Cell death induced by cytotoxic CD8 + T cells is immunogenic and primes caspase-3–dependent spread immunity against endogenous tumor antigens

Abstract

Background
Elimination of cancer cells by some stimuli like chemotherapy and radiotherapy activates anticancer immunity after the generation of damage‐associated molecular patterns, a process recently named immunogenic cell death (ICD). Despite the recent advances in cancer immunotherapy, very little is known about the immunological consequences of cell death activated by cytotoxic CD8 ⁺ T (Tc) cells on cancer cells, that is, if Tc cells induce ICD on cancer cells and the molecular mechanisms involved.

Methods
ICD induced by Tc cells on EL4 cells was analyzed in tumor by vaccinating mice with EL4 cells killed in vitro or in vivo by Ag-specific Tc cells. EL4 cells and mutants thereof overexpressing Bcl-X L or a dominant negative mutant of caspase-3 and wild-type mice, as well as mice depleted of Tc cells and mice deficient in perforin, TLR4 and BATF3 were used. Ex vivo cytotoxicity of spleen cells from immunized mice was analyzed by flow cytometry. Expression of ICD signals (calreticulin, HMGB1 and interleukin (IL)-1β) was analyzed by flow cytometry and ELISA.

Results
Mice immunized with EL4.gp33 cells killed in vitro or in vivo by gp33-specific Tc cells were protected from parental EL4 tumor development. This result was confirmed in vivo by using ovalbumin (OVA) as another surrogate antigen. Perforin and TLR4 and BATF3-dependent type 1 conventional dendritic cells (cDC1s) were required for protection against tumor development, indicating cross-priming of Tc cells against endogenous EL4 tumor antigens. Tc cells induced ICD signals in EL4 cells. Notably, ICD of EL4 cells was dependent on caspase-3 activity, with reduced antitumor immunity generated by caspase-3–deficient EL4 cells. In contrast, overexpression of Bcl-X L in EL4 cells had no effect on induction of Tc cell antitumor response and protection.

Conclusions
Elimination of tumor cells by Ag-specific Tc cells is immunogenic and protects against tumor development by generating new Tc cells against EL4 endogenous antigens. This finding helps to explain the enhanced efficacy of T cell-dependent immunotherapy and provide a molecular basis to explain the epitope spread phenomenon observed during vaccination and chimeric antigen receptor (CAR)-T cell therapy. In addition, they suggest that caspase-3 activity in the tumor may be used as a biomarker to predict cancer recurrence during T cell-dependent immunotherapies.

Full text

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Jaime- SanchezP, etal. J Immunother Cancer 2020;8:e000528. doi:10.1136/jitc-2020-000528
Open access
Cell death induced by cytotoxic CD8+ T
cells is immunogenic and primes
caspase-3–dependent spread immunity
against endogenous tumor antigens
Paula Jaime- Sanchez,1 Iratxe Uranga- Murillo,1 Nacho Aguilo,1,2,3 Soa C Khouili,4
Maykel A Arias,5 David Sancho,4 Julian Pardo 6,7
To cite: Jaime- SanchezP,
Uranga- MurilloI, AguiloN,
etal. Cell death induced
by cytotoxic CD8+ T cells is
immunogenic and primes
caspase-3–dependent spread
immunity against endogenous
tumor antigens. Journal for
ImmunoTherapy of Cancer
2020;8:e000528. doi:10.1136/
jitc-2020-000528
►Additional material is
published online only. To view
please visit the journal online
(http:// dx. doi. org/ 10. 1136/ jitc-
2020- 000528).
Accepted 01 March 2020
For numbered afliations see
end of article.
Correspondence to
Dr Julian Pardo;
pardojim@ unizar. es
Original research
© Author(s) (or their
employer(s)) 2020. Re- use
permitted under CC BY- NC. No
commercial re- use. See rights
and permissions. Published by
BMJ.
ABSTRACT
Background Elimination of cancer cells by some
stimuli like chemotherapy and radiotherapy activates
anticancer immunity after the generation of damage‐
associated molecular patterns, a process recently named
immunogenic cell death (ICD). Despite the recent advances
in cancer immunotherapy, very little is known about the
immunological consequences of cell death activated
by cytotoxic CD8+ T (Tc) cells on cancer cells, that is, if
Tc cells induce ICD on cancer cells and the molecular
mechanisms involved.
Methods ICD induced by Tc cells on EL4 cells was analyzed
in tumor by vaccinating mice with EL4 cells killed in vitro or
in vivo by Ag- specic Tc cells. EL4 cells and mutants thereof
overexpressing Bcl- XL or a dominant negative mutant of
caspase-3 and wild- type mice, as well as mice depleted of
Tc cells and mice decient in perforin, TLR4 and BATF3 were
used. Ex vivo cytotoxicity of spleen cells from immunized
mice was analyzed by ow cytometry. Expression of ICD
signals (calreticulin, HMGB1 and interleukin (IL)-1β) was
analyzed by ow cytometry and ELISA.
Results Mice immunized with EL4.gp33 cells killed in
vitro or in vivo by gp33- specic Tc cells were protected
from parental EL4 tumor development. This result was
conrmed in vivo by using ovalbumin (OVA) as another
surrogate antigen. Perforin and TLR4 and BATF3-
dependent type 1 conventional dendritic cells (cDC1s)
were required for protection against tumor development,
indicating cross- priming of Tc cells against endogenous
EL4 tumor antigens. Tc cells induced ICD signals in
EL4 cells. Notably, ICD of EL4 cells was dependent on
caspase-3 activity, with reduced antitumor immunity
generated by caspase-3–decient EL4 cells. In contrast,
overexpression of Bcl- XL in EL4 cells had no effect on
induction of Tc cell antitumor response and protection.
Conclusions Elimination of tumor cells by Ag- specic
Tc cells is immunogenic and protects against tumor
development by generating new Tc cells against EL4
endogenous antigens. This nding helps to explain the
enhanced efcacy of T cell- dependent immunotherapy and
provide a molecular basis to explain the epitope spread
phenomenon observed during vaccination and chimeric
antigen receptor (CAR)- T cell therapy. In addition, they
suggest that caspase-3 activity in the tumor may be used
as a biomarker to predict cancer recurrence during T cell-
dependent immunotherapies.
BACKGROUND
In the past few years, a major understanding
of the regulation of cancer immunity has
allowed to develop new immunotherapy
approaches that have shown unprecedented
efficacy against aggressive bad prognosis
cancers.1 2 Natural killer (NK) cells and cyto-
toxic CD8+ T (Tc) cells are the key respon-
sible for the elimination of transformed cells
during both cancer immunosurveillance and
immunotherapy.3–6 However, meanwhile NK
cells eliminate stressed tumor cells and/or
cells that have downregulated major histo-
compatibilty complex (MHC)- I molecules,7
activation of Tc cells strictly depends on the
recognition of antigens presented by MHC- I.
