Cytokine-armed vaccinia virus promotes cytotoxicity toward pancreatic carcinoma cells via activation of human intermediary CD56dimCD16dim natural killer cells

Abstract

Pancreatic ductal adenocarcinoma (PDAC) remains a particularly aggressive disease with few effective treatments. The PDAC tumor immune microenvironment (TIME) is known to be immune suppressive. Oncolytic viruses can increase tumor immunogenicity via immunogenic cell death (ICD). We focused on tumor-selective (vvDD) and cytokine-armed Western-reserve vaccinia viruses (vvDD-IL2 and vvDD-IL15) and infected carcinoma cell lines as well as patient-derived primary PDAC cells. In co-culture experiments, we investigated the cytotoxic response and the activation of human natural killer (NK). Infection and virus replication were assessed by measuring virus encoded YFP. We then analyzed intracellular signaling processes and oncolysis via in-depth proteomic analysis, immunoblotting and TUNEL assay. Following the co-culture of mock or virus infected carcinoma cell lines with allogenic PBMCs or NK cell lines, CD56⁺ NK cells were analyzed with respect to their activation, cytotoxicity and effector function. Both, dose- and time-dependent release of danger signals following infection were measured. Viruses effectively entered PDAC cells, emitted YFP signals and resulted in concomitant oncolysis. The proteome showed reprogramming of normally active core signaling pathways in PDAC (e.g., MAPK-ERK signaling). Danger-associated molecular patterns were released upon infection and stimulated co-cultured NK cells for enhanced effector cytotoxicity. NK cell subtyping revealed enhanced numbers and activation of a rare CD56^dimCD16^dim population. Tumor cell killing was primarily triggered via Fas ligands rather than granule release, resulting in marked apoptosis. Overall, the cytokine-armed vaccinia viruses induced NK cell activation and enhanced cytotoxicity toward human PDAC cells in vitro. We could show that cytokine-armed virus targets the carcinoma cells and thus has great potential to modulate the TIME in PDAC.

Keywords: immune modulation; immunogenic cell death; modified vaccinia virus; natural killer cells; proteome analysis.

FIGURE 1

Oncolytic vvDD viruses infect PDAC cells and induce expression of YFP, cytokine transgenes and apoptosis. (A) Fluorescent YFP signals in PANC‐1 and BxPC‐3 cells of intracellular vaccinia virus following 24 h infection at MOI1. Scale bar = 10 μm. (B) YFP signals (flow cytometry) of PDAC cell lines following vvDD infection. Replicate data presented as mean, min/max. (C) At same conditions, patient‐derived PDAC cell cultures (n = 6) are efficiently infected with modified Western‐reserve (vvDD) and cytokine‐armed vaccinia virus (vvDD‐IL2 and vvDD‐IL15). Replicate data presented as mean, min/max. (D) Plaque titration assay shows a time‐dependent increase in virus titer following infection of PANC‐1 and BxPC‐3 at 24, 48, and 72 h. (E) The expression of GPI (rigid peptide linker) anchored IL2 was measured via flow cytometry, while IL15 was detected in the supernatant by ELISA. Replicate data presented as mean, min/max. (F) TUNEL assay indicated apoptosis of infected PANC‐1 and BxPC‐3 cells (MOI1) that peaked at 48 h post‐infection. In parallel the viability of carcinoma cells decreased time dependent as shown by MTS‐assay. Scale bars = 50 μm. (G) Western blot analyses shows increased expression of pro‐apoptotic BCL‐2 and BAD and down‐regulation of cell cycle checkpoint P53 (top). Proteomic analyses confirmed these observations (below).

