ArticleInflammasomeswithin Hyperactive Murine Dendritic
Cells Stimulate Long-Lived T Cell-Mediated Anti-
tumor ImmunityGraphical AbstractHighlightsd Hyperactive dendritic cells display enhanced ability to
migrate to lymph nodes
d Hyperactive dendritic cells retain inflammasome activity in
the dLN
d Hyperactive cDC1s induce long-lived CD8+ T-cell-mediated
anti-tumor immunity
d Hyperactivating stimuli eradicate tumors that are resistant to
anti-PD1 therapyZhivaki et al., 2020, Cell Reports 33, 108381
November 17, 2020 ª 2020 The Author(s).
https://doi.org/10.1016/j.celrep.2020.108381Authors
Dania Zhivaki, Francesco Borriello,
Ohn A. Chow, ..., Caroline L. Sokol,
Ivan Zanoni, Jonathan C. Kagan
Correspondence
jonathan.kagan@childrens.harvard.edu
In Brief
Inflammasome activation in dendritic
cells (DCs) leads to pyroptosis or
hyperactivation. Zhivaki et al. show that in
contrast to pyroptotic DCs, hyperactive
DCs stimulate durable anti-tumor
immunity that eradicates established
tumors. These protective responses are
intrinsic to cDC1 cells and depend on DC
hypermigration and on the
inflammasome-dependent cytokine IL-
1b.ll
OPEN ACCESS
llArticle
Inflammasomes within Hyperactive
Murine Dendritic Cells Stimulate Long-Lived
T Cell-Mediated Anti-tumor Immunity
Dania Zhivaki,1 Francesco Borriello,2,9 Ohn A. Chow,3 Benjamin Doran,6,7,8 Ira Fleming,6,7,8 Derek J. Theisen,4
Paris Pallis,5 Alex K. Shalek,6,7,8 Caroline L. Sokol,3 Ivan Zanoni,1,2 and Jonathan C. Kagan1,10,*
1Harvard Medical School and Division of Gastroenterology, Boston Children’s Hospital, Boston, MA, USA
2Harvard Medical School and Division of Immunology, Boston Children’s Hospital, Boston, MA, USA
3Center for Immunology & Inflammatory Diseases, Division of Rheumatology, Allergy & Immunology, Massachusetts General Hospital,
Harvard Medical School, Boston, MA 02114, USA
4Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA
5Department of Immunology, Harvard Medical School, Boston, MA 02115, USA
6Department of Chemistry, Institute for Medical Engineering and Sciences (IMES), Koch Institute for Integrative Cancer Research, MIT,
Cambridge, MA 02142, USA
7Ragon Institute of MGH, Harvard, and MIT, Cambridge, MA 02139, USA
8Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
9Department of Translational Medical Sciences and Center for Basic and Clinical Immunology Research (CISI), University of Naples Federico
II, Naples, Italy
10Lead Contact
*Correspondence: jonathan.kagan@childrens.harvard.edu
https://doi.org/10.1016/j.celrep.2020.108381SUMMARYCentral to anti-tumor immunity are dendritic cells (DCs), which stimulate long-lived protective T cell re-
sponses. Recent studies have demonstrated that DCs can achieve a state of hyperactivation, which is asso-
ciated with inflammasome activities within living cells. Herein, we report that hyperactive DCs have an
enhanced ability to migrate to draining lymph nodes and stimulate potent cytotoxic T lymphocyte (CTL) re-
sponses. This enhanced migratory activity is dependent on the chemokine receptor CCR7 and is associated
with a unique transcriptional program that is not observed in conventionally activated or pyroptotic DCs. We
show that hyperactivating stimuli are uniquely capable of inducing durable CTL-mediated anti-tumor immu-
nity against tumors that are sensitive or resistant to PD-1 inhibition. These protective responses are intrinsic
to the cDC1 subset of DCs, depend on the inflammasome-dependent cytokine IL-1b, and enable tumor ly-
sates to serve as immunogens. If these activities are verified in humans, hyperactive DCs may impact immu-
notherapy.INTRODUCTION
Central to our understanding of protective immunity to infection
and cancer are dendritic cells (DCs), which patrol the tissues of
the body (Alvarez et al., 2008). DCs survey the environment for
threats to the host through the actions of pattern recognition re-
ceptors (PRRs), which recognize microbial products or host-en-
coded molecules indicative of tissue injury (Janeway and Medz-
hitov, 2002; Brubaker et al., 2015).Microbial ligands for PRRs are
classified as pathogen-associated molecular patterns (PAMPs),
whereas host-derived PRR ligands are damage-associated mo-
lecular patterns (DAMPs) (Matzinger, 2002).
Upon detection of PAMPs, PRRs unleash signaling pathways
that shift DC activities from a non-stimulatory (naive) state to an
‘‘activated’’ state (Inaba et al., 2000; Mellman and Steinman,
2001). Active DCs have a life expectancy of a few days and are
equipped to prime T cells and boost antigen-specific T cell re-Ce
This is an open access article under the CC BY-Nsponses. As such, numerous strategies have been undertaken
to promote DC activation via Toll-like receptor (TLR) agonists
to drive protective anti-tumor immunity (Sabado et al., 2017).
Notably, TLRs alone do not upregulate all the signals needed
by DCs to promote T cell-mediated immunity. Members of the
interleukin-1 (IL-1) family of cytokines are regulators of T cell dif-
ferentiation, long-lived memory T cell generation, and effector
function (Ben-Sasson et al., 2009, 2013; Garlanda et al., 2013;
Jain et al., 2018; Lee et al., 2019). The expression of IL-1b is
induced by TLRs, but this cytokine lacks an N-terminal secretion
signal and is therefore not released from cells via the conven-
tional biosynthetic pathway (Garlanda et al., 2013). Rather, IL-
1b accumulates in an inactive state in the cytosol.
To induce IL-1b release, most cells require a second signal
that stimulates pyroptosis. Pyroptosis is a regulated process
that results from the actions of inflammasomes, which are supra-
molecular organizing centers (SMOCs) that assemble in thell Reports 33, 108381, November 17, 2020 ª 2020 The Author(s). 1
C-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
ll
OPEN ACCESS Articlecytosol (Kagan et al., 2014; Lu et al., 2014). Inflammasome as-
sembly is commonly stimulated upon detection of PAMPs or
DAMPs by cytosolic PRRs (Lamkanfi and Dixit, 2014; Kieser
and Kagan, 2017). Pyroptosis leads to the release of IL-1 family
members, thereby providing signals to T cells that TLRs cannot
offer. Despite this gain in activity, in terms of promoting IL-1b
release, pyroptotic cells are dead and lose the ability to partici-
pate in the days-long process needed to stimulate and differen-
tiate naive T cells in the draining lymph node (dLN) (Mempel et al.,
2004). Indeed, stimuli that promote pyroptosis, such as the vac-
cine adjuvant alum (Eisenbarth et al., 2008; Kool et al., 2008a),
are weak inducers of T cell-mediated protective immunity (Mar-
rack et al., 2009).
We reasoned that the ideal strategy to stimulate robust T cell im-
munity would be to combine the benefits of activated and
pyroptotic DCs, whereby DCs would have the ability to release
IL-1b while maintaining viability. We recently identified a
new activation state of DCs that displays these attributes.
WhenDCs are exposed toPAMPs (e.g., TLR ligands) and a collec-
tion of oxidized phospholipids released from dying cells (DAMPs),
the cells achieve a long-lived state of ‘‘hyperactivation’’ (Zanoni
et al., 2016, 2017). The collection of oxidized lipids is known as ox-
PAPC (oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phos-
phorylcholine), and individual components such as PGPC (1-pal-
mitoyl-2-glutaryl-sn-glycero-3-phosphocholine) can induce a
hyperactive state in bone-marrow-derived DCs (BMDCs) (Zanoni
et al., 2016). Hyperactive cells display the activities of activated
DCs, in terms of cytokine release (e.g., tumor necrosis factor alpha
[TNF-a]), but they have also gained the ability to release IL-1b over
the course of several days (Zanoni et al., 2017).
Mechanisms underlying the hyperactive state of DCs have
been defined, as oxPAPC binds and stimulates the cytosolic
PRR caspase-11 (Zanoni et al., 2016). Caspase-11 binding re-
sults in the activation of NLRP3 (NLR family pyrin domain con-
taining 3) and the assembly of an inflammasome that does not
lead to pyroptosis, but rather leads to the release of IL-1b from
living cells. IL-1b release from hyperactive cells is mediated by
the pore-forming protein gasdermin D (Kayagaki et al., 2015;
Aglietti et al., 2016; Liu et al., 2016; Evavold et al., 2017). The
impact of hyperactive cells on adaptive immunity is poorly
defined.
Herein, we report that oxidized phospholipids that hyperacti-
vate DCs induce strong cytotoxic T lymphocyte (CTL) re-
sponses, which endow hyperactive DCs with the ability to
mediate long-term protective anti-tumor immunity, even when
a complex antigen source is used (e.g., tumor cell lysates). Hy-
peractivation-mediated protection ismediated by the cDC1 sub-
set of DCs, which depend on inflammasome-dependent IL-1b
release and on an enhanced ability to migrate to the dLN. These
findings establish the physiological importance of the hyperac-
tive state of DC1s in protective immunity to cancer.
