In 2020, lung cancer was the second most diagnosed cancer in the world and was the leading cause of cancer death. It is estimated that 2.2 million new cases occurred in the world in 2020 (1).

Radiation therapy is the most commonly used treatment modality for non-small-cell lung cancer (NSCLC) patients. It is administered for curative purposes as a solo treatment or in combination with surgery and/or chemotherapy for patients with early-stage or locally advanced stage (stage I-III) NSCLC. It can also be used as palliative treatment to prolong life and improve quality of life for patients with distant metastases (stage IV). Curative radiotherapy options are stereotactic body radiation therapy (SBRT) and concurrent chemoradiation therapy (CCRT). During the recent years, however, a number of novel treatment modalities has been developed such as check-point inhibitors and several tyrosine kinase inhibitors targeting specific genetic alterations.

The lung is a complex organ consisting of at least 40 different types of cells with distinct functions. This complexity is associated with impaired regeneration potential, and consequently, the lung is the organ most exposed to damage following various forms of radiation therapy (2–4). The risk of developing radiation-induced lung injury limits effective high-dose thoracic radiation therapy for early-stage and locally advanced NSCLC patients (5) (5). The reported incidence of radiation pneumonitis (RP) after SBRT for NSCLC varies from 2% to 47% (6–11), and the incidence after CCRT varies from 5% to 40% (12–17).

The tumour microenvironment (TME) plays an important role in tumour growth (18) and in the outcome of treatment, including affecting resistance to cancer treatment (19). TME of a solid tumour consists of tumour cells, local cells, infiltrating nontumour cells, molecules present in the vicinity of these cells and cells comprising the blood and lymph vessels. Radiotherapy affects cancer cells and the TME, in particular the tumour blood vessels and cells of the immune system.

Radiation induces reactive oxygen species and reactive nitrogen species (ROS and NGS), which cause damage to mitochondrial DNA and to the alveolar–capillary barrier, both of which are sensitive to the effects of ionizing radiation (20–22). One of the many effects of radiation is increased vascular permeability and exudation of proteins into the alveolar space, causing the apoptosis of alveolar type-I pneumocytes. This triggers an influx of inflammatory cells (e.g., neutrophils, macrophages, and lymphocyte subsets) from the peripheral and pulmonary vasculature that infiltrate the damaged lung. These cells are further activated by ROS and NGS as well as danger-associated molecular patterns (DAMPs), leading to the release of various inflammatory molecules (23–26) and contributing to altered tissue remodeling, fibrogenesis and local and systemic inflammation associated with the development of complications to radiotherapy (27, 28) and poor survival (29, 30). The radiation-induced release of cytokines and related molecules the first 24 hours after radiation might be an important contributor to RP (31–34).

While clinical outcome like survival is the most important parameter when evaluating novel treatment options, biomarkers are of importance in order to predict treatment responses and risk categories and even more importantly, to select correct treatment options and to discover pathways that are not modulated by the current treatment modalities.

The inflammatory response in the lungs causing the development of RP and fibrosis is still not well understood. The regulation and importance of the different inflammatory and immune-related mediators in the TME are at present not clear. In the present study, we examined the serum parameters of the activation of different leukocyte subsets, including myeloperoxidase (MPO) as a marker of neutrophil activation; soluble CD25 (sCD25), soluble T cell immunoglobulin mucin domain-3 (sTIM-3) and soluble programmed cell death 1 (sPD-1) as markers of T cell activation and exhaustion; and sCD14 and sCD163 as markers of monocyte/macrophage activation. We also analyzed the levels of the homeostatic chemokines CCL21 and CCL19 as mediators of lymphocyte trafficking. These markers were evaluated in relation to RP and survival after curative radiotherapy for early-stage and locally advanced NSCLC.

The present study evaluated serum leukocyte activation markers in NSCLC cancer patients in relation to RP and mortality. Whereas we found no associations with neutrophil and monocyte activation markers, which are traditionally thought to reflect acute and, to some degree, chronic (monocyte) inflammation, we found increased levels and different temporal profiles according to radiation treatment of the T cell activation marker sCD25, T cell exhaustion marker sTIM-3 and chemotactic T cell signal CCL21 in patients who developed RP. Moreover, sTIM-3 and, in particular, sCD25 predicted mortality independent of other demographics, including RP and the mode of radiation treatment. Our study suggests that the development of adverse outcomes in non-small-cell lung cancer patients is linked to T cell activation and exhaustion.

T cell activation is typical in both cancer progression (36), among others, in NSCLC (37, 38) and pneumonitis (39) due to persistent T cell receptor stimulation and is accompanied by the expression of inhibitory receptors such as PD-1 and TIM-3 (40–42) and T cell dysfunction (40). At the same time, radiotherapy modulates several immunological processes: revelation of antigens, activation of T lymphocytes, recruitment and accumulation of T cells in the tumour, and acknowledgement and killing of tumour cells by T lymphocytes. Our finding of increased levels of T cell activation and exhaustion markers in patients who developed RP indicates an active role in RP by likely overstimulation of T cells.

