Radiotherapy and Immunotherapy: A Beneficial Liaison

REVIEWS
Radiotherapy and immunotherapy:

abeneficial liaison?
Ralph R.Weichselbaum1, Hua Liang1, Liufu Deng1 and Yang-Xin Fu2
Abstract | Investigations into the interaction between radiotherapy and the host immune system
have uncovered new mechanisms that can potentially be exploited to improve the efficacy of
radiotherapy. Radiation promotes the release of danger signals and chemokines that recruit
inflammatory cells into the tumour microenvironment, including antigen-presenting cells that
activate cytotoxic Tcell function. By contrast, radiation can attract immunosuppressive cells into
the tumour microenvironment. In rare circumstances, the antitumour effect of radiotherapy has
been observed outside of the radiation field, known as the abscopal effect. This phenomenon is
proposed to have an immune origin and indicates that local radiotherapy elicits systemic effects.
Herein, we highlight data that provide new mechanistic explanations for the success or failure of
radiotherapy, and postulate how the combination of immune-modulation and radiation could tip
the balance of the host immune response to promote cure. We use the concept of radiationinduced tumour equilibrium (RITE) as a starting point to discuss the mechanistic influence of
immune-checkpoint therapies on radiotherapy efficacy.
Department of Radiation and

Cellular Oncology and The
Ludwig Center for Metastasis
Research, The University of
Chicago, 5758 South
Maryland Avenue, BOX9006,
Chicago, Illinois 60637, USA.
2
Department of Pathology,
University of Texas
Southwestern Medical Center,
6000 Harry Hines Boulevard,
Dallas, Texas 752359072,
USA.
1
Correspondence to R.R.W.
rrw@radonc.uchicago.edu
doi:10.1038/nrclinonc.2016.211
Published online 17 Jan 2016
Radiotherapy is often used as a curative treatment for

localized cancer or isolated metastasis, and as a palliative
treatment in patients with widespread disease; overall,
approximately 5060% of patients with cancer receive
radiotherapy 1,2. Radiotherapyused alone or in combi
nation with conservative surgery or chemotherapyis
a highly effective treatment for earlytointermediate
stage cancers, including head and neck, breast, prostate,
skin, rectal, anal, lymphoid, endometrial, and cervical
tumours3. However, some tumours, such as glioblas
toma or very large stageIV tumours, are resistant to the
therapeutic effects of ionizing radiation4.
The concept of dose fractionation, that is, delivering
small doses of ionizing radiation each day, was prompted
by the desire to obviate normal-tissue toxicity. The work
of Regaud5 and Coutard6,7 shaped modern radiation
treatment schedules for most primary tumours that
leverage dose fractionation. In general, curative frac
tionated radiotherapy for localized cancers is delivered
in relatively small daily fractions (doses of 1.52.2Gy
per day) for a total dose of 6080Gy, depending on the
tumour type and the radiation sensitivity of the sur
rounding normal tissues. Larger daily doses (3Gy per
day) have been used in shorter fractionated treatment
schedules (3weeks versus 67weeks) in the treatment
of some cancers, when tumour control is equivalent to
that achieved with the typical daily schedule and in situ
ations when normal tissue damage is not prohibitive8.
Moreover, very large doses can be delivered in one to five
fractions using stereotactic body radiotherapy (SBRT),

an approach that takes advantage of improvements in
imaging, radiation delivery techniques, and the ability
to account for organ motion in real-time2. These doses
usually range from 20Gy delivered in one to three treat
ments to 34Gy delivered in one treatment 9. SBRT is
most-commonly used to treat brain and bone metasta
sis, as well as localized lung cancer. The recognition of
oligometastasis as a clinical state has led to the adoption
of SBRT to treat metastasis from many cancers at differ
ent anatomical locations with curative intent 1018. Doses
of 510Gy delivered in five to ten fractions using SBRT
techniques is known as hypofractionated radiotherapy.
Ablative and/or hypofractionated radiation has been
used to treat patients with localized prostate or pancre
atic cancers, as well as some patients with many metas
tases, in order to shorten treatment times and increase
thepalliative effects of radiation1924.
Data from many laboratories indicate that local
radiation produces systemic, immune-mediated anti
tumour and, potentially, antimetastatic effects2539.
Additionally, the combination of local radiotherapy
and immune-modulation can augment local tumour
control and cause distant (abscopal) antitumour effects
through increased tumour-antigen release and antigen-
presenting cell (APC) cross-presentation, improved
dendritic-cell (DC) function, and enhanced Tcell
priming 27,31,37,4045. On the other hand, ionizing radi
ation can also generate chemotactic signals that recruit
NATURE REVIEWS | CLINICAL ONCOLOGY
ADVANCE ONLINE PUBLICATION | 1
.
d
e
v
r
e
s
e
r
s
t
h
g
i
r
l
l
A
.
e
r
u
t
a
N
r
e
g
n
i
r
p
S
f
o
t
r
a
p
,
d
e
t
i
m
i
L
s
r
e
h
s
i
l
b
u
P
n
a
l
l
i
m
c
a
M
7
1
0
2
REVIEWS
macrophages, cytotoxic Tcells, and suppressive cells,
such as regulatory T (Treg) cells and myeloid-derived sup
Radiotherapy not only exerts direct cytotoxic effects on tumour cells, but also
pressor cells (MDSCs), as well as the efflux of immune
reprogrammes the tumour microenvironment to exert a potent antitumour immune
cells, such as DCs that are important APCs37,55,5961 (FIG.1).
response
An increasing body of evidence indicates that radio
Tumour-cell proliferation and cell death due to Tcell cytotoxic killing coexist in
therapy can augment innate and adaptive immune
irradiated tumours, resulting in stable disease that might provide a window of
responses against tumours, thereby decreasing immuno
opportunity for immune-modulation
suppression and potentiating the responsiveness of
Radiotherapy enhances antitumour immunity, but also induces immunosuppressive
tumours to radiation3237,6266. One common hypothesis
responses
is that localized radiotherapy creates an inflammatory
The combination of immunotherapy and radiotherapy presents a multimodal
microenvironment in the context of immunogenic cell
treatment approach that involves stimulating and suppressing various pathways
death, which is characterized by the release of tumour
antigens and danger-associated molecular patterns
(DAMPs), such as calreticulin, ATP and high-mobility
several myeloid-cell types with distinct roles in Tcell group protein B1 (HMGB1) 60,6771 ; furthermore,
suppression35,4649. Schaue and McBride50 have described increased production and recruitment of proinflam
the radiation-induced immune response, in general, as matory cytokines, such as interleukin1 (IL1), trans
having Janus-like opposing faces, as well as the conflict forming growth factor (TGF), fibroblast growth
ing yin and yang actions of Tcellsin pathogenesis factor (FGF), and tumour necrosis factor (TNF), as
and protection. Similarly, Zitvogel and Kroemer 51 note well as NACHT, LRR and PYD domains-containing
that ionizing radiation can both subvert and reinstate protein 3 (NALP3)-inflammasome activation and sig
tumour immunosurveillance.
nalling, are also proposed to have a complex role in the
host response to ionizing radiation45,7277. Radiotherapy
Irradiation and host immune responses
can also induce the expression of chemokines, such as
Host immune status. A study in a mouse fibrosarcoma CXC-motif chemokine 9 (CXCL9), CXCL10, CXCL11
model by Stone etal.52 established that the host immune and CXCL16, resulting in chemotaxis of Tcells into
status determines the efficacy of radiation-induced the tumour microenvironment 78,79. Preclinical studies
antitumour effects. Lee etal.31 subsequently demon have established that radiotherapy-induced liberation of
strated that Tcells are required for ablative radiation- tumour antigens and stimulation of maturation signals
induced tumour regression following irradiation with drive the migration of APCs to the draining lymph node,
1520Gy; mouse melanoma B16 tumours implanted where Tcell priming is augmented to initiate a systemic
into immunocompetent hosts responded to high-dose response29,31. Furthermore, localized radiotherapy induces
radiation, whereas tumours grown in nude mice (that antigen release and cross-presentation by DCs (such as
lack Tcells and some Bcells) or those in wild-type hosts CD11c+CD11b+ APCs) in the tumour microenvironment,
in which cytotoxic CD8+ Tcells had been depleted, did which can orchestrate tumour eradication following
not respond to ionizing radiation31. Data also indicated radiation, with or without immune-modulation30,31,39,80.
that radiation promoted antigen-specific Tcell prim Sharabi and co-authors39 confirmed the importance
ing in this model31; however, paclitaxel or dacarbazine of cross-presentation of tumour antigens by MHC
chemotherapy can suppress Tcell priming and abol classII (MHCII)-expressing APCscompared with
ish radiation-induced tumour regression. These data direct presentation via MHCI on tumour cellsusing
emphasize that the timing and type of chemotherapy MHC Iknockout mice. Research groups at the US
combined with radiation warrants careful consider National Cancer Institute (NCI) have reported that radi
ation to avoid unwanted immunosuppressive effects. ation changes the phenotype of tumour cells, resulting in
Immunostimulatory chemotherapysuch as cyclo upregulation of cell-surface molecules and expansion of
phosphamide treatment, which induces differentiation the cellular peptide pool81,82, which broadens the antigens
of type17Thelper (TH17) cells in patients with various available for presentation83, thereby rendering tumours
advanced-stage solid tumourshas been reported to more susceptible to Tcell-mediated antitumour effects.
enhance antitumour immune responses when used in
Multiple studies indicate that presence of tumour-
combination with radiotherapy 53. In general, reports infiltrating lymphocytes, especially effector Tcells,
from studies in different tumour models highlight the before therapy is correlated with better survival in
importance of CD8+ Tcell infiltration and function in patients with many types of cancers and under differ
contributing to the effects of radiotherapy 39,5459.
ent treatment regimens8489. Anitei et al.88 reported
thatthe density of the tumour immune infiltrate (that
Radiation enhances immune responses. Substantial is, the total number of CD3+ Tcells and cytotoxic CD8+
work over the past three decades has expanded our Tlymphocytes) is associated with disease-free and over
understanding of how the immune systemand, in all survival in patients treated with chemoradiotherapy
particular, Tlymphocytesparticipates in the host for rectal c ancer. We speculate that radiotherapy induces
response to tumour irradiation2529,31,37,38,55. Localized the release of chemokines that subsequently enrich the
radiation initiates cell death and the production and Tcell infiltrate, and enhances priming of pre-existing
release of cytokines and chemokines into the tumour or newly infiltrating Tcells, which provides a positive
microenvironment, which leads to infiltration of DCs, immunological outcome.
Key points
2 | ADVANCE ONLINE PUBLICATION
www.nature.com/nrclinonc
.
d
e
v
r
e
s
e
r
s
t
h
g
i
r
l
l
A
.
e
r
u
t
a
N
r
e
g
n
i
r
p
S
f
o
t
r
a
p
,
d
e
t
i
m
i
L
s
r
e
h
s
i
l
b
u
P
n
a
l
l
i
m
c
a
M
7
1
0
2
REVIEWS
Natural killer (NK) cells are lymphocytes that are a
critical component in host surveillance against tumours90.
Engagement of the receptor NKG2D activates NK cells91,
and irradiation increases the expression of NKG2D lig
ands in several human cancer cell lines92. Exploration of
Tumour
cell mass
Radiotherapy
Initiates cell death
Release of DNA and RNA
into the cytoplasm
RNA triggers TLRs

and RLRs
DNA triggers cGAS

to release cGAMP
Activation of the STING
signalling pathway
Activates transcription of
the type I IFN gene
Activates transcription of
the type I IFN gene
Production of IFN
Essential for DC activation and function, DCs in turn prime T cells
Mediates recruitment and eector function of CD8+ T cells
Activates pro-death signalling in tumour cells
Essential for maturation and activation of APCs and T cells
PD-L1 induced by inammatory cytokines, including IFN
Prolonged IFN signalling stimulates tumour prosurvival mechanisms
NKcell-based therapies in research or clinical trials has

been increasingly reported9396, with treatment strategies
including immune-checkpoint blockade using antibodies
to programmed cell-death protein 1 (PD1) or cyto
toxic Tlymphocyte-associated protein 4 (CTLA4)93,97,
and adoptive transfer of NK cells engineered to express
chimeric antigen receptors (CARs) that are specific for
tumour antigens96. In a mouse 4T1 spontaneous metas
tasis model, invariant NK Tcell deficiency not only limi
ted spontaneous lung metastasis, but also enhanced the
antimetastatic effects of radiation and anti-CTLA4 anti
body treatment 93. Ames etal.95 reported that, in a mouse
solid tumour model, irradiation followed by adoptive
transfer of NKcells that had been activated exvivo could
eliminate cancer stem-like cells, and this approach pro
longed survival compared with radiation therapy alone.
The role of NKcells in cancer immunotherapy has been
elegantly reviewed byCarotta97.
DCs are myeloid-derived cells that are affected pro
foundly by alterations to the tumour microenvironment
resulting from irradiation. Chemokines that attract anti
gen-specific Tcells and DCs are released within the irradi
ated tumour 29,31,60,62,98101; the Lord lab and o
thers29,39,54,62
have demonstrated that irradiation increases the levels
of tumour-associated DCs, enhances the mobilization of
these cells into draining lymph nodes, augments DC mat
uration, and increases the ability of DCs to cross-present
antigens and prime Tcells; antigen-specific effector Tcells
that were primed by DCs in the draining lymph nodes
then infiltrate tumours. In the case of tumour antigens
with a strong priming effect, tumour-associated DCs and
stromal antigen-presentation (by CD11b+ cells) in irradi
ated tumours are required and sufficient for o
ptimal
Tcell-mediated cytotoxicity 30.
Initiates cytokine and chemokine production

in the tumour environment
3
4
Inltration of
Dendritic cells
Macrophages
Cytotoxic T cells
Treg cells
MDSCs
Eux of immune cells

T cells
DCs
MDSCs
Cytokines
Treg cells build up in the

tumour microenvironment
and secrete TGF and IL-10,
which both suppress eector
T-cell activation and also
stimulate the suppressive
functions of MDSCs
MDSCs suppress T-cell

activation and promote
tumour regrowth
Initiates release of tumour antigens

Initiates release of DAMPs (calreticulin, ATP, HMGB1)
Activation of APCs
5
6
Initiates recruitment of IL-1B, TGF, FGF, TNF and NLRP-3

Generates chemotactic signals that recruit several myeloid cell
populations with distinct roles in T-cell suppression
Figure 1 |Radiation-induced effects on tumour cells.