FIGURE 2

Proteomic configuration of pancreatic carcinoma cells following modified and cytokine‐armed oncolytic virus infection (A) Proteomic analyses of 178 viral proteins in infected Panc‐1 cells presented as cluster blot with triplicate z‐score values, demonstrating the intracellular active viral machinery for protein transcription, cytoplasmatic replication and encapsidation. (B) Whole proteome (left) and phosphosite PCA analyses on infected Panc‐1 cells (triplicate) with vvDD, vvDD‐IL2, vvDD‐IL15 compared to mock. (C) Volcano blot of regulated proteins (left) and phosphosites (right) in vvDD, vvDD‐IL2 and vvDD‐IL15 infected PANC‐1 cells. (D) Cluster blot with triplicate z‐score values of proteins involved in cell cycle regulation/apoptosis. (E) Cluster blot with triplicate z‐score values on proteins involved in MAPK–ERK signaling.

FIGURE 3

Virus infection changes MHC‐I expression and other proteins involved in immune regulation and immunogenicity. (A) Surface expression of MHC‐I and MICA/‐B in mock and vvDD infected cells was negatively regulated solely in BxPC‐3 cells. (B) Surface expression in primary PDAC cells show stable MHC‐I expression in primary, patient derived PDAC cells. (C) Cluster blot with triplicate z‐score values of proteins involved in the antigen presenting machinery (APM). (D) Surface expression of PD‐L1 and CD155 across PDAC cell lines. (E) Up‐regulation of PD‐L1 in selected, vvDD infected primary PDAC cells. (F) Cluster blot with triplicate z‐score values of proteins involved in IFN‐γ signaling.

FIGURE 4

Induction of NK cell mediated cytotoxicity following cytokine‐armed vaccinia virus. (A) Screening of PDAC cell lines stained with crystal violet following 48 h co‐culture with NK92 revealed BxPC3 and PANC‐1 cells as sensitive candidates for NK cell mediated cytotoxicity. (B) Co‐culture with human PBMC indicated high tolerance with little if any activation of NK cells (CD69⁺ among gated CD3⁻CD56⁺ human NK cells). (C) vvDD infected cells induced NK cell activation as evidenced by shift toward CD69⁺ expression. (D) Co‐culture of infected tumor cells with human PBMCs revealed induced expression of INF‐γ in CD3⁻CD56⁺ NK cells. (E) A previously unappreciated immature NK cell population with presence of CD56^dimCD16^dim marker expression develops after 48 h co‐culture with infected tumor cells. (F) The CD56^dimCD16^dim subset was characterized by high fraction of CD69⁺, CD107a⁺, NKp46⁺ and NKp30⁺ cells when compared to CD56^dimCD16⁺. (G) Dose dependent CFSE labeled K‐562 suspension cells with a large fraction of late‐apoptotic cells after 48 h. Scale bar = 100 μm. (H) Cytotoxicity assay using untreated carcinoma cells (BxPC‐3 and PANC‐1) co‐cultured with or without PBMC after 48 h. (I) Cytotoxicity assay using infected carcinoma cells (BxPC‐3 and PANC‐1) in co‐culture with PBMC, compared to carcinoma cells without PBMC and mock.

FIGURE 5

FasL but not perforin up‐regulation correlates with NK cell mediated cytotoxicity following oncolysis. (A) Intracellular perforin granules from PBMC derived NK cells following 24 and 48 h co‐culture with PANC‐1, BxPC‐3, and K‐562 cells. (B) Granzyme B and perforin release from NK cells measured from supernatant via Luminex assay following co‐culture with mock or vvDD treated carcinoma cell lines. (C) Co‐culture of NK cells with vvDD infected tumor cells induces FasL expression on NK cells. (D) Infected carcinoma cells (vvDD infected) release ATP into supernatant in dose‐dependent manner. (E) Infected carcinoma cells (vvDD, vvDD‐IL2, and vvDD‐IL15 infected) transport calreticulin to the cell surface with dose‐dependence. 20× magnification, scale bar = 100 μm. On the right shown the fraction of calreticulin surface expression of infected carcinoma cells (vvDD, vvDD‐IL2, and vvDD‐IL15 infected) by FACS analysis.