Background
A variety of immunotherapeutic agents are being used to treat advanced malignancies and CTLA-4 and PD-1/PD-L1 T cell checkpoint blocking antibodies are currently the most common approach. Efficient tumor control by immunotherapies relies on robust CD8+ cytotoxic T lymphocyte (CTL) activity [
1‐
3] and these immune checkpoint blocking (ICB) antibodies release the inhibitory pathways restraining the action of CTLs. While the most effective immunotherapies in development seek to generate, promote, or stimulate tumor-specific CTLs, tumors often induce an immunosuppressive microenvironment that allows them to evade immune cell killing [
4]. A major mechanism of tumor-induced immunosuppression is the recruitment and/or induction of CD4+ regulatory T cells (T
REGS) within the tumor microenvironment [
5,
6].
T
REGS are a suppressive subset of CD4+ T cells important for preventing autoimmunity [
7]. These cells are characterized by expression of the high affinity IL-2 receptor, CD25, and the transcription factor forkhead box p3 (Foxp3) [
8]. T
REGS can be naturally derived in the thymus (nT
REG), or they can be induced in the periphery from naïve CD4+ precursors (iT
REG) [
5,
9,
10]. Several cancer types are known to contain high levels of T
REGS that facilitate escape from immune surveillance [
11‐
13]. To maintain an immunosuppressive microenvironment tumor cells have been reported to recruit peripheral T
REGS as well as induce conversion of CD4+ conventional T cells (T
CONV) into T
REGS within the tumor [
13‐
17]. Though nT
REG and iT
REG cells both have suppressive function, iT
REGS reportedly have less stable Foxp3 expression due to partial demethylation of CpG motifs within the
foxp3 locus [
18]. Functionally, T
REGS are capable of inhibiting the proliferation and killing activity of CTLs through several mechanisms including: [a] secretion of transforming growth factor-β1(TGF-β1) and IL-10, [b] metabolic disruption through CD39 and CD73 [
19], or [c] contact-dependent inhibition via cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), lymphocyte activation gene 3 (LAG-3), and programmed death ligand 1 (PD-L1) signaling [
20,
21].
Ionizing radiation (IR) remains a common treatment modality for most cancer types and is often used in combination with cancer immunotherapy-based strategies when radiation alone is insufficient to eradicate advanced disease [
22]. Interestingly, radiation has been shown to enhance anti-tumor immune responses by several mechanisms. Research in our lab, and others, has shown that tumor cells exposed to doses within the hypofractionated range of radiation increase the expression of several cell surface proteins on tumor cells that are important for immune attack. Major histocompatibility (MHC) class I, death receptors (Fas/CD95 and TRAIL/CD253), and effector T cell costimulatory molecules (OX40L and 4-1BBL) exhibit increased expression on tumor cells surviving radiation [
23‐
26]. Expression of these molecules subsequently promotes increased sensitivity to killing by CTLs [
27,
28]. Induction of immunogenic cell death (ICD) is another mechanism of immune enhancement by radiation that results in stimulation of antigen presenting cells that can promote and drive an adaptive anti-tumor immune response [
29]. In addition to local tumor control via DNA damage and cell death, radiation treatment can cause abscopal effects that result in immune control of tumors that are outside of the irradiated field [
30,
31]. This phenomenon is being seen more and more frequently with the increased use of radiation in combination with immunotherapies [
32,
33].
While much has been reported on the impact of IR on tumor cells, the impact of radiation on the frequency, phenotype, and suppressive function of regulatory immune cells such as T
REGS is less well studied. Several murine studies have shown that T
REGS are more radioresistant than other lymphocyte populations, however, it is less clear what effect radiotherapy (RT) has on the phenotype and function of human T
REGS [
34,
35]. Moreover, functional studies in mice have been contradictory. Studies by Qu et al found no difference in the suppressive function of T
REGS from radiation treated mice compared to control mice, in contrast, Balogh et al and Billiard et al both reported decreased functional activity of irradiated T
REGS [
36‐
38]. In addition, studies by Muroyama et al and Kachikwu et al reported increased T
REG numbers in locally irradiated tumors compared to control mice, in vivo [
39,
40]. However, Cao et al (2009) and Liu et al observed decreased frequencies of human T
REGS irradiated in vitro and murine T
REGS following whole body irradiation in vivo, respectively [
41,
42]. Many factors could contribute to the different outcomes reported among these studies, including differences in radiation dose used, time of evaluation after radiation, local irradiation versus whole body irradiation, and tumor-bearing versus non-tumor bearing model systems.
