In Vivo Priming of Peritoneal Tumor-Reactive Lymphocytes With a Potent Oncolytic Virus for Adoptive Cell Therapy

Abstract

Adoptive cell therapy (ACT) using autologous tumor infiltrating lymphocytes (TIL) achieves durable clinical benefit for patients from whom these cells can be derived in advanced metastatic melanoma but is limited in most solid tumors as a result of immune escape and exclusion. A tumor microenvironment (TME) priming strategy to improve the quantity and quality of TIL represents an important tactic to explore. Oncolytic viruses expressing immune stimulatory cytokines induce a potent inflammatory response that may enhance infiltration and activation of T cells. In this study, we examined the ability of an attenuated oncolytic vaccinia virus expressing IL15/IL15Rα (vvDD-IL15/Rα) to enhance recovery of lavage T cells in peritoneal carcinomatosis (PC). We found that intraperitoneal (IP) vvDD-IL15/Rα treatment of animals bearing PC resulted in a significant increase in cytotoxic function and memory formation in CD8⁺ T cells in peritoneal fluid. Using tetramers for vaccinia virus B8R antigen and tumor rejection antigen p15E, we found that the expanded population of peritoneal CD8⁺ T cells are specific for vaccinia or tumor with increased tumor-specificity over time, reinforced with viral clearance. Application of these vvDD-IL15/Rα induced CD8⁺ T cells in ACT of a lethal model of PC significantly increased survival. In addition, we found in patients with peritoneal metastases from various primary solid tumors that peritoneal T cells could be recovered but were exhausted with infrequent tumor-reactivity. If clinically translatable, vvDD-IL15/Rα in vivo priming would greatly expand the number of patients with advanced metastatic cancers responsive to T cell therapy.

Keywords: CD8+ T cells; IL-15; adoptive cell therapy (ACT); oncolytic virus; solid tumor.

Figure 1

Oncolytic virus-based vaccinia virus expressing IL15/IL15Rα (vvDD-IL15/Rα) expression leads to enhanced cytotoxic T cell activation. (A) Experimental design: Immunocompetent B6 mice were inoculated IP with 5.0x10⁵ MC38-luc cells (day −9), randomized and treated with intraperitoneal (IP) injection of PBS, vvDD or vvDD-IL15/Rα at a dose of 5.0x10⁷ pfu/mouse (day 0). (B) Representative in vivo bioluminescence pictures of tumor burden on day 0 and day 10 following treatment. (C) vvDD-IL15/Rα prolongs survival in lethal peritoneal carcinomatosis with high tumor burden. (D) Intratumoral mouse IL15/IL15-Rα-complex was elevated 3 days following IP injection of vvDD-IL15/Rα. (E–H) Three days after IP therapy, mRNA expression in tumor tissue by qPCR analysis showed elevated levels of cytotoxic proteins, perforin (E) and granzyme B (F) after vvDD-IL15/Rα treatment. Levels of Th1 cytokines, IFN-γ (G) and TNF-α (H), were increased in response to virus treatments relative to PBS. (I–K): Lavage CD8⁺ T cells were isolated and directly analyzed via flow cytometry 3 days following treatment. (I) IFN-γ showed highest intracellular abundance in CD8⁺ T cells derived from vvDD-IL15/Rα treated animals. (J) Intracellular expression of granzyme B was significantly increased in lavage CD8⁺ T cells after vvDD-IL15/Rα injection. (K) The costimulatory molecule CD137 (4-1BB) showed increased surface expression in lavage CD4⁺ and CD8⁺ T cells in the vvDD-IL15/Rα treatment group. N=14. All values presented as mean ± SEM. *p < 0.05. **p < 0.01. ***p < 0.001. ****p < 0.0001. Data are combined from two independent experiments.

Figure 2

Vaccinia virus expressing IL15/IL15Rα (vvDD-IL15/Rα) treatment leads to enrichment of memory CD8⁺ T cells with an activated phenotype. Immunocompetent B6 mice were inoculated IP with 5.0x10⁵ MC38-luc cells (day −9), randomized and treated with intraperitoneal (IP) injection of PBS, vvDD or vvDD-IL15/Rα at a dose of 5.0x107 pfu/mouse (day 0). Ten days following i.p. oncolytic virotherapy, peritoneal CD8⁺ T cells were isolated via peritoneal lavage and characterized via flow cytometry (in addition to day 3, 6, 8, and 14 for figure A). (A) Regional delivery of vvDD-IL15/Rα greatly enhanced and prolonged peritoneal CD8⁺ T cell infiltration on day 6 to 14 following virotherapy. (B) The amount of peritoneal central memory (Tcm, CD44⁺ CD62L⁺) and effector memory (Tem, CD44⁺ CD62L^-) T cells is increased 10 days following vvDD-IL15/Rα injection. (C) Representative flow cytometry charts of memory CD8⁺ T cell subsets. vvDD-IL15Rα treatment resulted in increased Ki67 (D), CXCR3 (E), CD122 (F), Tim3^-PD1^int (G), CD103 (H), and CD44⁺PD-1⁺ (I) staining compared to vvDD or PBS 10 days following injection. PD-1^high (J), TIGIT⁺ (K), and Tim-3⁺ (L) staining were not increased after vvDD-IL15/Rα treatment compared to vvDD or PBS treatment. Representative flow cytometry charts of PD-1 expression by treatment group are depicted in Supplemental Figure 2. N=8–12. All values presented as mean ± SEM. *p < 0.05. **p < 0.01. ***p < 0.001. ****p < 0.0001. Data are combined from two independent experiments.