Thus, the presence of tumor- mutated genes
raising new epitopes/antigens (neoantigens)
together with proper inflammatory signals
leading to efficient antigen cross- presentation
by dendritic cells (DCs) are prerequisites
for the generation of an effective Tc cell-
mediated anticancer response.8 Accordingly,
response to immunotherapy treatments that
rely on T cell immunity, such as checkpoint
antibody therapy, is limited to cancers that
express immunodominant mutations that
raise an optimal T cell response.
As an alternative to overcome this limita-
tion, in recent years, it has been described
that specific chemotherapy drugs and irra-
diation, in addition to kill cancer cells, have
the capacity to prime anticancer immune
responses against antigens released from
dying cells, a concept known as immuno-
genic cell death (ICD).9–11 This process
enhances the elimination of cancer cells
and generates immune memory against the
tumor antigens, reducing the chance of
cancer recurrence.10 12 This ability is related
to the activation of danger signaling path-
ways evoking emission of damage- associated
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Open access
molecular patterns (DAMPs) leading to inflamma-
tion, which favor DC maturation and antigen presenta-
tion.10 13–15 ICD increases the immunogenicity of tumor
antigens that per se might present a lower antigenic poten-
tial and would not induce an efficient T cell response. In
recent years, some molecular determinants that dictate
the immunogenic characteristics of dying cells have been
clarified.16 Apoptosis, ferroptosis, pyroptosis or necro-
ptosis are among the different modalities of programmed
cell death that can be immunogenic.17–20
Several molecules involved in the generation of eat- me
and danger signals leading to ICD in cancer cells have
been found such as exposure of calreticulin on the cell
membrane, release of Hsp70 or HMGB1, autophagy and
production of interleukin (IL)-1β or type I interferon
(IFN).16 At present, the relative importance of each of
these signals is not clear and different ICD signals have
been described in cells dying under different stimuli.
Thus, to find out if cancer cells eliminated by a specific
stimulus undergo ICD, the generation of anticancer
immunity and tumor development should be analyzed in
vivo in animals previously immunized with dead cancer
cells.
Despite the extensive studies focused on the mecha-
nisms involved in tumor cell recognition by T cells, the
molecular determinants that regulate cell death induced
by Tc cells on cancer cells, a key event for successful cancer
immunotherapy, are less characterized. Paradoxically,
little is known about the immunogenic characteristics
of cancer cell death induced by immunotherapy. Specif-
ically, whether cell death induced by Tc cells is immuno-
genic and, if so, which are the mechanisms responsible
for ICD induced by Tc cells?
Tc cells mainly use two pathways to kill cancer cells,
death ligands (ie, tumor necrosis factor (TNF)-α, fas
ligand and TNF- related apoptosis inducing ligand
(TRAIL)) or granule exocytosis.21 22 The later consists
of the release of a pore- forming protein perforin (perf)
that delivers a family of serine- proteases, the granzymes
(gzms) into the cytosol of target cells. Gzms are the ulti-
mate responsible (mainly gzmB) for cancer cell elimina-
tion. Apart from regulating target cell death, it was shown
that gzms are involved in antigen cross- presentation of
dying cells by somehow regulating DC phagocytosis.23 In
a recent work, we have shown that Tc cells efficiently elim-
inate in vivo tumor cells expressing pro- tumorigenic and/
or anti- apoptotic mutations.24 These results provided
the molecular basis to explain the efficacy of immuno-
therapy against multidrug- resistant bad prognosis cancer.
However, these findings raise a new question to predict
refractoriness and/or cancer relapse after immuno-
therapy: is Tc cell mediated- elimination of cancer cells
immunogenic? And if so, what is the impact of mutations
in cell death/prosurvival pathways on the immunoge-
nicity of cancer cell death induced by Tc cells?
Here we have employed the EL4 lymphoma mouse
model and different cancer vaccination strategies based
on the gp33 and OVA tumor Ag models to explore the
immunogenic characteristics of cell death induced by Tc
cells on cancer cells and the mechanisms involved in this
process both in vitro and in vivo. Our results show that
T cell cytotoxic activity on tumor cells induces ICD and
promotes a protective immune response in vivo, priming
the generation of new CD8+ Tc cell responses against
endogenous tumor antigens. Importantly, this response
is able to reduce tumor development in mice challenged
with a second tumor. Additionally, our results show that
expression of active caspase-3 is key for ICD induced by
CD8+Tc cells.
METHODS
Mouse strains
Inbred C57BL/6 (B6) and mouse strains deficient for
perf (perfKO) and TLR4 (TLR4KO), bred on the B6 back-
ground, were maintained at the Center for Biomedical
Research of Aragon (CIBA) and analyzed for their geno-
types as described.25 Mouse strains deficient for BATF3
(BATF3KO) on the C57BL/6 background were kindly
given by Dr David Sancho (Research Center for Cardio-
vascular Diseases Carlos III, Madrid, Spain). Mice from
both sexes and 8–10 weeks of age were used.
Cell lines, cell culture and reagents
EL4, EL4.DNC3 and EL4.Bcl- XL cells were cultured in
Roswell Park Memorial Institute (RPMI) medium with
5% heat- inactivated FBS at 37°C. EL4 cells overexpressing
Bcl- XL or expressing a dominant negative mutant of
caspase-3 (Cys285Ala; DNC3; Genscript)26 were gener-
ated by lentiviral infection employing the pBABE- puro
vector containing the cDNA of the proteins and the
psPAX and pMD2.G vectors containing the HIV-1 Gag
and Pol and VSV Env proteins, respectively. Transfected
cell clones were selected by limiting dilution, employing
conditioned medium and puromycin as antibiotic of
selection.
Mouse vaccination and isolation of ex vivo CD8+Tc cells
Mice were immunized with LCMV- WE intraperitoneal
(105 pfu) in 200 μL of RPMI 2% heat- inactivated FBS. On
day 8 postinfection, CD8+ cells were positively selected
from spleen using α-CD8- MicroBeads (Miltenyi Biotec,
Germany) and a MACS- cell separation system and resus-
pended in RPMI 5% heat- inactivated FBS before use in
cytotoxic assays. Purity of selected CD8+ cells was assessed
by fluorescence- activated cell sorting (FACS) staining and
found to be between 95% and 98%.