Ionizing radiation initiates: (1) cell death, (2) cytokine and
chemokine production in the tumour microenvironment,
(3)release of tumour antigens, (4) release of DAMPs,
(5)recruitment of IL1, TGF, FGF, TNF, and NLRP3, and
(6)the generation of chemotactic signals that recruit several
myeloid cell populations with distinct roles in T-cell
suppression. These primary events are the catalysts for an
elaborate succession of processes. Cell death (1) causes the
release of DNA and RNA into the cytoplasm leading to the
production of IFN. Cytokine and chemokine production (2)
triggers the infiltration of DCs, macrophages, cytotoxic Tcells,
regulatory Tcells, and MDSCs, as well as the efflux of immune
cells. The release of DAMPs (4) activates APCs. These complex
and sometimes conflicting events are reflective of the struggle
between host immune response and tumour prosurvival
mechanisms. APCs, antigen-presenting cells; cGAMP, cyclic
GMP-AMP; cGAS, cyclic GMP-AMP synthase; DAMPs,
damage-associated molecular patterns; DC, dendritic cell;
FGF, fibroblast growth factor; HMGB1, high mobility group
protein B1; IFN, interferon; IL1, interleukin1; IL10,
interleukin 10; MDSC, myeloid-derived suppressor cells;
NLRP3; NACHT, LRR and PYD domains-containing protein 3;
PDL1, programmed cell death 1 ligand 1; RLRs, RIGIlike
receptors; STING, stimulator of interferon genes; TGF,
transforming growth factor; TLRs, Toll-like receptors; TNF,
tumour necrosis factor; Treg cells, regulatory Tcells.
Nature Reviews | Clinical Oncology

.
d
e
v
r
e
s
e
r
s
t
h
g
i
r
l
l
A
.
e
r
u
t
a
N
r
e
g
n
i
r
p
S
f
o
t
r
a
p
,
d
e
t
i
m
i
L
s
r
e
h
s
i
l
b
u
P
n
a
l
l
i
m
c
a
M
7
1
0
2
REVIEWS
Activating immunosuppressive immune responses. The
tumour microenvironment comprises various inhibitory
immune cells that include Treg cells, macrophages, and
MDSCs86, as well as other stromal cells, such as vascu
lar endothelial cells, pericytes, and cancer-associated
fibroblasts.
Tregcells are a subset of CD4+ Tcells and are charac
terized by expression of the transcription factor forkhead
box P3 (FOXP3); these cells are critical to regulation of
both inflammation and autoimmunity. Tregcells accu
mulate in the tumour microenvironment and secrete the
cytokines TGF and IL10, which suppress effector-Tcell
activation and also stimulate the suppressive functions of
MDSCs35,102. A number of reports have indicated that
Tregcell numbers increase in tumours and immune organs
in response to localized or wholebody irradiation39,103,104,
which might reflect an intrinsic radioresistance of these
cells, as well as a fast regeneration rate when whole-body
low-dose radiation is administered105,106. Sharabi etal.39
reported that radiation induced an increase in intra
tumoural Tregcell numbers, but not in the abundance
of Treg cells within draining lymph nodes. Findings have
indicated, however, that Langerhans cells (a subset of
DCs found in the skin and mucosa), which are intrin
sically highly radioresistant, can stimulate expansion of
Tregcells when they migrate into draining lymph nodes
after wholebody irradiation107. Persa etal.104 found that
expression of CTLA4 in Tregcells increased over the
3days following whole-body radiation in an experi
mental setting. Moreover, increased TGF and IL10
production by Tregcells after various doses of radiother
apy has been reported104. These findings suggest that
Tregcells in tumour-bearing hosts develop enhanced
immunosuppressive properties following radiotherapy.
Indeed, the results of several clinical trials have indi
cated that the presence of highly suppressive Tregcells in
the circulation might represent a heightened immuno
suppressive environment induced by chemoradiother
apy, at least transiently, in patients with head and neck
or cervical cancer, or glioblastoma108110. Thus, targeting
Treg cells and/or their immunosuppressive effector mol
ecules, TGF and CTLA4, might be crucial to reversing
immunosuppression51,111,112. Another important factor to
consider when planning radiotherapy is to avoid certain
organs, such as the skin, in order to limit strong immuno
suppressive responses51. Of note, however, skin cancer is
highly curable with radiotherapy, and the functions of
Langerhans cells following radiotherapy in humans are
highly complex 113.
MDSCs contribute to tumour progression by promot
ing the survival of tumour cells, and via stimulation of
angiogenesis, tumour-cell invasion of adjacent tissues,
and metastasis114. Two subsets of MDSCs are widely
recognized: immature polymorphonuclear cells, which
are pathologically activated neutrophils (PMN-MDSC;
defined as CD11b+Ly6Ghi cells in mice), and monocytic
cells, which are pathologically activated inflammatory
monocytes (MMDSC, characterized as CD11b+Ly6Chi
cells in mice). In mice, MDSCs are more-generally
characterized as CD11b+Gr1+ cells, with Gr1 identi
fying a mixture of Ly6C+ and Ly6G+ populations115,116.
Interactions of these inhibitory immune cell types or

the growth factors and/or cytokines they produce cause
suppression of effector Tcell function and tumour
angiogenesis, and promote tumour progression 117.
Major factors implicated in MDSC-mediated immuno
suppression include expression of arginase (ARG1),
inducible nitric oxide synthase (iNOS), TGF, and
IL10, and induction of Tregcells115,118120. The importance
of angiogenesis in tumorigenesis and tumour-cell sur
vival was established in 1971 by Judah Folkman121, and
angiogenesis is now accepted as a hallmark of cancer
progression. MDSCs, tumour-associated macrophages
(TAMs), and other immune cells, are essential for
tumourvascularization116,122.
Evidence suggests that MDSCs also have a requisite
role in chemoresistance and radioresistance. Massagu
and colleagues123 have reported that the production of
CXCL1/2 by breast cancer cells attracts MDSCs, which
secrete pro-survival S100A8/9 proteins that rescue cells
from the effects of cytotoxic chemotherapy. Thus, MDSCmediated resistance of cancer cells to cytotoxic therapies is
a direct process that occurs through promotion of tumourcell survival, and also an indirect process caused by inhib
ition of Tcell responses116. MDSCs (CD11b+Gr1+ cells,
representing both PMN-MDSCs and MMDSCs) are rap
idly recruited into the tumour stroma following localized
radiotherapy (within around 3days)124,125. Several groups
have reported that, in murine models, local and systemic
MDSC numbers decrease drastically 714days after a
single high dose of ionizing radiation37,59,126. However,
this observation might reflect the differential kinetics of
inflammation in various tumour models, as well as dif
ferences in the radiation doses used and the timings of
the analyses116,127. Of note, while MDSC recruitment is
an immediate effect of radiation, the delayed reduction
of MDSC numbers could be an indirect effect of host
adaptive immune response (as discussed below).
Traditionally, TAMs have been classified into M1
(activated, tumour-cell-killing) and M2 (alternatively
activated, tumour-promoting) subtypes with opposing
functions in tumours128,129. Whether this demarcation is
accurate remains unclear, owing to the observed diversity
in the TAM populations present in different tumour types,
as well as overlapping gene-expression signatures. Instead,
TAMs are now generally classified as immunostimulatory
or immunoregulatory on the basis of assessing specific
molecules responsible for the differentiation and various
phenotypes of TAMs130. Inoue etal.131 reported that radi
ation alters the expression levels of chemokines produced
by TAMs, thereby altering the regulation of Tcell infil
tration. Moreover, depletion of TAMs using clodronate-
encapsulated liposomes can reverse immunosuppression
and promote radiotherapy efficacy. For example, in a
mouse melanoma model, Meng etal.47 noted that deple
tion of TAMs before irradiation increases the antitumour
effects of radiotherapy, either when given as a large single
dose (20Gy) or at 2Gy in 10 fractions (102Gy); these
data from mouse models indicate that the TAMs are
predominantly immunosuppressive. By contrast, Klug
etal.132 reported that, in a pancreatic tumour model,
low-dose (2Gy) radiation induced iNOS expression in
.
d
e
v
r
e
s
e
r
s
t
h
g
i
r
l
l
A
.
e
r
u
t
a
N
r
e
g
n
i
r
p
S
f
o
t
r
a
p
,
d
e
t
i
m
i
L
s
r
e
h
s
i
l
b
u
P
n
a
l
l
i
m
c
a
M
7
1
0
2
REVIEWS
TAMs to enable an iNOS+/M1 phenotypic switch, which
is required for CD8+ Tcell recruitment and function. In
orthotopic models, radiotherapy-induced recruitment of
F4/80+ macrophages limits the efficacy of radiation133, and
radiation therapy can induce myeloid-derived tumour-
infiltrating macrophages to suppress immune responses
against pancreatic tumours in mice as a result of increased
production of macrophage-stimulating colony factor1
(CSF1) by tumour cells134. These conflicting results reflect
the complexity and plasticity of macrophage develop
ment and transformation at different stages of tumori
genesis135,136. In the clinical setting, high expression of the
M2 macrophage marker CD163 in the tumour stroma of
patients with breast cancer is associated with decreased
survival137. Further investigation into how radiotherapy
affects TAM function isneeded.
Fractionation and immune-cell infiltration. Fractionated
radiotherapy regimens are designed to exploit vulnerabil
ities in DNA-repair and cell-cycle processes; however, the
fraction size and appropriate timing to achieve optimal
antitumour effects remains to be determined. As such, this
continues to be an active area of both preclinical and clin
ical investigation. Dewan etal.138 compared an ablative
120Gy dose to a hypofractionated 38Gy schedule,
or to more-fractionated dosing of 56Gy in combina
tion with a monoclonal antibody targeting CTLA4. The
hypofractionated regimen (38Gy) was more effective
in inducing immune infiltration and an abscopal effect
than the single, ablative dose (20Gy) of radiation. The
investigators noted that radiation enriched tumours for
functionally active CD8+ Tcells and was effective at medi
ating regression in subcutaneously implanted tumours.
Verbrugge and coauthors139 investigated the effects of
antiCD137 and antiCD40 antibodies combined with
a single dose of 12Gy irradiation compared with either
44Gy or 45Gy dosing in a mouse orthotopic breast
cancer model. They found that CD8+ cells positive for
PD1 expression and targeting of this inhibitory immune
checkpoint with antiPD1 antibodies were required for
combination treatment with antiCD137 antibodies and
radiation to be effective. Notably, tumour control after
treatment with 44Gy or 54Gy radiation in combina
tion with antiCD137 and antiPD1 antibodies was not
as effective as that observed with a single dose of 12Gy
plus these antibodies. More recently, Filatenkov etal.59
compared a single dose of 30Gy radiation with 103Gy,
or 30Gy+103Gy schedules in mouse colon cancer
models. The results indicated that only single-dose radia
tion (30Gy) led to a decrease in MDSCs and an increase in
CD8+ Tcell infiltration into tumours59. Collectively, these
results and data from a study by Lee etal.31 indicate that
both ablative and fractionated doses of radiation could be
effective in tumour control depending on the experimen
tal system studied and the approach to Tcell modulation
used in combination with radiation140.
The role of interferon. How activation of innate immu
nity by radiation results in robust Tcell responses is
unclear. Burnette etal.62 reported that typeI interferon
(IFN) is essential for bridging innate and adaptive
immunity for tumour regression: innate immunity is

implicated in initiating a radiation-induced response in a
typeI IFNdependent manner62, and the typeI IFN signal
ling in response to irradiation is essential for DC activa
tion and function, which, in turn, is responsible for Tcell
priming and tumour control62,141. One study revealed that
typeI IFN mediates the recruitment and effector func
tion of CD8+ Tcells38. IFN can, however, have opposing
effects on tumour-cell survival and antitumour immu
nity. For example, in response to radioinduction of IFN
in mouse models, pro-death signalling is activated in
tumour cells, resulting in maturation and activation of
APCs and Tcells, and improved therapeutic responses,
whereas prolonged IFN signalling can stimulate tumour
pro-survival mechanisms resulting in the selection of
radioresistant tumour clones142144. The duality of prodeath and pro-survival effects of typeI IFN signalling in
tumour cells and the importance of the host response in
terms of IFN expression levels, activation strength, tim
ing of expression, and regulatory mechanisms make the
tumour microenvironment exquisitely complex 145. In
summary, typeI IFN signalling in innate immune cells,
such as DCs, is essential for their function to prime and
activate Tcells. In the setting of ablative radiotherapy,
typeI IFNs improve antigen cross-presentation and Tcell
function.
The STING signalling pathway. TypeI IFN is induced
by activation of Toll-like receptor (TLR) signalling
pathways via either myeloid differentiation primary
response 88 (MYD88) or TIR-domain-containing adap
tor-inducing IFN (TRIF); however, neither protein
is essential for radiation-induced tumour regression146.
Studies have revealed a unique pathway for typeI IFN
induction through sensing of cytosolic DNA. Stimulator
of interferon genes (STING) is an endoplasmic-reticu
lum-associated protein that can activate transcription
of the typeI IFN gene, via a STING/TBK/IRF3/NFB
signal transduction pathway 147,148. Upon sensing of cyto
plasmic DNA, STING is activated by cyclic GMP-AMP
(cGAMP), a product of cyclic GMP-AMP synthase
(cGAS)149,150. STING is essential for host protection
against DNA pathogens, as well as for inducing adap
tive immune responses to DNA vaccines151153. Activity
of the STING-signalling pathway in host DC cells was
reported to be essential to antitumour immunity in a
mouse regressing-tumour model146,154. In parallel, STING
was found to be essential for a radiation-induced anti
tumour response in established tumours146. In vitro and
invivo, STING is not only required to induce typeI IFN
production, but can also promote an antigen-specific
Tcell response following radiotherapy. When cocul
tured with irradiated tumour cells, STING-deficient DCs
lose the ability to produce typeI IFN and prime Tcells.
Activation of the DNA-sensing/IFN-induction path
way mediated by STING in DCs is greatly enhanced by
irradiating the tumour (invivo) or by coculturing DCs
with irradiated tumour cells (invitro), which highlights
the precipitating role of radiotherapy in triggering the
STING pathway. The importance of STING-mediated
DNA-sensing in host antitumour response was further
.
d
e
v
r
e
s
e
r
s
t
h
g
i
r
l
l
A
.
e
r
u
t
a
N
r
e
g
n
i
r
p
S
f
o
t
r
a
p
,
d
e
t
i
m
i
L
s
r
e
h
s
i
l
b
u
P
n
a
l
l
i
m
c
a
M
7
1
0
2
REVIEWS
verified by the observation of substantial tumour regres
sion in response to cGAMP administration in combina
tion with radiotherapy 146. Thus, ionizing radiation not
only kills tumour cells directly, but also promotes innate
immunity as well as adaptive immune responses via the
STING-mediated DNA-sensing pathway.
Microenvironment and tumour response