RESULTS
Oxidized Phospholipids Induce a State of
Hyperactivation in cDC1s and cDC2s
Several studies have assessed phagocyte hyperactivation, but
those focused on DCs have used monocyte-like BMDCs that2 Cell Reports 33, 108381, November 17, 2020were generatedwith granulocyte-macrophage colony-stimulating
factor (GM-CSF) (Chen et al., 2014; Gaidt et al., 2016; Wolf et al.,
2016; Zanoni et al., 2016; Monteleone et al., 2018). To determine if
conventional DCs (cDCs) can achieve a hyperactive state, we
used DCs that were differentiated from BM using Fms-like tyro-
sine kinase 3 ligand (FLT3L). FLT3L-DCs were primed with LPS
(lipopolysaccharide) and then treated with the oxidized phospho-
lipids oxPAPC or PGPC. Alternatively, FLT3L-DCs were stimu-
lated with the traditional activation stimulus LPS or were primed
with LPS and treated with the pyroptotic stimulus alum. LPS treat-
ment did not induce IL-1b release from DCs, whereas pyroptotic
DCs released IL-1b into the extracellular space (Figure 1A, left
panel). As expected, IL-1b secretion was co-incident with cell
death in pyroptotic DCs, as assessed by the release of the cyto-
solic enzyme lactate dehydrogenase (LDH) (Figure 1A, middle
panel). Stimulation with the hyperactivating stimuli LPS plus
PGPC induced dose-dependent IL-1b secretion from DCs in the
absence of LDH release (Figures 1A and S1A). Dose responses
of DCs treated with various concentrations of LPS and PGPC
identified conditions that were used in this study, where maximal
levels of IL-1b release correlated with inflammatory markers in
DCs (e.g., TNF-a secretion) (Figure S1A). We noted a cell-type-
specific behavior of the oxPAPC mixture, as compared to the
pure component PGPC. In contrast to PGPC, which induced
robust IL-1b release from all DCs, oxPAPC was a strong inducer
of IL-1b release from GMCSF-DCs but a weak inducer of IL-1b
release fromFL3TL-DCs (Figures 1A, S1A, andS1B). IL-1b release
from pyroptotic or hyperactive DCs was in both cases dependent
on the inflammasome components NLRP3 and caspases 1 and
11 (Figure 1A). Similar behaviors of DCs were observed when
DCs were primed with the unmethylated cytosine-phosphate-
guanine (CpG) DNA (Figure S1C).
cDCs are divided into two major subsets: cDC1s and cDC2s.
Of these subsets, cDC1s are uniquely capable of antigen cross-
presentation and can prime naive CD8+ T cells, but also CD4+
T cells (Cancel et al., 2019; Theisen et al., 2019; Ferris et al.,
2020). In contrast, cDC2s activate Th2 and Th17 immunity. To
determine if the behavior of hyperactive DCs extends to cDC1
or cDC2 subsets, we isolated cDC1s or cDC2s from FLT3L-
differentiated DCs (Figure S1D). Similar to the behavior of bulk
FLT3L-derived DCs, treatment with LPS plus PGPC led to
TNF-a and IL-1b release from FLT3L-cDC1s and cDC2s in the
absence of cell death (Figure 1B). oxPAPC exhibited toxic effects
on cDC1s, but not cDC2s (Figure 1B). Based on the uniformity of
DC responses to PGPC, we focused much of our subsequent
work on the activities of PGPC as an inducer of cDC1 and
cDC2 hyperactivation.
Hyperactive cDCs Display a Hypermigratory Phenotype
We observed that hyperactive cDC1s stimulated with LPS plus
PGPC displayed highly extended membrane protrusions, as
compared to naive or active DCs, which suggested potential
changes in migratory activity (Figure 1C). RNA sequencing of
cDC1s and cDC2s, stimulated with LPS alone or with LPS plus
PGPC, revealed upregulated gene signatures that includemigra-
tion-related clusters involved in DC chemotaxis, actin polymeri-
zation, and DC migration (GEO: GSE156159) (Figure 1D; Table
S1). Interestingly, in addition to the common signatures shared
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Article OPEN ACCESS
A
B
C
D
Figure 1. Oxidized Phospholipids Induce a State of Hyperactivation in cDC1s and cDC2s
(A and B) BMDCs, cDC1s, or cDC2s (as indicated) were either left untreated (‘‘None’’) or treated with indicated stimuli. IL-1b and TNF-a release wasmonitored by
ELISA. Cell death was measured by LDH release. Means and SDs from three replicates are shown, and data are representative of at least three independent
experiments.
(C) Immunofluorescence staining of phalloidin (green) and DAPI (blue) for cDC1s. Scale bars: 10 mm (upper panel) and 20 mm (lower panel).
(D) cDC1s and cDC2s were either left untreated (‘‘None’’) or treated as indicated prior to RNA sequencing (n = 3 mice). Shown is the expression of mouse Gene
Ontology modules. Color of dot represents the average relative increase or decrease of log-normalized expression from randomized modules of same number
genes. Size of dot shows percent of module expressed by samples. p values of < 0.05 (*), < 0.01 (**), < 0.001 (***), or, % 0.0001 (****) are indicated.
Cell Reports 33, 108381, November 17, 2020 3
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OPEN ACCESS Articleby active and hyperactive DCs, hyperactive cDC1s and cDC2s
upregulated an exclusive gene signature that we named
‘‘curated cell migration module,’’ since this gene set mainly en-
compasses genes whose products control cell migration (e.g.,
Rhob, Rhoc, Rac1, Arpin, L1cam) (Figures 1D and S2A; Table
S1). These data prompted an exploration of DC motility. We
tracked the motility of single cells and the collective motion of
naive, active, or hyperactive DCs stimulated with LPS plus
PGPC on a glass surface. A measurement of straightness index,
the total cell displacement from its starting point compared to
the distance traveled by the cell, revealed that hyperactive
DCs exhibited the highest movement and directionality, as
compared to their naive or active counterparts (Figure 2A; Videos
S1, S2, and S3). Furthermore, we noted that LPS plus PGPC—
and to a lesser extent, LPS plus oxPAPC—strongly induced
the upregulation of the chemokine receptor CCR7 in living
cDC1 and cDC2 subsets as compared to their naive, active,
and pyroptotic counterparts (Figures 2B and S2B). CCR7 upre-
gulation coincides with migratory capacity of DCs from the
skin to the dLN (Martı́n-Fontecha et al., 2003; Ohl et al., 2004; Al-
varez et al., 2008). To determine if hyperactive DCs displayed an
enhanced ability to migrate to the dLN, we performed an adop-
tive cell transfer of carboxyfluorescein succinimidyl ester
(CFSE)-labeled CD45.2+ FLT3L-DCs that were previously un-
treated, pre-treated with LPS for 15 h, or primed with LPS for
3 h then treated with PGPC or oxPAPC or alum for 12 h. Live
DCs were injected subcutaneously (s.c.) into wild-type (WT)
CD45.1 mice. Fifteen hours post-DC injection, we measured
the absolute number of fluorescent DCs recovered from the
adjacent skin dLN as CD45.2+ CFSE+ among CD11c+ live cells.
Notably, DCs that were stimulated with LPS plus PGPC ex-
hibited the strongest ability to emigrate to the dLN (Figure 2C).
Hyperactive DCs that lack CCR7 demonstrated a reduced
migratory capacity to the skin dLN (Figure 2C). Nlrp3
/
 DCs
maintained a strong emigration capacity from the skin to the
skin dLN, indicating that hyperactivation-mediated hypermigra-
tory capacity is independent of inflammasome activity (Fig-
ure 2C). In summary, these data demonstrate that in addition
to releasing IL-1b while maintaining viability, hyperactive DCs
display a hypermigratory phenotype that enables their enhanced
accumulation in adjacent dLN.
To assess whether hyperactive DCs retained the ability to pro-
duce IL-1b after migration to the dLN, we sorted from the skin
dLN WT or Nlrp3
/
 hyperactive DCs as CD11c+ CD45.2+
CFSE+ live cells (Figures S2C and S2D) and resident DCs as
CD11c+ CFSEneg DCs. We then measured IL-1b in the extracel-
lular media 24 h post-sorting without any further stimulation.
Resident DCs or Nlrp3
/
 DCs stimulated with LPS plus PGPC
thatmigrated to the dLNwere unable to secrete IL-1b (Figure 2D).
Conversely, WT hyperactive DCs that migrated to the dLN
secreted IL-1b in the absence of LDH release (Figure 2D). To
further assess their inflammasome activity, we determined the
percentage of hyperactive DCs in the dLN that harbor ASC
(Apoptosis-associated speck-like protein containing a CARD)
specks, a hallmark of inflammasome activation. We sorted hy-
peractive DCs or resident DCs based on differential staining of
CD45.1 and CD45.2. We used two strategies to detect ASC
specks in DCs: we stained endogenous ASC in hyperactive4 Cell Reports 33, 108381, November 17, 2020WT or Nlrp3
/
 DCs that migrated to the dLN and in resident
DCs immediately post-sorting (Figure 2E), or we used FLT3L-
DCs frommice that constitutively express ASC-citrine transgene
(Tzeng et al., 2016) (Figure S2E). We found that 40% of WT hy-
peractive DCs sorted from the dLN harbored ASC specks, as de-
tected by confocal microscopy, while 1% of Nlrp3
/
 or resident
DCs harbored ASC specks in the dLN (Figure 2E). Furthermore,
20% of ASC-citrine DCs stimulated with LPS plus PGPC that
have migrated to the dLN harbored perinuclear ASC specks,
as well as ASC specks located in the long DC protrusions (Fig-
ure S2E). Overall, these results establish that hyperactivating
stimuli induce inflammasome-dependent IL-1b release from hy-
peractive DCs that display an enhanced ability tomigrate to dLN.
Hyperactive DCs Potentiate CTL Responses in an
Inflammasome-Dependent Manner
RNA sequencing indicated that FLT3L-cDC1s and cDC2s upre-
gulated many genes involved in positively regulating CD8+ T cell
responses (Figure S3A; Table S1). To address whether hyperac-
tive DCs can enhance antigen-specific CD8+ T cell responses,
we s.c. transferred DCs that were treated as described above
and loaded with ovalbumin (OVA), then measured endogenous
OVA-specific CD8+ T cells in the dLN 7 days post-DC injection.
Active and hyperactive DCs were able to uptake fluorescent
OVA (OVA-FITC) to a similar extent, whereas pyroptotic DCs ex-
hibited reduced OVA internalization capacity (Figure S3B). In
addition, we found that active and hyperactive DCs exhibited
enhanced cross-presentation of the OVA-derived peptide SIIN-
FEKL on H2kb molecules following OVA protein internalization,
as compared to their naive or pyroptotic counterparts (Fig-
ure S3C). Accordingly, when we injected OVA-loaded FLT3L-
DCs into WT mice, we observed that hyperactive DCs induced
the highest frequency and absolute number of SIINFEKL+
CD8+ T cells in the skin dLN, as assessed by H2kb-restricted
SIINFEKL (OVA 257–264) tetramer staining (Figures 3A and
S3D). The enhanced CD8+ T cell responses mediated by hyper-
active DCs were dependent on inflammasomes, since the injec-
tion of Nlrp3
/
 DCs treated with LPS plus PGPC induced weak
OVA-specific CD8+ T cell responses (Figures 3A and S3D).
