Distinct roles but cooperative effect of TLR3/9 agonists and PD-1 blockade in converting the immunotolerant microenvironment of irreversible electroporation-ablated tumors

Abstract

Irreversible electroporation (IRE) is a new cancer ablation technology, but methods to improve IRE-induced therapeutic immunity are only beginning to be investigated. We developed a mouse model bearing large primary (300 mm³) and medium distant (100 mm³) EG7 lymphomas engineered to express ovalbumin (OVA) as a nominal tumor antigen. We established experimental protocols including IRE alone and IRE combined with Toll-like receptor (TLR)3/9 agonists (poly I:C/CpG) (IRE + pIC/CpG), PD-1 blockade (IRE + PD-1 blockade), or both (IRE + Combo) to investigate therapeutic effects on primary and distant EG7 tumors and conversion-promoting effects on the immunotolerant tumor microenvironment (TME). We demonstrated that IRE alone simulated very weak OVA-specific CD8⁺ T cell responses and did not inhibit primary tumor growth. IRE + pIC/CpG synergistically stimulated more efficient OVA-specific CD8⁺ T cell responses and primary tumor growth inhibition than IRE + PD-1 blockade. IRE + pIC/CpG played a major role in the modulation of immune cell profiles but a minor role in the downregulation of PD-L1 expression in the TME and vice versa for IRE + PD-1 blockade. IRE + Combo cooperatively induced potent OVA-specific CD8⁺ T cell immunity and rescued exhausted intratumoral CD8⁺ T cells, leading to eradication of not only primary tumors but also untreated concomitant distant tumors and lung metastases. IRE + Combo efficiently modulated immune cell profiles, as evidenced by reductions in immunotolerant type-2 (M2) macrophages, myeloid-derived suppressor-cells, plasmacytoid dendritic cells, and regulatory T cells and by increases in immunogenic M1 macrophages, CD169⁺ macrophages, type-1 conventional dendritic cells, and CD8⁺ T cells, leading to conversion of immunotolerance in not only primary TMEs but also untreated distant TMEs. IRE + Combo also showed effective therapeutic effects in two breast cancer models. Therefore, our results suggest that IRE + Combo is a promising strategy to improve IRE ablation therapy in cancer.

Keywords: CD8+ T-cell response; IRE ablation; PD-1-blockade; TLR3/9-agonists; antitumor immunity.

Fig. 1. An enhanced immunotolerant TME is associated with tumor progression.

A Representative image of EG7 tumors of different sizes. B Representative hematoxylin and eosin (H&E) staining of tissue sections of small, medium, and large EG7 tumors. Black arrows, areas of focal necrosis in the center of the large tumor; red arrow, amplified tumor necrosis. C Tumor-derived single-cell suspensions (TSCSs) were enzymatically prepared from tissues of EG7 tumors of different sizes. Cell samples were stained with a cocktail of antibodies against a combination of molecular markers and then analyzed by flow cytometry with progressive gating strategies. The last sets of representative flow cytometry plots show quantitative measurements of various immune cell subsets. The relative quantification of (i) M1/M2 macrophage ratio calculated as % MHCII⁺ M1 macrophages/% MHCII^- M2 macrophages in total CD11b⁺F4/80⁺ macrophages; (ii) % Treg cells calculated as CD4⁺Foxp3⁺ Treg cells^/total CD4⁺ T cells; (iii) % MDSCs and (iv) % pDCs in tumor-infiltrating host CD45.1⁺ cells calculated as % CD11b⁺CD45.1⁺ cells in upper square × % Gr1⁺Ly6G⁺ cells in lower square) and % CD11b^-CD11c^- cells in upper square × % B220⁺CD137⁺ cells in lower square, respectively, are described in the Methods and Supplementary Figure S1. D Flow cytometric analysis of PD-L1 expression in M2 macrophages, MDSCs, and tumor cells. The gray line represents control isotype antibody staining. MFI, mean fluorescence intensity. Flow cytometry plots representing one of two independent experiments (4-5 replicates each) are presented as the mean ± SEM. E The average percentages of CD45.2⁺ EG7 tumor cells and CD45.1⁺ tumor-infiltrating immune cells in the TME of large tumors and (F) the average percentages of CD11b⁺F4/80⁺MHCII^- M2 macrophages, CD4⁺Foxp⁺ Treg cells, Gr1⁺Ly6G⁺ MDSCs and B220⁺CD317⁺ pDCs in CD45.1⁺ tumor^-infiltrating immune cells were measured based upon Fig. 1C and Supplementary Figure S1 (n = 5/group). *P < 0.05, **P < 0.01 by one-way ANOVA with Tukey’s test. ns, not significant

Fig. 2. IRE ablation induces tumor cell apoptosis but weak OVA-specific CD8⁺ T cell responses and is ineffective in inhibiting tumor growth.