In the tumour microenvironment, irradiation induces
stromal, immunological and vascular changes that are
essential for a tumour response155. In the context of
oncogenic evolution resulting from responses to the host
immune system, tumours have several possible fates:
elimination, equilibrium, dormancy, and escape. The
concepts of tumour dormancy and immunological equi
librium were proposed by Lewis Thomas156 and further
extended by Schreiber and colleagues157, who first intro
duced the paradigm that the oncogenic process involves
sequential neoplastic progression158. Elimination clearly
involves complete eradication of tumour cells; however,
the distinction between equilibrium and dormancy
is less well-defined because it is difficult to discern
when one state ends and the other begins. In a mouse
model, Schreiber and co-authors158 demonstrated that
the immune response eliminates most cells in nascent
tumours, but, as the immune system becomes less effec
tive at removing neoplastic cells, tumours enter a state
of equilibrium; following further immunosuppression
orchestrated by tumour and host factors, tumours
then escape from immune suppression owing to loss of
immune potency. Historically, tumour relapse 510years
after chemoradiotherapy has been attributed to tumour
dormancy, whereas long-term progression-free survival
in patients with palpable tumours after therapy cessation
has been assumed to reflect equilibrium. Tumour dor
mancy is an older concept that involves a dampening
of immune processes, whereas equilibrium relates to a
dynamic process of maintaining a balanced state159. The
concept of tumour escape involves tumour evasion of
immune-mediated killing through a number of possi
ble mechanisms160162, including immunosenescence
(age-associated deterioration of the immune system),
acquired immune resistance, or the formation and cul
tivation of an immunosuppressive milieu that precipi
tates immune tolerance (that is, deletion or suppression
of self-reactive/tumour-specific Tcells)159,163166. Thus,
therapeutic strategies that aim to modulate tumour-
induced immune suppression are c rucial for improving
outcomes167.
Tumour dormancy and equilibrium. In 1980, Suit and
Walker 168 noted that the presence of a palpable tumour
and the rate of tumour regression at the end of radio
therapy was not predictive of cure in both preclinical
models and patients. Remarkably, they reported that in
immunogenic experimental tumour models, the degree
of tumour regression was closely correlated with per
manent cure; however, in animal models with weakly
or non-immunogenic tumours, no correlation between
outcome and tumour resolution at the end of treatment
was observed. Hence, these researchers suggested that
the immune system might have a key role in suppressing

tumours that remain palpable at the end of treatment,
and in radiation-mediated cure, in general, because the
calculated radiotherapy doses required for cure in ani
mal tumour models were higher than the actual doses
used in the clinic. Some tumours that do not respond
to radiotherapy rarely regress (such as glioblastoma) or
relapse quickly following regression (such as small-cell
lung cancer), although a surprising number of patients
relapse locally (in the radiation field) at relatively long
time periods after complete regression (or excision and
radiation), as demonstrated in clinical trials. For exam
ple, in women with breast cancer, up to 50% of relapses
occur >5years after radiotherapy 169. This observation is
also true for subsets of men treated with radiotherapy
for prostate cancer 170. A smaller percentage of patients
with ocular melanoma171 or head and neck cancer 172
also develop late relapses. Conversely, some patients
with palpable or visible tumours at the completion of
radiotherapy are cured with no further treatment.
Whether these differing clinical responses reflect a
short or prolonged state of clinical or subclinical equi
librium or dormancy is unclear. One view has been that
radiotherapy eliminates most tumour cells, but rare
radioresistant clones or a small fraction of tumour cells
remain and regrow over a period of time, albeit very
slowly. In this model, the extent of initial tumour-cell
killing is proportional to the time to tumour relapse, and
the involvement of host nontumour cells is not consid
ered. An alternative or complementary explanation is
that a state of equilibrium or dormancy exists that is gov
erned, at least in part, by external factors, such as tumour
angiogenesis or the state of the host immunesystem.
Radiation-induced tumour equilibrium. To investigate
the relative roles of both intrinsic tumour radiosensitiv
ity and the immune system, we have studied radiationinduced tumour equilibrium and dormancy, and the
contribution of adaptive and innate immunity to these
processes164. Specifically,we investigated the four possi
ble types of tumour response to radiotherapy using two
model systemsTUBO (HER2positive breast can
cer) and B16 (melanoma). Tumours that exhibited no
response to radiotherapy were deemed to have made an
early escape from immunosurveillance (non-respon
sive). In responsive tumours, regression was observed
as early as 10days post-radiation; stable tumours were
classified as those that regressed and remained stable
(and palpable) over an arbitrary 3460day observation
period; finally, late relapse occurred when formerly
stable tumours regrew at any point after the 60day
time point. In both tumour models, diverse responses
to radiation were observed. In TUBO tumours, 45%
(87/193) were deemed stable, but some of these stable
tumours eventually regrew (considered a late relapse).
A lower percentage of tumours were either not respon
sive to irradiation, or they completely regressed (part
of the responsive group). This spectrum of responses
mimics what has been observed in patients undergoing
radiotherapy. The tumours that responded differently to
irradiation (nonresponsive/early escape compared with
.
d
e
v
r
e
s
e
r
s
t
h
g
i
r
l
l
A
.
e
r
u
t
a
N
r
e
g
n
i
r
p
S
f
o
t
r
a
p
,
d
e
t
i
m
i
L
s
r
e
h
s
i
l
b
u
P
n
a
l
l
i
m
c
a
M
7
1
0
2
REVIEWS
Control
EE
R
Late relapsed
Stable
Survival cells (fraction)
1.0
0.5
0.0
Non-RT
2 Gy
Radiation dose
5 Gy
Figure 2 | Host immune responses, not the radiosensitivity of cancer cells, correlate
Nature Reviews | Clinical Oncology
with efficacy of radiation therapy (RT). We investigated the four possible types of
tumour response to radiotherapy using a TUBO (HER2positive breast cancer) mouse
model system164. Tumour cells had similar levels of sensitivity to radiation, despite
differential early tumour responses to RT invivo. NonRT control, EE (early escape), and R
(responsive) tumours were removed for analysis 8days after RT (or sham treatment); late
relapsed and stable tumours were removed for analysis at 21days post RT. After harvesting,
TUBO tumours were digested using collagenase and DNase to generate cell suspensions.
The cells were then treated exvivo with 0, 2, or 5Gy radiation and were cultured for 7days,
and an invitro clonogenic assay was performed. The data from the clonogenic assays are
displayed as the fraction of cells surviving after radiation treatment, compared with
number of colonies formed by the non-irradiated control cells from each type of tumour.
responsive at early time points (as early as 10days), or

stable versus late relapse) were surgically removed and
digested into single-cell suspensions. The plated TUBO
tumour-cell suspensions received 2, 5 or 10Gy irradi
ation exvivo and were assessed in clonogenic assays. The
plated cells derived from tumours that responded to irra
diation differentially invivo displayed indistinguishable
radiosensitivity exvivo (FIG.2). This finding suggested
that intrinsic cellular radiosensitivity does not account
for differences in tumour regrowth in some mouse mod
els, and possibly the same conclusion is true for some
human cancers. Of note, regions ofcell death coexisted
proximately with an abundance of CD8+ Tcells in irradi
ated stable tumours, and depletion of CD8+ Tcells led
to regrowth of the stable tumours. These results indicate
that CD8+Tcells might mediate tumour cell death in
TUBO tumours, and that radiation-induced tumour
equilibrium (RITE) might be a balance between cell
birth (indicated by the presence of cells positive for the
proliferation marker Ki67), and cell death (indicated
by terminal deoxynucleotidyl transferase dUTP nick
end-labelling (TUNEL)-positivity) that is mediated
principally by CD8+ Tcells (FIG.3). CD8+ Tcells medi
ate RITE through IFN; accordingly, neutralization of
IFN can break this equilibrium, leading to tumour
regrowth164. The finding that RITE exists in stable mouse
tumours, and perhaps in patients with nonprogressing
tumours, suggests that therapy-induced equilibrium
between cancer-cell division and killing by the host
immune system dictates the status of disease. These
data support the feasibility of treating stable disease by
tipping the balance (equilibrium) in favour of host anti
tumour immunity (host immune responses to cancer
therapy have recently been reviewed in thisjournal167).
In 2003, Zou and co-workers173,174 reported that

expression of programmed cell death 1 ligand 1 (PDL1)
in the tumour microenvironment led to Tcell exhaus
tion. Furthermore, Verbrugge etal.139 reported that,
in irradiated tumours, CD137 expression on tumour-
associated CD8+ Tcells was elevated, but was largely
restricted to a subset of Tcells with high expression of
PD1, whereas no increase in PDL1 expression was
noted on tumour cells after irradiation. Data from mul
tiple tumour models have indicated that PDL1 expres
sion is inducible in tumour cells or in host immune cells
(mainly DCs)37,175,176. We found that PDL1 expression
levels in stable tumours were elevated164. When PDL1
was blocked in mice harbouring stable tumours, most
of the tumours regressed, which further confirmed that
activation or restoration of antitumour immunity can
shift the balance towards eradication of nonprogress
ing tumours164. Notably, in a clinical trial involving 277
patients with any of a range of different cancer types177,
PDL1 expression on both tumour cells and immune
cells was a potent predictor of response to antiPDL1
therapy.
In a different study in a mouse model175, radiother
apy and delayed PD1/PDL1 therapy did not achieve
the same tumour-response effects compared with those
observed after administration of an antiPDL1 antibody
on the same day as radiotherapy. However, in the RITE
model, only tumours that initially respond to radiation
which enhances effector Tcell functionand subse
quently reach equilibrium (stable disease) were selected
for PDL1 blockade164. The goal of PDL1 blockade
in this setting was to shift the Tcells out of a state of
equilibrium. Although confirmation is required in the
clinical setting, we hypothesize that the RITE model is
applicable to some patients with radiotherapy-induced
stable tumours that do not progress for a long period of
time; such patients might have tumour resolution with
the use of immune-checkpoint blockade or immuno
modulators before the tumours relapse or enter the
escape phase. We note, however, that mouse models
are heterogeneous, and in our RITE model, not all mice
developed PDL1 expression leading toRITE.
Combining radiation and immunomodulation

The major clinical successes in the nascent field of
radioimmunomodulation are the result of the advent
of immune-checkpoint inhibitors. The immune system
has several checkpoint pathways that function to stimu
late or inhibit activation of some Tcell processes. Thus,
Chen and Han178 have cautioned against a simplistic
view of checkpoint inhibition, noting that the PD1
PDL1 pathway primarily regulates ongoing inflam
matory activity, whereas the CTLA4 pathway regulates
autoreactive Tcell responses. Over the past decade,
research into checkpoint-based therapies has evolved
from establishing efficacy to elucidating mechanisms
and improving patient selection. In an overview of sev
eral reports on immune-checkpoint therapies177,179182,
Wolchok and Chan183 noted that patients with a good
immune score and pre-existing tumour-specific Tcells
had better outcomes following immune-checkpoint
.
d
e
v
r
e
s
e
r
s
t
h
g
i
r
l
l
A
.
e
r
u
t
a
N
r
e
g
n
i
r
p
S
f
o
t
r
a
p
,
d
e
t
i
m
i
L
s
r
e
h
s
i
l
b
u
P
n
a
l
l
i
m
c
a
M
7
1
0
2
REVIEWS
a
2.5
**
Relative apoptosis
2.0
1.5
1.0
0.5
0.0
CD8+
Ki67+
Area
Figure 3 | Tumour-cell division coexists with apoptosis

in stable
tumours.
Nature
Reviews
| Clinical Oncology
a|Immunohistochemical assays of slides containing sequential slices of tumours from
mice that remained stable after radiotherapy, stained with antiKi67, anti-TUNEL (scale
bar: 50m), and antiCD8 antibodies; Ki67, TUNEL, and CD8 are markers of cell
proliferation, apoptosis, and cytotoxic Tcells, respectively. Analysis of the stain patterns
reveals that apoptotic cells are colocalized with CD8 Tcells, whereas proliferative cells
are generally located at distinct sites. b | Quantification of the relative intensity of TUNEL
staining in areas of the tumour containing CD8+ and Ki67+ cells. Three or four high-power
fields were counted per mouse (n=3). **P=0.001.
therapy. Vanpouille-Box and colleagues184 suggested

that, in patients who would otherwise not respond to
immune-checkpoint inhibitors, localized radiotherapy
can induce tumour-specific Tcells, thereby promoting
responses to these therapies.
Inhibition of the CTLA4 checkpoint. Inhibitors of
the CTLA4 pathway, such as ipilimumab, have shown
encouraging results in the treatment of patients with
cancer. CTLA4 functions as an immunosuppres
sor by increasing the signal intensity required for
CD8+ Tcells to engage target cells in the tumour 185,186.
Demaria etal.187 reported that treatment of mouse 4T1
primary mammary carcinomas with 12Gy irradiation
and CTLA4 blockade inhibited the formation of lung
metastases; they also noted that this therapeutic effect
required CD8+, but not CD4+, Tcells. A subsequent
study from the same group indicated that a hypofrac
tionated regimen (38Gy) plus anti-CTLA4 therapy
was more effective than either modality alone (as well
as single dose radiotherapy, or 56Gy radiation plus
CTLA4 blockade) in inducing an immune infiltrate and
an abscopal effect in a mouse model138. Furthermore,
results of early clinical studies of anti-CTLA4 antibodies
have demonstrated tumour regression and stable-disease
responses, and improved overall survival, primarily in
patients with melanoma, but also those with lymphoma,
or prostate or renal cancer 188196.
Anti-CTLA4 therapies have been tested in combi