Similar weak T cell responses were observed upon injection of
Ccr7
/
 hyperactive DCs (Figures 3A and S3D). These data indi-
cate that hyperactive DCs enhance the generation of antigen-
specific CD8+ T cell responses in an inflammasome- and
CCR7-dependent manner. In accordance with these data,
CD8+ T cells sorted from the dLN displayed the highest fre-
quency of IFNg expression when isolated from mice that
received hyperactive DCs (Figure S3E). Thus, as compared to
stimuli that promote pyroptosis (alum) or DC activation (LPS), hy-
peractivating stimuli elicit the most robust antigen-specific CD8+
T cell responses examined.
IL-1b promotes the reactivation of pre-committed effector
T cells, thereby enhancing their cytokine production (Jain et al.,
2018). To assess the effect of the direct interaction between hy-
peractive DCs and effector CD8+ T cells, we performed co-cul-
ture assays using differentially stimulated FLT3L-DCs that were
also loaded with OVA protein. These DCs were exposed to
OVA-specific CD8+ T cells that were sorted from the spleen of
mice previously immunized s.c. with OVA antigen emulsified in
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Article OPEN ACCESS
A
B C D
E
Figure 2. Hyperactive cDCs Display a Hypermigratory Phenotype
(A) FLT3L-derived BMDCs were either left untreated or treated with indicated stimuli. Spider plots depict individual cell trajectories from an origin point (0;0) from
four regions of interest. Each trajectory line represents one cell (n = 30–50 cells). Straightness index and mean velocity were calculated (right panels).
(B) cDC1s or cDC2s were either left untreated or treated as indicated. The mean fluorescence intensity (MFI) of surface CCR7 (among CD11c+ live cells) was
measured by flow cytometry. Means and SDs from three replicates are shown, and data are representative of at least three independent experiments.
(C) The absolute number of CD45.2+ CFSE+ among CD11c+ live cells was calculated by flow cytometry. Means and SDs from five mice are shown, and data are
representative of at least three independent experiments.
(D and E) Hyperactive DCs that migrated to the skin dLN were sorted as CD11c+ CD45.2+ CFSE+ live cells. Alternatively, resident myeloid cells from the skin dLN
were sorted as CD11c+ CD45.1+ CFSEneg live cells. (D) Cells were cultured in media for 24 h, and IL-1b and LDH release were measured. Means and SDs from
three independent experiments are shown. (E) DCs were stained with the markers indicated and examined by confocal microscopy. Scale bar: 5 mm on
representative images (left panel). Quantification of the percent of cells containing ASC specks (right panel). DCx: DC injection.
In (C)–(E), BMDCs were either left untreated or treated with the stimuli indicated. Cells were stained with CFSE and injected subcutaneously into CD45.1 mice. At
15–18 h post-DC injection, skin dLNs were dissected. p values of < 0.05 (*), < 0.01 (**), < 0.001 (***), or % 0.0001 (****) are indicated.
Cell Reports 33, 108381, November 17, 2020 5
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OPEN ACCESS Article
A B
C
D E
Figure 3. Hyperactive DCs Potentiate CTL Responses in an Inflammasome-Dependent Manner
(A) FLT3L-DCs were either left untreated (DCNone) or treated as indicated. 1 3 10e6 live BMDCs were incubated with OVA protein and injected s.c. into mice.
Seven days post-DC injection, the absolute number of SIINFEKL+ CD8+ T cells in the skin dLN was measured by flow cytometry. Means and SDs of five mice are
shown.
(B andC)WTmicewere immunizedwith OVA protein. Seven days post-immunization, total CD8+ T cells were isolated from the spleen and co-cultured at a ratio of
10:1 with DCs pretreated with indicated stimuli. DCs were loaded with a serial dilution of OVA protein prior to co-culture. Five days post-coculture, supernatants
were collected, and cells were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin in the presence of monensin for intracellular IFNg staining.
(B) The percentage of SIINFEKL+ IFNg+ T cells wasmeasured by flow cytometry. (C) IFNg and IL-2 secretion wasmeasured by ELISA.Means and SDs from three
replicates are shown, and data are representative of at least two independent experiments.
(D and E) Mice were injected s.c. on the upper right back with 3 3 105 of B16OVA cells. Seven days post-tumor cell injection, 1 3 106 of FLT3L-DCs of the
genotypes indicated were treated as in (A) and incubated with B16OVA WTLs, then injected s.c. on the left flank into tumor-bearing recipient mice. Mice
received two subsequent DC injections. The percentage ofmice survival wasmeasured (n = 5–6mice per group, and n = 10 forWTDCLPS+PGPC). p values of < 0.05
(*), < 0.01 (**), < 0.001 (***), or % 0.0001 (****) are indicated.
6 Cell Reports 33, 108381, November 17, 2020
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Article OPEN ACCESSincomplete Freund’s adjuvant to generate a resident population
of antigen-specific cells. In contrast to naive, active, or pyrop-
totic DCs, hyperactive DCs strongly stimulated the expansion
of OVA-specific CD8+ T cells—as assessed by SIINFEKL
tetramer staining—which highly expressed intracellular IFNg
(Figure 3B). Accordingly, total CD8+ T cells produced higher
levels of the cytokines IFNg and IL-2 when co-cultured with hy-
peractive DCs as compared to naive or active DCs (Figure 3C).
Alum-treated pyroptotic DCs failed to stimulate CD8+ T cell
effector responses (Figure 3C).
The enhanced ability of hyperactive DCs to stimulate CD8+
T cell effector functions was dependent on inflammasomes,
since the co-culture of Nlrp3
/
 DCs pre-treated with LPS plus
PGPC abrogated the enhanced OVA-specific CD8+ T cell re-
sponses seen with WT hyperactive DCs (Figures 3B and 3C).
Conversely, the ability of hyperactive DCs to stimulate strong
CD8+ T cells responses in vitro was independent of CCR7, since
Ccr7
/
 hyperactive DCs retain their ability to produce IL-1b
from living cells (Figure S3F). As such, Ccr7
/
 hyperactive
DCs also induced CD8+ T cells to produce similar levels of
IFNg and IL-2 as WT hyperactive DCs (Figure 3C). Collectively,
these data indicate that hyperactive DCs not only enhance de
novo antigen-specific CD8+ T cell generation in vivo, but also
have the ability to reactivate and maintain the effector function
of pre-committed CD8+ T cells in an inflammasome-dependent
manner.
Hyperactive DCs Induce Long-Lived Anti-tumor
Immunity in an Inflammasome-Dependent Manner
We reasoned that hyperactivating stimuli may be particularly
useful in the context of cancer immunotherapy. We considered
the possibility that hyperactive DCs would allow us to bypass
the need for neo-antigen identification and permit the use of
whole-tumor cell lysates (WTLs) as an antigen source.WTLs pro-
vide a spectrum of mutated and aberrantly expressed tumor-
specific antigens (TSAs) and enable the generation of a broad
repertoire of T cells specific to tumor-associated antigens.
To determine if conditions that hyperactivate DCs are suffi-
cient to confer anti-tumor immunity, we performed an adoptive
transfer of FLT3L-DCs into mice harboring OVA-expressing
B16 tumors (B16OVA). To this end, tumor-bearing mice received
s.c. injections with differentially stimulated DCs that were loaded
with B16OVAWTLs. Hyperactive DCs induced a complete rejec-
tion of B16OVA tumors in 90%of tumor-bearingmice (Figure 3D),
and 75% of survivor mice rejected a lethal re-challenge with
B16OVA cells, which remained tumor-free for more than
100 days post-tumor inoculation (Figure 3D). DCs that were acti-
vated with LPS alone and pulsed with B16OVA WTLs provided
minimal protection from B16OVA-induced lethality.
The anti-tumor activity of hyperactive DCs was dependent on
inflammasomes in these cells, as Nlrp3
/
 FLT3L-DCs that were
treated with WTLs, LPS plus PGPC minimally impacted survival
rate (Figure 3D). Hyperactive DC entry to the dLN was crucial for
anti-tumor protection, since Ccr7
/
 DCs treated with WTLs,
LPS plus PGPC did not induce tumor rejection (Figure 3D). To
further confirm the role of intrinsic inflammasome activation in
hyperactive DCs for anti-tumor immune induction, B16OVA tu-
mor-bearing Casp1/11
/
 mice were injected with either WT-naive, active, or hyperactive DCs loaded with WTLs. We found
that naive or active DCs induced a minor tumor rejection in
Casp1/11
/
 mice. In contrast, WT hyperactive DCs induced tu-
mor rejection in 75% of Casp1/11
/
 tumor-bearing mice (Fig-
ure 3E). This protection was abolished when Casp1/11
/
 DCs
treated with WTLs, LPS plus PGPC were injected into Casp1/
11
/
 tumor-bearing mice. The same trends were observed
when injecting WT or Nlrp3
/
 DCs treated with WTLs, LPS
plus PGPC into Nlrp3
/
-deficient tumor-bearing mice (Fig-
ure S3G). These data indicate that hyperactive DCs are sufficient
to establish durable anti-tumor immunity and confirm that DC
migration into the dLN and inflammasomes within DCs are
essential for the anti-tumor activity of hyperactive DCs.
Hyperactivating Stimuli Enhance Memory T Cell
Generation and Potentiate Antigen-Specific IFNg
Responses in an Inflammasome-Dependent Manner
We examined if hyperactivating stimuli have a pro-inflammatory
effect on endogenous DCs in vivo. To test this possibility, mice
were immunized s.c. with OVA alone, OVA plus an activating
stimulus (LPS), or OVA plus a hyperactivating stimulus (LPS
plus oxPAPC or PGPC). Seven and 40 days post-immunization,
T effector (Teff), Teff memory (TEM), and T central memory (TCM)
cell generation in the dLN was assessed (Sallusto et al., 1999).
Seven days post-immunization, hyperactivating stimuli were
found to be superior to activating stimuli at inducing CD8+ Teff
cells (Figures 4A, upper panel, S4A, and S4B). At this time point,
hyperactivating stimuli also induced the highest abundance of
CD8+ TEM (Figures 4A, middle panels, S4A, and S4B). Forty
days post-immunization, ample TCM cells were observed in
mice exposed to hyperactivating stimuli, whereas these cells
were less abundant in mice immunized with OVA alone or with
LPS (Figure 4A, lower panels). Teff cells were conversely more
abundant in mice immunized with OVA plus LPS, as compared
to mice immunized with OVA plus oxPAPC or PGPC 40 days
post-immunization. These data indicate that hyperactivating
stimuli enhance the magnitude and rate of effector and memory
T cell generation. When total CD8+ T cells were isolated from
mice immunized with hyperactivating stimuli and co-cultured
with B16OVA cells, CD8+ T cells exhibited enhanced degranula-
tion activity as compared with T cells that were isolated from
mice immunized with OVA alone or OVA plus LPS (Figure 4B).