To more specifically extend these observations towards clinically relevant tumor immunity we sought to determine the impact of hypofractionated doses of radiation on induced human TREGS, as these are most likely to accumulate at tumor sites. We first assessed the direct effect of radiation on the viability and expression of Foxp3 in both nTREG and iTREG cells. We also evaluated the impact of radiation on the suppressive function of iTREGS and the expression of molecules associated with TREG functional activity: CD25, CTLA-4, LAG-3, CD39, CD73, and PD-L1. Our data reveal that radiation induces similar levels of death among human nTREGS and iTREGS, but that less death occurs in TREGS as compared to CD4+ TCONV cells. We also found that radiation decreases expression of Foxp3 in both types of TREG cells but that Foxp3 expression is more robustly reduced by radiation in iTREGS. Additionally, we show that iTREG cell phenotype is directly modulated by radiation and that these cells are functionally less suppressive following radiotherapy.
Discussion
RT is a common treatment modality for cancer and is well documented to enhance antitumor immune responses by modulating tumor phenotypes, making them more susceptible to killing by CTLs [
60]. The activity of CTLs, however, can be limited by suppressive T
REGS. The effect of radiation directly on T
REG biology remains controversial and there are very few reports evaluating human T
REG cells. While T cells are known to be sensitive to high doses of radiation, the increased use of lower radiation doses per fraction, such as those used during hypofractionated radiotherapy, necessitates the need to elucidate the effects of radiation on T cells surviving RT exposure. In this study, we compared the effect of radiation treatment on human natural and induced T
REG cell viability and Foxp3 expression. We show that irradiated human nT
REG and iT
REG cells are more viable than irradiated CD4+ T
CONV cells, and that CD4 + CD25+ T
REG cells exhibit decreased expression of Foxp3 after exposure to ionizing radiation. We then extended our studies to further examine how the phenotype and function of iT
REG cells are impacted by radiation as these are likely the cells that accumulate in advanced cancers during immune escape. We demonstrate that molecules associated with T
REG suppressive function are differentially modulated by radiation and that the suppressive function of iT
REGS is inhibited.
Results here, using human cells, are in line with previous reports in mice demonstrating that T
REG cells are more resistant to radiation-induced cell death compared to CD4+ T
CONV cells [
34,
44]. Additionally, we found that this resistance exists in both nT
REG and iT
REG cells (Fig.
1). Curiously, these results contrast those reported in a previous study assessing human T
REGS [
45] exposed to much lower doses of radiation (0.94 Gy and 1.875 Gy). The authors reported significantly more cell death in T
REGS as compared to CD4+ T
CONV cells [
45]. Radiation decreases human CD4+ T cell viability in a dose-dependent manner [
61] and cells exposed to 5 Gy of radiation exhibit a robust decrease in live cells not detected in cells treated with ≤2 Gy. Therefore, it is plausible that human T
REGS are relatively more resistant to higher doses (> 2 Gy) of radiation since low dose radiation (≤ 2 Gy) did not induce significant death in CD4+ T
CONV cells. In the current study we selected doses, above 2 Gy, that would be relevant to those given per fraction during cancer therapy with hypofractionated RT. Moreover, most studies demonstrating the ability of RT to serve as an adjuvant for anti-tumor immunity point towards a benefit from moderate doses, around 5–12 Gy, being superior than lower 2 Gy fractions.
Much of what is known about the impact of radiation on T
REGS has been derived from murine models and both increased and decreased T
REG frequencies have been reported following radiation [
39‐
42]. In studies evaluating T
REG frequency in mice, the use of whole-body versus local radiation treatment appears to have a profound effect on the number of T
REGS detected. Mice treated with low-dose total body irradiation (1.25 Gy) exhibited a decrease in the frequency and total number of nodal CD4 + Foxp3+ T
REG cells [
42], while mice that received local irradiation (10 Gy and 20 Gy) were found to increase the proportion of tumoral and splenic T
REGS [
39,
40]. It is difficult to know if the changes observed are due to the direct effect of radiation on T
REG cells themselves or due to changes induced in tumor cells or another immune cell type in the irradiated area. It is also unclear if the changes are due to T
REG cell death, redistribution to another location, or a change in the phenotypic markers used to identify the cells. Our current study, demonstrating the differential sensitivities between CD4+ T cell subsets, support the idea that in vivo observations showing increases in T
REGS post-RT may be detecting a decrease in the frequency of conventional CD4+ T cells which are more sensitive to RT than T
REGS. Overall, though characterization of immune cell frequencies can provide useful information, details about the functional status of the cells, and expression of suppressive molecules, in diverse tumor model systems would be more informative. Reports of this nature have been limited. Muroyama et al recently reported that isolated tumor and splenic T
REGS retained their suppressive function 7 days following local irradiation in B16/F10 tumor-bearing mice [
39]. Whether there was an earlier window of time during which T
REG function was suppressed was not explored.
