Blocking Feedback Immunosuppression of Antigen Presentation in Brain Tumor During Oncolytic Virotherapy with oHSV-mshPKR

Abstract

Glioblastoma (GBM) is the most frequent malignant brain tumor. We recently discovered that oncolytic herpes simplex virus engineered to disable tumor-intrinsic protein kinase R (PKR) signaling (oHSV-shPKR) could increase oHSV oncolysis and antitumor immune response. However, in this study, we show that disabling tumor-intrinsic PKR signaling can also induce the activation of the indoleamine 2,3-dioxygenase (IDO) signaling pathway. Both GBM tumor progression and oHSV intratumoral therapy increased infiltration of IDO+CD11c+ dendritic cells (DC) into the tumor. The coculture of oHSV-infected human GBM neurospheres with monocyte-derived DCs (MoDC) dramatically increased IDO signaling activation in MoDCs through type-I IFN signaling. Addition of IDO inhibitor (indoximod) in the coculture significantly increased MoDC activation and reduced the consumption of tryptophan. Combining indoximod and oHSV significantly inhibited tumor growth and induced antigen-specific CD8+ T-cell activation. These results suggest that inhibition of the IDO pathway could significantly block feedback immunosuppression during oncolytic virotherapy of GBM.

Fig. 1. oHSV-mshPKR induces cell cycle G2/M arrest and inhibits tumor cell growth.

(a-b) Bulk mRNA-seq data from GSC005 cells infected with oHSV-mshPKR or oHSV-shCtl for (a) GO pathways for cell cycle, cell growth (green arrows) and cell killing (red arrows), and DNA damage response (DDR, yellow) represented as enrichment score of DEGs. (b). Human GBM neurospheres GBM12 and GBM28 infected with oHSV-shPKR and p-H2AX was analyzed by western blot assay. (c) Heatmap of pathways related to cell cycle G2/M arrest and cell growth pathway. (d) GSC005 cells were infected with oHSV-mshPKR or oHSV-shCtl (MOI=0.05) for 72hr. Cell cycle was analyzed by anti-BrdU and 7-AAD staining. n=3, * p < 0.01. (e-f) Western blot analysis of cell cycle regulator protein p90RSK, H3 and HSV1-ICP4 in GSC005 and GBM28 cells infected with oHSV-mshPKR or oHSV-shCtl. S= S phase arrest. M=M phase arrest.

Fig. 2. Increased IDO⁺CD11c cells infiltration in mouse GBM model with intratumoral treatment of oHSV-mshPKR.

(a-b). 005-OVA tumor-bearing mice were intratumorally treated with 2e5 oHSV-mshPKR. Flow cytometry analysis of antigen specific T cells in untreated (day 17), and oHSV-mshPKR-treated, tumor-free mice (day 35) using OVA^257-264 tetramer staining of CD8 T cells in the brain (a). n=3, * p < 0.01. CD69 and OVA^257-264 CD8 T cells in the tumor, cervical lymph node (CLN) and spleen of the tumor-free mice after oHSV-mshPKR treatment were analyzed by flow cytometry (c-d). n=3, * p < 0.01. (e-f). Flow cytometry analysis of IDO expression in brain and spleen CD11c cells from non-tumor-bearing and 005-tumor bearing mice. n=4, * p < 0.01. (g-i). Analysis of IDO in 005 tumor with or without intra-tumor oHSV-mshPKR treatment (day 5 after treatment) by bulk mRNA-seq of whole tumor(g), or qRT-PCR of IDO mRMA (h) and flow cytometry analysis of mean fluorescence intensity (i) of IDO in tumor-infiltrating CD11c cells. n=3, * p < 0.01.

Fig. 3. Interferon signaling is responsible for IDO upregulation in human MoDCs cocultured with supernatant from oHSV-shPKR-infected GBM neurospheres and 1-MT could reverse immunosuppression in DCs in vitro.

(a-c). Human CD14+ monocytes-derived dendritic cells (MoDCs) were treated with 100U/ml IFNγ with or without GBM28 neurospheres culture supernatant for 16hr. IDO expression in MoDCs was analyzed using flow cytometry. n=3, * p < 0.01. (d-g). Human MoDCs were cocultured with supernatant from oHSV-shCtl or oHSV-shPKR-infected GBM28 neurospheres, with or without addition of anti-IFNAR1 antibody (2μg/ml) for 48hr. IDO expression in MoDCs was quantified using flow cytometry analysis. n=3, * p < 0.01. (h-i). Human MoDcs were cocultured with supernatant from oHSV-shPKR-infected GBM28 with or without addition of 1-MT (200mM) for 24hr. (GBM=GBM28, oHSV=oHSV-shPKR). CD80 expression in MoDCs was analyzed by flow cytometry (h). n=3, * p < 0.01. Tryptophan metabolism in the coculture was quantified using tryptophan assay kit (Sigma) and Kynurenine ELISA kit (Biomatik) (i). n=3, * p < 0.01. (i). Mouse bone-marrow derived dendritic cells (BMDCs) were cocultured with supernatant from oHSV-mshPKR-infected GSC005 neurospheres with or without addition of 1-MT (200mM) for 24hr. CD80 expression in BMDCs was analyzed by flow cytometry (j). n=3, * p < 0.01.

Fig. 4. 1-MT increases anti-tumor efficacy of oHSV-mshPKR in murine GSC005 glioblastoma model.

C57BL/6 mice were intracranially inoculated with 1e5 GSC005 neurospheres and on day 7 tumor-bearing mice were intratumorly treated with 2e5 ifu oHSV-mshPKR with or without 1-MT (4mg/L in drink water, d.w.) (a). Tumor-bearing mice survival was monitored (b, n=10, * p = 0.02). Day 28, the surviving tumor-bearing mice were analyzed for immune cells infiltration in tumor by flow cytometry (c-d) and immunofluorescence (e-f). Bar, 100 μm. oHSV=oHSV-mshPKR. n=5, * p < 0.01. ns=no statistical significance. (g) C57BL/6 mice were intracranially inoculated with 1e5 GSC005 neurospheres and on day 7 tumor-bearing mice were intratumorly treated with 2e5 ifu oHSV-mshPKR with 1-MT (4mg/L in drink water, d.w.) (Combo treatment). Anti-CD4, anti-CD8 or anti-NK1.1 antibodies were i.p. injected into some of the tumor-bearing mice with the Combo treatment. Tumor-bearing mice survival was monitored (n=9, * p < 0.05).

Fig. 5. 1-MT increases antigen specific anti-tumor immune response in GSC005-OVA glioblastoma model treated with oHSV-mshPKR.

C57BL/6 mice were intracranially inoculated with 1e5 GSC005-OVA neurospheres and on day 7 tumor-bearing mice were intratumorly treated with 2e5 ifu oHSV-mshPKR with or without 1-MT (4mg/L in drink water, d.w.) (a). Tumor-bearing mice survival was monitored (a n=10, * p = 0.03). Day 28, the surviving tumor-bearing bearing mice were analysis of immune cells infiltration in tumor by flow cytometry (b-c). n=5, * p < 0.01. (d)Graphical model of this study: oHSV-shPKR (oHSV-mshPKR) infected GBM cells induce oncolysis, DDR and tumor cell growth inhibition, which not only activate anti-tumor immune response, but also promote interferon mediated feedback IDO upregulation in antigen presentation cells. Combination of IDO inhibitor, indoximod, significantly increase oHSV-mshPKR anti-tumor immune response.