Furthermore, the increase in the frequency of Teff cells 7 days
post-immunization correlated with the enhanced IFNg re-
sponses of CD8+ T cells that were isolated from the dLN of
mice immunized with OVA plus hyperactivating stimuli (Fig-
ure 4C). These results indicate that hyperactivating stimuli
potentiate CTL effector functions.
To assess the antigen specificity of individual T cells that result
from a s.c. immunization with distinct activation stimuli, mice
were injected as described above. Seven days post-immuniza-
tion, CD8+ T cells were isolated from the skin dLN and re-stimu-
lated ex vivo with naive BMDCs loaded with OVA. We found that
OVA with hyperactivating stimuli generated the highest fre-
quency of SIINFEKL tetramer+ IFNg+ responses upon CD8+
T cell re-stimulation with OVA (Figure S4C). NLRP3 activity was
required for the hyperactivation-induced enhancement of anti-
gen-specific responses by CD8+ T cells (Figure S4C).Cell Reports 33, 108381, November 17, 2020 7
ll
OPEN ACCESS Article
A B Figure 4. Hyperactivating Stimuli Induce
Robust CTL Responses
(A) Mice were injected s.c. on the right flank with
OVA either alone or as indicated. Seven or 40 days
post-immunization, T cells were isolated from the
dLN. (A) The percentage of Teff cells as
CD44lowCD62Llow, TEM cells as CD44hiCD62Llow,
and TCM cells as CD44hiCD62Lhi are represented
among CD3+ CD8+ live cells.
(B and C) CD8+ T cells were sorted from the dLN
7 days post-immunization, then (B) treated either
with PMA plus ionomycin, or co-cultured with
C B16OVA cells (target cells). CD8+ T cells degranu-
lation was assessed bymonitoring the percentage of
CD107a+. (C) CD8+ T cells were cultured with BMDC
loaded (or not) with a serial dilution of OVA protein
starting from 1000 mg/ml. IFNg secretion was
measured by ELISA. Means and SDs of fivemice are
shown.
(D) Seven days post-immunization, the percentage
of Teff, TEM, TCM, and T naive cells in the skin dLN
was measured by flow cytometry.
(E) The percentage of SIINFEKL+ among CD8+ live
T cells in the dLN (left panel) or in the spleen (right
panel) was measured by flow cytometry.
E (F and G) Total CD8
+ T cells were sorted from the
dLN and (F) co-cultured with untreated BMDCs
loaded (or not) with OVA for 7 days at a ratio of 1:10
(DC: T cell). IFNg secretion was measured by ELISA.
(G) CD8+ T cells were co-cultured with B16OVA cells
(target cells) at ratio of 1:3 (effector: target). The
percentage of LDH release was measured from
D B16OVA-CD8+ T cells co-culture and normalized to
the LDH released from B16OVA cells or CD8+ T cells
cultured separately. Means and SDs from five mice
F G are shown.
In (D)–(F), CD45.1 mice were irradiated then recon-
stituted with mixed BM of the genotypes indicated.
Six weeks post-reconstitution, chimera mice were
injected with DTx 3 times a week for a total of 9 DTx
injections. Chimeric mice were then immunized s.c.
on the right flank with OVA with LPS plus
PGPC. p values of < 0.05 (*), < 0.01 (**), < 0.001 (***),
or % 0.0001 (****) are indicated.Furthermore, pyroptotic stimuli (LPS plus alum) were the
weakest inducers of antigen-specific responses (Figure S4C).
No significant differences in IFNg were observed among SIIN-
FEKL-negative T cells between immunizations (Figure S4C).
Collectively, these data highlight that hyperactivating stimuli
induce strong antigen-specific CD8+ T cell responses.
Inflammasome Activation in DCs Potentiates Antigen-
Specific CTL Responses
To determine the role of inflammasome activation in endogenous
DCs inmediating hyperactivation-based activities, we generated
mixed chimeras in mice as previously described (Zanoni et al.,
2013). To this end, CD45.1 mice were irradiated and reconsti-
tuted with mixed BM using 80% BM cells isolated from
Zbtb46DTR mice mixed with 20% BM cells isolated from WT,
Nlrp3
/
, or Casp1/11
/
 mice from a CD45.2 background (Fig-8 Cell Reports 33, 108381, November 17, 2020ure S4D). Six weeks post-reconstitution, the efficacy of reconsti-
tution was above 92% efficiency (Figure S4D). Reconstituted
mice were then injected intraperitoneally (i.p.) with diphtheria
toxin (DTx) to deplete Zbtb46+ cDCs, giving rise to mice that har-
bor either WT or inflammasome-deficient (Nlrp3
/
 or Casp1/
11
/
) DCs. Chimera mice were s.c. immunized with OVA plus
LPS plus PGPC. Seven days later, CD8+ T cell responses from
the skin dLN were assessed. We found that the abundance of
Teff CD8+ T cells was reduced in the chimera mice harboring
DCs that cannot become hyperactive (Nlrp3
/
 and Casp1/
11
/
 chimera mice), as compared to chimera mice harboring
WT DCs (Figure 4D). Furthermore, the frequency of SIINFEKL+
CD8+ T cells in the dLN or the spleen was reduced in mice
harboring Nlrp3
/
 and Casp1/11
/
 DCs (Figures 4E and
S4E). CD8+ T cells sorted from chimera mice harboring WT
DCs produced the highest amounts of IFNg (Figure 4F) and
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Article OPEN ACCESS
A Figure 5. Hyperactivating Stimuli Induce Dura-
ble Prophylactic Anti-tumor Immunity in an IL-
1b-Dependent Manner
(A) Mice were injected s.c. on the right flank with PBS
(unimmunized), with B16OVA cell WTLs alone
(‘‘None’’), or with LPS, or B16OVAWTLs plus LPS and
oxPAPC or PGPC. Fifteen days post-immunization,
mice were challenged s.c. on the left upper back with
33 105 of B16OVA cells. One hundred fifty days later,
tumor-free mice were re-challenged s.c. with 53 105
of B16OVA cells. (A) Tumor growth (left panel) and
mice survival (middle panel) was monitored every
2 days. The percentage of tumor-free mice 300 days
post-tumor inoculation is indicated (right panel) (n =
8–15 mice per group).
(B and C) Tumors were harvested at the endpoint of
B C D tumor growth, and (B) the percentages of tumor
infiltrating CD3+CD4+ and CD3+CD8+ T cells among
CD45+ live cells were assessed by flow cytometry. (C)
Tumor-infiltrating T cells were sorted then stimulated
in the presence of anti-CD3 and anti-CD28 dyna-
beads. IFNg release was measured by ELISA (n = 4
mice per group).
(D) Mice were either left untreated (unimmunized) or
were immunized s.c. on the right flank with B16OVA
WTLs plus the stimuli indicated. Fifteen days post-
immunization, mice were challenged with 3 3 105
B16OVA cells s.c. on the left upper back. The per-
E F centage of survival is monitored every 2 days (n = 5
mice per group).
(E and F) Mice were either left untreated (unimmu-
nized) or were immunized s.c. on the right flank with
B16OVAWTLs (E) or with (F) MC38OVAWTLs or OVA
alone or in combination with the treatments indicated.
Fifteen days post-immunization, mice were chal-
lenged with (E) 3 3 105 of viable B16OVA cells or (F)
53 105MC38OVA cells s.c. on the left upper back. (E)
Ninety days later, tumor-free mice were re-chal-
lenged with 53 105 B16OVA cells s.c. on the back. (F)
Fifty days later, tumor-free mice were re-challenged
s.c. with 1 3 106 MC38OVA cells. Survival was
monitored every 2 days (n = 3–5 mice per group). p
values of < 0.01 (**) is indicated.exhibited higher CTL ability, as compared to CD8+ T cells from
mice lacking inflammasome-competent DCs (Figure 4G). In
summary, these data demonstrate that (1) endogenous DCs
can achieve a state of hyperactivation in vivo and potentiate
CTL responses, and (2) inflammasomes within endogenous
DCs are crucial for hyperactivation-mediated CTL responses.
Hyperactivating Stimuli Stimulate T Cell Responses
That Confer Long-Term Anti-tumor Immunity
To address the possibility that hyperactivating stimuli can adju-
vant WTLs and mediate strong CTL responses in vivo, mice
were immunized on the right flank with WTLs alone, WTLs mixed
with the activating stimulus LPS, or the hyperactivating stimuli
LPS plus oxPAPC or LPS plus PGPC. The source of the WTLs
was B16OVA cells. Fifteen days post-immunization, mice were
challenged s.c on the left upper back with the parental
B16OVA cells. Unimmunized mice or mice immunized with
WTLs alone did not exhibit any protection, and all mice suc-
cumbed to tumor growth (Figure 5A). Similarly, WTLs plus LPSimmunizations offered minimal protection (Figure 5A). In
contrast, WTL immunizations in the presence of LPS plus ox-
PAPC induced a delay in the tumor growth and resulted in a
strong protection against subsequent lethal re-challenge with
parental B16OVA tumor cells (Figure 5A). Tumors from mice
immunized with LPS plus oxPAPC contained a large abundance
of CD4+ and CD8+ T cells, as compared to LPS immunizations
(Figure 5B). Moreover, when equal numbers of T cells from these
tumors were stimulated ex vivowith anti-CD3 and anti-CD28, ox-
PAPC-based immunizations resulted in intra-tumoral T cells that
secreted the highest amounts of IFNg (Figure 5C). Thus, the su-
perior restriction of tumor growth induced by hyperactivating
stimuli coincided with inflammatory T cell infiltration into the
tumor.
Notably, the protective phenotypes of oxPAPC were super-
seded by those elicited by PGPC. WTL immunizations in the
presence of LPS plus PGPC led to 100% of mice being tumor-
free for 150 days post-tumor challenge. Thesemice rejected a le-
thal re-challenge with B16OVA cells and remained tumor-freeCell Reports 33, 108381, November 17, 2020 9
ll
OPEN ACCESS Article300 days post-initial tumor challenge (Figure 5A). Interestingly,
not all NLRP3 agonists conferred anti-tumor immunity, as LPS
plus alum was unable to adjuvant WTLs to protect against tumor
growth (Figure 5D). These findings underscore the importance of
hyperactive DCs (not pyroptotic DCs) in the induction of anti-tu-
mor immunity.