Our data reveal a significant decrease in the expression of Foxp3 in human T
REG cells 48 h post treatment. This decrease was observed in both nT
REG and iT
REG cells but was more profound in iT
REGS, particularly among CD25
hi cells (Fig.
2e). We narrowed our focus to human TGF-β1-induced T
REGS as these are likely to be most similar to the tumor-induced T
REGS that accumulate during tumor progression and immune escape. Though iT
REGS downregulated Foxp3, they did not alter their expression of surface CD4. In addition, the loss of Foxp3 did not appear to be due to conversion to another CD4+ T
H subtype as we did not detect an increase in the T
H1 or T
H2-associated transcription factors T-bet or GATA3 (Fig.
3). Though nT
REG and iT
REG cells both have suppressive function, iT
REGS have been reported to have less stable Foxp3 expression due to partial demethylation of CpG motifs within the
foxp3 locus [
18]. Demethylation of the
foxp3 locus yields the gene accessible to the binding of numerous transcription factors [
62]. Radiation can alter the epigenetic enzymes associated with specific gene promotors in cancer cells [
63,
64] and has been reported to alter DNA methylation, both globally and in a gene-specific manner [
65]. Thus, it seems reasonable that radiation could be altering the epigenetic state of the
foxp3 locus, and we would expect iT
REGS to be more sensitive to these changes since the region is already partially methylated. Confirmation of this mechanism warrants further investigation. Alternatively, binding of STAT5 to the T
REG-specific demethylated region (TSDR) within the conserved noncoding sequence 2 (CNS2) has been shown to stabilize Foxp3 expression [
66], and blockade of the JAK3/STAT5 signaling pathway has been demonstrated to downregulate Foxp3 expression in both human and murine T
REG cells [
67]. It is possible that radiation alters the expression of STAT5, however, we did not observe any change in phosphorylated STAT5 following radiation treatment (unpublished data) suggesting that radiation-induced regulation of Foxp3 may be independent of STAT5. The CNS2 region, however, is bound by several other transcription factors in addition to STAT5 [
62], and it is possible that radiation modulates the expression of these other factors causing the subsequent reduction in Foxp3 expression.
Beyond Foxp3, expression of CD25, CTLA-4, CD39, CD73, and LAG-3 are commonly associated with T
REG phenotype. Additionally, the presence of PD-L1 has been detected on both human [
21] and mouse T
REGS [
68], as well as on TGF-β1-induced T
REGS [
69]. To our knowledge, the effect of radiation on the expression of many of these molecules in human T
REGS has not been characterized. T
REG cells are commonly defined as being Foxp3+ and CD25
hi and we found that CD4 + CD25
hi cells were the most significantly reduced following treatment with radiation (Fig.
4). This cell population also exhibited a significant decrease in CTLA-4. This observation is particularly noteworthy because it suggests that, as Foxp3 regulated genes, the reduction in CD25 and CTLA-4 expression may be directly tied to the reduction of Foxp3 expression. Moreover, there was no significant reduction in the expression of the other non-Foxp3 genes associated with T
REG phenotype that we evaluated (CD39, CD73, and PD-L1) (Figs.
4 and
5).
We did detect a moderate increase in CD73 and the expression of both CD39 and CD73 can be increased by TGF-β [
70,
71]. Though CD73 is expressed intracellularly in humans, surface expression can be induced upon activation with high-dose IL-2 therapy [
72]. However, the cells examined in our study exhibited reduced expression of the IL-2 receptor and are likely less responsive to IL-2. Thus, the moderate increase we detected in the expression of CD73 could indicate that there is increased production of TGF-β from the cells. However, the fact that we saw no increase in CD39 expression, which is also sensitive to TGF-β, suggests that another mechanism of regulation may be occurring. Hypofractionated doses of radiation have been shown to increase expression of type I interferon pathway genes associated with an inflammatory signature [
73,
74]. While these observations have been made in the context of the whole tumor microenvironment, our data may reveal that radiation can directly alter the cytokines secreted from T cells, which may then modulate CD73 expression.
LAG-3 expression has also been reported to be regulated by Foxp3 [
57], however, it can also be expressed in CD4 + Foxp3-negative cells indicating that it is not strictly dependent on Foxp3 for expression [
59]. In our experiments, we were surprised to observe a significant increase in LAG-3 expression by radiation as opposed to the decreased expression of Foxp3, CD25, and CTLA-4 (Fig.
