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. 2021 Feb;28(1-2):98-111.
doi: 10.1038/s41417-020-0189-4. Epub 2020 Jul 7.

Oncolytic virus promotes tumor-reactive infiltrating lymphocytes for adoptive cell therapy

Mathilde Feist  1   2 , Zhi Zhu  1 , Enyong Dai  1   3 , Congrong Ma  1 , Zuqiang Liu  1   2 , Esther Giehl  1   2 , Roshni Ravindranathan  1 , Stacy J Kowalsky  1 , Natasa Obermajer  1 , Udai S Kammula  1 , Andrew J H Lee  1 , Michael T Lotze  1 , Zong Sheng Guo  4 , David L Bartlett  5   6
Affiliations

Oncolytic virus promotes tumor-reactive infiltrating lymphocytes for adoptive cell therapy

Mathilde Feist et al. Cancer Gene Ther. 2021 Feb.

Abstract

Adoptive cell therapy (ACT) using tumor-specific tumor-infiltrating lymphocytes (TILs) has demonstrated success in patients where tumor-antigen specific TILs can be harvested from the tumor, expanded, and re-infused in combination with a preparatory regimen and IL2. One major issue for non-immunogenic tumors has been that the isolated TILs lack tumor specificity and thus possess limited in vivo therapeutic function. An oncolytic virus (OV) mediates an immunogenic cell death for cancer cells, leading to elicitation and dramatic enhancement of tumor-specific TILs. We hypothesized that the tumor-specific TILs elicited and promoted by an OV would be a great source for ACT for solid cancer. In this study, we show that a local injection of oncolytic poxvirus in MC38 tumor with low immunogenicity in C57BL/6 mice, led to elicitation and accumulation of tumor-specific TILs in the tumor tissue. Our analyses indicated that IL2-armed OV-elicited TILs contain lower quantities of exhausted PD-1hiTim-3+ CD8+ T cells and regulatory T cells. The isolated TILs from IL2-expressing OV-treated tumor tissue retained high tumor specificity after expansion ex vivo. These TILs resulted in significant tumor regression and improved survival after adoptive transfer in mice with established MC38 tumor. Our study showcases the feasibility of using an OV to induce tumor-reactive TILs that can be expanded for ACT.

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Conflict of interest statement

Conflicts of Interests: MF, ZL, ZSG and BLB filed a patent partly based on this work. Other authors declare that they have no conflict of interest.

