†These authors contributed equally.
Academic Editor: Gustavo Caetano-Anollés
While the primary purpose of radiotherapy (RT) is the
elimination of cancer cells by inducing DNA-damage, considerable evidence emerges
that anti-neoplastic effects extend beyond mere tumor cell killing. These
secondary effects are based on activation of dendritic cells (DCs) via induction
of antitumoral immune reactions. However, there is an ongoing debate whether or
not irradiation of the DCs themselves may negatively affect their maturation and
functionality. Human monocytes were irradiated with different
absorbed doses (1
Dendritic cells (DCs) are professional antigen-presenting cells (APCs) which
play a crucial role in initiating tumor immunity. Immature DCs (iDCs) reside in
peripheral tissue, where they are activated and matured upon encounter of
pathogens. Phagocytosed antigens such as bacteria, viruses or damaged (tumor)
tissue are processed by the immunoproteasome in the iDC and ultimately presented
as epitopes on major histocompatibility complex (MHC) molecules expressed by the
DC. The so-called monocyte-derived dendritic cells (MO-DC) are generated from
Radiotherapy (RT) is one of the main pillars of oncological therapy. Primary
purpose of RT is elimination of cancer cells by inducing DNA-damage that either
causes induction of tumor cell death or inhibition of the proliferating capacity
of these cells in the high dose region. However, considerable evidence emerges,
that antineoplastic effects extend beyond these mechanisms. Furthermore,
controversial data on the effects of radiation dose (low dose RT with doses
On the molecular level, high dose irradiation has been shown to upregulate stress proteins, which can function as neoantigens, activating APC on the one hand , but has also been shown to massively kill blood cells, such as lymphocytes after whole-body irradiation in vivo [14, 15] and to reduce DC function in vitro . Regarding low dose irradiation, results are also controversial showing stimulated expression of APC and increased IL-12 levels [16, 17] but also a decrease of T-cell proliferation due to reduced APC and T-cell interaction . Additionally, most of the DC-mediated processes depend on their state of differentiation, maturation and migration capacity, which might all be influenced by irradiation. These above-mentioned secondary effects may have the potential to contribute to anti-tumor responses in a local, but also systemic manner via activation of the immune system outside of the irradiated volume (abscopal effects) [19, 20].
Despite of the well understood physical aspects of particle therapy, radiobiology and its clinical relevance regarding activation of the immune system are still scarcely understood and we did not find any data describing direct effects on immune cells like DCs. Particle therapy consists not only of low-linear energy transfer (LET) RT with protons but also heavier high-LET ions like carbon ions (C12). Although often assumed to be a low-LET treatment, the LET of protons is heterogeneous, with values up to 10 times that of photons over the last 2 mm of the beam range (5 to 20 keV/um) at the edge of the spread-out Bragg peak (SOBP) . High LET has the potential to intensely damage cells due to DNA damage and may therefore induce higher proportions of cell death. These effects are well-known in tumor cells and therefore we hypothesized that particle therapy of dendritic cells might also decrease their function.
In this study we investigated for the first time the effects of different irradiation types (photon, proton and carbon ion RT) and dose concepts (low-dose, normofractionated and hypofractionated/ablative RT) on the phenotype and functionality of MO-DCs.
Photon irradiation was performed with a biological cabinet x-ray irradiator
(XRAD 320 Precision X-ray Inc., N. Bradford, CT, MO, USA) with 320 kV and 12.50
mA and a 110 cGy/min dose rate at absorbed doses of 1
Since relative biological effectiveness (RBE)-values are dose-dependent,
biological dose estimations were made according to the local effect model (LEM)
IV in pre-experimental calculations and a mean RBE of 1.2 for protons  and
2.5 for C12  was used. Therefore, physical absorbed doses of 1
To investigate whether irradiated monocytes are able to differentiate into DCs,
we irradiated CD14-positive PBMCs on day 0 (-4) with different radiation doses.