nation with other treatments, including vaccines197201,
granulocyte-macrophage colony-stimulating factor
(GMCSF)197,202,203, other checkpoint inhibitors204,205,
radiotherapy 184,206,207 and chemotherapy 195,208212. For
example, a phaseIII trial (CA184043)207 has been per
formed to assess radiotherapy followed by ipilimumab
or placebo treatment (up to 2days later) in patients
with metastatic castration-resistant prostate cancer that
had progressed after docetaxel chemotherapy; while
the difference in overall survival between the two arms
was not statistically significant, the authors report that
post-hoc analyses of subgroups revealed a trend toward
improved overall survival on the ipilimumab study
arm (P=0.053) and significance in patients with bone
metastasis207. Demaria etal.186 reported that, in preclin
ical models, combined radiotherapy and antiCTLA4
treatment is efficacious owing to stress-induced NKG2D
(natural-killer group 2, member D) expression on
tumour cells that survive irradiation, which makes them
susceptible to NKcell-mediated cytotoxicity.
Inhibition of the PD1PDL1 checkpoint. PDL1 (the
ligand of PD1) is undetectable in most normal tissues
and is inducible in various cell types using inflammatory
cytokines213, especially typeI145 and typeII IFN214. PDL1
expression has been observed in a wide variety of solid
malignancies, and might be a dominant mechanism of
immunosuppression in some tumours37,174,215. Indeed,
the presence of PDL1 in the tumour is an important
predictor of patient responses to PD1/PDL1 block
ade. Inhibition of the PD1/PDL1 pathway on Tcells
has been associated with potent antitumour activity in
mouse tumour models and in clinical trials216229.
Building upon previous tumour equilibrium experi
ments, Deng etal.37 used a mouse flank tumour model
to evaluate the efficacy and mechanisms of action of
treatment with a single high dose of radiation and an
antiPDL1 antibody; substantial tumour regression was
observed in the combined treatment group. A dramatic
reduction of MDSCs (CD11b+Gr1+) was observed in
tumours treated with radiotherapy and antiPDL1
therapy, which was associated with increased CD8+
Tcell infiltration and priming 37. Conversely, when
CD8+ Tcells were depleted in the host, MDSC levels
recovered37. MDSC-mediated suppression of Tcell func
tion in autoimmune disease, viral infection, and cancer
progression is well-established, and MDSCs have been
associated with resistance to chemotherapy 116,230. Closer
examination of the interaction between Tcells and
MDSCs by immunofluorescence staining has revealed
that, in tumours treated with combined radiation and
antiPDL1 therapy, most of the remaining MDSCs were
localized adjacent to CD8+ Tcells and stained positive
for cleaved caspase 3, an apoptosis marker 37. In vitro
cytotoxicity assays revealed that activated CD8+ Tcells
can kill MDSCs directly 37: cytokines produced by acti
vated Tcells, especially TNF, can lead to apoptosis of
MDSCs. By contrast, although IFN production is cor
related with the activity of Tcells, IFN did not mediate
the induction of MDSC death in coculture assays37. The
.
d
e
v
r
e
s
e
r
s
t
h
g
i
r
l
l
A
.
e
r
u
t
a
N
r
e
g
n
i
r
p
S
f
o
t
r
a
p
,
d
e
t
i
m
i
L
s
r
e
h
s
i
l
b
u
P
n
a
l
l
i
m
c
a
M
7
1
0
2
REVIEWS
connection between Tcells, TNF, and MDSCs was veri
fied invivo: tumour control induced by radiotherapy and
antiPDL1antibody treatment was completely abro
gated in mice in which TNF was neutralized, or in which
MDSCs were depleted37; expression of exogenous TNF in
tumours via an adenoviral vector restored the antitumour
response, leading to tumour regression. Nevertheless,
data regarding the capacity of TNF to determine the fate
of MDSCs in tumours remain unclear, probably reflect
ing differences in the tumour models used and pheno
typic differences in the genetic plasticity of MDSCs231,232.
Another possibility is that PDL1 expression on MDSCs
not only contributes to their immunosuppressive capa
city, but also protects MDSCs from Tcell-mediated cyto
toxicity. This hypothesis is supported by the finding that
inhibition of PDL1 led to restoration of Tcell function
and susceptibility of MDSCs to Tcell-mediated apop
tosis, leading to an imbalance in favour of an immune
response (Tcells) over immunosuppression (MDSCs)37.
Dahan etal.233 noted that the reduction of myeloid-cell
populations in the tumour microenvironment following
treatment with antiPDL1 antibodies was governed by
both FcR-dependent and FcR-independent mechan
isms in an MC38 mouse model. In this study 233, engage
ment of activatory FcRs mediated the reduction of
immunosuppressive CD11b+F4/80+ cell numbers in the
tumour microenvironment and potentiated antiPDL1
treatment.
These reports suggest that, in preclinical models,
inhibition of the PD1PDL1 checkpoint combined
with radiotherapy liberates T cells from immuno
suppression, which in turn positively alters the tumour
microenvironment owing to killing of suppressive cells
via cytokine secretion. Several mouse tumour models
have been used to demonstrate the synergistic effect of
radiotherapy and immunotherapy via checkpoint inhib
ition in solid tumours. In an orthotopic brain tumour
mouse model, Zeng etal.234 showed that stereotactic
radiation combined with antiPD1 antibody therapy
markedly increased survival compared with either treat
ment alone. Higher levels of CD8+ Tcells and lower
Tregcell infiltration were observed in tumours treated
with combination therapy than in those treated with
the monotherapies234. Vanpouille-Box and co-authors45
reported improved survival in a mouse tumour model
after treatment with antiPD1 antibodies combined
with TGF blockade plus radiation-induced vaccination,
compared with radiation-induced vaccination plus TGF
blockade alone. Radiation-induced vaccination involves
conversion of the tumour into an insitu vaccine by
inducing immunogenic death of cancer cells using radi
ation, in order to promote a pro-immunogenic tumour
microenvironment and thereby improve the priming of
tumour-specific Tcells235. Notably, the results indicate
that TGF controls the ability of radiotherapy to induce
this insitu state45. Data from Sharabi etal.39 indicate that
radiation and antiPD1 therapy increases memory CD8+
Tcell numbers. Although NK cells might contribute to
local tumour control, CD8+ Tcells have been shown
to be required for the antitumour effects produced by
combined r adiotherapy and antiPDL1 antibodies175.
A preliminary preclinical report from the Drake lab

indicates that radiotherapy combined with antiPD1
antibody treatment can result in primary tumour con
trol and an abscopal effect 236. More-recent data from
the same group indicate that this therapy combination
results in induction of endogenous antigen-specific
immune responses, resulting in improved local control
in single tumour models of melanoma or breast carci
noma42; however, no experiments on the abscopal effect
were reported42. Dovedi etal.237 subsequently noted that
the timing of antiPDL1 blockade is crucial; concurrent
radiation and antiPDL1 treatment, but not sequential
treatment, resulted in long-term tumour control.
Currently, over a dozen clinical trials are evaluating
antiPD1 and antiPDL1 antibodies in combination
with radiation for cancer treatment, but results are not
yet available (TABLE1). Some encouraging results have
been reported in clinical trials evaluating antiPD1
or antiPDL1 antibodies alone for cancer immuno
therapy 181,205,215,220,238. For example, Tumeh et al. 181
reported that proliferation of CD8+ Tcells in the tumour
was associated with therapeutic response in patients
with metastatic melanoma undergoing antiPD1 ther
apy alone. In a phaseIII trial of the antiPD1 antibody
nivolumab in patients with previously untreated mela
noma, 72.9% of patients achieved 1year overall survival,
compared with 42.1% of those who received standard
dacarbazine chemotherapy 220. Combination check
point-blockade treatments are also being tested. For
instance, Larkin etal.205 have reported the results of a trial
in which treatment with nivolumab alone or nivolumab
combined with ipilimumab increased progression-free
survival compared with ipilimumab monotherapy.
The abscopal effect. The abscopal effect, first described
in the 1950s by Mole239, refers to regression or dis
appearance of lesions outside of the irradiated field. This
phenomenon has rarely been observed in routine clinical
practice when radiation therapy is administered alone240.
With the advent of immune modifiers, however, this
effect has been increasingly reported in both preclini
cal models and patients27,31,37,40,4345,138,241,242. Demaria and
colleagues27 were the first to propose that the abscopal
effect is an immune-driven phenomenon, indicating that
local radiotherapy produces systemic effects. Demaria,
Formenti and colleagues138,187 subsequently reported that
localized radiotherapy and CTLA4 blockade inhibited
the development of lung metastases, and that hypo
fractionated radiation plus CTLA4 blockade resulted
in an abscopal effect in a mouse tumour model. Deng
etal.37 reported that local radiotherapy and systemic
PDL1 blockade also augmented Tcell responses in the
primary tumour and at distant sites in a mouse model.
Similarly, radiotherapy and antiPD1 antibody treat
ment has reportedly produced abscopal effects in mouse
models of melanoma, renal-cell carcinoma, adenocarci
noma, and other carcinoma models44,45. Of note, Park
etal.44 demonstrated that radiotherapy and antiPD1
treatment resulted in almost complete regression of the
primary tumours treated and a 66% reduction in distant
tumours via abscopal responses.
.
d
e
v
r
e
s
e
r
s
t
h
g
i
r
l
l
A
.
e
r
u
t
a
N
r
e
g
n
i
r
p
S
f
o
t
r
a
p
,
d
e
t
i
m
i
L
s
r
e
h
s
i
l
b
u
P
n
a
l
l
i
m
c
a
M
7
1
0
2
REVIEWS
Table 1 |Clinical studies of PD1/PDL1 immune-checkpoint blockade in combination with radiotherapy
ClinicalTrials.gov
identifier; year
opened; sponsor
Drug
Type of cancer
Estimated
enrolment; phase
and end point
classification
Interventions
Outcome/
results
Pidilizumab
(developed as
an antiPD1
antibody; true
target unknown)
Diffuse intrinsic
pontine glioma
(paediatric)
PhaseI: 6 phaseII:
15; safety/ efficacy
study
Following RT (no details provided),

biweekly pidilizumab (phaseI), plus
weekly CTX (phaseII)
None;
estimated
completion
April 2019
NCT02289209; 2014;
Pembrolizumab
University of Maryland, (antiPD1
with MSD.
antibody)
Locoregional
inoperable
recurrence or
secondprimary
SCCHN
48; phaseII safety/

efficacy study
Reirradiation with 1.2Gy for 5days

per week for 5weeks, followed by
pembrolizumab every 3weeks
None;
estimated
completion
Dec. 2018
NCT02298946; 2014;
NCI (USA)
AMP224 (a PD1
inhibitor) Fc fusion
protein
Colorectal cancer
with liver metastases
17; phaseI safety/

efficacy study
Arm 1: 8Gy SBRT to liver metastases

and CTX on day 0, followed by AMP224
on day 1 and then every 2weeks
Arm 2: As in arm 1, but with SBRT with
8Gy on days -2, -1, and 0
None;
estimated
completion
Nov. 2017
NCT02303366; 2014;
Peter MacCallum
Cancer Centre
Pembrolizumab
Oligometastatic
breast cancer
15; phaseI safety

study
SABR at 20Gy to at least one metastasis

None;
(max. of five), followed by pembrolizumab estimated
every 3weeks
completion
Sept. 2020
NCT02303990;
2014; Abramson
Cancer Center of
the University of
Pennsylvania
Pembrolizumab
Metastatic cancer
70; phaseI
treatment study
Hypofractionated RT and pembrolizumab None;

(no details of regimen provided)
estimated
completion
Feb. 2017
NCT02383212; 2015;
Regeneron
REGN2810
(antiPD1
antibody)
Advanced
malignancies
60; phaseI safety

study
REGN2810 alone or with

hypofractionated and/or CTX (no details
of regimen provided)
None;
estimated
completion
Oct. 2018
NCT02407171; 2015;
Yale University
Pembrolizumab
Metastatic
melanoma, NSCLC
60; phaseI/phaseII
safety/ efficacy
study
Pembrolizumab every 2weeks after SBRT

(30Gy in 3 or 5 fractions, or 10Gy in 1
fraction)
None;
estimated
completion
Dec. 2018
NCT02434081; 2015;
European Thoracic
Oncology Platform,
with BMS and the
Frontier Science
Foundation
Nivolumab
(antiPD1
antibody)
Stage III NSCLC
43; phaseII safety/

efficacy study
Nivolumab after standard

chemoradiotherapy
Not open for

enrolment;
estimated
completion
Aug. 2019
NCT02560636; 2015;
Royal Marsden NHS
Foundation, Institute
of Cancer Research,
NIHR (UK), with MSD
Pembrolizumab
Invasive (group A) or
metastatic (group B)
bladder cancer
34; phaseI safety

study
Pembrolizumab after hypofractionated

RT (no details of schedule provided)
Not open for

enrolment;
estimated
completion
Jan. 2019
NCT02587455; 2015;
Royal Marsden NHS
Foundation, Institute
of Cancer Research,
NIHR (UK), with MSD
Pembrolizumab
Lung cancer
48; phaseI safety

study
Pembrolizumab after standard palliative

low-dose or high-dose radiotherapy
Not open for

enrolment;
estimated
completion
Jan. 2018
NCT02599779; 2015;
Sunnybrook Health
Sciences Centre, with
MSD and Ozmosis
Research
Pembrolizumab
Metastatic renal cell

cancer (refractory
to approved
first-line therapy
with sunitinib or
pazopanib)
35; phaseII safety/

efficacy study
Pembrolizumab every 3weeks, with SBRT

given at the time of progression or before
the 2nd course of pembrolizumab
Not open for

enrolment;
estimated
completion
Jan. 2019
NCT02608385; 2015;
University of Chicago
Pembrolizumab
NSCLC; advanced
solid tumours
138; phaseI safety

study
Pembrolizumab every 3weeks after SBRT

to metastases (3 or 5 fractions depending
on location of metastases)
None;
estimated
completion
Dec. 2017.
PD1 inhibitors
NCT01952769; 2014;
Hadassah Medical
Organization
.
d
e
v
r
e
s
e
r
s
t
h
g
i
r
l
l
A
.
e
r
u
t
a
N
r
e
g
n
i
r
p
S
f
o
t
r
a
p
,
d
e
t
i
m
i
L
s
r
e
h
s
i
l
b
u
P
n
a
l
l
i
m
c
a
M
7
1
0
2
REVIEWS
Table 1 (cont.) |Clinical studies of PD1/PDL1 immune-checkpoint blockade in combination with radiotherapy
ClinicalTrials.gov
identifier; year
opened; sponsor
Drug
Type of cancer
Estimated
enrolment; phase
and end point
classification
Interventions
Outcome/
results
NCT02621398; 2015;
Rutgers Cancer
Institute of New Jersey,
Rutgers University,
with NCI (USA)
Pembrolizumab
NSCLC
30; phaseI safety

study
Pembrolizumab every 3weeks,

starting 26weeks after initiating
chemoradiotherapy, 2weeks before
the end of chemoradiotherapy, or
at the start of chemoradiotherapy;
chemoradiotherapy comprises 3DCRT
or IMRT for 5days a week over 6weeks,
and paclitaxel and carboplatin on days 1,
8, 15, 22, 29, and 36.
Not open for

enrolment;
estimated
completion
Sept. 2019
NCT02642809; 2015;
Washington University
School of Medicine,
with MSD
Pembrolizumab
Metastatic
oesophageal cancers
(initial treatment)
15; phase I safety/

efficacy study
Pembrolizumab every 3weeks,

started within 1week ( 3days) after
brachytherapy (8Gy x 2, with 710days
between fractions)
Not open for

enrolment;
estimated
completion
Jan. 2019
NCT02400814;
2015; University of
California, Davis,
with NCI (USA) and
Genentech
Atezolizumab
(antiPDL1
antibody)
Stage IV NSCLC
45; phaseI safety/