Ex vivo cytotoxicity assays
Target cells were preincubated with the LCMV- derived
peptide gp33 (Neosystem Laboratoire) and MACS-
enriched ex vivo virus immune CD8+ T cells were stained
with CellTracker Green (CTG; Invitrogen). Effector
and target cells were incubated at different ratios
depending on the conditions (10:1, 7:1, 3:1, 1:1 (effec-
tor:target)) at 37°C. In some experiments, unselected
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immune splenocytes from immunized mice were incu-
bated with fluorescently labeled target cells at 100:1
ratio. Subsequently, phosphatidyl serine (PS) exposure
on plasma membrane (Annexin V staining) and incor-
poration of 7- AAD were measured by three- color flow
cytometry in the target population with a FACSCalibur
(BD Pharmingen) and CellQuests software described
previously.27
IL-1β release in cell culture supernatants was quanti-
fied using a Ready- SET- Go ELISA Set from eBioscience.
HMGB1 release in cell culture supernatants was quanti-
fied using a kit from Finetest Biotechnolgy. Calreticulin
exposure on plasma membrane was measured by flow
cytometry using a specific antibody anti- mouse calretic-
ulin from Abcam (clon EPR3924, PE).
Generation of mouse bone marrow–derived dendritic cells
DCs were generated from bone marrow cells using
wild- type (wt) C57BL/6 mice, in RPMI 1640 medium
containing 10% of FCS serum, 100 U/mL of penicillin/
streptomycin, 50 mM of 2- ME and 10% of supernatant
of X63Ag8653 cell cultures as source of GM- CSF (Zal et
al, 1994) (DC medium). Cells were cultured on 100 mm
petri dishes (1×106 cells/10 mL DC culture medium). On
days 3 and 5, the cell medium was refreshed. On day 7,
supernatants contained cells, which showed differenti-
ated morphology and expressed the DC markers CD11c+,
MHC- II low and CD40 low, confirming their identity as
immature DCs. For their maturation, these DCs were
incubated with LPS 1 μg/mL for 20 hours.
Tumor development
Non- pulsed or gp33- pulsed EL4 cells were inoculated
intraperitoneally or subcutaneously in mice following the
different protocols described. For pulsed cells, EL4 cells
were incubated with 100 nM gp33 or 1 μM OVA peptide
for 1 hour at 37°C and washed before inoculation. In
some experiments, mice were injected with 100 μg of anti-
CD8β mAb (clon H35-17.2) or the same amount of rat
isotype control before injecting tumor cells.
Subcutaneous tumor development was analyzed by
measuring tumor volumes every second day. Volume
was calculated using the equation formula W x L x H,
where W, L and H represent the width, length and height
of the tumor. Mice were sacrificed when they reach the
humane endpoint as established by the Animal Ethics
Committee (volume larger than 0.5 cm3 or presenting
signs of ulceration).
Statistical analysis
Statistical analysis was performed using GraphPad Prism
software. The difference between means of unpaired
samples was performed using two- way analysis of variance
(ANOVA) with Bonferroni’s post- test or using unpaired
t- test as indicated. Survival curves were compared using
log- rank test (Mantel- Cox). The results are given as the CI
with p values and are considered significant when p<0.05.
RESULTS
EL4 cells killed by Ag-specic CD8+ Tc cells express ICD
signals
We have previously shown that Ag- specific CD8+ cytotoxic
T cells (CD8+ Tc cells) eliminate EL4 lymphoma cells in
vitro and in vivo, independently of the apoptotic mito-
chondrial pathway, caspases, necroptosis and pyroptosis
by a mechanism involving granule exocytosis.24 This was
demonstrated using different cell lines expressing anti-
apoptotic mutations. Thus, we now aimed to analyze if the
target cells killed in vitro by CD8+ Tc cells express some of
the molecular immunogenic determinants and the effect
of the cell death mutations indicated above. To this aim,
we focused on some danger signals previously described
to participate in ICD induced by other anticancer treat-
ments like chemotherapy and radiotherapy: calreticulin
membrane exposure (CRT) and IL-1β and HMGB1
release.28–30 CRT is an ‘eat- me’ signal that contributes
to the uptake of dying cells by DCs. The nuclear protein
HMGB1 is released when nuclear membrane is disrupted,
acting as a danger signal. IL-1β is a well- known inflamma-
tory cytokine.
As shown in figure 1A, and confirming previous find-
ings,24 ex vivo gp33- specific Tc cells induced the same
level of cell death in parental EL4 cells as in the EL4.
DNC3 and EL4.Bcl- XL mutant cells, being most cells
eliminated at 3:1 effector:target ratio. A summary of the
gating strategy shown in online supplementary figure 1.
As previously found,24 cell death is delayed in the pres-
ence of Q- VD- OPh (figure 1A); thus, we required to
increase effector:target ratio to 7:1 to get similar levels of
dead cells to be used in the immunization protocols later
on. It should be noted that at this time point (20 hours)
all cells are positive for AAD, indicating that they have
permeabilized plasma membrane. However, this effect
is due to secondary necrosis observed in culture since
at earlier time points (1–2 hours) most cells present an
apoptotic phenotype, presenting PS translocation in
the absence of membrane permeabilization.24Next, we
analyzed the indicated ICD signals (figure 1B–D). In
order to compare the results in conditions with the same
level of cell death, a higher effector:target ratio was used
when Q- VD was included. CRT exposure was analyzed in
7AAD negative cells to prevent staining of intracellular
CRT (online supplementary figure 1). CD8+ Tc killing
dramatically increased CRT exposure in EL4.gp33 cells
compared with EL4 control cells, and this was prevented
if killing was performed in the presence of Q- VD or using
target cells expressing the caspase-3 mutant, EL4DNC3.
gp33 (figure 1B). In contrast, EL4 target cells overex-
pressing Bcl- XL(EL4Bcl- XL.gp33) exposed CRT at the
same level as EL4.gp33 cells following Tc cell killing. In
all cases, CRT exposure was higher than the level of cell
death (see figure 1A) and it was significantly reduced in
the presence of QVD or in EL4DNC3 cells, even when
cell death was not affected (see figure 1A), confirming
that CRT exposure is not a consequence of membrane
permeabilization during cell death.
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Open access
Figure 1 EL4.gp33 cells killed by antigen- specic Tc cells express immunogeneic cell death signals. (A) EL4 cells and mutants
thereof (Bcl- xL and DNC3) were incubated with ex vivo gp33- specic CD8+ T cells from virus- immunized C57BL/6 mice in the
presence of the gp33 Ag for 20 hours at the indicated effector:target ratios. Subsequently, phosphatydilserine exposure on
plasma membrane and cell membrane integrity were measured by three- color ow cytometry using Annexin- V and 7- AAD as
described in the Methods section. Data are represented as mean±SD of four independent experiments using eight mice in total.