Figures

Figure 1.
Figure 1.. Oncolytic VVs elicit tumor-specific antitumor CD8+ T cell response in the tumor tissue.
Ten days after viral treatments, subcutaneous MC38 tumors and/or splenocytes were collected and single cell suspensions were made followed by magnetic separation. Then isolated CD8+ T cells or splenocytes were tested by IFN-γ ELISPOT assay. (A). CD8+ T cells isolated from tumors (n = 4 – 5 mice/group) were either left unstimulated or challenged with γ-irradiated MC38 tumor cells for 24 h. Results were shown as individual data points (number of spots in each well) and bars (means ± standard deviation) of IFN-γ+ CD8+ T cells from each mouse evaluated in triplicate. Data from one experiment representing 2 independent experiments are shown. (B). Tumor-specificity of the OV-induced CD8+ TILs (n = 7 mice/group). Data are from one experiment representative of 3 independent experiments. For multiple group comparison, One-way ANOVA was used. ***p < 0.001; ****p < 0.0001. (C). MC38 tumor cell reactivity of splenocytes by IFN-γ ELISPOT assay. Splenocytes from treated mice were either left unstimulated or challenged with γ-irradiated MC38 tumor cells for 24 h. (D). MC38 tumor cell reactivity of isolated CD8+ TILs by IFN-γ ELISPOT assay. One experiment representative of 2 independent experiments is shown (n = 4–5 mice/group). Student’s t-test was used to analyze the statistical significance for data presented in panels A, C and D. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Figure 2.
Figure 2.. vvDD-IL2 treatments increase the number of TILs.
The tumor cell inoculation and tumor harvest were as described previously. Tumors were collected, fixed and stained for Hoechst (blue), CD3 (green), CD4 (red) and CD8 (white). A. Representative immunofluorescence image of one sample from each group (n = 5). For the representative panel, a single representative field was cropped from the original nine field acquisition. Scale bar 25 μm. B. Summary of the percentage of CD3+ T cells and CD3+CD4+ and CD3+CD8+ T cells per area. Student’s t-test was used to analyze the statistical significance. ***p < 0.001.
Figure 3.
Figure 3.. Immune status in the TME and spleen post virus treatment.
B6 mice were inoculated subcutaneously with 5.0e5 MC38 tumor cells. When tumors reached 5 × 5 mm (about day 9) they were treated intratumorally with PBS, vvDD, vvDD-IL-2 at a dose of 1.0e8 PFU. The mice were sacrificed 10 days post viral treatment and primary tumors and splenocytes were collected and analyzed by flow cytometry to determine the activation and immunosuppressive markers in (A) splenocytes, (B) TILs and (C) subsets of CD8+ T cells in the TILs. Tn for naïve T cells; Tcm for central memory T cells, and Tem for effector memory T cells. The percentages are over total CD8+ T cells, or over total CD4+ T cells for Treg cells. * p < 0.05, ** p < 0.01, *** p < 0.001; **** p < 0.0001.
Figure 4.
Figure 4.. OV-induced TILs can be cultured and expanded ex vivo and retain their tumor specificity.
(A). Tumor specificity of the expanded TILs. TILs from each individual mouse have been cultured for 4 days and then tested by IFN-γ ELISPOT assay. (B, C). Analysis of 4–1BB upregulation on (B) CD4+ and (C) CD8+ T cells by flow cytometry. As previously described, T cells were either left unstimulated (medium) or challenged with γ-irradiated MC38 tumor cells or γ-irradiated B16 tumor cells or naïve splenocytes from non-tumor-bearing B6 mouse in duplicate. After 24 h the cells have been stained for flow cytometry analysis for CD3, CD4, CD8, 4–1BB. Results are shown as individual data points (percentage of CD8+4–1BB+ T cells and CD4+4–1BB+ T cells) and bars (means ± standard deviation) of T cells from each mouse evaluated in duplicate. Data are presented as summary from 2 out of 5 independent experiments (n = 3–4 mice/group). For multiple group comparison One-way ANOVA was used. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Figure 5.
Figure 5.. Analysis of the vvDD-IL2 induced TILs after ex vivo expansion.
Prior to ACT, samples (T cells from naïve spleens: n = 4; TILs: n = 6) of the ex vivo cultured and expanded TILs were tested for tumor reactivity using IFN-γ ELISpot assay and co-culture assays for 4–1BB expression analyzed by flow cytometry as described previously. (A). Schema of experimental procedure. (B, C). Shown are (B) the ELISpot plate and (C) calculated IFN-γ+ spots per 1.0e6 T cells. (D, E). The cultured and expanded TILs were analyzed for cell surface 4–1BB expression of (D) CD4+ and (E) CD8+ T cells by flow cytometry. For multiple group comparison One-way ANOVA was used. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Figure 6.
Figure 6.. ACT of oncolytic virus-induced TILs led to significant therapeutic efficacy in mice bearing peritoneal carcinomatosis of MC38 colon cancer.
B6 mice were intraperitoneally inoculated with 5 × 105 MC38-luc cancer cells followed in 7 days, these mice were imaged for tumor growth and randomly grouped (n = 7 mice/group). Prior to ACT, mice in the treated groups received 5 Gy of sublethal irradiation. Grouped mice were intraperitoneally injected with PBS, naïve T cells, or vvDD-IL2 induced and ex vivo expanded TILs. All treated mice received exogenous IL-2 (1.0e5 IU/mouse, i.p. injection once every ~12 h for 3 days). One experiment representative of two independent experiments is shown (A, B). (A). Tumor growth has been monitored by live animal imaging 9 days and 17 days post treatment. (B). Radiance data quantified for two time points at days 9 and 17 post ACT. Student’s t-test was used to analyze the statistical significance. The variance was similar between the groups in this experiment. (C). The long-term survival of tumor-bearing mice was monitored by Kaplan-Meier analysis. These data represent one of the two independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001. (D). Summary of median survival in the different treatment groups. This experiment was performed 2 times.
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