To analyze the differentiation to iMO-DCs and mMO-DCs, cells were labeled with
specific monoclonal antibodies for characteristic surface markers. Non-specific
binding was assessed using appropriate isotype controls. Mean fluorescence
intensity (MFI) was measured via flow cytometry. Antibodies used were: CD14 PE,
#clone HB15e (eBiosciences); CD80 PerCP-eFlour710, #clone 2D10.4
(eBiosciences); CD83 PE, #clone HB15e (eBiosciences); CD86 Pacific blue, #clone
GL-1 (Bioledgend); CD209 PE-Cy7, clone# eB-h209 (eBiosciences); HLA-DR APC,
clone# L243 (Bioledgend). iMO-DCs were defined as CD14
Gating strategy. Cells are gated for alive cells (FSC-A vs.
SSC-A) and single cells (FSC-A vs. FSC-H). The population of HLA-DR
Different functional assays have been performed to distinct timepoints. Fig. 2 gives an overview of the experimental setup.
Overview of the experimental setup. Radiation was performed on
the day of the CD14
To analyze phagocytotic capacity of iMO-DCs and mMO-DCs cells were incubated
with Fluorescein isothiocyanate-labled (FITC-labeled) Dextran (Sigma) (1 mg/mL)
for 60 minutes at 37
In order to analyze the migrational ability of iDCs, modified Boyden chamber
assays were performed: Polycarbonate membranes with 8-
Cell supernatants were harvested on day 11 from in vitro culture experiment on generating CD14 derived MO-DC. Cells supernatants were analyzed for IL-12 Cytokine levels using an IL-12 ELISA kit (Coud-Clone Corp, TX, USA).
Data is displayed as means +/– standard deviations (SD). Comparisons between two
groups were performed using Student’s t-test or Wilcoxon rank test
(software: SPSS 24, IBM Corporation, NY, USA). Asterisk in figures indicates
statistical significance (p
Irradiation of monocytes on day 0 with photons, protons and carbon ions mostly
showed no significant change in the expression profile of characteristic surface
markers of iMO-DCs (CD14
Effect of irradiation on the amount of iMO-DCs and mMO-DCs.
(A) Effect of irradiation on the amount of iMO-DC generated from
Phagocytosis activity of MO-DCs was analyzed by measuring the uptake of
FITC-labeled Dextran by MO-DCs. Uptake of FITC-labled Dextran by iMO-DCs was
analyzed at 37
Uptake of FITC-labeled Dextran did not show significant differences between irradiated MO-DCs compared to untreated MO-DCs. Moreover, no significant differences between FITC-labeled Dextran uptake could be detected within the three treatment groups (Photons/C12/Protons) of irradiated MO-DCs (Fig. 4).
Measurement of phaygocytic capacity of iMO-DCs measured on day 7
using FITC-labeled Dextran. CD14
Migration of iMO-DCs was analyzed on day 7. Compared to untreated control,
migratory capacity was significantly decreased after 1
Effect of irradiation on migratory capacity of iMO-DC.
IL-12 secretion was measured using supernatants from MO-DC differentiation set up. IL-12 cytokine levels were non-significantly different comparing supernatants harvested from untreated MO-DCs to supernatants harvested from treated MO-DCs. Furthermore, no significant differences of IL-12 levels were detected within supernatants harvested form the different treatment groups (MO-DCs irradiated with Photons, C12, Protons) compared to untreated control (Fig. 6).
IL-12 Cytokine levels in cell supernatants of mDC measured on
day 11. CD14
Although extensive research is carried out in the field of radiation oncology regarding the local tumor tissue or the surrounding normal tissue effects, the influence of irradiation on immune activation due to DCs is not satisfactorily investigated so far. Especially the influence of different doses and fractionation as well as different radiation types is unclear.
Most of preclinical experiments are performed using single RT doses only, thus
comparability with the clinically used concepts is disputable. Strength of our
experiments is that we used different doses: low dose, high dose and a
normofractionated dose regimen. Since antigen contact with DC might occur in
different places, radiation doses on DCs might differ considerable. For example,
in the tumor itself the RT dose can be very high like in hypofractionated
concepts like stereotactic body radiotherapy (SBRT) and is then applied in a
single dose or in a small number of fractions (e.g., 1
Our results demonstrate that irradiation with different dose regimens and RT techniques did not negatively influence the phenotype and differentiation of iMO-DCs to mDCs, which is supported by the data of Merrick et al. , where no significant alteration of surface markers was seen after RT with 2, 8 and 30 Gy.