(B–D) EL4 cells and mutants thereof (Bcl- XL and DNC3) were incubated with ex vivo gp33- specic CD8+ T cells from virus-
immunized C57BL/6 mice in the presence of the gp33 Ag for 20 hours at 1:1 effector:target ratio. A higher effector:target ratio
(3:1) was used when QVD (30 µM) was included. Non- pulsed EL4 cells incubated with Tc cells and gp33- EL4 cells alone were
used as controls. (B) Calreticulin exposure was analyzed by ow cytometry as indicated in the Methods section. (C, D) HMGB1
and interleukin (IL)-1β was measured in cell supernatants by ELISA. Data are represented as the mean±SD of four independent
experiments, where *p<0,1; **p<0,01; ***p<0001, analyzed by unpaired t- test.
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Open access
HMGB1 was significantly increased in supernatant
from EL4.gp33 cells incubated with gp33- specific Tc
cells compared with that from EL4 cells (figure 1C). In
addition, HMGB1 was not released from activated Tc
cells, confirming its specific release from dying EL4.
gp33 cells. In contrast with CRT exposure, HMGB1
release was independent of caspase-3 inhibition or
mutation.
IL-1β in supernatant from EL4.gp33 cells incubated
with gp33- specific Tc cells was also increased compared
with control cells, gp33- specific Tc cells or non- pulsed
EL4 cells incubated with gp33- specific Tc cells. IL-1β
production was reduced by QVD, likely due to inflam-
matory caspase inhibition. In contrast, IL-1β concentra-
tion was not reduced in EL4DNC3 or EL4.Bcl- XL cells.
These results show that Ag- specific CD8+Tc cells
induce three main hallmarks of ICD in tumor dying
EL4 cells, CRT exposure and HMGB1 and IL-1β release
and indicate that these signals are differentially regu-
lated by caspases and caspase-3.
Immunization with EL4.gp33 cells killed by gp33-specic Tc
cells generates CD8+ T cell-mediated protection against EL4
tumor development
Despite the ability of Tc cells to induce ICD signals in
target cells, this finding does not imply that this process
is able to induce anticancer immunity against dying
cancer cells. To decipher whether elimination of cancer
cells by Tc cells is actually immunogenic, we analyzed if
tumor elimination by Tc cells resulted in the priming
of the host immune system against endogenous tumor
antigens different from those involved in the cytotoxic
killing of the tumor by the gp-33- specific Tc cells. We set
up a model of whole- cell tumor vaccination employing
gp33- pulsed EL4 cells (EL4.gp33) killed by gp33-
specific Tc cells ex vivo as indicated in figure 1A and
subsequently used to immunize C57Bl/6 (B6) wt mice.
This model is summarized in figure 2A. For simplicity,
we will refer to cells killed under this protocol as EL4.
gp33TcLCMV. As control, one group was inoculated with
phosphate- buffered saline (PBS) -and another one was
inoculated with the same amount of activated gp33-
specific Tc cells alone (TcLCMV group) in order to
analyze if immunomodulatory cytokines produced by
Tc cell might contribute to tumor immunogenicity.
After 7 days, mice were challenged with parental EL4
cells in the right flank, and tumor development was
monitored. As shown in figure 2B, tumor growth was
delayed and survival was longer in mice immunized with
EL4.gp33TcLCMV cells, compared with control groups
(figure 2B). In addition, we found a slight but signifi-
cant increase in CD4+ and CD8+ infiltrating T cells in
mice immunized with EL4.gp33TcLCMV cells compared
with PBS control (figure 2C). However, CD8+ T cells
also increased in mice inoculated with TcLCMV cells
alone, which were not protected from tumor devel-
opment, indicating that increased infiltration is not
sufficient for protection. These results indicate that
immunization with EL4.gp33TcLCMV cells generates a
protective immune response against parental EL4 cells.
In addition, the data indicate that the potential immu-
nomodulatory effect of activated Tc cells is not sufficient
to provide any protection against tumor development.
To find out whether the secondary protective
response is dependent on anti- tumoral CD8+ Tc cells
generated against EL4 endogenous Ags, we immunized
mice (following the protocol indicated in figure 2A) in
which CD8+ T cells had been depleted or mice lacking
the cytotoxic molecule perforin (perfKO). While mice
immunized with EL4.gp33TcLCMV cells developed signifi-
cantly smaller tumors and survived significantly longer
than control mice, perfKO mice or those lacking CD8+
Tc cells lost this protection (figure 1D). Similar to
the results obtained in figure 2C, immunization with
EL4.gp33TcLCMV cells significantly increased the % of
CD3+/CD4+ T cells and CD3+/CD8+ T cells in wt mice
(figure 2E). There were not significant differences
between wt and and perfKO mice, further indicating
that the absence of tumor protection in perfKO mice
was not due to a reduction in the generation/infiltra-
tion of CD8+ Tc cells. In conclusion, protective response
to EL4 secondary challenge is dependent on CD8+ T
cell perforin- dependent killing activity against EL4 cells
after immunization with dying EL4.gp33TcLCMV cells.
BATF3-dependent dendritic cells and TLR4 are required for
priming of new EL4-specic CD8+ Tc
To demonstrate that new CD8+ Tc cells are primed
against endogenous antigens expressed on EL4 cells, we
analyzed whether blockade of Ag cross- priming affected
protection against EL4 tumor development after vacci-
nation with EL4.gp33TcLCMV cells. BATF3- deficient
mice, which lack cDC1s specialized in tumor Ag cross-
presentation,31 32 and TLR4- deficient mice, which
lack a pathway of sensing HMGB1 involved in antigen
processing for cross- presentation,28 were immunized
following the protocol indicated in figure 2A. Notably,
protection to EL4 secondary challenge conferred by
immunization with EL4.gp33TcLCMV cells was lost in
TLR4KO and BATF3KO mice (figure 3A). This result
suggests the priming of new CD8+ T cells against endog-
enous EL4- derived Ags by cDC1s and using a TLR4-
dependent pathway for improved Ag cross- presentation.
Subsequently, to confirm CD8+ Tc cell cross- priming,
we analyzed the generation of specific CD8+ Tc cells
against EL4 endogenous Ags in wt and TLR4KO and
BATF3KO mice. Since EL4 endogenous tumor Ags are
not known, it is not possible to analyze the generation
of Ag- specific CD8+ T cells by conventional multimer
staining.