A Diagram illustrating the design of the IRE ablation experiment. B Experimental setup for IRE treatment. C Schematic diagram showing the placement of the IRE device electrode in a tumor (8–9 mm in diameter) during IRE ablation. D Representative H&E staining of tumor tissue sections collected at 3 days post IRE ablation. Arrows indicate areas of massive apoptosis in IRE-treated tumors. Arrowheads indicate the surrounding tumor tissues. E Blood cells collected from the tail vein of IRE-treated or naïve control mice were stained with OVA-specific PE-Tetramer and a FITC-conjugated anti-CD8 antibody and analyzed by flow cytometry. OVA-specific CD8⁺ T cells were defined as CD8 and tetramer double-positive cells. The value in each panel represents the percentage of OVA-specific CD8⁺ T cells in the total CD8⁺ T cell population. **P < 0.01 by two-tailed Student t test. F Tumor-bearing mice were monitored for tumor growth post IRE ablation. ns, not significant by two-way ANOVA with Tukey’s test. Flow cytometry or tumor growth plots representing one of two independent experiments are presented as the mean ± SEM (n = 5/group)

Fig. 3. IRE combined with PD-L1 blockade and TLR3/9 agonists results in potent OVA-specific CD8⁺ T cell responses and antitumor immunity.

A Schematic diagram of IRE ablation combined with PD-1 blockade (anti-PD-L1 Ab) and/or TLR3/9 agonists (pIC/CpG) in six protocols: (i) IRE, (ii) IRE + anti-PD-L1, (iii) IRE + pIC, (iv) IRE + CpG, (v) IRE + pIC/CpG, (vi) IRE + Combo and (vii) Combo alone (treatment schedules described in the Methods). B Schematic illustrating IRE device electrode placement and pIC/CpG injection points (red color) in the tumor (8–9 cm in diameter) during IRE ablation. C Blood cells collected from the tail vein of mice treated with each of the above six different protocols (n = 4/group) were stained with OVA-specific PE-Tetramer and a FITC-conjugated anti-CD8 antibody and analyzed by flow cytometry. The value in each panel represents the percentage of OVA-specific CD8⁺ T cells in the total CD8⁺ T cell population. **P < 0.01 by one-way ANOVA with Tukey’s test. D Tumor-bearing mice were monitored for tumor growth or regression. *P < 0.05, **P < 0.01 by two-way ANOVA with a post hoc Tukey’s test. Tumor-bearing mice were also monitored for mouse survival post IRE ablation. Kaplan–Meier survival analysis for the same experiments (n = 12/group) with the log-rank test. **P < 0.01. E Tumor growth curves of IRE + Combo-treated tumors with and without depletion of CD8⁺ cells using anti-CD8 and control antibodies^. Tumor growth or regression was monitored. Tumor-bearing mice were monitored for tumor growth post IRE + Combo ablation. **P < 0.01 by two-way ANOVA with Tukey’s test. Tumor growth plots representing one of two independent experiments are presented as the mean ± SEM (n = 4/group)

Fig. 4. IRE combined with PD-L1 blockade and TLR3/9 agonists modulates immune cell profiles in the TME.