efficacy study
Atezolizumab every 3weeks, with

SBRT 23 times per week (with 4096hr
between fractions) over 1.52weeks
(total of 5 fractions) beginning on
day 1 of the first or third course of
atezolizumab
Not open for

enrolment;
estimated
completion
Jan. 2019
NCT02584829; 2015;
Fred Hutchinson
Cancer Research
Center, and NCI (USA),
with EMD Serono
Avelumab
(antiPDL1
antibody)
Metastatic
Merkel-cell
carcinoma
20; phaseI/phaseII
safety/ efficacy
study
Avelumab every 2weeks for 6months,

with either RT or IFN, or RT or IFN plus
ACT. RT and IFN given within 710days
after completion of 13 doses of
avelumab, and ACT performed 25days
after RT or IFN
None;
estimated
completion
Jan. 2019
NCT02463994; 2015;
University of Michigan
Cancer Center, with
the University of
Washington
Atezolizumab
Metastatic NSCLC
12; phase 0
treatment study
Atezolizumab every 3weeks after

hypofractionated image-guided RT (no
details provided)
None;
estimated
completion
July 2020
NCT02525757;
2015; MD Anderson
Cancer Center, with
Genentech
Atezolizumab
Lung cancer,
including non-small
cell
40; phaseII safety/

efficacy study
Arm 1: Standard carboplatin and

paclitaxel chemotherapy plus RT
(6066Gy in 2Gy fractions 5days a
week) for 67weeks, followed by at
least 2 cycles of atezolizumab starting
34weeks later (for up to 1year)
Arm 2: Chemoradiotherapy (as
in arm 1) plus atezolizumab for
67weeks, followed by a rest period of
34weeks, during which only 1 dose
of atezolizumab will be given. After
the rest period, atezolizumab plus
chemotherapy will be continued for 2
cycles, followed by atezolizumab alone
for up to 1year.
Not open for

enrolment;
estimated
completion
Dec. 2018
NCT02336165; 2015;
Ludwig Institute for
Cancer Research,
the Cancer Research
Institute, New York,
and Cure Brain Cancer
Foundation, Australia,
with MedImmune
Durvalumab
(antiPDL1
antibody)
Glioblastoma
108; phaseII
safety/efficacy
study
Durvalumab every 2weeks either alone,

with standard RT, or with bevacizumab
(anti-VEGF antibody) at two different
doses
None;
estimated
completion
July 2017
PD1 inhibitors (cont.)
PDL1 inhibitors
An advanced search of ClinicalTrials.gov performed in December 2015 for PD1 and radiation and Interventional Studies retrieved (34 records), and the
same search using PDL1 and radiation retrieved 29 records. These were reviewed for inclusion in this summary Table. Studies were excluded if they did not
include administration of radiation. 3D CRT, three-dimensional conformal radiation therapy; BID, twice per day; CTX: cyclophosphamide; IFN, interferon ;
IMRT, intensity-modulated radiation therapy; MSD, Merck, Sharp and Dohme; NCI, National Cancer Institute; NIHR, National Institute for Health Research;
NSCLC: non-small-cell lung cancer; PD1, programmed cell-death protein 1; PDL1, programmed cell death1 ligand 1; RT, radiotherapy; SABR, stereotactic
ablative body radiosurgery; SBRT, stereotactic body radiotherapy; SCCHN, squamous cell carcinoma of the head and neck; VEGF, vascular endothelial
growthfactor.
.
d
e
v
r
e
s
e
r
s
t
h
g
i
r
l
l
A
.
e
r
u
t
a
N
r
e
g
n
i
r
p
S
f
o
t
r
a
p
,
d
e
t
i
m
i
L
s
r
e
h
s
i
l
b
u
P
n
a
l
l
i
m
c
a
M
7
1
0
2
REVIEWS
In the clinical setting, Postow etal.241 reported that
palliative radiotherapy (28.5Gy in three fractions deliv
ered over 7days) given concurrently with maintenance
ipilimumab treatment in a patient with melanoma caused
regression of the targeted lesion as well as marked absco
pal effects. Since this report was published, several case
reports have highlighted similar findings in patients
treated with ipilimumab243247. In the first clinical trial
testing a treatment regimen designed to generate abscopal
responses, comprising radiotherapy and GMCSF, 27% of
patients with metastatic solid tumours experienced such
an effect 43. In preclinical studies, cell lines derived from
human metastatic melanoma tumours that relapsed after
radiotherapy plus CTLA4 blockade were injected into
PDL1 knockout mice and treated with radiotherapy and
anti-CTLA4 with or without antiPDL1176; the PDL1
knockout mice were not only sensitive to the combination
of radiotherapy and anti-CTLA4, but they also achieved
superior antitumour response to the triple combination
treatment of radiotherapy, anti-CTLA4 and antiPDL1.
These data indicate that anti-CTLA4 and anti
PDL1work through nonredundant m
echanisms to
liberate Tcells from immunosuppression176.
Although Demaria, Formenti and colleagues27,138,187
were the first to suggest that Tcell effector function
within the irradiated tumour microenvironment is a
driver of the abscopal effect, Kaminski etal.248 postu
lated that cytokine release was also a major mechanism,
and advocated for consideration of similarities with the
bystander effect as a means to further elucidate associ
ated mechanisms of action. Supporting this hypothesis,
Deng etal.37 found that the production of TNF by acti
vated Tcells in the irradiated tumour microenviron
ment led to direct elimination of MDSCs locally, and
a concomitant reduction in systemic MDSC numbers.
Furthermore, this finding complements the clinical
observation from the Wolchok group241 that a patient on
an immune-checkpoint inhibitor (CTLA4) undergoing
palliative radiotherapy experienced abscopal regression
of a distant unirradiated tumour, which was preceded
1. Orth,M. etal. Current concepts in clinical radiation
oncology. Radiat. Environ. Biophys. 53, 129 (2014).
2. Hoskin,P.J. & Bhattacharya,I.S. Protons and more:
state of the art in radiotherapy. Clin. Med. (Lond.) 14,
s61s65 (2014).
3. Bast,R.C. Holland-Frei Cancer Medicine 9th edn
(John Wiley & Sons, 2016).
4. Kufe,D.W. etal. Cancer Medicine 6th edn (BC Decker,
2003).
5. Regaud,C. The influence of the duration of irradiation
on the changes produced in the testicle by radium.
Int.J.Radiat. Oncol. Biol. Phys. 2, 565567 (1977).
6. Coutard,H. Principles of x ray therapy of malignant
diseases. Lancet 224, 18 (1934).
7. Coutard,H. The results and methods of treatment of
cancer by radiation. Ann. Surg. 106, 584598 (1937).
8. Whelan,T.J. etal. Long-term results of
hypofractionated radiation therapy for breast cancer.
N.Engl. J.Med. 362, 513520 (2010).
9. Adebahr,S. etal. LungTech, an EORTC phase II trial
ofstereotactic body radiotherapy for centrally located
lung tumours: a clinical perspective. Br. J.Radiol. 88,
20150036 (2015).
10. Hellman,S. & Weichselbaum,R.R. Oligometastases.
J.Clin. Oncol. 13, 810 (1995).
11. Okunieff,P. etal. Stereotactic body radiation therapy
(SBRT) for lung metastases. Acta Oncol. 45, 808817
(2006).
immediately by a sharp reduction in the proportion of

MDSCs in the peripheral blood mononuclear cell popu
lation. These results indicate that decreasing MDSC
levels could be a critical step in achieving a shift towards
induction of optimal immune response via combina
tion treatment with radiation and immune-checkpoint
inhibitors.
Collectively, these observations indicate that the
molecular and cellular events generating the abscopal
effect are the result of a cellular feedback mechanism
involving effector Tcells within the irradiated tumour
microenvironment, which occurs several days follow
ing local radiotherapy and subsequent to APC migration
and Tcell activation in the draining lymph nodes. Thus,
the abscopal effect could be modulated by tipping the
balance between positive immune-regulators (of Tcell
function) and negative regulators (of the local and sys
temic suppressive microenvironment) in order to elicit
a strong immune response.
Conclusions
Current evidence indicates that radiotherapy can, via
diverse mechanisms, invoke both local and systemic
immune responses, which can either support tumourcell survival or promote tumour-cell death. The equi
librium between these effects of radiation is postulated
to have a key role in determining the ultimate outcome
of treatment. Thus, enhancing innate and adaptive
immunity by combining radiotherapy and immuno
therapy is a crucial strategy to improve patient survival.
Current paradigms of radioimmunotherapy are based
on results from animal models, which are inherently
limited in their applicability to the clinical setting, as
well as observations of responses in patients, and pre
liminary data from trials of combination immunother
apy and radiotherapy. These data, although interesting,
do not yet justify implementation in clinical practice;
robust hypothesis-testing clinical trials are required to
determine the appropriate approach to integrating these
modalities.
12. Milano,M.T., Philip,A. & Okunieff,P. Analysis of

patients with oligometastases undergoing two or more
curative-intent stereotactic radiotherapy courses.
13. Salama,J.K. etal. Stereotactic body radiotherapy for
multisite extracranial oligometastases: final report of a
dose escalation trial in patients with 1 to 5 sites of
metastatic disease. Cancer 118, 29622970 (2012).
14. Fumagalli,I. etal. A single-institution study of
stereotactic body radiotherapy for patients with
unresectable visceral pulmonary or hepatic
oligometastases. Radiat. Oncol. 7, 164 (2012).
15. Corbin,K.S., Hellman,S. & Weichselbaum,R.R.
Extracranial oligometastases: a subset of metastases
curable with stereotactic radiotherapy. J.Clin. Oncol.
31, 13841390 (2013).
16. Kao,J. etal. Concurrent sunitinib and stereotactic
body radiotherapy for patients with oligometastases:
final report of a prospective clinical trial. Target Oncol.
9, 145153 (2014).
17. Decaestecker,K. etal. Surveillance or metastasisdirected Therapy for OligoMetastatic Prostate cancer
recurrence (STOMP): study protocol for a randomized
phaseII trial. BMC Cancer 14, 671 (2014).
18. Niibe,Y. etal. Stereotactic body radiotherapy results
for pulmonary oligometastases: a two-institution
collaborative investigation. Anticancer Res. 35,
49034908 (2015).
19. Norkus,D. etal. A randomized hypofractionation

dose escalation trial for high risk prostate cancer
patients: interim analysis of acute toxicity and
qualityof life in 124 patients. Radiat. Oncol. 8, 206
(2013).
20. Loblaw,A. etal. Prostate stereotactic ablative body
radiotherapy using a standard linear accelerator:
toxicity, biochemical, and pathological outcomes.
Radiother. Oncol. 107, 153158 (2013).
21. Viani,G.A. etal. Acute toxicity profile in prostate
cancer with conventional and hypofractionated
treatment. Radiat. Oncol. 8, 94 (2013).
22. Aluwini,S. etal. Hypofractionated versus
conventionally fractionated radiotherapy for patients
with prostate cancer (HYPRO): acute toxicity results
from a randomised non-inferiority phase 3 trial.
Lancet. Oncol. 16, 274283 (2015).
23. Anand,A.K. etal. Hypofractionated stereotactic
body radiotherapy in spinal metastasis - with or
without epidural extension. Clin. Oncol. 27, 345352
(2015).
24. Crane,C.H. Hypofractionated ablative radiotherapy
for locally advanced pancreatic cancer. J.Radiat. Res.
http://dx.doi.org/10.1093/jrr/rrw016 (2016).
25. Hellstrom,K.E., Hellstrom,I., Kant,J.A. &
Tamerius,J.D. Regression and inhibition of sarcoma
growth by interference with a radiosensitive Tcell
population. J.Exp. Med. 148, 799804 (1978).
.
d
e
v
r
e
s
e
r
s
t
h
g
i
r
l
l
A
.
e
r
u
t
a
N
r
e
g
n
i
r
p
S
f
o
t
r
a
p
,
d
e
t
i
m
i
L
s
r
e
h
s
i
l
b
u
P
n
a
l
l
i
m
c
a
M
7
1
0
2
REVIEWS
26. North,R.J. Radiation-induced, immunologically
mediated regression of an established tumor as an
example of successful therapeutic immunomanipulation.
Preferential elimination of suppressor Tcells allows
sustained production of effector Tcells. J.Exp. Med.
164, 16521666 (1986).
27. Demaria,S. etal. Ionizing radiation inhibition of
distant untreated tumors (abscopal effect) is immune
mediated. Int. J.Radiat. Oncol. Biol. Phys. 58,
862870 (2004).
28. Demaria,S., Bhardwaj,N., McBride,W.H. &
Formenti,S.C. Combining radiotherapy and
immunotherapy: a revived partnership. Int. J.Radiat.
Oncol. Biol. Phys. 63, 655666 (2005).
29. Lugade,A.A. etal. Local radiation therapy of B16
melanoma tumors increases the generation of tumor
antigen-specific effector cells that traffic to the tumor.
J.Immunol. 174, 75167523 (2005).
30. Zhang,B. etal. Induced sensitization of tumor stroma
leads to eradication of established cancer by Tcells.
J.Exp. Med. 204, 4955 (2007).
31. Lee,Y. etal. Therapeutic effects of ablative radiation
on local tumor require CD8+ Tcells: changing
strategies for cancer treatment. Blood 114, 589595
(2009).
32. Burnette,B., Fu,Y.X. & Weichselbaum,R.R.
Theconfluence of radiotherapy and immunotherapy.
Frontiers Oncol. 2, 143 (2012).
33. Frey,B. etal. Induction of abscopal anti-tumor
immunity and immunogenic tumor cell death by
ionizing irradiation - implications for cancer therapies.
Curr. Med. Chem. 19, 17511764 (2012).
34. Formenti,S.C. & Demaria,S. Combining radiotherapy
and cancer immunotherapy: a paradigm shift.
J.NatlCancer Institute 105, 256265 (2013).
35. Burnette,B. & Weichselbaum,R.R. Radiation as
animmune modulator. Semin. Radiat. Oncol. 23,
273280 (2013).
36. Demaria,S. & Formenti,S.C. Radiotherapy effects
onanti-tumor immunity: implications for cancer
treatment. Frontiers Oncol. 3, 128 (2013).
37. Deng,L. etal. Irradiation and antiPDL1 treatment
synergistically promote antitumor immunity in mice.
J.Clin. Invest. 124, 687695 (2014).
38. Lim,J.Y., Gerber,S.A., Murphy,S.P. & Lord,E.M.
Type I interferons induced by radiation therapy
mediate recruitment and effector function of CD8+
Tcells. Cancer Immunol. Immunother. 63, 259271
(2014).
39. Sharabi,A.B. etal. Stereotactic radiation therapy
augments antigen-specific PD1mediated antitumor
immune responses via cross-presentation of tumor
antigen. Cancer Immunol. Res. 3, 345355
CIR140196 (2015).
40. Seetharam,S. etal. Enhanced eradication of local and
distant tumors by genetically produced interleukin12
and radiation. Int. J.Oncol. 15, 769773 (1999).
41. Formenti,S.C. etal. Abscopal response in irradiated
patients: results of a proof of principle trial.
Int.J.Radiat. Oncol. Biol. Phys. 72, S6S7 (2008).
42. Sharabi,A.B., Tran,P.T., Lim,M., Drake,C.G.
&Deweese,T.L. Stereotactic radiation therapy
combined with immunotherapy: augmenting the
roleof radiation in local and systemic treatment.
Oncol. (Williston Park) 29, 331340 (2015).
43. Golden,E.B. etal. Local radiotherapy and
granulocyte-macrophage colony-stimulating factor
togenerate abscopal responses in patients with
metastatic solid tumours: a proofofprinciple trial.
Lancet. Oncol. 16, 795803 (2015).
44. Park,S.S. etal. PD1 restrains radiotherapy-induced
abscopal effect. Cancer Immunol. Res. 3, 610619
(2015).
45. Vanpouille-Box,C. etal. TGF Is a master regulator
ofradiation therapy-induced antitumor immunity.
Cancer Res. 75, 22322242 (2015).
46. Bunt,S.K. etal. Reduced inflammation in the tumor
microenvironment delays the accumulation of myeloidderived suppressor cells and limits tumor progression.
Cancer Res. 67, 1001910026 (2007).
47. Meng,Y. etal. Blockade of tumor necrosis factor
signaling in tumor-associated macrophages as a
radiosensitizing strategy. Cancer Res. 70, 15341543
(2010).
48. Ahn,G.O. etal. Inhibition of Mac1 (CD11b/CD18)
enhances tumor response to radiation by reducing
myeloid cell recruitment. Proc. Natl Acad. Sci. USA
107, 83638368 (2010).
49. Chiang,C.S. etal. Irradiation promotes an m2
macrophage phenotype in tumor hypoxia.
FrontiersOncol. 2, 89 (2012).
50. Schaue,D. & McBride,W.H. T lymphocytes and