Thus, we tested ex vivo the anti- tumoral activity of
spleen cells from control and immunized mice against
EL4 cells. Spleen cells from wt mice immunized with
EL4.gp33TcLCMV cells showed increased killing capacity
of EL4 cells compared with spleen cells from non-
immunized control, TcLCMV immunized mice or
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Figure 2 EL4.gp33 cells killed ex vivo by gp33- specic Tc cells generate protection against EL4 tumors. (A) EL4 cells were
incubated with ex vivo gp33- specic CD8+ T cells from virus- immunized C57BL/6 mice in the presence of the gp33 Ag for
20 hours at an effector:target ratio 3:1. After this time, cell cultures were collected and used to immunize C57B/L6 mice
intraperitoneal at day 0 and day 7 (EL4.gp33TcLCMV=gp33- EL4.gp33 cells+gp33 Tc cells). In the gp33- Tc cell group (TcLCMV),
mice were immunized with the equivalent number of gp33- specic Tc cells (1.5×106 cells). As control, mice were immunized
with PBS. At day 14, the three groups were inoculated with 2×105 EL4 cells in the right ank. (B) Tumor development was
monitored over 25 days as described in the Methods section. The data correspond to 10 mice from two independent
experiments, where ***p<0001.Two- way analysis of variance (ANOVA), with Bonferroni post- test and log- rank test (Mantel- Cox)
in the survival graph. (C) In some mice, tumors were removed and TIL composition was analyzed by ow cytometry using the
indicated cell markers. The data correspond to ve mice from two independent experiments, where *p<0,1; **p<0,01; analyzed
by unpaired t- test. (D) The same experiment as shown in (A) was performed but employing wild- type (WT) and perfKO mice
or mice depleted of CD8+ T cells using an anti- CD8β monoclonal antibody (days 13, 17, 21 and 25). The data correspond
to 10 mice from two independent experiments, where ***p<0001. Two- way ANOVA, with Bonferroni post- test and log- rank
test (Mantel- Cox) in the survival graph. (E) In some mice, tumors were removed and TIL composition was analyzed by ow
cytometry using the indicated cell markers. The data correspond to ve mice from two independent experiments, where *p<0,1;
**p<0,01. Analyzed by unpaired t- test.
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Figure 3 BATF3 and TLR4 are required for the generation of cancer immunity after immunization with dead EL4 cells. (A) EL4
cells were incubated with ex vivo gp33- specic CD8+ T cells from virus- immunized C57BL/6 mice in the presence of the gp33
Ag for 20 hours at an effector:target ratio 3:1. After this time, cell cultures were collected and used to immunize wild- type (wt),
TLR4KO and BATF3KO mice at day 0 and day 7. As control, mice were immunized with PBS. On day 14, mice were inoculated
with 2×105 EL4 cells in the right ank. Tumor development was monitored over 25 days as described in the Methods section.
The data correspond to 10 mice from two independent experiments, where ***p<0001. Two- way analysis of variance (ANOVA),
with Bonferroni post- test and log- rank test (Mantel- Cox) in the survival graph’s. WT, TLR4KO and BATF3KO mice were immunized
with gp33- pulsed EL4 dead cells as indicated in (A). On day 10, splenocytes from these mice were isolated and incubated at
an effector:target ratio 100:1 with EL4 cells in the presence or absence of the viral peptide GP33. After 18 hours, PS exposure
on plasma membrane was measured by three- color ow cytometry using Annexin- V. Data are represented as the mean±SD of
three independent experiments using six mice in total, where *p<0,1; analyzed by unpaired t- test.
EL4.gp33TcLCMV- immunized mice deficient in TLR4 or
BATF3 (figure 3B). These results show that BATF3 and
TLR4- dependent cross- priming to dying EL4.gp33TcLCMV
generates an effective Tc response to endogenous EL4
Ags that is dependent on granule exocytosis (figure 2).
GP33-SPECIFIC CD8+TC KILLING IN VIVO ALSO PROTECTS
AGAINST EL4 SECONDARY CHALLENGE
To demonstrate whether Ag- specific CD8+Tc cell killing
in vivo was immunogenic, we employed a clinical rele-
vant immunotherapy protocol consisting of vaccination
with DCs loaded with gp33 Ag (DCgp33) (figure 4A). This
protocol has shown good efficacy in vivo n mice using
other antigen models.33 A summary of the protocol used
is depicted in figure 4A. Immunization with mature
DCgp33 conferred a large protection against EL4.gp33
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Figure 4 Vaccination with dendritic cells (DCs) pulsed with gp33 induces Tc cell- dependent protection against EL4.gp33
tumors. Upper panels: C57BL/6 WT, perfKO mice or mice depleted of CD8+ T cells using an anti- CD8β monoclonal antibody
(days 5, 9, 13 and 17) were inoculated with PBS (control) or with 2×106 mature DC alone (DC) or incubated with gp33 peptide
(DCgp33) intraperitoneal on day 6, the three groups were inoculated with 2.5×105 gp33- EL4 cells in the right ank. Tumor
development was monitored over 50 days as described in the Methods section. The data correspond to 12 mice from three
independent experiments, where ***p<0.001. Two- way analysis of variance with Bonferroni post- test and log- rank test (Mantel-
Cox).
tumor development compared with mice non- immunized
or those immunized with DCs matured but without gp33
(figure 4B). Protection against EL4.gp33 tumor develop-
ment was completely abrogated when CD8+ T cells were
depleted or in perfKO mice (figure 4C). These results
indicate that immunization with mature DCgp33 generates
a CD8+ T cell- dependent and perf- dependent protec-
tion against EL4 tumor development, showing that this
model is suitable to analyze immunogenicity of cell death
induced by CD8+ Tc cells in vivo.
Next, we analyzed if the elimination of EL4.gp33
cells in vivo in mice immunized with DCgp33 (from now
on EL4.gp33TcDC cells) generated a protective response
against parental EL4 tumors. Mice inoculated with
mature DCgp33 developed EL4 tumors and reach the
endpoint on day 15 (DCgp33, figure 5A), similar to control
non- immunized mice (figure 3A). In contrast, EL4 tumor
development was largely reduced and survival increased
in mice immunized following the protocol EL4.gp33TcDC
(figure 5A). This tumor protection was lost on CD8+ T
cell depletion, showing that elimination of parental EL4
cells was dependent on the generation of new CD8+ Tc
cells specific against EL4 endogenous antigens. A similar
result was found, when the same protocol was performed,
but using the OVA antigen instead of gp33. In this case,
elimination of EL4OVA cells in vivo in mice immunized
with DCOVA generated a protective response that signifi-
cantly delayed the growth of parental EL4 tumors (online
supplementary figure 2), indicating that immunization
against endogenous EL4 tumors was independent of the
antigen model.