A Diagram illustrating the experimental setup for analyzing intratumoral immune cell subsets of B6.1 mice bearing primary tumors (8–9 mm in diameter) at 3 days post IRE ablation. Single-cell suspensions were enzymatically prepared from primary tumor tissues at 3 days post IRE ablation. Cell samples were stained with a cocktail of antibodies and then analyzed by flow cytometry with progressive gating strategies. B The last sets of representative flow cytometry plots show quantitative measurements of various immune cell subsets. The relative quantitation of (i) the M1/M2 macrophage ratio calculated as % MHCII⁺ M1 macrophages/% MHCII^- M2 macrophages in total CD11b⁺F4/80⁺ macrophages; (ii) % M169 macrophages calculated as CD169⁺CD11b⁺F4/80⁺ M169 macrophages/total CD11b⁺F4/80⁺ macrophages; (iii) % cDC1s calculated as CD8⁺CD103⁺CD11c⁺ cDC1s/total CD11c⁺ DCs; (iv) % Treg cells calculated as CD4⁺Foxp3⁺ Treg cells/total CD4⁺ T cells; and (v) % MDSCs and (vi) % pDCs in tumor-infiltrating host CD45.1⁺ cells calculated as % CD11b⁺CD45.1⁺ cells in upper square × % Gr1⁺Ly6G⁺ cells in lower square and % CD11b^-CD11c^- cells in upper square × % B220⁺CD137⁺ cells in lower square, respectively, is described in the Methods and Supplementary Figure S1. * p < 0.05, **P < 0.01 by one-way ANOVA with Tukey’s test. C Cell samples were also stained with a cocktail of antibodies and then analyzed by flow cytometry. PD-L1 expression in gated M2 macrophages, MDSCs, and tumor cells were analyzed by flow cytometry. * p < 0.05, **P < 0.01 by one-way ANOVA with Tukey’s test. D Immunohistochemistry (IHC) analysis of frozen primary tumor tissue sections. Representative micrographs of IHC staining for MDSCs. The above data representing one of two independent experiments are presented as the mean ± SEM (n = 5/group)

Fig. 5. IRE + Combo treatment modulates immune cells and cytokines in the blood and promotes CD8⁺ T cells in IRE + Combo-treated tumor tissues and the tumor-draining lymph nodes.

A Diagram illustrating the experimental setup for different analyses. B TSCSs were enzymatically prepared from primary tumor tissues collected from mice treated with IRE, Combo or IRE + Combo at 3 days post IRE. Cell samples were stained with a cocktail of antibodies and analyzed by flow cytometry. The expression of IDO and arginase-1 in gated M2 macrophages, MDSCs and tumor cells was analyzed by flow cytometry, as described in the Methods and Supplementary Figure S1. The gray line represents control isotype antibody staining. MFI, mean fluorescence intensity. C Mouse blood was collected at 3 days post IRE ablation, and blood monocytes purified by Ficoll-Hypaque density gradient centrifugation were stained with a cocktail of antibodies. The relative quantitation of MDSCs calculated as % CD11b⁺Gr1⁺Ly6G⁺ MDSCs in total monocytes and the ratio of M1/M2 macrophages calculated as the amount of MHCII⁺CD11b⁺F4/80⁺ M1 macrophages/the amount of MHCII^-CD11b⁺F4/80⁺ M2 macrophages were determined by flow cytometry. D Quantification of TGF-β, IL-2, and IFN-γ in mouse sera collected on day 3 post IRE ablation. E TSCSs were enzymatically prepared from primary tumor tissues collected at 3 days post IRE + Combo or IRE ablation. Cell samples were stained with a cocktail of antibodies and analyzed by flow cytometry with progressive gating strategies as described in the Methods and Supplementary Figure S1. The last sets of representative flow cytometry plots show quantitative measurement of CD4⁺ and CD8⁺ T cells in total tumor-infiltrating host CD45.1⁺ cells by gating CD3⁺CD45.1⁺ T cells for measurement of CD4⁺ and CD8⁺ T cells, respectively. F IHC analysis of frozen primary tumor tissue sections. Representative micrographs of IHC staining for CD8⁺ T cells. G Purified CD8⁺ T cells from TSCSs were cultured in medium containing IL-2 and OVAI or the unrelated Mut1 peptide for three days and then counted in a T cell proliferation assay. H Purified CD8⁺ T cells from TSCSs were measured for their cytotoxic effect in a cell cytotoxicity assay, in which activated CD8⁺ T cells and GranToxiLux-labeled EG7 or EL4 tumor cells were used as effector (E) and target (T) cells, respectively. The percentage of positive fluorogenic granzyme-B substrate cleavage (GBSC⁺) cells was measured at the indicated E:T (10:1 and 2:1) ratios. I Single-cell suspensions prepared from the tumor-draining lymph nodes (TDLNs) at 7 days post primary tumor treatment with IRE + Combo or IRE ablation. Cell samples were stained with a cocktail of antibodies and analyzed by flow cytometry with progressive gating strategies as described in the Methods and Supplementary Figure S2. The last sets of representative flow cytometry plots show quantitative measurement of the percentage of CD8⁺CD103⁺ cDC1s in total DCs by analysis of gated CD11c⁺MHCII⁺ DCs and quantitative measurement of the percentage of IFN-γ⁺TNF-α⁺ (double-positive) CD8⁺ effector T cells in total CD3⁺ T cells by analysis of gated CD3⁺CD8⁺ T cells. J T cell memory recall responses. Mice with complete eradication of IRE + Combo-treated primary tumors for 30 days or naïve mice as a control were i.v. boosted with recombinant rLmOVA bacteria. Blood cell samples were collected from the tail vein and stained with OVA-specific PE-Tetramer and a FITC-conjugated anti-CD8 antibody, and OVA-specific CD8⁺ T cell responses were then analyzed by flow cytometry on day 4 after the booster immunization. Each bar represents an average of 4 mice/group. Error bars indicate the mean ± SEM. *P < 0.05, **P < 0.01 by two-tailed Student t test. One representative experiment out of two independent experiments is shown