Similar findings were made when we replaced LPS with
monophosphoryl lipid A (MPLA), an FDA-approved TLR4 ligand
(Kundi, 2007; Didierlaurent et al., 2009; Paavonen et al., 2009).
All mice immunized with MPLA plus PGPC remained tumor-free
for 90 days post-tumor B16OVA challenge, and most mice re-
jected a lethal re-challenge with parental cells (Figure 5E).
Similar results were observed in a colon adenocarcinoma
MC38OVA model (Figure 5F). These anti-tumor responses
were dependent on IL-1b, since neutralizing IL-1b at the time
of immunization abolished the protection of mice against tumor
growth (Figures 5E and 5F). Interestingly, WTLs were superior at
inducing long-term protective immunity after tumor challenge,
as compared to immunizations with OVA (Figure 5F). The
inability of single antigens to strongly protect from cancer is
consistent with work demonstrating the value of using
multiple neo-antigens in cancer vaccines (Castle et al., 2012;
Ott et al., 2017).
Amongmemory T cell subsets, T resident memory (TRM) cells,
defined by the expression of CD103 and CD69 (Mami-Chouaib
et al., 2018), accumulate at the tumor site in various cancer tis-
sues and correlate with a favorable clinical outcome (Webb
et al., 2014; Djenidi et al., 2015; Park et al., 2019). We examined
the presence of TRM cells at the tumor injection site, as well as
the immunization skin biopsies in the survivormice that were pre-
viously immunized with the hyperactivating stimuli LPS plus
PGPC. Two hundred days post-tumor inoculation, CD8+ CD69+
CD103+ TRM cells were enriched at the site of tumor injection
but were scarce at the immunization site in all survivor mice (Fig-
ures S5A and S5B). WTLs and hyperactivating stimuli may there-
fore generate TRM cells that control local tumor cell growth. To
investigate this possibility, we monitored the cytolytic activity of
TRM cells from survivor mice. Circulating memory CD8+ T cells
and TRM cells were isolated from the spleen or the skin adipose
tissue 200 days post-immunization from survivormice that previ-
ously received hyperactivating stimuli. Cytolytic activity, as as-
sessed by LDH release, was only observed when CD8+ T cells
weremixedwith B16OVA or B16 cells, whereas no killing of unre-
lated CT26 cells was observed (Figure S5C), highlighting the an-
tigen-specific nature of hyperactivation-induced T cell re-
sponses. To further determine if TRM or circulating memory
T cells are sufficient to protect against tumor progression, CD8+
T cells were transferred from survivor mice into naive mice and
subsequently challenged with the parental tumor cell line used
as the initial immunogen. Transfer of CD8+ TRM or circulating
CD8+ T cells from survivor mice into naive recipients conferred
protection from a subsequent tumor challenge, with the TRM
subset playing a dominant protective role (Figure S5D). Transfer
of both T cell subsets from survivormice into naivemice provided
100% protection of recipient mice from subsequent tumor chal-
lenges (Figure S5D). These collective data indicate that PGPC-
based hyperactivating stimuli confer protection by inducing
strong circulating and resident anti-tumor CD8+ T cell responses.10 Cell Reports 33, 108381, November 17, 2020Hyperactivating Stimuli Protect against Established
Tumors That Are Resistant to Anti-PD1 Therapy
To determine if hyperactivating stimuli could be harnessed as a
cancer immunotherapy, we examined anti-tumor responses in
mice that harbored a growing tumor prior to any treatment. For
these studies, rather than using parental in vitro cultured tumor
cells as an antigen source, ex vivo WTLs were generated using
syngeneic tumors from unimmunized mice, in which 10-mm har-
vested tumors were dissociated and then depleted of CD45+
cells. Mice were inoculated s.c. with tumor cells on the left upper
back. When tumors reached a size of 3–4 mm, tumor-bearing
mice were left untreated (unimmunized) or received an injection
on the right flankwith ex vivoWTLs and LPS plus PGPC. Interest-
ingly, hyperactivation-based therapeutic injections induced du-
rable tumor eradication in a range of tumors, such as B16OVA
and B16F10 melanoma models, in MC38OVA and CT26 colon
cancer tumor models and in Lewis Lung Carcinoma model
(LLC1) (Figures 6A–6E and S6A). The efficacy of the immuno-
therapy was dependent on IL-1b in all the tested models, since
the neutralization of IL-1b abolished protection conferred by hy-
peractivating stimuli plus ex vivoWTLs (Figures 6A–6E and S6A).
In addition, CD8+ T cells were crucial for protection against
immunogenic tumor models such as B16OVA or MC38OVA tu-
mors, whereas CD4+ and CD8+ T cells were both required for
protection against less immunogenic tumors such as CT26,
B16F10, and LLC1 (Figures 6A–6E and S6A) (Mosely et al.,
2017). Interestingly, hyperactivation-based immunotherapy
was as efficient as anti-PD-1 therapy in the immunogenic
B16OVA model, but more efficient in tumor models that are
insensitive to anti-PD-1 treatment such as CT26, B16F10, and
LLC1 (Figures 6A–6E and S6A).
The LLC1 cell line is associated with an absence of tumor-infil-
trating CTL (Lechner et al., 2013). To determine if hyperactivating
stimuli enhance CTL responses in LLC1 tumors, mice harboring
a tumor of 4 mm were immunized as described in Figure 6E. Tu-
mor weight was measured for each mouse (Figure S6A) and
dissociated, and immune cells were quantified. When mice
were treated with anti-PD-1 antibodies, CD8+ tumor-infiltrating
lymphocyte (TIL) abundance was similar to TIL abundance in
the tumors of unimmunized mice, which is around 1%–2% of
TILs (Figures 6F and S6C). In addition, we detected low levels
of CD8+ TRM cells in the tumormicroenvironment (TME) of unim-
munized mice or in mice treated with anti-PD1 (Figures 6F and
S6C). Thus, anti-PD-1 treatment had a minimal effect on
enhancing anti-LLC1 CD8+ T cell responses. Interestingly,
mice immunized with WTLs and LPS plus PGPC induced a
strong increase in the frequency of CD8+ TILs (Figure 6F, left
panel), and most of these T cells displayed a TRM phenotype
(Figures 6F, middle panel, and S6C). The high abundance of
CD8+ TRM cells positively correlated with the ability of CD45+
TILs to secrete IFNg upon CD3/CD28 stimulation, as compared
to anti-PD1 treatment (Figure 6F, right panel), and with a higher
tumor rejection rate (Figure 6E). Moreover, hyperactivation-
mediated enhanced CD8+ T cell responses were dependent on
IL-1b, since the immunization of mice with WTLs and LPS plus
PGPC in the presence of neutralizing anti-IL-1b antibodies in-
hibited tumor rejection (Figure 6E), and CTL responses were
abrogated (Figure 6F). Similar to conditions of IL-1b inhibition,
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Article OPEN ACCESS
A B Figure 6. Hyperactivating Stimuli Eradicate
Established Tumors That Are Resistant to
Checkpoint Inhibitors
Mice of the indicated genotypes were inoculated
subcutaneously on the left upper back with (A) 53
105 of MC38OVA cells, (B) 3 3 105 B16OVA cells,
(C) 33 105 B16-F10 cells, (D) 33 105CT26 cells, or
(E and F) 3 3 105 LLC1 cells. In (A)–(E), when tu-
mors reached 3–4 mm in size, mice were either left
untreated (unimmunized) or were injected s.c. on
the right flank with WTLs plus LPS and PGPC with
or without neutralizing anti-IL-1b antibodies, anti-
CD4, anti-CD8a antibodies, or (F) IL-RA. Mice
received two boost injections with WTLs and LPS
C D E plus PGPC. In (B)–(E), alternatively, tumor-bearing
mice were injected with anti-PD-1 antibody. The
percentage of survival is indicated (n = 10-12 mice
per group). In (F), LLC1 tumors were harvested at
the endpoint of tumor growth. The percentage of
CD8+ TILs (left panel) among CD3+CD45+ live cells
and CD69+CD103+ TRM cells among CD8+ TILs
(middle panel) were measured by flow cytometry.
CD45+ live TILs were cultured for 48 h on anti-
CD28 and anti-CD3 coated plates. IFNg was
measured by ELISA (right panel) (n = 5 mice per
group). p values of < 0.05 (*), < 0.01 (**), < 0.001
(***), or % 0.0001 (****) are indicated.
F
no protection was conferred to Nlrp3
/
 or Casp1/11
/
 mice
bearing LLC1 tumors (Figures 6F and S6B). Furthermore, IL-1
signaling was crucial for anti-tumor protection, as IL-1 receptor
antagonist (IL-RA) injection abrogated hyperactivation-mediated
CD8+ TRM cell infiltration into the tumor (Figures 6F and S6A)
and reduced IFNg-producing T cells in the TME (Figure 6F). As
a consequence, in the absence of IL-1b signaling, LLC1 tumor
rejection was abrogated (Figures 6E and S6A).
Thus, hyperactivating stimuli strongly potentiate CTL re-
sponses in the TME of non-immunogenic tumors in an inflamma-
some- and IL-1b-dependent manner and induce tumor rejection
in models that are resistant to checkpoint immunotherapy.
Hyperactivating Stimuli Protect against Lung
Metastasis
We next wondered whether hyperactivation-based immuno-
therapy can induce distal protection against tumor growth. WeCell RusedaB16 lungmetastasismodel inwhich
mice were intravenously (i.v.) injected with
B16F10cells.Sevendayspost-tumorcells
injection, mice were left untreated or
received a s.c. immunization with WTL in-
jection alone, WTLs and LPS, or WTLs
and LPS plus PGPC. We found that WTL
injection alone or in combination with
LPS provide some protection against tu-
mor lung colonization inmice. This protec-
tionwas enhancedwhenmice received an
immunization withWTLs and the hyperac-
tivating stimuli LPS plus PGPC (Fig-
ure S6D). These observations indicatethat the anti-tumor responses observed can contribute to sys-
temic protection.
Endogenous Hyperactive DCs Stimulate Durable Anti-
tumor T Cell Immunity in an Inflammasome-Dependent
Manner
To test whether endogenous DCs can initiate hyperactivation-
mediated anti-tumor responses, we used Zbtb46DTR mice in
which cDCs are depleted by DTx injection. When tumors
reached 4 mm in size, Zbtb46DTR or WT mice were immunized
with B16OVA WTLs and LPS plus PGPC. We found that in
contrast to WT mice, which rejected tumors in 90% of mice,
Zbtb46DTR mice, which lack DCs, were unable to reject tumors
(Figure 7A). These data confirm that DCs are the initiator of hy-
peractivation-mediated anti-tumor protection.