normal tissue responses to radiation. Frontiers Oncol.
2, 119 (2012).
51. Zitvogel,L. & Kroemer,G. Subversion of anticancer
immunosurveillance by radiotherapy. Nat. Immunol.
16, 10051007 (2015).
52. Stone,H.B., Peters,L.J. & Milas,L. Effect of host
immune capability on radiocurability and subsequent
transplantability of a murine fibrosarcoma. J.Natl
Cancer Institute 63, 12291235 (1979).
53. Viaud,S. etal. Cyclophosphamide induces
differentiation of Th17 cells in cancer patients.
CancerRes. 71, 661665 (2011).
54. Gupta,A. etal. Radiotherapy promotes tumor-specific
effector CD8+ Tcells via dendritic cell activation.
J.Immunol. 189, 558566 (2012).
55. Demaria,S. & Formenti,S.C. Role of T lymphocytes
intumor response to radiotherapy. Frontiers Oncol. 2,
95 (2012).
56. Gerber,S.A. etal. IFN- mediates the antitumor
effects of radiation therapy in a murine colon tumor.
Am. J.Pathol. 182, 23452354 (2013).
57. Gerber,S.A. etal. Radio-responsive tumors
exhibitgreater intratumoral immune activity than
nonresponsive tumors. Int. J.Cancer 134, 23832392
(2014).
58. Yoshimoto,Y. etal. Radiotherapy-induced anti-tumor
immunity contributes to the therapeutic efficacy of
irradiation and can be augmented by CTLA4 blockade
in a mouse model. PLoS ONE 9, e92572 (2014).
59. Filatenkov,A. etal. Ablative tumor radiation can
change the tumor immune cell microenvironment to
induce durable complete remissions. Clin. Cancer Res.
21, 37273739 (2015).
60. McBride,W.H. etal. A sense of danger from radiation.
Radi. Res. 162, 119 (2004).
61. Schaue,D., Kachikwu,E.L. & McBride,W.H.
Cytokines in radiobiological responses: a review.
Radiat. Res. 178, 505523 (2012).
62. Burnette,B.C. etal. The efficacy of radiotherapy relies
upon induction of type I interferon-dependent innate
and adaptive immunity. Cancer Res. 71, 24882496
(2011).
63. Liu,C. etal. Gamma-ray irradiation impairs dendritic
cell migration to CCL19 by down-regulation of CCR7
and induction of cell apoptosis. Int. J.Biol. Sci. 7,
168179 (2011).
64. Manda,K., Glasow,A., Paape,D. & Hildebrandt,G.
Effects of ionizing radiation on the immune system
with special emphasis on the interaction of dendritic
and Tcells. Frontiers Oncol. 2, 102 (2012).
65. Foulds,G.A., Radons,J., Kreuzer,M., Multhoff,G.
&Pockley,A.G. Influence of tumors on protective
anti-tumor immunity and the effects of irradiation.
66. Park,B., Yee,C. & Lee,K.M. The effect of radiation on
the immune response to cancers. Int. J.Mol. Sci. 15,
927943 (2014).
67. Zitvogel,L., Kepp,O. & Kroemer,G. Decoding cell
death signals in inflammation and immunity. Cell 140,
798804 (2010).
68. Krysko,D.V. etal. Immunogenic cell death and
DAMPs in cancer therapy. Nat. Rev. Cancer 12,
860875 (2012).
69. Krysko,O., Love Aaes,T., Bachert,C.,
Vandenabeele,P. & Krysko,D.V. Many faces of
DAMPs in cancer therapy. Cell Death Dis. 4, e631
(2013).
70. Obeid,M. etal. Calreticulin exposure dictates the
immunogenicity of cancer cell death. Nat. Med. 13,
5461 (2007).
71. Golden,E.B. etal. Radiation fosters dose-dependent
and chemotherapy-induced immunogenic cell death.
Oncoimmunology 3, e28518 (2014).
72. Hallahan,D.E., Spriggs,D.R., Beckett,M.A.,
Kufe,D.W. & Weichselbaum,R.R. Increased tumor
necrosis factor mRNA after cellular exposure to
ionizing radiation. Proc. Natl Acad. Sci. USA 86,
1010410107 (1989).
73. Vlodavsky,I. etal. Extracellular matrix-resident
growth factors and enzymes: possible involvement
intumor metastasis and angiogenesis.
CancerMetastasis Rev. 9, 203226 (1990).
74. Hallahan,D.E. etal. Tumor necrosis factor gene
expression is mediated by protein kinase C following
activation by ionizing radiation. Cancer Res. 51,
45654569 (1991).
75. Apetoh,L. etal. Toll-like receptor 4dependent
contribution of the immune system to anticancer
chemotherapy and radiotherapy. Nat. Med. 13,
10501059 (2007).
76. Fu,Y. etal. Resveratrol inhibits ionising irradiationinduced inflammation in MSCs by activating SIRT1
and limiting NLRP3 inflammasome activation.
Int.J.Mol. Sci. 14, 1410514118 (2013).
77. Pasi,F., Paolini,A., Nano,R., Di Liberto,R. &
Capelli,E. Effects of single or combined treatments
with radiation and chemotherapy on survival and
danger signals expression in glioblastoma cell lines.
Biomed Res. Int. 2014, 453497 (2014).
78. Matsumura,S. etal. Radiation-induced CXCL16
release by breast cancer cells attracts effector Tcells.
J.Immunol. 181, 30993107 (2008).
79. Meng,Y. etal. Ad. Egr-TNF and local ionizing radiation
suppress metastases by interferon--dependent
activation of antigen-specific CD8+ Tcells. Mol. Ther.
18, 912920 (2010).
80. Taieb,J. etal. A novel dendritic cell subset involved in
tumor immunosurveillance. Nat. Med. 12, 214219
(2006).
81. Chakraborty,M. etal. External beam radiation of
tumors alters phenotype of tumor cells to render
themsusceptible to vaccine-mediated Tcell killing.
Cancer Res. 64, 43284337 (2004).
82. Garnett,C.T. etal. Sublethal irradiation of human
tumor cells modulates phenotype resulting in
enhanced killing by cytotoxic T lymphocytes.
CancerRes. 64, 79857994 (2004).
83. Reits,E.A. etal. Radiation modulates the peptide
repertoire, enhances MHC classI expression, and
induces successful antitumor immunotherapy.
J.Exp.Med. 203, 12591271 (2006).
84. Characiejus,D. etal. Prognostic significance of
peripheral blood CD8highCD57+ lymphocytes in
bladder carcinoma patients after intravesical IL2.
Anticancer Res. 31, 699703 (2011).
85. Characiejus,D. etal. Prediction of response in cancer
immunotherapy. Anticancer Res. 31, 639647 (2011).
86. Fridman,W.H., Pages,F., Sautes-Fridman,C. &
Galon,J. The immune contexture in human tumours:
impact on clinical outcome. Nat. Rev. Cancer 12,
298306 (2012).
87. Liu,S. etal. CD8+ lymphocyte infiltration is an
independent favorable prognostic indicator in basallike breast cancer. Breast Cancer Res. 14, R48
(2012).
88. Anitei,M.G. etal. Prognostic and predictive values
ofthe immunoscore in patients with rectal cancer.
Clin.Cancer Res. 20, 18911899 (2014).
89. Dushyanthen,S. etal. Relevance of tumor-infiltrating
lymphocytes in breast cancer. BMC Med. 13, 202
(2015).
90. Morvan,M.G. & Lanier,L.L. NK cells and cancer: you
can teach innate cells new tricks. Nat. Rev. Cancer 16,
719 (2015).
91. Bauer,S. etal. Activation of NK cells and Tcells by
NKG2D, a receptor for stress-inducible MICA. Science
285, 727729 (1999).
92. Kim,J.Y. etal. Increase of NKG2D ligands and
sensitivity to NK cell-mediated cytotoxicity of
tumorcells by heat shock and ionizing radiation.
Exp.Mol. Med. 38, 474484 (2006).
93. Pilones,K.A. etal. Invariant natural killer Tcells
regulate breast cancer response to radiation and
CTLA4 blockade. Clin. Cancer Res. 15, 597606
(2009).
94. Gaipl,U.S. etal. Kill and spread the word: stimulation
of antitumor immune responses in the context of
radiotherapy. Immunotherapy 6, 597610 (2014).
95. Ames,E. etal. Enhanced targeting of stem-like solid
tumor cells with radiation and natural killer cells.
96. Hermanson,D.L. & Kaufman,D.S. Utilizing chimeric
antigen receptors to direct natural killer cell activity.
Frontiers Immunol. 6, 195 (2015).
97. Carotta,S. Targeting NK cells for anticancer
immunotherapy: clinical and preclinical approaches.
Frontiers Immunol. 7, 152 (2016).
98. Balkwill,F. Cancer and the chemokine network.
Nat.Rev. Cancer 4, 540550 (2004).
99. Cummings,R.J., Mitra,S., Foster,T.H. & Lord,E.M.
Migration of skin dendritic cells in response to ionizing
radiation exposure. Radiat. Res. 171, 687697
(2009).
100. Moravan,M.J., Olschowka,J.A., Williams,J.P. &
OBanion,M.K. Cranial irradiation leads to acute and
persistent neuroinflammation with delayed increases
in Tcell infiltration and CD11c expression in C57BL/6
mouse brain. Radiat. Res. 176, 459473 (2011).
101. Yang,X. etal. Targeting the tumor microenvironment
with interferon-beta bridges innate and adaptive
immune responses. Cancer Cell 25, 3748 (2014).
.
d
e
v
r
e
s
e
r
s
t
h
g
i
r
l
l
A
.
e
r
u
t
a
N
r
e
g
n
i
r
p
S
f
o
t
r
a
p
,
d
e
t
i
m
i
L
s
r
e
h
s
i
l
b
u
P
n
a
l
l
i
m
c
a
M
7
1
0
2
REVIEWS
102. Facciabene,A., Motz,G.T. & Coukos,G. TRegulatory
cells: key players in tumor immune escape and
angiogenesis. Cancer Res. 72, 21622171 (2012).
103. Kachikwu,E.L. etal. Radiation enhances regulatory
Tcell representation. Int. J.Radiat. Oncol. Biol. Phys.
81, 11281135 (2011).
104. Persa,E., Balogh,A., Safrany,G. & Lumniczky,K. The
effect of ionizing radiation on regulatory Tcells in health
and disease. Cancer Lett. 368, 252261 (2015).
105. Balogh,A. etal. The effect of ionizing radiation on the
homeostasis and functional integrity of murine splenic
regulatory Tcells. Inflamm Res. 62, 201212 (2013).
106. McFarland,H.I. etal. Regulatory Tcells in
irradiation-induced immune suppression. PLoS ONE 7,
e39092 (2012).
107. Price,J.G. etal. CDKN1A regulates Langerhans cell
survival and promotes Treg cell generation upon
exposure to ionizing irradiation. Nat. Immunol. 16,
10601068 (2015).
108. Fadul,C.E. etal. Immune response in patients with
newly diagnosed glioblastoma multiforme treated
withintranodal autologous tumor lysate-dendritic
cellvaccination after radiation chemotherapy.
J.Immunother. 34, 382389 (2011).
109. Schuler,P.J. etal. Effects of adjuvant
chemoradiotherapy on the frequency and function
ofregulatory Tcells in patients with head and neck
cancer. Clin. Cancer Res. 19, 65856596 (2013).
110. Qinfeng,S. etal. In situ observation of the effects
oflocal irradiation on cytotoxic and regulatory
Tlymphocytes in cervical cancer tissue. Radiat. Res.
179, 584589 (2013).
111. Bouquet,F. etal. TGF1 inhibition increases the
radiosensitivity of breast cancer cells invitro and
promotes tumor control by radiation invivo.
Clin.Cancer Res. 17, 67546765 (2011).
112. Schaue,D., Xie,M.W., Ratikan,J.A. & McBride,W.H.
Regulatory Tcells in radiotherapeutic responses.
113. Formenti,S.C., Demaria,S., Barcellos-Hoff,M.H. &
McBride,W.H. Subverting misconceptions about
radiation therapy. Nat. Immunol. 17, 345 (2016).
114. Condamine,T., Ramachandran,I., Youn,J.I. &
Gabrilovich,D.I. Regulation of tumor metastasis by
myeloid-derived suppressor cells. Annu. Rev. Med. 66,
97110 (2015).
115. Marvel,D. & Gabrilovich,D.I. Myeloid-derived
suppressor cells in the tumor microenvironment:
expect the unexpected. J.Clin. Invest. 125,
33563364 (2015).
116. Kumar,V., Patel,S., Tcyganov,E. & Gabrilovich,D.I.
The nature of myeloid-derived suppressor cells in
thetumor microenvironment. Trends Immunol. 37,
208220 (2016).
117. Quail,D.F. & Joyce,J.A. Microenvironmental
regulation of tumor progression and metastasis.
Nat.Med. 19, 14231437 (2013).
118. Movahedi,K. etal. Identification of discrete
tumor-induced myeloid-derived suppressor cell
subpopulations with distinct Tcell-suppressive activity.
Blood 111, 42334244 (2008).
119. Fridlender,Z.G. etal. Polarization of tumor-associated
neutrophil phenotype by TGF-: N1 versus N2 TAN.
Cancer Cell 16, 183194 (2009).
120. Lindau,D., Gielen,P., Kroesen,M., Wesseling,P. &
Adema,G.J. The immunosuppressive tumour network:
myeloid-derived suppressor cells, regulatory Tcells
andnatural killer Tcells. Immunology 138, 105115
(2013).
121. Folkman,J. Tumor angiogenesis: therapeutic
implications. N.Engl. J.Med. 285, 11821186 (1971).
122. Weis,S.M. & Cheresh,D.A. Tumor angiogenesis:
molecular pathways and therapeutic targets. Nat. Med.
17, 13591370 (2011).
123. Acharyya,S. etal. A CXCL1 paracrine network links
cancer chemoresistance and metastasis. Cell 150,
165178 (2012).
124. Crittenden,M.R. etal. Expression of NFB p50 in
tumor stroma limits the control of tumors by radiation
therapy. PLoS ONE 7, e39295 (2012).
125. Xu,J. etal. CSF1R signaling blockade stanches tumorinfiltrating myeloid cells and improves the efficacy of
radiotherapy in prostate cancer. Cancer Res. 73,
27822794 (2013).
126. Crittenden,M.R. etal. The peripheral myeloid
expansion driven by murine cancer progression is
reversed by radiation therapy of the tumor. PLoS ONE
8, e69527 (2013).
127. Vatner,R.E. & Formenti,S.C. Myeloid-derived cells in
tumors: effects of radiation. Semin. Radiat. Oncol. 25,
1827 (2015).
128. Sica,A. etal. Macrophage polarization in tumour

