Oncolytic HSV-IL27 expression improves CD8 T cell function and therapeutic activity in syngeneic glioma models

Abstract

Background: Malignant gliomas (MGs) are the most common primary brain malignancies and are considered universally fatal. Oncolytic herpes simplex viruses (oHSVs) are promising immunotherapeutics capable of selectively lysing cancer cells, eliciting antitumor immunity, and providing local delivery of immune-activating transgenes. Interleukin 27 (IL-27) is a pleiotropic cytokine capable of enhancing tumor-reactive cytotoxic T lymphocyte (CTL) function while also possessing neuroprotective properties. We hypothesized that IL-27 expression by oHSV would enhance CTL function and improve antiglioma therapeutic activity.

Methods: We developed an oHSV that expresses IL-27 (C027). The antiglioma efficacy of C027 was tested in three syngeneic orthotopic glioma models derived from both chemical (CT-2A) and genetic (SB28, KR158) glioma lines. Spectral flow cytometry was used to assess immunophenotypic and functional changes in the tumor infiltrates and systemically. To further investigate the C027-related CTL activity, we employed in vivo cell-specific depletion and IL-27 blockade alongside in vitro T cell stimulation assays. Local and systemic antitumor memory was evaluated by both orthotopic and flank tumor rechallenge of C027-treated long-term survivors.

Results: C027 significantly prolonged survival in syngeneic orthotopic glioma models derived from both chemical (CT-2A) and genetic (KR158, SB28) glioma lines. In the CT-2A model, IL-27-expressing oHSV treatment was associated with increased intratumoral multifunctional effector CTLs and functional T cell populations systemically. Mechanistically, both CD8 T cells and IL-27 were required for the C027 survival benefit in vivo and IL-27 enhanced CTL function in vitro. C027-treated mice that survived their initial tumors had local and systemic antiglioma memory rejecting tumors on rechallenge.

Conclusions: Our findings demonstrate that IL-27 expression by oHSV significantly improves antiglioma therapeutic efficacy, enhances CTL effector function, and induces durable immune memory. Thus, IL-27-oHSV may provide a promising therapeutic approach for MGs.

Keywords: Central Nervous System Cancer; Cytokine; Immunotherapy; Oncolytic virus; T cell.

Figure 1. C134-mIL27 (C027) prolongs survival in syngeneic orthotopic murine glioma models. (A) Schematic illustration of C134 and C027. C134 is a second-generation oHSV with deletion of the γ134.5 genes and insertion of the human cytomegalovirus (HCMV) IRS1 gene in the UL3/UL4 intergenic region. C027 was generated from C134 by insertion of bicistronic copies of murine IL-27 (mIL27) in the deleted γ134.5 regions under the MND promoter. (B) mIL27 (p28) secretion from C027 or C134 infected (MOI=1) CT-2A cell supernatants at 24-hour and 48-hour postinfection measured by ELISA. (C) Viral replication in CT-2A cells infected with C027, C134, or WT HSV (MOI=1) demonstrating viral replication kinetics. Experimental design schematics and representative Kaplan-Meier survival curves in (D) CT-2A (n=11 per cohort), (F) KR158 (n=5–10 per cohort), and (H) SB28 (n=10 per cohort) immunocompetent syngeneic murine glioma models (log-rank test of significance) after treatment with saline or equivalent PFU (1×10⁷) of C027 or C134 (parental control). In vivo viral recovery from murine brain tumors (E) CT-2A, (G) KR158, and (I) SB28 at 2 days post-treatment with C134 (n=3) or C027 (n=3). For B, C, E, G, and I, data are mean±SEM with each shape representing one replicate or animal. Statistical analyses were performed using two-way analysis of variance with Holm-Šídák’s correction for multiple comparisons (B), unpaired two-tailed Student’s t-test (E, G, and I), or log-rank tests of significance (D, F, and H). IC, intracranial; IL, interleukin; MOI, multiplicity of infection; oHSV, oncolytic herpes simplex virus; PFU, plaque-forming unit; Tx, treatment.

Figure 2. IL-27-oHSV-related tumor infiltrating leukocyte immunophenotypic changes. Immunophenotyping of CT-2A murine gliomas treated with vehicle, C134, or C027. Tumors were collected and immune cells were isolated for spectral flow cytometry analysis 12 days post-treatment. (A) Experimental designs schematic. (B) FlowSOM metaclusters overlaid on opt-SNE of all concatenated samples and by treatment cohort using the spectral flow cytometry panel. Each dot represents a single cell. (C) Volcano plots comparing abundance of immune metaclusters between vehicle (gray), C134 (pink), and C027 (teal). Gray line represents a p value cut-off of 0.05 by EdgeR analyses with statistically significant clusters indicated with cluster labels in volcano plots. N=4 mice per group. DC, dendritic cell; FC, fold change; FDR, false discovery rate; FOL-B, follicular B cell; GC B, germinal center B cell; GzB, granzyme B; IC, intracranial; ICS, intracellular cytokine staining; IFNγ, interferon gamma; mMDSC, monocytic myeloid derived suppressor cell; MZ-B, marginal zone B cell; NK, natural killer; NKT, natural killer T; oHSV, oncolytic herpes simplex virus; PFU, plaque-forming unit; TNFα, tumor necrosis factor alpha; Treg, regulatory T cell.