Since specific immune responses against TAA depend on the ability of DCs to
internalize antigen and migrate to lymph nodes for further T-cell activation, we
subsequently tested phagocytic and migrational capacities. Firstly, we could
demonstrate that phagocytic and migrational capacities iMO-DCs are not negatively
altered after irradiation and secondly that high-LET RT with protons and C12 did
also not negatively influence functionality of iMo-DCs, despite our initial
hypothesis. Furthermore, after proton irradiation with 5
Only one other published paper regarding photon irradiation of human DCs was found which reported partially contradictory results regarding differentiation and functionality: Cao et al.  showed that CD86 expression, which is a marker for mMO-DCs, and their functional capacity, measured by T-cell activation is downregulated by irradiation. However, they did not investigate detailed phagocytic or migrational capacity as well as secreting of the immunostimulatory cytokine IL-12 of DCs. An additional factor that decreases comparability of these results, is the fact that they used DCs generated from PBMCs obtained from patients. with multiple sclerosis. It is not unlikely that in this setting, in vitro generated DCs are negatively impaired in their functional capacity due to the underlying autoimmune disease. A comparison with DCs derived from healthy donors is lacking and results should therefore be taken with precaution.
We also investigated wether irradiation alters IL-12 cytokine production of mMO-DCs and found no decrease in IL-12 production, supporting the hypothesis of preserved functional ability of DCs after RT. Thus, our overall results regarding phenotype and functional capacities of DCs after irradiation are supported by the data given from Merrick et al. . However, the experimental set up lacks a positive control, e.g., experiments on irradiated DCs stimulated with an IL-12 inducing agent. Within the given experimental set up, incapability of the maturation medium used in this experimental set up to induce IL-12 secretion of mature DCs cannot be excluded.
One possible limitation of our analysis is certainly the artificial in vitro situation which cannot fully represent the situation in vivo including the interaction with the microenvironment. Furthermore, while we did show a possible preserved IL-12 secretion of irradiated mMO-DCs, we did not investigate the direct interaction between DCs and T-cells which represents the final activation step in immune response and anti-cancer cell activity. Overall, the model used for DCs generation represents an artificial model thus in vitro generated Mo-DCs might differ in their functionality from in vivo Mo-DCs. Moreover, this work cannot exclude an impact of irradiation on other subsets of DC (cDCs, pDCs). Our data however support the notion that DCs play an important role in the radiation associated immune response in cancer therapy: for example, in combination experiments of photon irradiation with toll like receptor agonists 7 (TLR7) we and other groups have shown in several tumor models including pancreatic cancer, colon cancer, sarcoma and lymphomas that DC activity is essential to convey a strong immunogenic antitumor effect of radiation [27, 28, 29]. Nevertheless, DCs that are present in cancer patients may have a different sensitivity to radiation than DCs generated with cells of healthy subjects given another limitation of the experiments performed in this research work.
To our current knowledge, no research group has ever investigated effects of particle therapy (e.g., proton and/or C12) on DC function. Although it is known that particle therapy offers beneficial characteristics regarding higher LET and higher relative biological effectiveness and therefore induces cancer cell killing more than conventional photon RT, our results indicate, that particle therapy might have a rather stimulatory effect on immune cells like DCs, as we demonstrated in migration assays for proton irradiation (Fig. 5).
All together our data show that irradiation with photons, protons and carbon ions do not markedly alter the phenotypic maturation of MO-DCs, nor does irradiation alter their basic functionality including phagocytosis, migration ability and cytokine secretion. The data support the notion that DCs can play a robust role in the immune response after radiotherapy of cancer.
In summary, our results show that different fractionation regimens and doses do not negatively impact differentiation and functionality of DCs and therefore we assume that their potential for inducing an adaptive immune response is not significantly impaired. Although using a higher LET, particle therapy with protons and carbon ions did not reduce DC function compared to photon RT, but might be stimulatory regarding migrational capacity of iMO-DCs.
LK, AH and LO performed data collection. LK and AH were responsible for writing and original draft preparation. LK, AH and JH-R performed the statistical analysis. LK, AH, PEH, JD and SR conceived of the analysis and participated in its design and coordination. SB performed radiobiological dose calculations for particle therapy. All the authors were responsible for data interpretation, participated in manuscript revisions, and approved the final manuscript.
This research received no external funding.
The authors declare no conflict of interest.
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.