We reasoned that inflammasomes within endogenous hyper-
active DCs are crucial to mediate long-lived anti-tumoreports 33, 108381, November 17, 2020 11
ll
OPEN ACCESS Article
A B
Immunization Boost Boost Immunization Boost Boost
WTL+LPS+PGPC WTL+LPS+PGPC  WTL+LPS+PGPC B16OVA WTL+LPS+PGPC WTL+LPS+PGPC  WTL+LPS+PGPCB16OVA
inoculation inoculation
D0 D7 D14 D21 D0 D7 D14 D21
Boost#1 Zbtb46DTR Immunized with Boost#1 DTR
Zbtb46DTR +DTx WTL+LPS+PGPC Zbtb46 + WTImmunization Boost#2 Zbtb46DTR+ Nlrp3-/- Immunized with
Immunization Boost#2 Zbtb46DTR+ Casp1/11-/- WTL+LPS+PGPC
100 100 Zbtb46DTR+ Ccr7-/-
75 75
50 50
25 25
0 0
0 20 40 60 80 0 20 40 60 80
Days post tumor inoculation Days post tumor inoculation
C Immunized (or not) Boost Boost D
B16OVA WTL+LPS+PGPC WTL+LPS+PGPC WTL+LPS+PGPC
inoculation dLN Tumor Spleen
**
18 WT
D24 Immunized withD0 D10 D17 15 Batf3-/- WTL+LPS+PGPC
12
Boost#1 9
Immunization Boost#2 WT  -/- No immunization 6Batf3 3 *
WT Immunized with
100 Batf3-/- WTL+LPS+PGPC 0
75 dLN Tumor Spleen
20
50 15 * WT Immunized with
10 Batf3-/- WTL+LPS+PGPC25 5
0
0 20 40 60 0.5
Days post tumor inoculation ns0.0 *
E
cDC1 injection cDC1 injection cDC1 injection
B16OVA
inoculation F No DCx 
dLN Tumor Spleen WT cDC1naive
20 WT cDC1active
D0 D7 D14 D21 ** ** WT cDC1Hyperactive
15 Nlrp3-/- cDC1Hyperactive
100 10 Ccr7-/- cDC1Hyperactive
No cDC1 injection * *
75 WT cDC1naive 5
WT cDC1active Batf3-/- *** **
WT cDC1hyperactive recipients 0
50 Nlrp3-/- cDC1hyperactive3
25 Ccr7
-/- cDC1hyperactive
dLN Tumor Spleen
8 **
0 *
0 20 40 60 80 6 *
Days post tumor inoculation
4
2 *** ns
0
Figure 7. Hyperactive cDC1s Can Use Complex Antigen Sources to Stimulate T-Cell-Mediated Anti-tumor Immunity
(A) Zbtb46DTRmice were s.c. injected with B16OVA cells. Mice were either injected with DTx every other day for four consecutive injections, or mice were injected
with PBS. Seven days post-tumor injection, all mice were immunized with B16OVAWTLs plus LPS and PGPC, followed by two boost injections. The percentage
of mice survival is indicated (n = 10 mice per group).
(B) CD45.1 mice were irradiated then reconstituted with mixed BM from Zbtb46DTR mice plus either WT or Nlrp3
/
, Casp1/11
/
, or Ccr7
/
 mice. Six weeks
post-reconstitution, mouse chimeras were injected s.c. with B61OVA cells, then all mice received DTx 3 times a week for a total of 12 consecutive injections.
Seven days post-tumor inoculation, chimeric mice were immunized with B16OVAWTLs and LPS plus PGPC and received two boost injections. The percentage
of mice survival is indicated (n = 5 mice per group).
(legend continued on next page)
12 Cell Reports 33, 108381, November 17, 2020
% of survival % of survival % of survival
Frequency of Frequency of Frequency of Frequency of + % of survival
AAHAEINEA+ T cells SIINFEKL+ T cells AAHAEINEA  T cells SIINFEKL+ T cells
ll
Article OPEN ACCESSimmunity. To test this possibility, we generated BM chimeras as
described in Figures 4D and S4D. We reconstituted mice with a
mix of BM constituted of 80% BM from Zbtb46DTR mice and
20% BM from WT mice (in which the DC compartment can be
hyperactivated) or 80% BM from Zbtb46DTR mice and 20%
from inflammasome-deficient Nlrp3
/
 or Casp1/11
/
 mice.
Chimeric mice were s.c. implanted with B16OVA cells and then
injected with DTx to deplete Zbtb46+ DCs. When all tumors
reached 3 mm in size, mice were immunized with B16OVA
WTLs and hyperactivating stimuli LPS plus PGPC. We found
that hyperactivating stimuli were effective in inducing tumor
rejection only in mice that harbor a WT DC compartment. In
contrast, the anti-tumor protection was abrogated in mice
harboring inflammasome-deficient DCs (Figure 7B). Similar
studies were performed to assess the importance of endoge-
nous hyperactive DC migration. Mixed chimera mice were
generated using BM from Zbtb46DTR mice and WT or Ccr7
/

mice. We found that the ability of DCs to enter the dLN via
CCR7 is crucial for DCs to induce tumor rejection, as hyperacti-
vating stimuli were not able to protect mice lacking CCR7 in DCs
from tumor progression and lethality (Figures 7B and S7A).Hyperactive cDC1s Stimulate T-Cell-Mediated Anti-
tumor Immunity in an Inflammasome- and CCR7-
Dependent Manner
Finally, given the importance of the cDC1 subset in tumor rejec-
tion in other experimental contexts, we hypothesized that cDC1s
play a central role in inducing hyperactivation-mediated anti-tu-
mor protection. To test this possibility, we used Batf3
/
 mice
(which lack cDC1s) (Hildner et al., 2008). We immunizedBatf3
/

or WT tumor-bearing mice with LPS plus PGPC and WTLs. WT
mice rejected tumors in 100% of mice, whereas all Batf3
/

mice succumbed to tumor growth (Figures 7C and S7B). Thus,
cDC1s are required for hyperactivation-mediated anti-tumor im-
munity. In addition, while immunizedWTmice induced a high fre-
quency of OVA peptide-specific CD8+ and CD4+ T cells in the
TME and the skin dLN, immunized Batf3
/
 induced a slightly
reduced frequency of antigen-specific CD4+ T cells and no anti-
gen-specific CD8+ T cells in the TME (Figure 7D).
To further confirm the role of hyperactive cDC1s in anti-tumor
protection, we adoptively transferred naive, active, or hyperac-
tive cDC1s into Batf3
/
 mice. cDC1s were left naive or treated
with activating stimuli (LPS) or hyperactivating stimuli (LPS plus
PGPC). All cells were loaded with B16OVA WTLs before their
s.c. injection into Batf3
/
 mice that harbored a 3-mm tumor.
We found that only hyperactive cDC1s induced tumor rejection
in 100% of tumor-bearing mice (Figure 7E). Of note, hyperac-
tive cDC1 injections uniquely restored antigen-specific CD8+(C and D) WT or Batf3
/
mice were injected s.c with B16OVA cells. Seven days p
were immunized with B16OVA WTLs and LPS plus PGPC followed by two boos
group). (D) Twenty-one days post-tumor inoculation, the percentage of OVA-spec
mice per group).
(E and F) Batf3
/
mice were injected s.c on the right flank with B16OVA cells. Sev
were injected s.c. on the left flank with FLT3-derived naive cDC1s or active cDC1s
cDC1s were loaded with B16OVAWTLs for 1 h prior to their injection. (E) The perc
post-tumor inoculation, OVA-specific CD8+ T cells and CD4+ T cells were assesse
or < 0.001 (***) are indicated.T cells in Batf3
/
 mice in the tumor and the skin dLN (Figures
7F and S7C).
cDC1-mediated tumor rejection was dependent on inflamma-
somes, since the injection of Nlrp3
/
 cDC1s that were pre-
treated with LPS plus PGPC and loaded with WTLs did not
provide any anti-tumor protection and abrogated the ability of
hyperactive cDC1s to restore CD8+ T cell responses (Figures
7E, 7F, and S7D). Furthermore, cDC1 entry into the dLN played
a crucial role in mediating tumor rejection, since the adoptive
transfer of Ccr7
/
 cDC1s that were pre-treated with LPS plus
PGPC and loaded with B16OVAWTLs failed to initiate protective
anti-tumor immunity (Figures 7E and 7F).
Finally, to address whether the protective immune response is
mediated solely by the injected cDC1s or by endogenous DCs
capturing the antigen from the injected DCs, we generated full
chimeric mice in which the endogenous DC compartment could
be depleted prior to cDC1 injection. Six weeks post-reconstitu-
tion, mice were s.c. injected with B16OVA cells and subse-
quently injected with DTx. DTx injection specifically depleted
cDCs, whereas the abundance of macrophages was unaffected
(Figure S7E). When tumors reached 3–4 mm in all mice, differen-
tially stimulatedWT orNlrp3
/
 cDC1s that were pre-loadedwith
B16OVA WTLs were injected. We found that the anti-tumor
response was not significantly different between chimeric mice
that harbor and do not harbor resident DCs (Figure S7E), indi-
cating that the anti-tumor response generated were primarily
mediated by the injected cDC1s . In summary, this study illus-
trates the importance of hyperactive cDC1s for the establish-
ment of anti-tumor immunity.DISCUSSION
In this study, we have expanded the immunological activities of
hyperactive DCs. Not only are hyperactive DCs capable of
releasing IL-1b while maintaining viability, but they also display
enhanced migratory capacity to the adjacent dLN via CCR7.
Consequently, hyperactive DCs are the most potent stimulators
of T cell-mediated immunity that we have examined in mice.
