Rational Combination of Parvovirus H1 With CTLA-4 and PD-1 Checkpoint Inhibitors Dampens the Tumor Induced Immune Silencing

Abstract

The recent therapeutic success of immune checkpoint inhibitors in the treatment of advanced melanoma highlights the potential of cancer immunotherapy. Oncolytic virus-based therapies may further improve the outcome of these cancer patients. A human ex vivo melanoma model was used to investigate the oncolytic parvovirus H-1 (H-1PV) in combination with ipilimumab and/or nivolumab. The effect of this combination on activation of human T lymphocytes was demonstrated. Expression of CTLA-4, PD-1, and PD-L1 immune checkpoint proteins was upregulated in H-1PV-infected melanoma cells. Nevertheless, maturation of antigen presenting cells such as dendritic cells was triggered by H-1PV infected melanoma cells. Combining H-1PV with checkpoint inhibitors, ipilimumab enhanced TNFα release during maturation of dendritic cells; nivolumab increased the amount of IFNγ release. H-1PV mediated reduction of regulatory T cell activity was demonstrated by lower TGF-ß levels. The combination of ipilimumab and nivolumab resulted in a further decline of TGF-ß levels. Similar results were obtained regarding the activation of cytotoxic T cells. H-1PV infection alone and in combination with both checkpoint inhibitors caused strong activation of CTLs, which was reflected by an increased number of CD8⁺GranB⁺ cells and increased release of granzyme B, IFNγ, and TNFα. Our data support the concept of a treatment benefit from combining oncolytic H-1PV with the checkpoint inhibitors ipilimumab and nivolumab, with nivolumab inducing stronger effects on cytotoxic T cells, and ipilimumab strengthening T lymphocyte activity.

Keywords: H-1PV; immune cells; immunotherapy; ipilimumab; melanoma; nivolumab.

Figure 1

Expression of CTLA-4, PD-1, and PD-L1 on the surface of the melanoma cells Sk29Mel-1 and Mz7Mel after H-1PV infection using FACS analysis (n = 3). Histogram blots are illustrated in an example manner. Experiments were performed independently as triplicate. FACS, fluorescence-activated cell sorting.

Figure 2

Determination of DC maturation after co-culturing of iDCs with uninfected and H-1PV infected Sk29Mel-1 cells with or without the checkpoint inhibitors ipilimumab and nivolumab for 3 days. Surface expression of the maturation markers CD80, CD83, and CD86 was determined using FACS analysis (A) while release of IL-6, IFNγ, and TNFα in the supernatant is determined by ELISA (B–D). Experiments were performed independently as triplicate *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. iDC, immatured dendritic cells; mDC, matured dendritic cells; Ipi, ipilimumab; Nivo, nivolumab; ELISA, enzyme linked immunosorbent assay; FACS, fluorescence-activated cell sorting.

Figure 3

Determination of DC maturation after co-culturing of iDCs with uninfected and H-1PV infected Mz7Mel with or without the checkpoint inhibitors ipilimumab and nivolumab for 3 days. Results of three different experiments are shown. Surface expression of the maturation marker CD80, CD83 and CD86 was determined using FACS analysis (A) while release of IL-6, IFNγ, and TNFα in the supernatant is determined by ELISA (B–D). Experiments were performed independently as triplicate *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. iDC, immatured dendritic cells; mDC, matured dendritic cells; Ipi, ipilimumab; Nivo, nivolumab; ELISA, enzyme linked immunosorbent assay; FACS, fluorescence-activated cell sorting.

Figure 4

Determination of regulatory T cell (Treg) activity induced by H-1PV infected and uninfected Sk29Mel-1. Treg* functions as positive control and are activated with functional anti CD3 and anti CD28. Measurement of proportion of CD4+FoxP3+GranzymB+ cells by FACS analysis (A) and determination of the IL-10 (B) and TGF-ß (C) level in the supernatant (ELISA) with and without ipilimumab and nivolumab. Experiments were performed independently as triplicate *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. ELISA, enzyme linked immunosorbent assay; FACS, fluorescence-activated cell sorting.

Figure 5

Determination of regulatory T cell (Treg) activity induced by H-1PV infected and uninfected Mz7Mel. Treg* functioned as positive control and are activated with functional anti CD3 and anti CD28. Measurement of proportion of CD4⁺FoxP3⁺GranzymB⁺ cells by FACS analysis (A) and determination of the IL-10 (B) and TGF-ß (C) level in the supernatant (ELISA) with and without ipilimumab and nivolumab. Experiments were performed independently as triplicate *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. ELISA, enzyme linked immunosorbent assay; FACS, fluorescence-activated cell sorting.

Figure 6

Determination of CTL clone IVSB cytotoxicity induced by H-1PV after 2 days of co-culture with melanoma cells. Measurement of the proportion of CD3⁻CD8⁻CPD⁺PI⁺ cells by FACS analysis. (A) Co-culture with Sk29Mel cells and (B) with Mz7Mel with and without ipilimumab and nivolumab. Experiments were performed independently three times *P ≤ 0.05; **P ≤ 0.01; P ≤ 0.001. CPD, cell proliferation dye eFluor 670; PI, propidium iodide; CTL, human cytotoxic T lymphocytes; FACS, fluorescence-activated cell sorting.