To confirm that CD8+ Tc cells cross- primed against
dying EL4.gp33- derived antigens were the respon-
sible of EL4 tumor elimination, we employed an adop-
tive transfer protocol using CD8+T cells from wt and
BATF3KO mice, previously immunized following the
protocol EL4.gp33TcDC (figure 5B). Only those mice
transferred with CD8+ Tc cells from EL4.gp33TcDC immu-
nized wt mice showed protection against EL4 tumor
development compared with mice transferred with Tc
from wt mice immunized with DCgp33, or Tc coming
from BATF3- deficient mice independently of the immu-
nization stimulus (figure 5B). These results show that
in vivo elimination of EL4.gp33 cells by gp33 Ag- spe-
cific CD8+Tc cells is immunogenic and cross- primes a
CD8+ Tc cell response against endogenous EL4- derived
antigens, suggesting that is a general mechanism of
epitope spreading not only applicable to adoptive T cell
transfer but also to DC vaccination strategies for cancer
immunotherapy.
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Figure 5 EL4.gp33 cells killed in vivo after immunization with dendritic cells (DCs) pulsed with gp33 protects against EL4
tumor development. (A) C57BL/6 mice were inoculated with mature gp33- DCs. On day 6, the control group was inoculated with
PBS (control) and the rest of groups were inoculated with 2.5×105 gp33- EL4 cells in the right ank (EL4.gp33TcDC). On day 20, all
groups were inoculated with 2×105 EL4 cells in the left ank. In one group of mice CD8 cells were depleted employing an anti-
CD8β monoclonal antibody (days 19, 23, 27 and 31) (EL4.gp3TcDC anti- CD8). Tumor development was monitored over 50 days
as described in theMethods section. The data correspond to 12 mice from three independent experiments, where ***p<0.001.
Two- way analysis of variance (ANOVA) with Bonferroni post- test and log- rank test (Mantel- Cox). (B) C57BL/6 wt and BATF3KO
mice were inoculated with 3×106 mature DCs (LPS 1 µg/mL 20 hours) incubated with gp33 peptide via intraperitoneal. On day 6,
one group was inoculated with PBS (Tc wt DCsgp33, Tc BATF3KO DCsgp33) and the other group was inoculated with 5×105 gp33-
EL4 cells in the right ank (TC wt EL4.gp33TcDC, Tc BATF3KO EL4.gp33TcDC). Seven days later, mice were sacriced, CD8+ Tc cells
from the spleen and lymph nodes were enriched by MACS and transferred (6×106 cells) into C57BL/6 wt mice, who had been
inoculated with 1.5×105 EL4 cell. Tumor development was monitored over 20 days as described in the Methods section. The
data correspond to ve mice from one experiment, where **p<0.01; ***p<0.001. Two- way ANOVA with Bonferroni post- test and
log- rank test (Mantel- Cox).
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ROLE OF CASPASES AND THE MITOCHONDRIAL CELL DEATH
PATHWAY IN ICD INDUCED BY CD8+ TC CELLS
Finally, we wondered how the mutations on cell death
pathways analyzed in figure 1 in vitro would affect the
immune response against tumor Ags derived from
cancer cells eliminated by Tc cells in vivo. To this aim, we
analyzed the role of caspases and the mitochondrial cell
death pathway in the generation of immunity against EL4
tumor development after vaccination. The level of protec-
tion offered by EL4.gp33 cells killed in vivo after DCgp33
vaccination was much pronounced as compared with the
protocol employing EL4.gp33 killed in vitro (figures 5
and 2). However, in order to reduce the number or mice
and to study the mechanism in a more controlled way,
we followed the vaccination protocol with EL4.gp33
killed in vitro (EL4.gp33TcLCMV cells), using EL4.gp33
wt cells as well as mutants thereof overexpressing Bcl-
XL or expressing a dominant negative caspase-3 mutant
(DNC3). In addition, we used EL4.gp33 wt cells in which
caspases were blocked with the pan- inhibitor Q- VD- OPh.
Mice were immunized employing the cells killed in
figure 1A, following the protocol indicated previously
(figure 2). As shown in figure 6A, mice immunized
with EL4.gp33TcLCMV cells were protected against tumor
development and survived significantly longer than non-
immunized control mice. In contrast, protection disap-
peared in those mice immunized with EL4.gp33TcLCMV
cells that had been killed in the presence of Q- VD, indi-
cating that functional caspases are required to generate
protection against parental EL4 tumor development. A
similar result was found when EL4DNC3.gp33TcLCMV cells
were used. The protection observed after immunization
with EL4.gp33TcLCMV was lost when cells expressed the
caspase-3 mutant (EL4DNC3.gp33TcLCMV, figure 6B). In
contrast, mice immunized with EL4Bcl- XL.gp33TcLCMV
cells were protected and developed EL4 tumors signifi-
cantly smaller and survived significantly longer than non-
immunized control mice (figure 6B). Although tumors
were slightly bigger than mice immunized with EL4.
gp33TcLCMV, neither tumor growth nor mouse survival was
significantly different. Therefore, this result suggests that
the intrinsic apoptotic pathway is dispensable for CD8+
Tc- induced ICD in EL4 tumor model, which critically
depends on the presence of active caspase-3.
To confirm the role of caspase-3 in ICD induced by
CD8+Tc cells, we analyzed the generation of EL4- specific
CD8+ Tc cells after immunization with EL4.gp33 dead
cells. Splenocytes from mice immunized with EL4.
gp33TcLCMV showed increased killing of parental EL4
cells in vitro compared with splenocytes from control
mice or those immunized with EL4.gp33TcLCMV killed in
the presence of Q- VD or with EL4DNC3.gp33TcLCMV cells
(figure 6C). Cytotoxic activity of splenocytes from mice
immunized with EL4Bcl- XL.gp33TcLCMV cells was similar to
that from EL4.gp33TcLCMV immunized mice. These results
confirm that caspase-3, but not the intrinsic apoptotic
pathway, is required for ICD induced by CD8+ Tc cells,
including the generation of a protective anti- tumor CD8+
Tc cell response.
DISCUSSION
A major understanding of the regulation of CD8+ Tc cell-
mediated immunity in cancer has been key to successfully
treat mutated bad prognosis cancers with immune check-
point inhibitors or with modified CAR- T cells. However,
the number of patients benefiting from immunotherapy
is still relatively low and restricted to a small propor-
tion of some types of cancer. The factors contributing
to immunotherapy efficacy and tumor resistance and/
or relapse are poorly explored. We have recently shown
that antigen- specific Tc cells are able to eliminate tumor
cells expressing anti- apoptotic mutations conferring bad
prognosis and drug resistance, preventing tumor devel-
opment in vivo.24 However, it is unclear if these mutations
may affect tumor relapse. Here, employing two different
models of cancer vaccination in mice and the EL4
lymphoma model, we show that elimination of cancer
cells by antigen- specific CD8+ Tc cells ex vivo and in vivo
is immunogenic and generates a secondary protective
immune response against endogenous tumor antigens.