This study also highlights the differences in DC behavior when
stimulated with non-pyroptotic inflammasome stimuli versus py-
roptotic inflammasome stimuli. It is noteworthy that alum, a well-
defined inflammasome stimulus (Kool et al., 2008b), does not
exhibit the same activities as PGPC. Indeed, alum induces Th2
immunity (Kool et al., 2008a; Marichal et al., 2011; Oleszycka
et al., 2018). One possible reason for the lack of CTL-mediated
immunity by alum-treated DCs is based on our findings
that alum is a poor inducer of several signals necessary for
T cell priming. Consequently, these stimuli are weak inducersost-tumor inoculation, mice were either left untreated, or WT and Batf3
/
mice
t injections. (C) The percentage of mice survival is indicated (n = 10 mice per
ific CD8+ T cells and CD4+ T cells was assessed using tetramer staining (n = 5
en days post-tumor inoculation, mice were left untreated (no cDC1 injection) or
treatedwith LPS or with hyperactive cDC1s pretreated with LPS plus PGPC. All
entage of mice survival is indicated (n = 5 mice per group). (F) Twenty-one days
d using tetramer staining (n = 5mice per group). p values of < 0.05 (*), < 0.01 (**),
Cell Reports 33, 108381, November 17, 2020 13
ll
OPEN ACCESS Articleof anti-tumor immunity. In contrast, non-pyroptotic inflamma-
some activation in DCs induces protective anti-tumor immunity
by a process dependent on IL-1 and enhanced cell migration
to the dLN. It is likely that each of these activities is important
for hyperactive DC functions and contributes to the long-lived
T cell-mediated anti-tumor responses. Consistent with this
idea, blocking pyroptotic cell death in BMDCs enhances T cell
priming (McDaniel et al., 2020), which corroborates our finding
that pyroptotic DCs fail to participate in T cell priming and
reactivation.
cDC1s were critical for hyperactivation-mediated anti-tumor
responses, as hyperactivation-based immunization in Batf3
/

mice failed to reject transplanted tumors. This finding is in
accordance with studies showing that cDC1s are required for
anti-tumor T cell responses in other experimental contexts
(Hildner et al., 2008; Ferris et al., 2020). We found that the abil-
ity of cDC1s to achieve a hyperactive state increased antigen-
specific CD8+ T cell generation, intra-tumoral CTL trafficking,
and CTL responses that eradicate many types of tumors. All
of these activities were dependent on inflammasomes and
the enhanced migratory capacity of hyperactive cDC1s. Conse-
quently, as demonstrated in the LLC1 model, hyperactive
cDC1s can convert cold tumors into hot tumors, leading to
regression.
Breast cancer patients with a loss-of-function allele of
P2X7R, which is essential for activation of the NLRP3 inflam-
masome and IL-1b secretion, develop a more rapid metastatic
disease than individuals bearing the normal allele (Casares
et al., 2005; Ghiringhelli et al., 2009). Furthermore, oxaliplatin--
treated tumors activate the NLRP3 inflammasome and stimu-
late IL-1b production, which activates anti-tumor IFNg-produc-
ing CD8+ T cells (Ghiringhelli et al., 2009). If human DCs can
achieve a state of hyperactivation, our results may explain
why certain chemotherapeutic agents (e.g., oxaliplatin and an-
thracycline) induce tumor cell death and inflammasome-depen-
dent anti-tumor T cell immunity. Oxaliplatin is a robust stimu-
lator of reactive oxygen species (ROS) production, which can
oxidize membranes and create a complex mixture of oxidized
lipids that may include PGPC. It is possible that the protective
immunity induced by oxaliplatin or ROS inducers results from
the actions of hyperactive DCs that prime anti-tumor T cell
responses.
We have found that hyperactivating stimuli could be har-
nessed as an immunotherapy using complex mixtures of anti-
gen. WTL is an attractive source of antigens, as it alleviates
the need for neo-antigen identification. Despite the potential
benefits offered by WTL-based immunotherapies, prior work
in this area has yielded mixed results (Chiang et al., 2015).
Our finding that hyperactivating stimuli are capable of adjuvant-
ing WTLs to elicit anti-tumor immunity may explain the lack of
success in prior work, as our strategies of DC activation have
not previously been considered. On this latter point, it is note-
worthy that DC hyperactivating strategies can protect mice
from tumors that are sensitive or resistant to PD-1 blockade.
The full spectrum of tumors amenable to treatment by hyperac-
tivating stimuli is undefined, but these studies provide a
mandate to further explore the value of DC-centric strategies
in cancer immunotherapy.14 Cell Reports 33, 108381, November 17, 2020STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
d KEY RESOURCES TABLE
d RESOURCE AVAILABILITYB Lead Contact
B Materials Availability
B Data and Code Availability
d EXPERIMENTAL MODEL AND SUBJECT DETAILS
B Mouse strains, and Tumor cell lines
B Differentiation of GM-CSF- and FLT3L-BMDCs
d METHOD DETAILS
B Generation of full bone marrow chimeric mice for DC
injections
B Generation of full or mixed bone marrow chimeric mice
for immunizations
B Ligand and Chemical Reconstitution
B DC stimulation and T cell culture
B LDH Assay and ELISA
B Antigen uptake and peptide presentation assay
B DC injections for DC migration assay
B Microscopy Imaging
B Cell tracking and time-series analysis
B Flow cytometry and cell sorting
B Adoptive DC transfers
B DC and antigen-specific CD8+ T cell coculture
B In vivo immunization and T cell re-stimulation
B CD107a degranulation assay
B In vitro cytotoxicity assay
B Whole tumor lysates preparation
B Prophylactic immunization and tumor challenges
B Immunotherapeutic immunization and tumor chal-
lenges
B Tumor infiltrating T cells in the tumormicroenvironment
B In vivo immunization and B16-F10 pulmonary coloniza-
tion
B Skin biopsies and skin adipose tissue dissociation
B Adoptive T cell transfer
B RNA sequencing
d QUANTIFICATION AND STATISTICAL ANALYSISSUPPLEMENTAL INFORMATION
Supplemental Information can be found online at https://doi.org/10.1016/j.
celrep.2020.108381.
ACKNOWLEDGMENTS
We thank D. Mathis (Harvard), J. Lieberman (Harvard), and N. Joshi (Yale) for
advice on this work and the NIH Tetramer Core Facility for providing the OVA
peptide tetramers. This work was supported by NIH grants AI133524,
AI093589, AI116550, and P30DK34854 and Northwest Bio to J.C.K. D.Z.
was supported by the NIH T32 appointments AI007245-34 and AI007245-
35. A.K.S. was supported by the Pew-Stewart Scholars Program for Cancer
Research, a Sloan Fellowship in Chemistry, and the NIH (1DP2GM119419,
1U54CA217377, and 2RM1HG006193). C.L.S. was supported by
K08AI121421, MGH Transformative Scholar Award, and an AAAAI Foundation
Faculty Development Award. O.A.C. was supported by T32HL116275. I.Z. is
ll
Article OPEN ACCESSsupported by NIH grants AI121066, DK115217, and AI201700100. J.C.K. and
I.Z. hold Investigators in the Pathogenesis of Infectious Disease Awards from
the Burroughs Wellcome Fund.
AUTHOR CONTRIBUTIONS
D.Z. designed and performed experiments. D.J.T. consulted on cDC1 and
cDC2 differentiations. F.B. and I.Z. designed and generated bone-marrow
chimeramice and performed intravenous B16F10 cell injections for metastasis
experiments. O.A.C. and C.L.S. generated bone-marrow chimera mice. P.P.
analyzed in vitro cell tracking data. B.D., I.F., and A.K.S. performed and
analyzed RNA sequencing experiments. J.C.K. supervised all research. All au-
thors discussed results and commented on the manuscript.
DECLARATION OF INTERESTS
J.C.K. holds equity and consults for IFM Therapeutics, Quench Bio, and
Corner Therapeutics. A.K.S. reports compensation for consulting and/or
SAB membership from Merck, Honeycomb Biotechnologies, Cellarity, Reper-
toire ImmuneMedicines, Orche Bio, and Dahlia Biosciences. D.Z. holds equity
and consults for Corner Therapeutics. Boston Children’s Hospital has filed
patents related to the use of hyperactivating stimuli and hyperactive DCs in
the treatment of disease. J.C.K. serves on the advisory board of Cell Reports.