We could not, however, find any previous studies evaluating circulating levels of T cell activation and exhaustion markers in NSCLC or in response to RP. Herein, we found that changes in the levels of T cell markers in relation to RP occurred at different times according to radiation treatment. SBRT delivers the radiation dose to the pulmonary tumour more precisely than CCRT, allowing an escalation of SBRT treatment doses far beyond traditional conventional radiotherapy, damaging healthy lung tissue to a lesser extent and triggering RP more rarely and later than CCRT (11). While T cell exhaustion is mostly used in relation to chronic infections, we speculate that the enhanced early levels of sTIM-3 (i.e., within 3 months) in the CCRT group, without enhanced sCD25 levels, could reflect the effects of more acute RP, which is seen 1-3 months after CCRT (43–46), as a mechanism to prevent persistent and overshooting T cell activation. The more gradual increases in sTIM-3, sCD25 and CCL21 within the SBRT group correlate with the later onset of RP in this group occurring after 5-10 months (9–11, 47–49). Although PD-1, another T cell exhaustion marker, was not significantly associated with RP, the temporal trajectory was similar to sTIM-3 in the SBRT group but not in the CCRT group, possibly reflecting some different effects on T cell subsets of these radiation modalities. The concurrent increase in the T cell chemoattractant CCL21 in the SBRT group could potentially be linked to T cell migration or effects in regional lymph nodes (50).

As T cell exhaustion and dysfunction seem to be hallmarks of cancer progression (51) and are the targets of current immunotherapy (52), it is tempting to speculate that increased sTIM-3 and sCD25 could link RP and poor prognosis in lung cancer. However, while severe RP is associated with poor short-term outcome, prognosis in milder cases seems to be more dependent on underlying factors (53–55). Furthermore, we found that the association between sCD25, sTIM-3 and mortality was independent of both RP and the mode of radiotherapy. In patients with glioblastoma (56) and melanoma (57), only tumour-infiltrating, but not peripheral, T cells showed enhanced levels of exhaustion-associated inhibitory receptors.

Thus, our findings could potentially reflect immunological abnormalities in the TME, which has been associated with poor prognosis in lung cancer (58). This may also support double immunotherapy targeting both TIM-3 and PD-1, which has indeed been shown to improve antitumour T cell responses in preclinical models (59–61). Moreover, inflammation is a recognized hallmark in carcinogenesis (62). Macrophages are associated with chronic inflammation in cancer initiation and promotion and are also involved in tumour progression and metastasis (63). Tumour-associated macrophages correlate with poor prognosis (64). Radiotherapy changes the TME by attacking cancer cells, blood vessels and immune-related cells in the TME. Immunological changes occur in the affected tissue first and later become measurable in peripheral blood. The absence of neutrophil and monocyte activation markers after radiotherapy in our study suggests that those markers do not play a crucial role in the pathogenesis of RP.

We were unable to find studies evaluating serum levels of the monocyte/macrophage activation markers sCD14 or sCD163 or the neutrophil activation marker MPO in relation to survival in lung cancer patients. Enhanced tumour-associated CD163 expression by immunohistochemistry (IHC) was associated with poor survival in a small study (65), while a larger study (n=335) found no association between CD66b(+) neutrophils and CD163(+) macrophages and survival in lung cancer patients (66). Nevertheless, our data suggest that any potential activation of these cells in RP or in relation to disease progression is not reflected by circulating levels of activation markers.

Treatment with immune checkpoint inhibitors (ICIs) is a relatively new class of therapeutic agents that have shown impressive anticancer effects for a number of solid cancer types. ICI-induced pneumonitis is a rare but severe side effect and is occasionally fatal. Life-threatening pneumonitis has been reported in up to 2% of cases (67–69). The incidence of ICI-induced pneumonitis is higher in NSCLC than in other cancer types and in combined therapy with radiotherapy or chemotherapy (6.5%–10%) than in monotherapy (3%–4%) (70, 71). These findings can be useful in the study of ICI-induced pneumonitis pathogenesis.

The occurrence of RP after SBRT is lower than after CCRT (13, 14, 16, 72) and severe RP is quite uncommon (7, 8, 73). SBRT techniques allow minimizes the size of the planning target volume (PTV) and meaning the normal lung tissue in the target volume. The larger irradiation fields increase values of inflammation markers. In the present study, however, too few patients underwent conventionally fractioned radiotherapy alone in the CCRT group to make any reliable comparison.

In conclusion, our findings showing increased sCD25 and sTIM-3 in relation to RP and survival suggest that persistent T cell activation and exhaustion may contribute to progression and adverse outcomes in locally advanced NSCLC and support T cell-targeted treatment in this disorder.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by Regional Ethical Committee, REK nr. 2013/169/REK sør-øst D. The patients/participants provided their written informed consent to participate in this study.

Conception and design: JB, TU, PA, ÅH. Provision of study materials or patients: JB, M-BB, ÅH. Collection and assembly of data: JB, TU. Data analysis and interpretation: JB, TU, PA, ÅH, ARH, ML. Manuscript writing: JB, TU, PA, ÅH. Final approval of manuscript: All authors.

This research was funded by the Regional Health Authorities in Southeast Norway (grant 2015058), the Vestfold Hospital Trust (grant 197430) and an unrestricted grant from Boehringer Ingelheim Norway (grant 197430).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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