Importantly, specific mutation of the cell death execu-
tioner caspase-3, although did not affect cell killing and
elimination of primary tumors completely abrogated the
generation of protective CD8+ Tc cell response against
secondary tumor development. This finding indicates
that ICD induced by CD8+ Tc cells and the generation of
spread immunity against endogenous tumor antigens rely
on caspase-3- dependent apoptosis of EL4 cancer cells.
Although we have mainly used a model of tumor
antigen derived from the LCM virus, gp33 antigen, this
should not be a major limitation to extrapolate our find-
ings to other immunotherapies, like those employing
viral infections (oncolytic viruses) and others.34 First, ICD
induced by Tc cells has been confirmed in vivo using a
conventional approach consisting of vaccination with
peptide (gp33) pulsed bone marrow–derived DCs, which
has been previously shown to offer CTL priming and
tumor immunity.33 In addition, the affinity of the antigen
T cell receptor for viral gp33 is similar to that one for
‘real’ tumor antigens like MelA or gp100.35 36 This affinity
is even higher for engineered T cell receptors as in CAR- T
cells. Anyway, we have confirmed that our results are not
restricted to gp33- antigen since elimination of EL4.OVA
cells in mice vaccinated with OVA- pulsed bone marrow–
derived DCs significantly delayed the development of
parental EL4 tumors. Thus, the use of two different surro-
gate antigens supports the conclusion that our results
are not influenced by the selection of specific surrogate
tumor antigens. Moreover, a similar result in other tumor
models employing different tumor antigens has been
shown by Ignacio Melero’s group in an accompanying
paper (Cordeiro- Minute et al, to be added in production)
reinforcing that ICD induced by Tc cells is not restricted
by specific antigens.
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Figure 6 Role of caspases and Bcl- xL in the generation of immunity against EL4 antigens. (A) EL4 cells were incubated with
ex vivo gp33- specic CD8+ T cells from virus- immunized C57BL/6 mice in the presence of the gp33 Ag for 20 hours at an
effector:target ratio 3:1 (EL4.gp33TcLCMV Group) or at an effector:target ratio 7:1 in the presence of the pan- caspase inhibitor
Q- VD- OPh (30 µM; EL4.gp33TcLCMV + Q VD group). After this time, cell cultures were collected and used to immunize C57B/L6
mice via intraperitoneal. At day 0 and day 7, as control, mice were immunized with PBS. On day 14, the different groups were
inoculated with 2×105 EL4 cells in the right ank. Tumor development was monitored over 25 days as described in the Methods
section. The data correspond to 12 mice from three independent experiments, where ***p<0.001. Two- way analysis of variance
(ANOVA) with Bonferroni post- test and log- rank test (Mantel- Cox). (B) The same experiment as in (A) was performed but using
EL4 cells and the mutants thereof overexpressing Bcl- XL or DNC3. The data correspond to 12 mice from three independent
experiments, where **p<0.01; ***p<0.001. Two- way ANOVA with Bonferroni post test and log- rank test (Mantel- Cox). (C) wt mice
were immunized with the indicated gp33- pulsed EL4 dead cells killed as indicated in (A), with PBS (CTR) or with gp33- specic
Tc cells (TcLCMV). On day 10, splenocytes from these mice were isolated and incubated at an effector:taget ratio 100:1 with EL4
cells in the absence of the viral peptide gp33. After 18 hours, PS exposure on plasma membrane was measured by three- color
ow cytometry using Annexin- V. Data are represented as the mean±SD of three independent experiments, using six mice in
total, where *p<0.05, analyzed by unpaired t- test.
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12 Jaime- SanchezP, etal. J Immunother Cancer 2020;8:e000528. doi:10.1136/jitc-2020-000528
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The concept of immunogenic cell death raised from
the observation that cancer cells eliminated by specific
chemotherapy drugs or radiotherapy induced the gener-
ation of protective CD8+ Tc cells against antigens released
by dying tumor cells.9 10 14 15 20 This response contributed
to the elimination of the tumor cells and, in addition, it
was shown to prevent cancer recurrence.9 28 Since then,
several stimuli have been found to induce ICD on cancer
cells including irradiation, hyperthermia, infection or
cell starvation.16 However, and paradoxically, studies
addressing if cell death induced by CD8+ Tc cells is immu-
nogenic are scarce.
Protection correlates with the generation of well- known
ICD signals in dying cancer cells: calreticulin membrane
exposure and the release of HMGB1 and the proinflam-
matory cytokine IL-1β.16 However, several signals have
been associated to ICD induced by different stimuli. Thus,
analyzing the effect of in vivo immunization with dead
cells on tumor development is mandatory to analyze if
cell death induced by a specific stimulus is immunogenic.
Here an important question to genuinely identify ICD on
cancer cells is the potential direct in vivo immunomodu-
latory effects of the stimulus used to kill the cancer cells
used for the immunization. For example, different drugs,
commonly referred to as ICD inductors, are capable to
induce tumor cell death as well as to directly modulate
the immune system in vivo making it impossible to attri-
bute the observed biological effects solely to ICD, unless
they are removed before immunization. Activated Tc cells
also produce several immunomodulatory cytokines that
might contribute to the effects observed in vivo in our
study. Having in mind this possibility, our study included
different controls suggesting that a direct effect of Tc cells
is not the responsible of the protection observed during
vaccination with dead EL4 tumor cells. First, inoculation
of mice with activated Tc cells alone has no effect on
tumor development. On top of that, immunization with
the mixture of activated Tc cells and dead tumor cells,
killed in the absence of caspase-3 activity, did not confer
any protection against tumor development. An optimal
approximation would be to separate the cell debris from
Tc cells after the killing assay. However, this approxima-
tion is challenging and would likely change the immu-
nogeneic properties of cell debris like the presence of
soluble factors released by dying cells. Thus, being aware
of this difficult limitation to overcome the in vivo vaccina-
tion experiments strongly support for an immunogeneic
phenotype of cell death induced by CD8+ Tc cells.
We find herein that general caspase inhibition as well as
caspase-3 inhibition impairs immunogenicity and spread
immunity against endogenous tumor antigens. However,
the role of specific caspases seems to be dependent on
the stimuli used29 37 38 and likely the tumor cell type.
Our results show that, at least in EL4 lymphoma cells,
caspase-3 plays a key role in ICD induced by CD8+ Tc
cells since vaccination with dying EL4 cells expressing the
DN caspase-3 mutant does not stimulate the generation
of CD8+ Tc cells against endogenous EL4 antigens and
does not protect against secondary tumor development.
In contrast to caspase-3, the mitochondrial apoptotic
pathway does not seem to be involved in ICD induced by
CD8+ Tc cells since Bcl- XL overexpression had no effect.