progression. Semin. Cancer Biol. 18, 349355
(2008).
129. Mills,C.D. M1 and M2 macrophages: oracles of
health and disease. Crit. Rev. Immunol. 32, 463488
(2012).
130. Noy,R. & Pollard,J.W. Tumor-associated
macrophages: from mechanisms to therapy. Immunity
41, 4961 (2014).
131. Inoue,T. etal. CCL22 and CCL17 in rat radiation
pneumonitis and in human idiopathic pulmonary
fibrosis. Eur. Respir. J. 24, 4956 (2004).
132. Klug,F. etal. Low-dose irradiation programs
macrophage differentiation to an iNOS+/M1
phenotype that orchestrates effective Tcell
immunotherapy. Cancer Cell 24, 589602 (2013).
133. Shiao,S.L. etal. TH2polarized CD4+ T cells and
macrophages limit efficacy of radiotherapy. Cancer
Immunol. Res. 3, 518525 CIR140232 (2015).
134. Seifert,L. etal. Radiation therapy induces
macrophages to suppress immune responses
againstpancreatic tumors in mice. Gastroenterology
http://dx.doi.org/10.1053/j.gastro.2016.02.070
(2016).
135. Sica,A. & Mantovani,A. Macrophage plasticity and
polarization: invivo veritas. J.Clin. Invest. 122,
787795 (2012).
136. Schaue,D. etal. Radiation and inflammation.
Seminars Radiat. Oncol. 25, 410 (2015).
137. Medrek,C., Ponten,F., Jirstrom,K. & Leandersson,K.
The presence of tumor associated macrophages in
tumor stroma as a prognostic marker for breast
cancer patients. BMC Cancer 12, 306 (2012).
138. Dewan,M.Z. etal. Fractionated but not single-dose
radiotherapy induces an immune-mediated abscopal
effect when combined with anti-CTLA4 antibody.
Clin.Cancer Res. 15, 53795388 (2009).
139. Verbrugge,I. etal. Radiotherapy increases the
permissiveness of established mammary tumors to
rejection by immunomodulatory antibodies.
CancerRes. 72, 31633174 (2012).
140. Demaria,S. & Formenti,S.C. Radiation as an
immunological adjuvant: current evidence on dose
andfractionation. Frontiers Oncol. 2, 153 (2012).
141. Gupta,A. etal. Radiotherapy supports protective
tumor-specific immunity. Oncoimmunology 1,
16101611 (2012).
142. Weichselbaum,R.R. etal. An interferon-related gene
signature for DNA damage resistance is a predictive
marker for chemotherapy and radiation for breast
cancer. Proc. Natl Acad. Sci. USA 105, 1849018495
(2008).
143. Khodarev,N.N. etal. STAT1 pathway mediates
amplification of metastatic potential and resistance
totherapy. PLoS ONE 4, e5821 (2009).
144. Khodarev,N.N., Roizman,B. & Weichselbaum,R.R.
Molecular pathways: interferon/stat1 pathway:
role in the tumor resistance to genotoxic stress and
aggressive growth. Clin. Cancer Res. 18, 30153021
(2012).
145. Minn,A.J. Interferons and the immunogenic effects
ofcancer therapy. Trends Immunol. 36, 725737
(2015).
146. Deng,L. etal. STING-dependent cytosolic DNA
Sensing promotes radiation-induced type I interferondependent antitumor immunity in immunogenic
tumors. Immunity 41, 843852 (2014).
147. Ishikawa,H., Ma,Z. & Barber,G.N. STING regulates
intracellular DNA-mediated, typeI interferondependent innate immunity. Nature 461, 788792
(2009).
148. Tanaka,Y. & Chen,Z.J. STING specifies IRF3
phosphorylation by TBK1 in the cytosolic DNA
signaling pathway. Sci. Signal. 5, ra20 (2012).
149. Sun,L., Wu,J., Du,F., Chen,X. & Chen,Z.J. Cyclic
GMP-AMP synthase is a cytosolic DNA sensor that
activates the typeI interferon pathway. Science 339,
786791 (2013).
150. Li,X.D. etal. Pivotal roles of cGAS-cGAMP signaling
in antiviral defense and immune adjuvant effects.
Science 341, 13901394 (2013).
151. Watson,R.O., Manzanillo,P.S. & Cox,J.S.
Extracellular M.tuberculosis DNA targets bacteria
forautophagy by activating the host DNA-sensing
pathway. Cell 150, 803815 (2012).
152. Sharma,S. etal. Innate immune recognition of an
ATrich stem-loop DNA motif in the Plasmodium
falciparum genome. Immunity 35, 194207 (2011).
153. Coban,C. etal. Novel strategies to improve DNA
vaccine immunogenicity. Curr. Gene Ther. 11,
479484 (2011).
154. Woo,S.R. etal. STING-dependent cytosolic DNA

sensing mediates innate immune recognition of
immunogenic tumors. Immunity 41, 830842 (2014).
155. Barker,H.E., Paget,J.T., Khan,A.A. &
Harrington,K.J. The tumour microenvironment
afterradiotherapy: mechanisms of resistance and
recurrence. Nat. Rev. Cancer 15, 409425 (2015).
156. Thomas,L. Cellular and humoral aspects of the
hypersensitive states; a symposium held at the
NewYork Academy of Medicine (Hoeber, 1959).
157. Koebel,C.M. etal. Adaptive immunity maintains
occult cancer in an equilibrium state. Nature 450,
903907 (2007).
158. Schreiber,R.D., Old,L.J. & Smyth,M.J. Cancer
immunoediting: integrating immunitys roles in cancer
suppression and promotion. Science 331, 15651570
(2011).
159. Mittal,D., Gubin,M.M., Schreiber,R.D. &
Smyth,M.J. New insights into cancer immunoediting
and its three component phaseselimination,
equilibrium and escape. Curr. Opin. Immunol. 27,
1625 (2014).
160. Klein,E. Tumour immunology; escape mechanisms.
Ann. Inst. Pasteur 122, 593602 (1972).
161. Hyman,R. Genetic alterations in tumor cells
resultingin their escape from the immune response.
Cancer Chemother. Rep. 58, 431439 (1974).
162. Yutoku,M., Fuji,H., Grossberg,A.L. & Pressman,D.
An experimental model for evaluation of factors in
tumor escape from immunological attack. Cancer Res.
35, 734739 (1975).
163. Bruttel,V.S. & Wischhusen,J. Cancer stem cell
immunology: key to understanding tumorigenesis and
tumor immune escape? Frontiers Immunol. 5, 360
(2014).
164. Liang,H. etal. Radiation-induced equilibrium is a
balance between tumor cell proliferation and Tcellmediated killing. J.Immunol. 190, 58745881
(2013).
165. Liang,H., Deng,L., Burnette,B., Weichselbaum,R.R.
& Fu,Y.X. Radiation-induced tumor dormancy reflects
an equilibrium between the proliferation and T
lymphocyte-mediated death of malignant cells.
166. Schietinger,A. & Greenberg,P.D. Tolerance and
exhaustion: defining mechanisms of Tcell dysfunction.
Trends Immunol. 35, 5160 (2014).
167. Shaked,Y. Balancing efficacy of and host immune
responses to cancer therapy: the yin and yang effects.
Nat. Rev. Clin. Oncol. http://dx.doi.org/10.1038/
nrclinonc.2016.57 (2016).
168. Suit,H.D. & Walker,A.M. Assessment of the response
of tumours to radiation: clinical and experimental
studies. Br. J.Cancer. Suppl. 4, 110 (1980).
169. Early Breast Cancer Trialists Collaborative,G. etal.
Effect of radiotherapy after breast-conserving surgery
on 10year recurrence and 15year breast cancer
death: meta-analysis of individual patient data for
10,801 women in 17 randomised trials. Lancet 378,
17071716 (2011).
170. Pilepich,M.V. etal. Androgen suppression adjuvant
to definitive radiotherapy in prostate carcinomalongterm results of phaseIII RTOG 8531. Int. J.Radiat.
Oncol. Biol. Phys. 61, 12851290 (2005).
171. Char,D.H., Kroll,S., Phillips,T.L. & Quivey,J.M.
Lateradiation failures after iodine 125 brachytherapy
for uveal melanoma compared with charged-particle
(proton or helium ion) therapy. Ophthalmology 109,
18501854 (2002).
172. Chao,K.S. etal. Patterns of failure in patients
receiving definitive and postoperative IMRT for headand-neck cancer. Int. J.Radiat. Oncol. Biol. Phys. 55,
312321 (2003).
173. Curiel,T.J. etal. Blockade of B7H1 improves myeloid
dendritic cell-mediated antitumor immunity. Nat. Med.
9, 562567 (2003).
174. Zou,W. & Chen,L. Inhibitory B7family molecules in
the tumour microenvironment. Nat. Rev. Immunol. 8,
467477 (2008).
175. Dovedi,S.J. etal. Acquired resistance to fractionated
radiotherapy can be overcome by concurrent PDL1
blockade. Cancer Res. 74, 54585468 (2014).
176. Twyman-Saint Victor,C. etal. Radiation and dual
checkpoint blockade activate non-redundant immune
mechanisms in cancer. Nature 520, 373377 (2015).
177. Herbst,R.S. etal. Predictive correlates of response to
the antiPDL1 antibody MPDL3280A in cancer
patients. Nature 515, 563567 (2014).
178. Chen,L. & Han,X. AntiPD1/PDL1 therapy of human
cancer: past, present, and future. J.Clin. Invest. 125,
33843391 (2015).
.
d
e
v
r
e
s
e
r
s
t
h
g
i
r
l
l
A
.
e
r
u
t
a
N
r
e
g
n
i
r
p
S
f
o
t
r
a
p
,
d
e
t
i
m
i
L
s
r
e
h
s
i
l
b
u
P
n
a
l
l
i
m
c
a
M
7
1
0
2
REVIEWS
179. Gubin,M.M. etal. Checkpoint blockade cancer
immunotherapy targets tumour-specific mutant
antigens. Nature 515, 577581 (2014).
180. Powles,T. etal. MPDL3280A (antiPDL1) treatment
leads to clinical activity in metastatic bladder cancer.
Nature 515, 558562 (2014).
181. Tumeh,P.C. etal. PD1 blockade induces responses by
inhibiting adaptive immune resistance. Nature 515,
568571 (2014).
182. Yadav,M. etal. Predicting immunogenic tumour
mutations by combining mass spectrometry and
exome sequencing. Nature 515, 572576 (2014).
183. Wolchok,J.D. & Chan,T.A. Cancer: antitumour
immunity gets a boost. Nature 515, 496498 (2014).
184. Vanpouille-Box,C., Pilones,K.A., Wennerberg,E.,
Formenti,S.C. & Demaria,S. In situ vaccination by
radiotherapy to improve responses to anti-CTLA4
treatment. Vaccine 33, 74157422 (2015).
185. Ruocco,M.G. etal. Suppressing Tcell motility induced
by anti-CTLA4 monotherapy improves antitumor
effects. J.Clin. Invest. 122, 37183730 (2012).
186. Demaria,S., Pilones,K.A., Formenti,S.C. &
Dustin,M.L. Exploiting the stress response to
radiation to sensitize poorly immunogenic tumors to
anti-CTLA4 treatment. Oncoimmunology 2, e23127
(2013).
187. Demaria,S. etal. Immune-mediated inhibition of
metastases after treatment with local radiation and
CTLA4 blockade in a mouse model of breast cancer.
Clin. Cancer Res. 11, 728734 (2005).
188. Small,E.J. etal. A pilot trial of CTLA4 blockade with
human anti-CTLA4 in patients with hormone-refractory
prostate cancer. Clin. Cancer Res. 13, 18101815
(2007).
189. Yang,J.C. etal. Ipilimumab (antiCTLA4 antibody)
causes regression of metastatic renal cell cancer
associated with enteritis and hypophysitis.
J.Immunother. 30, 825830 (2007).
190. Weber,J.S. etal. Phase I/II study of ipilimumab for
patients with metastatic melanoma. J.Clin. Oncol. 26,
59505956 (2008).
191. Weber,J. etal. A randomized, double-blind, placebocontrolled, phaseII study comparing the tolerability
and efficacy of ipilimumab administered with or
without prophylactic budesonide in patients with
unresectable stage III or IV melanoma. Clin. Cancer
Res. 15, 55915598 (2009).
192. Ansell,S.M. etal. Phase I study of ipilimumab, an
anti-CTLA4 monoclonal antibody, in patients with
relapsed and refractory Bcell non-Hodgkin lymphoma.
Clin. Cancer Res. 15, 64466453 (2009).
193. Wolchok,J.D. etal. Ipilimumab monotherapy in
patients with pretreated advanced melanoma:
arandomised, double-blind, multicentre, phase2, doseranging study. Lancet. Oncol. 11, 155164 (2010).
194. ODay,S.J. etal. Efficacy and safety of ipilimumab
monotherapy in patients with pretreated advanced
melanoma: a multicenter single-arm phaseII study.
Ann. Oncol. 21, 17121717 (2010).
195. Hersh,E.M. etal. A phaseII multicenter study of
ipilimumab with or without dacarbazine in
chemotherapy-naive patients with advanced
melanoma. Investigat. Drugs 29, 489498 (2011).
196. Sharma,P., Wagner,K., Wolchok,J.D. & Allison,J.P.
Novel cancer immunotherapy agents with survival
benefit: recent successes and next steps.
Nat.Rev.Cancer 11, 805812 (2011).
197. Hodi,F.S. etal. Immunologic and clinical effects
ofantibody blockade of cytotoxic T lymphocyteassociated antigen 4 in previously vaccinated cancer
patients. Proc. Natl Acad. Sci. USA 105, 30053010
(2008).
198. Madan,R.A. etal. Ipilimumab and a poxviral vaccine
targeting prostate-specific antigen in metastatic
castration-resistant prostate cancer: a phase1 doseescalation trial. Lancet Oncol. 13, 501508 (2012).
199. Sarnaik,A.A. etal. Extended dose ipilimumab with a
peptide vaccine: immune correlates associated with
clinical benefit in patients with resected high-risk stage
IIIc/IV melanoma. Clin. Cancer Res. 17, 896906
(2011).
200. McDermott,D. etal. Efficacy and safety of ipilimumab
in metastatic melanoma patients surviving more than
2years following treatment in a phaseIII trial
(MDX01020). Ann. Oncol. 24, 26942698 (2013).
201. Jochems,C. etal. A combination trial of vaccine plus
ipilimumab in metastatic castration-resistant prostate
cancer patients: immune correlates. Cancer Immunol.
Immunother. 63, 407418 (2014).
202. Fong,L. etal. Potentiating endogenous antitumor
immunity to prostate cancer through combination
immunotherapy with CTLA4 blockade and GMCSF.

