Our study observed the time-sequential change in immune-related gene expressions after irradiation of glioblastoma cells. We mainly focused on speculating the optimal time points for combining radiotherapy and anti-PD-(L)1 treatment. We found that neutrophil-mediated immunity, antigen processing/cross-presentation to activate cytotoxic T cells, and radiation-induced-interferon-γ-related signals in tumor cells were highly upregulated at 24 hours after irradiation and maintained until 48 hours. It suggests that tumor antigen-specific T cells would infiltrate to tumor cells after 24 hours from radiation, and the PD-L1 increase in tumor cells by interferon-γ from the infiltrating T cell would take more than 24 hours after irradiation. Therefore, concurrent anti-PD-(L)1 therapy with daily fractionated radiotherapy might not be fully effective in glioblastoma because of the time required for re-infiltration of tumor antigen-specific T cells after the previous infiltrating T cells were killed by radiotherapy.
Radiation increases PD-L1 in tumor cells. Radiation increases interferon-γ by infiltrating T cell, and increased interferon-γ provokes tumor cells to generate PD-L1 to make T cells dysfunctional[10, 21, 22]. T cell-mediated-interferon-γ is thought to be the main mechanism behind increasing PD-L1 levels[22]. Also, radiation damages tumor cells, and as damage responses, cancer cells itself express PD-L1 without T cells via the base repair system[23], and the DNA double-strand break repair pathway[24]. Several studies, using a tumor cell line only, have reported increased PD-L1 expression post-irradiation[23, 24].
In our study, although interferon-γ signaling increased after irradiation, PD-L1 expression did not increase. There are two possible causes. First, we observed immune signal regulations only up to 48 hours after irradiation, which may have been a short time to detect an increase in PD-L1 by radiation-induced interferon-γ. Several in vitro and in vivo studies reported that interferon-γ increases approximately after 2 days (1–6 days) after irradiation[21, 23, 25, 26]. Interferon-γ may be produced by tumor cells after irradiation or by T cells recruited after antigen-presenting process. Interferon-γ generated by tumor cells recruits T cells, and interferon-γ produced by T cells increases PD-L1 in tumor cells. In our study, increased interferon-γ signaling was derived from tumor cells (not T cells). Therefore, the results of this study suggest that T cell infiltration induced by interferon-γ from tumor cells rarely occurs until at least 1–2 days after irradiation.
Second, since there was no T cell in our cell line study, the amount of PD-L1 might be less than when T cells are present. When T cells were inhibited, the amount of interferon-γ was reduced significantly[21]. Autocrine signaling by interferon-γ produced in tumor cells may increase PD-L1, but the release of interferon-γ by T cells is much more massive and is thought to play a pivotal role in PD-L1 expression. This result suggests that without T cell infiltration and interferon-γ secreted by T cells, anti-PD-(L)1 therapy may not be effective.
Recent trials that showed improved results when radiotherapy and immunotherapy were combined as an adjuvant (after radiotherapy) or neoadjuvant (before radiation) settings. The Pacific trial demonstrated a survival benefit from the adjuvant anti-PD-1 therapy after chemo-radiotherapy in stage III non-small-cell lung cancer[4]. Without surgery, chemo-radiotherapy would upregulate tumor antigen process and presentation and PD-L1 level in tumor cells would also be highly expressed after completing chemo-radiotherapy. This study demonstrated that patients with time interval between chemo-radiotherapy and immunotherapy less than 14 days showed favorable survival compared to those with time interval 14 days or more.
According to our study, significant upregulation of adaptive immunity-related tumor antigen presentation started at 24 hours after irradiation, suggesting that 14 days was enough time for infiltrating T cells in tumor cells. Also, as the chemo-radiotherapy-induced immune responses might be reduced over time, resulting in lower survival benefits for patients from immunotherapy.
A recent trials of neoadjuvant anti-PD-1 therapy before surgery in recurrent glioblastoma also showed a promising result[2]. In this study, anti-PD-1 was administered 14 days before surgery. Intratumoral T cells would be sufficiently activated by anti-PD-1 therapy before surgery. Based on this success, it would be also possible to try neoadjuvant immunotherapy for newly diagnosed glioblastoma.
On the other hands, recent two phase III trials which failed to meet the primary endpoint were concurrent anti-PD-1 therapy with (chemo)-radiotherapy in newly diagnosed glioblastoma after surgery (NCT02667587, NCT02617589)[1]. Without gross tumor tissue, the PD-L1 expression level might not be prominent than the Pacific trial. Moreover, daily radiotherapy may not provide sufficient time for new T cell infiltration to tumor cells.
Concurrent immuno-radiotherapy has another point to weaken the effectiveness of the combined therapy. Anti-PD-L1 therapy activates infiltrating T cells which are dysfunctional because of PD-L1 from tumor cells. Therefore, to make a success of anti-PD-(L)1 treatment, enough density of infiltrating tumor antigen-specific T cells is indispensable. Radiotherapy affects all cells in the target field. Moreover, lymphocytes are radio-sensitive. Therefore, after irradiation, it would require time to re-infiltrate new T cells in the tumor microenvironment.
PD-L1 expression was higher in the p53 mutant tumor than in the p53 wild type cancer, and the p53 mutant tumor was highly sensitive to the anti-PD-L1 therapy[27]. As the apoptotic process is not available in the p53 mutant cancer, anti-PD-(L)1 treatment might be a promising option for this disease[28].
Generally, radiation-induced TGF-ß1 signaling is associated with normal tissue injury and fibrosis[29]. TGF-ß signaling is now regarded as one of the possible mechanisms of immunotherapy resistance[20, 30–33]. Co-administration of anti-TGF-β1 and anti-PD-(L)1 (± radiotherapy) extended survival in a mouse model[20, 31]. In our study, 6 Gy single dose did not increase TGF-ß1 expression. Radiation protocol without TGF-ß1 increase and/or anti-TGF-β1 therapy might be a new strategy to improve survival in glioblastoma.
In our study we used various tumor cell lines. Further studies using tumor tissues such as heterogeneous tumor spheres or organoids or various preclinical and clinical studies would be helpful to deepen the understanding of the time-sequential change of immune response induced by radiotherapy. Administration of temozolomide, anti-PD-L1 treatment, and various doses/fractions of radiotherapies were not performed. As a time-sequential NGS study, however, this study provides a comprehensive and in-depth understanding of gene expression post-irradiation and offers a valuable recommendation for the optimal treatment protocol for combined radiotherapy and immunotherapy.
In conclusion, this study observed a time-sequential change in gene expression of glioblastoma cell line post-irradiation. Innate and adaptive immune signals were significantly upregulated at 24 hours post-irradiation and maintained until 48 hours. These results suggest that daily radiotherapy might not provide sufficient time for infiltrating immune cells into the tumor microenvironment. Therefore, anti-PD-(L)1 therapy against T cell-mediated PD-L1 in tumor cells might not be fully synergistic with daily radiotherapy treatment schedule.