BackgroundOne of the significant tumor immune escape mechanisms and substantial barrier for successful immunotherapy is tumor-mediated inhibition of immune response through cell-to-cell or receptor/ligand interactions. Programmed death receptor-1 (PD-1) interaction with its ligands, PD-L1 and PD-L2, is one of the important strategies that many tumors employ to escape immune surveillance. Upon PD-Ls binding to PD-1, T cell receptor (TCR) signaling is dampened, causing inhibition of proliferation, decreased cytokine production, anergy and/or apoptosis. Thus PD-Ls expression by tumor cells serves as a protective mechanism, leading to suppression of tumor-infiltrating lymphocytes in the tumor microenvironment. Lm-LLO immunotherapies have been shown to be therapeutically effective due to their ability to induce potent antigen-specific immune responses. However, it has been demonstrated that infection with Lm leads to up-regulation of PD-L1 on mouse immune cells that can inhibit effector T cells through PD-1/PD-L1 pathway.MethodsTherapeutic and immune efficacy of Listeria-based vaccine (Lm-LLO-E7) in combination with anti-PD-1 antibody was tested in E7 antigen expressing TC-1 mouse tumor model. Tumor growth, survival, as well as peripheral and tumor-infiltrating immune cell profiles after immunotherapy were assessed.ResultsHere we demonstrate that the combination of an Lm-LLO immunotherapy with anti-PD-1 antibody that blocks PD-1/PD-L1 interaction, significantly improves immune and therapeutic efficacy of treatment in TC-1 mouse tumor model. Importantly, we show that in addition to significant reduction of regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC) in both spleen and tumor microenvironment that are mediated solely by the Lm-LLO immunotherapy, the addition of anti-PD-1 antibody to the treatment results in significant increase of antigen-specific immune responses in periphery and CD8 T cell infiltration into the tumor. As a result, this combinational treatment leads to significant inhibition of tumor growth and prolonged survival/complete regression of tumors in treated animals.We also demonstrate that in vitro infection with Lm results in significant upregulation of surface PD-L1 expression on human monocyte-derived dendritic cells suggesting the translational capacity of this finding.ConclusionsOur findings demonstrate that combination of Lm-LLO-based vaccine with blocking of PD-1/PD-L1 interaction is a feasible approach with clinical translation potential that can lead to overall enhancement of the efficacy of anti-tumor immunotherapy.

Studies have shown that Listeria monocytogenes (Lm)-based vaccine expressing a fusion protein comprising truncated listeriolysin O (LLO) and human papilloma virus (HPV) E7 protein (Lm-LLO-E7) induced a decrease in regulatory T cells (Treg) and complete regression of established, transplanted HPV-TC-1 tumors in mice. However, how the Lm-based vaccine causes a decrease in Tregs remains unclear. Using a highly attenuated Lm dal dat ΔactA strain (LmddA)-based vaccine, we report here that the vector LmddA was sufficient to induce a decrease in the proportion of Tregs by preferentially expanding CD4+FoxP3− T cells and CD8+ T cells, by a mechanism dependent on and directly mediated by LLO. Episomal expression of a nonhemolytic truncated LLO in Lm (LmddA-LLO) significantly augmented the expansion, thus further decreasing Treg frequency. While adoptive transfer of Tregs compromised the antitumor efficacy of the LmddA-LLO-E7 vaccine, a combination of LmddA-LLO and an Lm-based vaccine expressing E7 protein (Lm-E7) induced complete regression against established TC-1 tumors. An engineered LLO-minus Lm expressing perfringolysin O (PFO) that enables the recombinant bacteria to exit from the phagolysosome without LLO confirmed that the adjuvant effect was dependent on LLO. These results suggest that LLO may serve as a promising adjuvant by preferentially inducing the expansions of CD4+FoxP3− T cells and CD8+ T cells, thus reducing the ratio of Tregs to CD4+FoxP3− T cells and to CD8+ T cells favoring immune responses to eradicate tumor.

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<p>PDF file - 312K, Figure S1. LmddA-LLO-E7 induces regression of established TC-1 tumors.</p>

<p>PDF file - 2785K, Figure S3. LmddA does not augment Lm-E7 antitumor activity.</p>

<p>PDF file - 312K, Figure S1. LmddA-LLO-E7 induces regression of established TC-1 tumors.</p>

<p>PDF file - 6597K, Figure S2. LmddA-LLO-E7 and Lm-E7 induce similar E7-specific CD8+ T-cell response.</p>