Cancer Res. 69, 609615 (2009).
203. Le,D.T. etal. Evaluation of ipilimumab in combination
with allogeneic pancreatic tumor cells transfected
witha GMCSF gene in previously treated pancreatic
cancer. J.Immunother. 36, 382389 (2013).
204. Wolchok,J.D. etal. Nivolumab plus ipilimumab in
advanced melanoma. N.Engl. J.Med. 369, 122133
(2013).
205. Larkin,J. etal. Combined nivolumab and ipilimumab or
monotherapy in untreated melanoma. N.Engl. J.Med.
373, 2334 (2015).
206. Slovin,S.F. etal. Ipilimumab alone or in combination
with radiotherapy in metastatic castration-resistant
prostate cancer: results from an open-label, multicenter
phaseI/II study. Ann. Oncol. 24, 18131821 (2013).
207. Kwon,E.D. etal. Ipilimumab versus placebo after
radiotherapy in patients with metastatic castrationresistant prostate cancer that had progressed after
docetaxel chemotherapy (CA184043): a multicentre,
randomised, double-blind, phase 3 trial. Lancet Oncol.
15, 700712 (2014).
208. Di Giacomo,A.M. etal. Ipilimumab and fotemustine
in patients with advanced melanoma (NIBITM1):
anopen-label, single-arm phase2 trial. Lancet Oncol.
13, 879886 (2012).
209. Reck,M. etal. Ipilimumab in combination with
paclitaxel and carboplatin as first-line therapy in
extensive-disease-small-cell lung cancer: results from
arandomized, double-blind, multicenter phase2 trial.
Ann. Oncol. 24, 7583 (2013).
210. Lynch,T.J. etal. Ipilimumab in combination with
paclitaxel and carboplatin as first-line treatment in
stage IIIB/IV non-small-cell lung cancer: results from a
randomized, double-blind, multicenter phaseII study.
J.Clin. Oncol. 30, 20462054 (2012).
211. Robert,C. etal. Ipilimumab plus dacarbazine for
previously untreated metastatic melanoma.
N.Engl.J.Med. 364, 25172526 (2011).
212. Weber,J. etal. Randomized phaseI pharmacokinetic
study of ipilimumab with or without one of two
different chemotherapy regimens in patients with
untreated advanced melanoma. Cancer Immun. 13, 7
(2013).
213. Lu,W. etal. Inflammation promotes oral squamous
carcinoma immune evasion via induced programmed
death ligand1 surface expression. Oncol. Lett. 5,
15191526 (2013).
214. Chen,J. etal. Interferon-gamma-induced PDL1
surface expression on human oral squamous
carcinoma via PKD2 signal pathway. Immunobiology
217, 385393 (2012).
215. Lu,J., Lee-Gabel,L., Nadeau,M.C., Ferencz,T.M. &
Soefje,S.A. Clinical evaluation of compounds targeting
PD1/PDL1 pathway for cancer immunotherapy.
J.Oncol. Pharm. Pract. 21, 451467 (2014).
216. Sakuishi,K. etal. Targeting Tim3 and PD1 pathways
to reverse Tcell exhaustion and restore anti-tumor
immunity. J.Exp. Med. 207, 21872194 (2010).
217. Brahmer,J.R. PD1targeted immunotherapy:
recentclinical findings. Clin. Adv. Hematol. Oncol. 10,
674675 (2012).
218. Topalian,S.L. etal. Safety, activity, and immune
correlates of antiPD1 antibody in cancer. N.Engl.
J.Med. 366, 24432454 (2012).
219. Ribas,A. Tumor immunotherapy directed at PD1.
N.Engl. J.Med. 366, 25172519 (2012).
220. Robert,C. etal. Nivolumab in previously untreated
melanoma without BRAF mutation. N.Engl. J.Med.
372, 320330 (2015).
221. Postow,M.A. etal. Nivolumab and ipilimumab versus
ipilimumab in untreated melanoma. N.Engl. J.Med.
372, 20062017 (2015).
222. Brahmer,J. etal. Nivolumab versus docetaxel in
advanced squamous-cell non-small-cell lung cancer.
N.Engl. J.Med. 373, 123135 (2015).
223. Borghaei,H. etal. Nivolumab versus docetaxel in
advanced nonsquamous non-small-cell lung cancer.
N.Engl. J.Med. 373, 16271639 (2015).
224. Brahmer,J.R. etal. Safety and activity of antiPDL1
antibody in patients with advanced cancer. N.Engl.
J.Med. 366, 24552465 (2012).
225. Topalian,S.L. etal. Survival, durable tumor remission,
and long-term safety in patients with advanced
melanoma receiving nivolumab. J.Clin. Oncol. 32,
10201030 (2014).
226. Hamid,O. etal. Safety and tumor responses with
lambrolizumab (antiPD1) in melanoma. N.Engl.
J.Med. 369, 134144 (2013).
227. Gajewski,T.F. etal. Cancer immunotherapy strategies
based on overcoming barriers within the tumor
microenvironment. Curr. Opin. Immunol. 25, 268276

(2013).
228. Weber,J.S. Current perspectives on immunotherapy.
Seminars Oncol. 41 (Suppl. 5), S1429 (2014).
229. Sui,X. etal. The anticancer immune response of
antiPD1/PDL1 and the genetic determinants of
response to antiPD1/PDL1 antibodies in cancer
patients. Oncotarget 6, 1939319404 (2015).
230. Gabrilovich,D.I., Ostrand-Rosenberg,S. & Bronte,V.
Coordinated regulation of myeloid cells by tumours.
Nat. Rev. Immunol. 12, 253268 (2012).
231. Sade-Feldman,M. etal. Tumor necrosis factor- blocks
differentiation and enhances suppressive activity of
immature myeloid cells during chronic inflammation.
Immunity 38, 541554 (2013).
232. Zhao,X. etal. TNF signaling drives myeloid-derived
suppressor cell accumulation. J.Clin. Invest. 122,
40944104 (2012).
233. Dahan,R. etal. FcRs modulate the anti-tumor
activityof antibodies targeting the PD1/PDL1 axis.
CancerCell 28, 285295 (2015).
234. Zeng,J. etal. AntiPD1 blockade and stereotactic
radiation produce long-term survival in mice with
intracranial gliomas. Int. J.Radiat. Oncol. Biol. Phys.
86, 343349 (2013).
235. Formenti,S.C. & Demaria,S. Radiation therapy to
convert the tumor into an insitu vaccine. Int. J.Radiat.
Oncol. Biol. Phys. 84, 879880 (2012).
236. Sharabi,A. etal. Role of radiation therapy in inducing
antigen specific antitumor immune responses when
combined with antiPD1 checkpoint blockade:
Mechanism and clinical implications. Int. J.Radiat.
Biol. Oncol. Phys. 90, S1 (2014).
237. Dovedi,S.J. & Illidge,T.M. The antitumor immune
response generated by fractionated radiation therapy
may be limited by tumor cell adaptive resistance
andcan be circumvented by PDL1 blockade.
238. Ansell,S.M. etal. PD1 blockade with nivolumab
inrelapsed or refractory Hodgkins lymphoma.
N.Engl.J.Med. 372, 311319 (2015).
239. Mole,R.H. Whole body irradiation; radiobiology or
medicine? Br. J.Radiol. 26, 234241 (1953).
240. Siva,S., MacManus,M.P., Martin,R.F. & Martin,O.A.
Abscopal effects of radiation therapy: a clinical review
for the radiobiologist. Cancer Lett. 356, 8290 (2015).
241. Postow,M.A. etal. Immunologic correlates of the
abscopal effect in a patient with melanoma. N.Engl.
J.Med. 366, 925931 (2012).
242. Zeng,J., Harris,T.J., Lim,M., Drake,C.G. & Tran,P.T.
Immune modulation and stereotactic radiation:
improving local and abscopal responses.
BiomedRes.Int. 2013, 658126 (2013).
243. Hiniker,S.M., Chen,D.S. & Knox,S.J. Abscopal effect
in a patient with melanoma. New Engl. J.Med. 366,
2035 (2012).
244. Stamell,E.F., Wolchok,J.D., Gnjatic,S., Lee,N.Y.
&Brownell,I. The abscopal effect associated with
asystemic anti-melanoma immune response.
245. Golden,E.B., Demaria,S., Schiff,P.B., Chachoua,A.
&Formenti,S.C. An abscopal response to radiation
and ipilimumab in a patient with metastatic non-small
cell lung cancer. Cancer Immunol. Res. 1, 365372
(2013).
246. Grimaldi,A.M. etal. Abscopal effects of radiotherapy
on advanced melanoma patients who progressed after
ipilimumab immunotherapy. Oncoimmunology 3,
e28780 (2014).
247. Chandra,R.A. etal. A systematic evaluation of
abscopal responses following radiotherapy in patients
with metastatic melanoma treated with ipilimumab.
248. Kaminski,J.M. etal. The controversial abscopal effect.
Cancer Treat. Rev. 31, 159172 (2005).
Acknowledgements
The authors wish to thank Amy K. Huser at The University of

Chicago for her invaluable literature-research, writing, and
editing contributions to the historical passages of this Review,
and Table1 and Figure1. The work of R.R.W is supported in
part by NIH grant NCIR21CA19507501.
Author contributions
R.R.W., H.L., and L.D. wrote the manuscript. All authors

made substantial contributions to researching data for article, discussions of content, and review/editing of manuscript
before submission.
Competing interests statement
The authors declare no competing interests.
.
d
e
v
r
e
s
e
r
s
t
h
g
i
r
l
l
A
.
e
r
u
t
a
N
r
e
g
n
i
r
p
S
f
o
t
r
a
p
,
d
e
t
i
m
i
L
s
r
e
h
s
i
l
b
u
P
n
a
l
l
i
m
c
a
M
7
1
0
2

Radiotherapy and Immunotherapy: A Beneficial Liaison

Uploaded by

Copyright:

Available Formats

You might also like

Radiotherapy and Immunotherapy: A Beneficial Liaison

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Radiotherapy and Immunotherapy: A Beneficial Liaison

Uploaded by

Copyright:

Available Formats

REVIEWS

Radiotherapy and immunotherapy:

Department of Radiation and

Radiotherapy is often used as a curative treatment for

fractions using stereotactic body radiotherapy (SBRT),

NATURE REVIEWS | CLINICAL ONCOLOGY

ADVANCE ONLINE PUBLICATION | 1

2 | ADVANCE ONLINE PUBLICATION

RNA triggers TLRs

DNA triggers cGAS

NKcell-based therapies in research or clinical trials has

Initiates cytokine and chemokine production

Eux of immune cells

Treg cells build up in the

MDSCs suppress T-cell

Initiates release of tumour antigens

Initiates recruitment of IL-1B, TGF, FGF, TNF and NLRP-3

Figure 1 |Radiation-induced effects on tumour cells.

Nature Reviews | Clinical Oncology

ADVANCE ONLINE PUBLICATION | 3

Interactions of these inhibitory immune cell types or

4 | ADVANCE ONLINE PUBLICATION

immunity for tumour regression: innate immunity is

NATURE REVIEWS | CLINICAL ONCOLOGY

ADVANCE ONLINE PUBLICATION | 5

Microenvironment and tumour response

the immune system might have a key role in suppressing

6 | ADVANCE ONLINE PUBLICATION

Survival cells (fraction)

responsive at early time points (as early as 10days), or

In 2003, Zou and co-workers173,174 reported that

Combining radiation and immunomodulation

NATURE REVIEWS | CLINICAL ONCOLOGY

ADVANCE ONLINE PUBLICATION | 7

Figure 3 | Tumour-cell division coexists with apoptosis

therapy. Vanpouille-Box and colleagues184 suggested

Anti-CTLA4 therapies have been tested in combi

8 | ADVANCE ONLINE PUBLICATION

A preliminary preclinical report from the Drake lab

NATURE REVIEWS | CLINICAL ONCOLOGY

ADVANCE ONLINE PUBLICATION | 9

Following RT (no details provided),

48; phaseII safety/

Reirradiation with 1.2Gy for 5days

17; phaseI safety/

Arm 1: 8Gy SBRT to liver metastases

15; phaseI safety

SABR at 20Gy to at least one metastasis

Hypofractionated RT and pembrolizumab None;

60; phaseI safety

REGN2810 alone or with

Pembrolizumab every 2weeks after SBRT

Stage III NSCLC

43; phaseII safety/

Nivolumab after standard

Not open for

34; phaseI safety

Pembrolizumab after hypofractionated

Not open for