence induction in the λ-MYC model. Antibody therapy improved T-cell functions such as cytokine production, and long-time survivors were only observed in the presence of T cells. Yet, NK cells also had a pronounced effect on therapy-induced delay of tumor growth. Antibody treatment enhanced numbers, proliferation and IFN-γ expression of NK cells in developing tumors. The therapeutic effect was fully abrogated only after depletion of both, T cells and NK cells, or after ablation of either IFN-γ or TNF.

Conclusions: Tumor cell senescence may explain why patients responding to immune checkpoint blockade frequently show stable growth arrest of tumors rather than complete tumor regression. In the lymphoma model studied, successful therapy required both, tumor-directed T-cell responses and NK cells, which control, at least partly, tumor development through cytokine-induced tumor senescence.

Keywords: CTLA-4 antigen; hematologic neoplasms; immunotherapy; programmed cell death 1 receptor; tumor microenvironment.

Figure 1

Effect of ICB therapy on tumor growth in λ-MYC mice. Animals received anti-PD-1 and anti-CTLA-4 mAbs as described in the methods section or were left untreated. Other groups were additionally injected with IFN-γ-neutralizing and TNF-neutralizing mAbs during therapy. Up to 15 mice were included in each group. For P values see Materials and methods section. CTLA-4, cytotoxic T lymphocyte-associated protein-4; ICB, immune checkpoint blockade; IFN-γ, interferon-γ; PD-1, programmed cell death protein 1.

Figure 2

ICB-induced senescence in λ-MYC tumor cells. (A) Gating strategy for detection of intracellular SA-β-gal activity in B cells. Analysis of CD19⁺ cells from a wt mouse is shown as an example. (B) Senescence in CD19⁺ tumor cells derived from λ-MYC mice that were or were not treated with ICB mAbs. Part of the treated animals additionally received an IFN-γ-neutralizing or TNF-neutralizing mAb. The senescence index is defined as the x-fold amount of SSC-A⁺ C₁₂FDG⁺ B cells related to those from untreated λ-MYC animals. Four to 12 animals were included in each group. (C) Senescence induction in lymphoma cells from a λ-MYC mouse in vitro 24 hours after incubation with varying concentrations of IFN-γ and TNF. Means and SEM from quadruplicates. ICB, immune checkpoint blockade; IFN-γ, interferon-γ; mAbs, monoclonal antibodies; SA-β-gal, senescence-associated β-galactosidase; TNF, tumor necrosis factor.

Figure 3

Impact of ICB therapy on T-cell functions. (A) Numbers of CD4⁺ and CD8⁺ T cells, which are reduced in spleens of diseased λ-MYC mice, recover by ICB therapy. (B) The proliferative capacity of CD4⁺ and CD8⁺ T cells in spleens of tumor mice is restored after mAb treatment. Percentages of proliferating cells were measured after CD3/CD28 stimulation in vitro and related to those obtained without stimulation. (C) ICB therapy increases the expression of IFN-γ and TNF by CD4⁺ T cells. (D) In the absence of T cells, ICB therapy in λ-MYC mice fails to produce long-time survivors, but is still effective in delaying tumor development. Eleven to 15 mice were used in each group. CTLA-4, cytotoxic T lymphocyte-associated protein-4; ICB, immune checkpoint blockade; IFN-γ, interferon-γ; TNF, tumor necrosis factor.

Figure 4

Characterization of spleen NK cells from ICB-treated or untreated λ-MYC animals. (A) PD-1 and CTLA-4 expression on NK cells is increased in tumor-bearing animals as compared to wt mice. (B) The fraction of NK cells is augmented in spleens from λ-MYC mice that underwent ICB therapy. (C) NK cells from treated animals exhibit somewhat higher Ki-67 expression. (D) NK cells from treated animals show enhanced degranulation as indicated by increased CD107a surface levels after incubation with YAC-1 cells. (E) IFN-γ expression by NK cells, which becomes compromised during the course of tumor development, can partly be restored by ICB therapy. (F) IFN-γ expression by NK cells in the presence of YAC-1 cells in vitro is augmented when immune checkpoint inhibitors are added. CTLA-4, cytotoxic T lymphocyte-associated protein-4; ICB, immune checkpoint blockade; IFN-γ, interferon-γ; NK, natural killer; PD-1, programmed cell death protein 1; TNF, tumor necrosis factor.

Figure 5

Relevance of NK cells for the success of ICB therapy in λ-MYC lymphoma. Mice were treated with immune checkpoint inhibitors as described in Materials and methods section. Under simultaneous NK-cell depletion, tumor development is slightly delayed in comparison to the untreated controls, but the difference to this control group is no longer significant. Therapy is completely ineffective only in the absence of NK as well as T cells. Six to 15 mice were included in each group. CTLA-4, cytotoxic T lymphocyte-associated protein-4; NK, natural killer; PD-1, programmed cell death protein 1.