Figure 3

Vaccinia virus expressing IL15/IL15Rα (vvDD-IL15/Rα) treatment enhances CD8⁺ T cell reactivity to endogenous retroviral tumor-rejection antigen, p15E. 3, 6, 8, 10, and 14 days following i.p. oncolytic virotherapy with vvDD-IL15/Rα of late-stage MC38 tumor-bearing mice, peritoneal CD8⁺ T cells were analyzed by FACS using p15E (A, C) and B8R (B, D) tetramers. (A) Time course of p15E tetramer staining shows a marked increase in the number of peritoneal p15E Tetramer⁺ CD8⁺ T cells after vvDD-IL15/Rα treatment. (B) Time course of B8R staining shows a similar pattern, with slightly higher peak values. (C, D) When comparing the percentages of peritoneal p15E Tetramer⁺ CD8⁺ T cells, only vvDD-IL15/Rα induced a significant rise in comparison to PBS and vvDD treatment. The percentage of B8R Tetramer⁺ peritoneal CD8⁺ T cells per total peritoneal CD8⁺ T cells increases in response to vvDD and vvDD-IL15/Rα. (E–I) To validate the results from the tetramer experiments, we performed in vitro functional assays. Peritoneal CD8⁺ T cells, 10 days post treatment, were co-cultured with p15E_605-611-, B8R_20-27-, OVA_257-264-, β-gal_96-103-loaded, or unloaded irradiated splenocytes for 24 h and analyzed via IFN-γ-ELISPOT (E, F) or IFN-γ⁺ CD8⁺ T cells (G) and CD137(4-1BB)⁺ CD8⁺ T cells (H, I) via flow cytometry. All data represent the results of two independent experiments. All values presented as mean ± SEM. *p < 0.05. **p < 0.01. ***p < 0.001. ****p < 0.0001.

Figure 4

Enhanced anti-tumor reactivity of lavage CD8⁺ T cells following vaccinia virus expressing IL15/IL15Rα (vvDD-IL15/Rα) treatment correlates with improved survival after adoptive cell therapy (ACT). 10 days following i.p. oncolytic virotherapy with vvDD-IL15/Rα of late-stage MC38 tumor-bearing mice, reactivity of peritoneal CD8⁺ T cells was examined in vivo and in vitro 10 days following treatments. (A–C): MC38- and vvDD-specific reactivity was measured after co-culture for 24 h in vitro. (A) vvDD-IL15/Rα treatment increased MC38 reactivity as measured by IFN-γ production; and (B) 4-1BB⁺ staining. (C) The percent of exhausted cells (Tim-3⁺ PD-1^high) were decreased after co-culture with MC38 in response to vvDD-IL15/Rα treatment. (D) A higher percentage of the Tim-3^- PD-1^intermediate stained cells were present after vvDD-IL15/Rα in all co-culture conditions. (E) In ACT experiments, acceptor mice received 5.0x10⁵ MC38-luc cells 7 days prior to ACT and a preparatory regimen of sub-lethal whole-body irradiation (5 Gy) 12 h prior to ACT. 3,5x10⁵ total CD8⁺ T cells pooled per group were transferred IP, followed by IL-2 IP stimulation every 12 h six times. (F) Representative images of intraperitoneal (IP) tumor burden via in vivo bioluminescent imaging are displayed. Pictures were taken before adoptive transfer (D0), and 14 days post adoptive transfer (D14). (G) vvDD-IL15/Rα induced TIL increased long term survival of tumor-bearing mice as displayed by Kaplan-Meier survival curves. All data represent the results of two independent experiments with a minimum of 5 mice per treatment group, respectively. All values presented as mean ± SEM. *p < 0.05. **p < 0.01. ***p < 0.001. ****p < 0.0001.

Figure 5

Characterization of human peritoneal CD8⁺ and CD4⁺ T cells derived from peritoneal metastasized tumors. Peritoneal fluid was collected from 14 patients undergoing peritoneal surgery, and peritoneal lymphocytes were extracted and characterized. (A) Flowchart of flow cytometry gating to identify peritoneal CD4 and CD8 T cells. Single cells gating, dead cells exclusion via Zombie aqua staining. Subsequently CD3⁺ cells were separated into CD4⁺ or CD8⁺ cells. (B) Distribution of naïve T cells (Tn, CD45RO^- CCR7⁺), central memory T cells (Tcm, CD45RO⁺ CCR7⁺), effector memory T cells (Tem, CD45RO⁺ CCR7^-) and highly differentiated effector T cells (Temra, CD45RO^- CCR7^-) gated from CD8⁺ T cells. (C) Representative flow cytometry chart of peritoneal memory CD8⁺ T cell subsets. (D) Levels of 4-1BB and Ox40 on CD8⁺ T cells were analyzed. (E–H) Analysis of PD-1 and Tim-3 demonstrated the majority of CD8⁺ cells were PD-1 high and an average of 20% expressed Tim-3. Representative flow charts of PD-1 and Tim-3 expression are shown in (F, H) respectively. (I, J) (representative flow chart): The shared beta-subunit of the IL-2- and IL15-receptor (CD122) showed a mean expression in peritoneal CD8⁺ T cells of 29.1%. (K–M) Peritoneal CD8⁺ T cells from 7 patients were tested for secretion of cytotoxic IFN-γ by ELISA (K) and 4-1BB surface expression by flow (L, M) (representative images) after overnight coculture with correlating tumor digest. Monocytes were used as negative controls, CD3/CD28 dynabeads as a positive control. All values presented as mean ± SEM. *p < 0.05. **p < 0.01. ***p < 0.001. ****p < 0.0001.