Figure 3. C027 increases multifunctional CD8 effector T cells in the tumor microenvironment. CD3+T cell subanalyses of CT-2A murine gliomas treated with vehicle, C134, or C027. 12 days after treatment, tumors were collected and immune cells isolated for spectral flow cytometry analysis (refer to experimental schematic figure 2A). (A) FlowSOM metaclusters of CD3+T cells overlaid on opt-SNE embedding for all concatenated samples and by treatment cohort. Each dot represents a single cell. (B) Granzyme B (GzB), interferon-gamma (IFNγ), Ly6C, and CD69 expression overlaid on opt-SNE plots of samples concatenated by treatment cohort. Color gradient correlates with marker expression level. (C) Volcano plots comparing proportional CD3+T cell metaclusters differences between C027 (teal) and vehicle (gray) or C134 (pink). Gray line represents a p value cut-off of 0.05 by EdgeR analyses with statistically significant clusters indicated with cluster labels in the volcano plots. (D) Representative biaxial flow plots of IFNγ and GzB expression in tumor-infiltrating CD4 and CD8 T cells. (E) Frequencies of dual IFNγ+ GzB+ CD8+ and CD4+ T cells among total CD45+cells. Statistical analysis was performed by one-way analysis of variance with Holm-Šídák’s multiple comparisons test. N=4 mice per group. DN, dual CD4 and CD8 negative; EFF, effector; FC, fold change; FDR, false discovery rate; TNFα, tumor necrosis factor alpha; Treg, regulatory T cell; TRM, tissue-resident memory T cell.

Figure 4. C027 antiglioma efficacy is dependent on CD8 T cells and IL-27. (A) Experimental design schematic and Kaplan-Meier survival curve of orthotopic CT-2A-bearing mice depleted of CD8 T cells by intraperitoneal injection (days 5, 6, 7, 14, 21) of anti-CD8 or isotype control (n=7 per cohort). (B) Experimental design schematic and Kaplan-Meier survival curve of orthotopic CT-2A-bearing, C027-treated mice receiving intraperitoneal injection (days 7, 9, 11) of IL-27p28 neutralizing antibody or isotype control (n=6–10 per cohort). (C–D) CD3⁺ T cells were stimulated (αCD3/αCD28)±IL-27 (50 ng/mL) for 16 hours. (E–F) Splenocytes were stimulated with CT-2A conditioned media (Mock, C154, or C027) for 6 hours. (C, E) Representative flow plots of IFNγ and GzB expression by CD8⁺ T cells. (D, F) abundance of IFNγ⁺ and GzB⁺ CD8⁺ T cells. Data are mean±SEM with each shape representing one replicate. Statistical analyses were performed by log-rank tests of significance (A, B) or one-way analysis of variance with Holm-Šídák’s correction for multiple comparisons (D, F). CM, conditioned media; GzB, granzyme B; IFNγ, interferon gamma; IL, interleukin; i.p., intraperitoneal; OV, oncolytic herpes simplex virus; PFU, plaque-forming unit; stim, stimulated; Tx, treatment.

Figure 5. IL-27 expression by oHSV increases functional T cells systemically. Tumor-bearing mice were sacrificed 12 days post-treatment (vehicle, C134, or C027) and their splenocytes analyzed by spectral flow cytometry to assess systemic immunophenotypic changes. (A) Experimental designs schematic. (B) FlowSOM metaclusters overlaid on opt-SNE embedding using the spectral flow cytometry panel. Plots show total concatenated samples and by treatment cohort. Each dot represents a single cell. (C, D) Volcano plots comparing abundance of immune metaclusters between vehicle (gray), C134 (pink), and C027 (teal). Gray line represents a p value cut-off of 0.05 by EdgeR analyses with statistically significant clusters indicated with cluster labels in volcano plots. (E) Biaxial flow plot of Ly6C+CD8+ T cells concatenated by treatment cohort. Frequency of IFNγ+ GzB+ Ly6C+CD8+ T cells and IFNγ+ expression in Ly6C+CD8+ T cells. (F) Biaxial flow plot of CD44+CD4+ T cells concatenated by treatment cohort. Frequency of IFNγ+ TNFα+ CD44+ CD4+ T cells and IFNγ+ expression in CD44+CD4+ T cells. Statistical analyses were performed by one-way analysis of variance with Holm-Šídák’s correction for multiple comparisons. N=4 mice per group. FC, fold change; FDR, false discovery rate; GzB, granzyme B; IC, intracranial; ICS, intracellular cytokine staining; IFNγ, interferon gamma; IL-27, interleukin 27; MFI, mean fluorescence intensity; oHSV, oncolytic herpes simplex virus; PFU, plaque-forming unit; TNFα, tumor necrosis factor alpha; Treg, regulatory T cell.

Figure 6. C027-treated responders resist tumor regrowth on rechallenge suggesting the development of a durable immune memory response. (A) Schematic of experimental approach. CT-2A-bearing, C027-treated long-term survivors (LTS) were rechallenged intracranially alongside age-matched naïve controls. (B) Tumor growth curves approximated by bioluminescent imaging and (C) Kaplan-Meier survival curves of C027-LTS (teal) and naïve controls (black) (n=5 per cohort). (D) Schematic of experimental approach. CT-2A-bearing, C027-treated long-term survivors (LTS) were rechallenged in the flank alongside age-matched naïve controls. (E) Representative tumor growth curves and (F) Kaplan-Meier survival curves of C027-LTS (teal) and naïve controls (black) (n=3–5 per cohort). Statistical analyses were performed using log-rank tests of significance. BLI, bioluminescent imaging; oHSV, oncolytic herpes simplex viruses; PFU, plaque-forming unit.