Fig. 6. IRE + Combo induces an “abscopal” effect that eradicates distant tumors by converting the immunotolerant TME of the distant tumors.

A Schematic diagram illustrating the experimental design for the measurement of the “abscopal” effect. B6.1 mice bearing both primary (8–9 mm in diameter) and distant (6 mm in diameter) tumors were monitored for distant tumor regression post IRE + Combo, IRE ablation or Combo alone treatment. **P < 0.01 by two-way ANOVA with Tukey’s test. B Diagram displaying the experimental design for immune cell profiling, PD-L1 expression evaluation, and IHC analyses. C TSCSs were enzymatically prepared from distant tumor tissues on day 3 post IRE ablation of primary tumors. Cell samples were stained with a cocktail of antibodies and analyzed by flow cytometry. The last sets of representative flow cytometry plots show quantitative measurements of various immune cell subsets. The relative quantitation of (i) the M1/M2 macrophage ratio calculated as % MHCII⁺ M1 macrophages/% MHCII^- M2 macrophages in total CD11b⁺F4/80⁺ macrophages; (ii) % cDC1s calculated as CD8⁺CD103⁺CD11c⁺ cDC1s/total CD11c⁺ DCs; (iii⁾ % Treg cells calculated as CD4⁺Foxp3⁺ Treg cells^/total CD4⁺ T cells; ⁽iv) % MDSCs, (v) % pDCs and (vi) % CD4⁺ or CD8⁺ T cells in total tumor-infiltrating host CD45.1⁺ cells calculated as % CD11b⁺CD45.1⁺ cells in upper square × % Gr1⁺Ly6G⁺ cells in lower square % CD11b^-CD11c^- cells in upper square × % B220⁺CD137⁺ cells in lower square and % CD3⁺CD45.1⁺ cells in upper square × % CD4⁺ or CD8⁺ cells in lower square, respectively, is described in the Methods and Supplementary Figure S1. *P < 0.05, **P < 0.01 by two-tailed Student t test. D IHC analysis of frozen distant tumor tissue sections after IRE-Combo or IRE + Combo treatment of primary tumors. Representative micrographs of IHC staining for CD8⁺ T cells. E Flow cytometric analysis of PD-L1 expression in M2 macrophages, MDSCs, and tumor cells. The gray line represents control isotype antibody staining. MFI, mean fluorescence intensity. Tumor growth and flow cytometry plots representing one of two independent experiments are presented as the mean ± SEM (n = 5/group). *P < 0.05, **P < 0.01 by two-tailed Student t test

Fig. 7. IRE + Combo treatment of primary tumors inhibits lung tumor metastasis.

A Schematic diagram of the experimental design for assessing the antimetastatic activity of IRE + Combo treatment of primary tumors. Mice bearing small 7-day EG7 tumors or untreated control mice (n = 4 mice/group) were i.v. injected with BL6-10_OVA cells. Seven days later, IRE + Combo treatment was performed on the mice bearing primary EG7 tumors (8–9 mm in diameter). Mice were sacrificed 14 days after treatment, and lung tissues were collected. B Black metastatic BL6-10_OVA melanoma metastases in the lungs were counted. C Representative micrographs of H&E-stained tissue sections from lungs collected from control (untreated) and IRE + Combo-treated mice. One representative experiment out of two independent experiments is shown

Fig. 8. IRE + Combo treatment effectively eradicates tumors or significantly inhibits tumor growth in two mouse breast cancer models.

Mice bearing Tg1-1 or 4T1 breast cancer tumors (8–9 mm in diameter) were treated with IRE, Combo or IRE + Combo. Tumor-bearing mice treated with IRE or Combo alone were used as controls. Tumor-bearing mice were monitored for tumor growth or regression post IRE + Combo ablation. Tumor growth plots representing one of two independent experiments are presented as the mean ± SEM (n = 4/group). *P < 0.05, **P < 0.01 by two-way ANOVA with Tukey’s test