Further confirming that Tc cells induce ICD in tumor
cells, we show that the protective effect is loss in mice
deficient in TLR4 or in BATF3- dependent DCs. TLR4 is
a receptor for HMGB1, a DAMP that interacts with TLR4
in DCs for an efficient processing and cross- presentation
of antigens derived from cells killed by chemotherapy or
radiotherapy.28 39 Regarding BATF3- dependent cDC1s,
they are involved in phagocytosis and cross- presentation
of antigens from dying cells, contributing to protec-
tion against some but not all viral, bacterial and fungal
infections.31 40 A previous study found out that tumor
cells killed by OT1- specific Tc cells induced antigen
cross- presentation by DCs.23 However, that study did not
analyze whether this killing was immunogenic in the
tumor context and whether it conferred a protective
immune response against tumor development. Our data
show that cancer cells killed by Tc cells bear an ICD that
promotes cross- presentation by cDC1s, which are specif-
ically required for antitumor immunity following ICD
induction by Tc cells. These results concur with the essen-
tial role of cDC1s in tumor vaccination41 and its associ-
ation with improved overall survival in several tumors.42
Indeed, BATF3 is required for an efficient generation of
Tc cell immunity against primary immunogenic tumors.31
In addition, BATF3- dependent DCs modulate the efficacy
of different immunotherapy protocols including adoptive
T cell therapy or mAb against immune checkpoints.32 42 43
Our data show the requirement of cDC1 for generation
of an efficient protective immunity against endogenous
antigens expressed in dead tumor cells following Tc- in-
duced ICD. Remarkably, cDC1s are not essential for anti-
tumor immunity following other models of ICD.44
As indicated above, our results have been inde-
pendently confirmed using other cancer immunothera-
pies and different tumor models, including transgenic T
cell receptors and NK cells (Cordeiro- Minute et al, to be
added in production). Recent findings in clinical trials
employing T cell- based cancer immunotherapy support
the novelty, the conceptual advance and the potential
clinical relevance of our results. It has been recently
found that during CAR- T cell therapy in gastric cancer,
new T cell clones against tumor neoantigens are detected
in patients, a concept known as epitope spreading.45
Epitope spreading was also observed during cancer vacci-
nation in humans,46 although the molecular basis for this
phenomena remained unexplored. Our results provide
an explanation and the molecular mechanism involved in
that observation, enhancing our mechanistic knowledge
to design rational new vaccines and other T cell- based
therapies to overcome antigen loss or the absence of
known antigens, a problem commonly observed during
cancer vaccination and CAR- T cell therapy. These new
protocols may include approaches to overcome potential
tumor evasion strategies to counteract epitope spread.
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Jaime- SanchezP, etal. J Immunother Cancer 2020;8:e000528. doi:10.1136/jitc-2020-000528
Open access
CONCLUSIONS
Using a mouse in vivo model of cancer immunotherapy,
we have found that CD8+ Tc cells induce ICD on cancer
cells, generating spread immunity and a protective Tc
cell response against endogenous tumor antigens. The
mechanism involved depends on the presence of active
caspase-3 and is independent of the mitochondrial cell
death pathway. These findings indicate that ICD and
epitope spreading during cell death induced by CD8+ Tc
cells contribute to the efficacy of cancer immunotherapy.
Moreover, our findings suggest that mutations in caspase-3
or in pathways regulating its activity might increase the
risk of tumor refractoriness and/or recurrence after
T cell- based cancer immunotherapy. Clinical trials will
be required to test whether the presence of caspase-3
mutations and/or inhibitors like XIAP can be used as
biomarkers to predict relapse during immunotherapy.
Author afliations
1CIBA, Instituto de Investigacion Sanitaria Aragon, Zaragoza, Spain
2Microbiology, Preventive Medicine and Public Health, Medicine Faculty, University
of Zaragoza, Zaragoza, Spain
3CIBER Respiratory Diseases, Madrid, Spain
4Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid,
Spain
5Instituto de Carboquimica, Zaragoza, Spain
6Fundacion ARAID / IIS Aragon / CIBA, Universidad de Zaragoza, Zaragoza, Spain
7CIBER- BBN, Madrid, Spain
Acknowledgements The authors would like to acknowledge the use of Servicios
Cientíco Técnicos del CIBA (IACS- Universidad de Zaragoza) and Servicios Apoyo
Investigación de la Universidad de Zaragoza.
Contributors PJS designed and performed experiments and wrote the st draft
of the manuscript; IUM performed experiments; NA developed and characterized
EL4.Bcl- XL cells; MAA designed and performed experiments; DS and SCK
provided BATF3- decient mice and OT1 transgenic mice, designed and performed
experiments and wrote the manuscript; JP conceived and designed the original
study and wrote the manuscript. All authors revised and approved the last version
of the manuscript.
Funding Work in the JP laboratory is funded by Asociacion de Padres de
Niños con Cancer de Aragon (ASPANOA), FEDER (Fondo Europeo de Desarrollo
Regional, Gobierno de Aragón(Group B29_17R) and Ministerio de Ciencia,
Innovación e Universidades (MCNU), Agencia Estatal de Investigación (SAF2014-
54763- C2-1 and SAF2017‐83120‐C2‐1‐R). Predoctoral grants/contracts from
Fundación Santander/Universidad de Zaragoza (MA), and Gobierno de Aragon
(IUM, PJS) and a postdoctoral Juan de la Cierva Contract (MA). JP is supported
by ARAID Foundation. Work in the DS laboratory is funded by the CNIC, from
Ministerio de Ciencia, Innovación e Universidades (MCNU), Agencia Estatal de
Investigación and Fondo Europeo de Desarrollo Regional (FEDER) (SAF2016-
79040- R) and the European Research Council (ERC-2016- Consolidator Grant
725091). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the
MCNU and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence
(SEV-2015-0505).
Competing interests JP reports research funding from BMS and Gilead and
speaker honoraria from Gilead and Pzer.
Patient consent for publication Not required.
Ethics approval All experiments were performed in accordance with
FELASA guidelines under the supervision and approval of Comité Ético para la
Experimentación Animal (Ethics Committee for Animal Experimentation) from the
University of Zaragoza (number: PI33/13).
Provenance and peer review Not commissioned; externally peer reviewed.
Data availability statement All data relevant to the study are included in the
article or uploaded as supplementary information. All data are included in this
manuscript.
Open access This is an open access article distributed in accordance with the
Creative Commons Attribution Non Commercial (CC BY- NC 4.0) license, which
permits others to distribute, remix, adapt, build upon this work non- commercially,
and license their derivative works on different terms, provided the original work is
properly cited, appropriate credit is given, any changes made indicated, and the use
is non- commercial. See http:// creativecommons. org/ licenses/ by- nc/ 4. 0/.
ORCID iD
JulianPardo http:// orcid. org/ 0000- 0003- 0154- 0730
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