5d). Interestingly, chemo-radiation has been shown to increase the proportion of CD4 + LAG-3+ expressing cells in head and neck cancer patients [
75] demonstrating that this effect may be clinically relevant and detectable. It is possible that radiation is directly altering expression of this gene via epigenetic mechanisms as has been reported for expression of other immune regulatory genes (OX40L and 4-BBL) in irradiated tumor cells [
63]. Another possibility is that radiation is altering expression of the transcription factor early growth response gene 2 (Egr2) which has been shown to convert naïve CD4+ T cells into LAG-3-expressing T
REGS [
76]. Notably, these LAG-3-expressing T
REGS were characterized as being Foxp3-negative. Our study demonstrates that radiation induces a CD4 + Foxp3-negative T cell subset from CD4 + CD25
hiFoxp3+ iT
REGS (“ex-Foxp3+ cells”). While we did not detect conversion of cells towards a T
H1 or T
H2 subset it remains plausible that radiation treatment converts Foxp3+ iT
REGS to another regulatory T cell subset not evaluated here. LAG-3 expression has been reported to confer Foxp3+ regulatory T cells with greater suppressive capacity [
20,
56], however, we found that irradiated iT
REG cells were functionally less suppressive as compared to untreated cells (Fig.
6), despite a detectable increase in LAG-3 expression. This is in line with reports showing that Egr2-transduced CD4+ T cells, which express LAG-3 and IL-10, insufficiently suppressed proliferation of responder T cells in vitro [
76]. Subsequent in vivo studies, however, demonstrated that Egr2-transduced CD4+ T cells did have suppressive capacity which could suggest functional differences in the activity of LAG-3+ cells in vitro versus in vivo. This could indicate that signals, such as MHC Class II, from other immune cells are necessary to stimulate the full suppressive capacity of LAG-3+ T
REGS.
How modulation of LAG-3 expression on T cells could impact cancer immunotherapy approaches is worthy of further investigation. LAG-3 expression on CD4+ and CD8+ T
CONV cells is known to inhibit their expansion and effector function [
77,
78]. As a result, LAG-3 blocking antibodies are currently being tested pre-clinically and clinically, and recent studies have revealed that dual treatment with anti-LAG-3 and anti-PD-1 blocking antibodies can significantly enhance the proliferation of CD4+ and CD8+ T
CONV cells [
79]. Therefore, the combined use of radiotherapy and anti-LAG-3 blocking antibodies could greatly enhance the antitumor immune response. However, how LAG-3 signaling impacts T
REGS remains controversial. In a murine model of Type 1 diabetes, signaling through LAG-3 was shown to limit T
REG function [
80] and it is unclear if antagonistic antibodies that prevent LAG-3 signaling could enhance T
REG suppressive function at the same time that they are promoting effector T cell activity. Further studies are needed to elucidate the effect that LAG-3 antibodies have on iT
REG suppressive function, particularly when used in combination with radiotherapy.
Incorporation of immune-based strategies for the treatment of cancer is becoming increasingly more common in the clinic. Current use is most often for advanced disease where the tumor microenvironment has evolved to favor survival against immune attack. This selection often involves the accumulation of suppressive T
REGS that help cancer cells evade immune attack by CTLs. Radiotherapy (RT) has been shown to enhance tumor attack by T cells through multiple mechanisms. RT impacts diverse cells in the microenvironment (tumor cells, immune cells, stromal cells) but the effect of the therapy on each cell type has not been fully elucidated. This is challenging to fully interrogate in vivo
, where the impact on the independent cell types is difficult to isolate. A clearer understanding of the direct effects of radiation on suppressive subsets of immune cells can inform optimal strategies for incorporating RT to specifically serve immunotherapy strategies. In this study we found that radiation is capable of directly modulating the expression of Foxp3 and several suppressive surface molecules in human iT
REGS. Furthermore, radiation-induced changes resulted in significantly reduced functionality of induced T
REGS (Fig.
6). Whether this reduced activity is simply a consequence of the reduced IL-2 signaling capacity due to lower expression of CD25, or from the lower levels of CTLA-4 expression, will require further characterization. It would also be of interest to determine how radiation impacts levels of suppressive molecules that are secreted by T
REGS, such as TGF-β1 and IL-10, as well as how long this reduction in suppressive function is retained. Ongoing in vivo studies using radiation-treated tumor-bearing mice also demonstrate reduced T
REG numbers after local treatment with hypofractionated doses of RT. Even if only temporary, this reduction in T
REGS represents a window of opportunity during which CTL engagement with tumor cells can be manipulated. Given the conflicting observations regarding the role of LAG-3 on T
REG biology, future studies will need to determine the functional significance of increased LAG-3 post-RT to elucidate how LAG-3 antibodies in development can be used in combination with RT to most optimally enhance therapeutic efficacy.