Received: June 29, 2020
Revised: August 19, 2020
Accepted: October 22, 2020
Published: November 17, 2020
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Article OPEN ACCESSSTAR+METHODSKEY RESOURCES TABLEREAGENT or RESOURCE SOURCE IDENTIFIER
Antibodies
PE/Cyanine7 anti-mouse CD11c Antibody (clone N418) BioLegend 117318
PerCP anti-mouse I-A/I-E Antibody (clone M5/114.15.2) BioLegend 107624
APC anti-mouse CD40 Antibody (clone3/23) BioLegend 124612
FITC anti-mouse CD80 Antibody (clone 16-10A1) BioLegend 104706
PE anti-mouse CD69 Antibody (clone H1.2F3) BioLegend 104508
APC anti-mouse H-2Kb Antibody (clone AF6-88.5) BioLegend 116518
PE anti-mouse CD197 (CCR7) Antibody (clone 4B12) BioLegend 120106
PerCP-eFluor 710 anti-mouse SIRP alpha Antibody (clone P84) BD Biosciences 46-1721-82
PE/Cy7 anti-mouse CD24 Antibody (clone M1/69) BioLegend 101822
APC-eFluor 780 anti-mouse CD11c Antibody (clone N418) ThermoFisher 47-0114-82
VioletFluor 450 anti-Mouse MHC Class II (clone M5/114.15.2) VWR 75-5321-U100
Alexa Fluor 488 Rat Anti-Mouse CD45R (clone RA3-6B2) BD Biosciences 557669
Alexa Fluor 647 anti-Mouse CD64 a and b Alloantigens (clone X54-5/7.1) BD Biosciences 558539
PE anti-mouse F4/80 Antibody (clone BM8) ThermoFisher 12-4801-82
PerCP/Cy5.5 anti-mouse CD45.1 Antibody (clone A20) BioLegend 110728
APC anti-mouse CD45.2 Antibody (clone 104) BioLegend 109814
APC anti-mouse CD8a Antibody (clone 53–6.7) BioLegend 100712
PerCP/Cyanine5.5 anti-mouse CD107a (LAMP-1) Antibody BioLegend 121626
Brilliant Violet 510 anti-mouse CD4 Antibody (clone RM4-5) BioLegend 100559
PE/Cyanine7 anti-mouse/human CD44 Antibody (clone IM7) BioLegend 103030
Alexa Fluor 488 anti-mouse CD62L Antibody (clone MEL-14) BioLegend 104420
PerCP/Cyanine5.5 anti-mouse CD3 Antibody (clone 17A2) BioLegend 100218
Alexa Fluor 647 anti-mouse CD103 Antibody (clone 2E7) BioLegend 121410
PE anti-mouse CD69 Antibody (clone H1.2F3) BioLegend 104508
Brilliant Violet 711 anti-mouse CD45 Antibody (clone 30F11) BioLegend 103147
Alexa Fluor 488 anti-mouse IFNg Antibody (clone XMG1.2) BioLegend 505813
PE-conjugated H2K(b) SIINFEKL (OVA 257-264) NIH Tetramer Core Facility N/A
APC conjugated I-A(b) AAHAEINEA (OVA 329-337) NIH Tetramer Core Facility N/A
I-A(b) and H2K(b) associated with CLIP peptides PVSKMRMATPLLMQA NIH Tetramer Core Facility N/A
FITC anti-mouse CD8.1, Lyt-2.1 (clone CD8-E1) Accurate Chemicals DEV102-1-4/02
APC anti-mouse H-2Kb Antibody (Clone AF6-88.5) BioLegend 116518
PE anti-mouse H-2Kb bound to SIINFEKL Antibody (Clone 25-D1.16) BioLegend 141604
Ultra-LEAF Purified anti-mouse CD8a Antibody (clone 53-6.7) BioLegend 100746
Ultra-LEAF Purified anti-mouse CD4 Antibody (clone GK1.5) BioLegend 100442
LEAF Purified anti-mouse / rat IL-1b Antibody BioLegend 503504
Ultra-LEAF Purified anti-mouse CD279 (PD-1) (clone 29F.1A12) BioLegend 135247
IL-1R Antagonist Cayman Chemical 21349
PerCP/Cyanine5.5 anti-mouse TCR b chain Antibody BioLegend 109228
anti-Asc, pAb (AL177) Adipogen Cat# AG-25B-0006;
RRID: AB_2490440
Chicken anti-Rabbit IgG Secondary Antibody, AF488 Thermo Fisher A-21441
Alexa Fluor 647 Phalloidin Thermo Fisher A22287
Phalloidin-iFluor 488 Abcam ab176753
(Continued on next page)
Cell Reports 33, 108381, November 17, 2020 e1
ll
OPEN ACCESS Article
Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
Chemicals, Peptides, and Recombinant Proteins
E. coli LPS Serotype O55:B5-TLR grade Enzo Life Sciences ALX-581-013-L001
PGPC Cayman Chemical 10044
Monophosphoryl Lipid A (MPLA) Invivogen tlrl-mpla
oxPAPC Invivogen tlrl-oxp1
CpG ODN 1826 Invivogen tlrl-1826-1
Incomplete Freund’s Adjuvant Sigma Aldrich F550
Alhydrogel Invivogen vac-alu-250
Addavax MF-59 Invivogen vac-adx-10
phorbol 12-myristate 13-acetate (PMA) and ionomycin Biolegend 423301
Brefeldin A Solution (1,000X) Biolegend 420601
Dynabeads Mouse T-Activator CD3/CD28 ThermoFisher 11456D
Ovalbumin, Alexa Fluor 488 Conjugate ThermoFisher O34781
EndoFit Ovalbumin Invivogen vac-pova
Dextran, Alexa Fluor 488; 10,000 MW ThermoFisher D22910
Recombinant Murine IL-2 Peprotech 212-12
Critical Commercial Assays
Mouse IL-1b ELISA Kit Thermofisher 88-7013-86
Mouse TNFa ELISA Kit Thermofisher 88-7324-88
Mouse IFN gamma ELISA Kit Thermofisher 88-7314-77
Mouse IL-2 ELISA Kit Thermofisher 88-7024-88
CyQUANT LDH Cytotoxicity Assay Thermofisher C20300
CD45 (TIL) MicroBeads, mouse Miltenyi Biotec 130-110-618
Tumor Dissociation Kit, mouse Miltenyi Biotec 130-096-730
CD8a (Ly-2) MicroBeads, mouse Miltenyi Biotec 130-117-044
CD4 (L3T4) MicroBeads, mouse Miltenyi Biotec 130-117-043
LIVE/DEAD Violet Viability Kit Thermofisher L34958
CellTrace CFSE Cell Proliferation Kit Thermofisher C34554
BD Cytofix/Cytoperm BD Biosciences 554714
RNeasy Plus Micro Kit QIAGEN 74034
Deposited Data
Expression profiling by high throughput RNA sequencing GEO accession numbers GSE156159
Experimental Models: Cell Lines
B16.F10 cell line Arlene Sharpe Laboratory N/A
B16.F10 OVA cell line Arlene Sharpe Laboratory N/A
MC-38 OVA cell line Arlene Sharpe Laboratory N/A
CT26 cell line Jeff Karp Laboratory N/A
LLC1 cell line ATCC ATCC CRL-1642
Experimental Models: Organisms/Strains
C57BL/6J The Jackson Laboratory 000664
B6N.129S2-Casp1tm1Flv/J (Casp1/11 
/
) The Jackson Laboratory 016621
B6.129S6-Nlrp3tm1Bhk/J (NLRP3 
/
) The Jackson Laboratory 021302
B6 Ly5.1 The Jackson Laboratory 002014
B6(Cg)-Zbtb46tm1(HBEGF)Mnz/J (Zbtb46DTR) The Jackson Laboratory 019506
B6.Cg-Gt(ROSA)26Sortm1.1(CAG-Pycard/mCitrine*,-CD2*)Dtg/J (ASC-citrine) The Jackson Laboratory 030744
B6.129S(C)-Batf3tm1Kmm/J The Jackson Laboratory 013755
B6.129P2(C)-Ccr7tm1Rfor/J The Jackson Laboratory 006621
B6.SJL-PtprcaPepcb/BoyCrl (Ly5.1) Charles River Laboratories 494
(Continued on next page)
e2 Cell Reports 33, 108381, November 17, 2020
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Article OPEN ACCESS
Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
C57BL/6J Charles River Laboratories 027
BALB/c The Jackson Laboratory 000651
Software and Algorithms
GraphPad Prism 7.0 GraphPad Software N/A
FlowJo (v10.3.0) FlowJo N/A
Microsoft Excel Microsoft N/A
Zen 2 Blue edition Carl Zeiss Microscopy N/A
Fiji / ImageJ version 2.0.0 https://imagej.net/Fiji N/A
Other
gentleMACS Dissociator Miltenyi Biotec 130-093-235
Large Cell Columns Miltenyi Biotec 130-042-401
MACSmix Tube Rotator Miltenyi Biotec 30-090-753
Cytation Cell Imaging reader BioTek instrument N/A
5 mm Glass Diameter | Poly-D-Lysine Coated plates Mattek P96GC-1.5-5-FRESOURCE AVAILABILITY
Lead Contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Jonathan
C. Kagan (jonathan.kagan@childrens.harvard.edu).
Materials Availability
This study did not generate new unique reagents.
Data and Code Availability
The expression profiling by high throughput RNA sequencing generated during this study is available at GEO repository. The records
have been assigned GEO accession numbers GSE156159.
EXPERIMENTAL MODEL AND SUBJECT DETAILS
Mouse strains, and Tumor cell lines
Female six- to eight-week old C57BL/6J, casp1/-11
/
 mice, Nlrp3
/
, Casp11
/
, Batf3
/
, Ccr7
/
, B6 Ly5.1 (expressing the
CD45.1 allele), Zbtb46DTR mice bearing the human diphtheria toxin receptor under zinc finger and BTB domain containing 46,
R26-CAG-ASC-citrine and BALB/cmice were purchased from Jackson Labs. Purchasedmice were allowed to acclimate to the Bos-
ton Children’s Hospital (BCH) housing facility for at least oneweek. For chimeramice experiments, four week old CD45.1 andC57BL/
6Jwere used. In some experiments CD45.1, andC57BL/6were purchased fromCharles river andwere housed inMassGeneral Hos-
pital (MGH) animal facility. In all experiments, mice were randomly assigned to experimental groups. All experimental procedures
were approved by the institutional animal care and use committee at BCH (IACUC 18-09-3796R) and MGH (IACUC 2005N000209
and 2014N000227). For syngeneic tumor models in C57BL/6J, two melanoma cell lines were used; the parental cell line: B16.F10
and an OVA expressing cell line B16.F10OVA from Dr. Arlene Sharpe (Harvard Medical School). For Lewis lung carcinoma model,
LLC1 cells (obtained from ATCC) were used. For a syngeneic colorectal model, MC38 cell line expressing OVA derived from
C57BL/6J murine colon adenocarcinoma cells were used and obtained from Dr. Arlene Sharpe (Harvard Medical School). For a syn-
geneic colon cancer model in BALB/c mice, CT26 cell line was used, and obtained from Dr. Jeff Karp (Harvard Medical School).
These cells were cultured in DMEM containing 10% FBS, Penicillin and Streptomycin (Pen+Strep), and supplements of L-glutamine
and sodium pyruvate. This media is referred to below as complete DMEM. Cell lines expressing the OVA protein were cultured in
complete DMEM supplemented with puromycin (2 mg/ml).
Differentiation of GM-CSF- and FLT3L-BMDCs
B16 cell lines producing GM-CSF or FLT3L were cultured for approximatively 6 days in complete IMDM containing 10% FBS, Peni-
cillin and Streptomycin (Pen+Strep) and supplements of L-glutamine and sodium pyruvate. This media is referred to below as com-
plete IMDM. Supernatants were cleared of cellular debris by spinning at 400 x g for 5 min. Pooled supernatants from several cultureCell Reports 33, 108381, November 17, 2020 e3
ll
OPEN ACCESS Articleflasks were combined and passed through a 0.22 mm filter. GM-CSF and FLTL3L conditioned supernatants were aliquoted and
frozen at
20C. Leg bones were removed frommice, cut with scissors and flushed with sterile PBS pH 7.4 via syringe. Bonemarrow
suspension was passed through a cell strainer, resuspended in media consisting of complete IMDM with 10% of either GM-CSF or
FLT3L conditioned media, then plated at 1x106 bone marrow cells per untreated 10 cm dish. Plates were fed with 5mL of additional
conditioned media on day 3 of differentiation. The efficiency of differentiation was monitored by flow cytometry using BD Fortessa
and was routinely above 80%. Differentiated cells were used for subsequent assays on day 7 for GM-CSF-DCs and day 8 for FLT3L-
DCs. BMDCs were washed with PBS and re-plated in complete IMDM at a concentration of 1x106 cells/ml in a final volume of 100 ml.
METHOD DETAILS
Generation of full bone marrow chimeric mice for DC injections
To generate bone marrow chimeric mice using Zbtb46DTR and WT mice, four week old CD45.1+ female mice were irradiated with
1000 rads prior to their injection with bone marrow. Bone marrow was harvested from 8-11-week-old Zbtb46DTR or WT mice by me-
chanical disruption using amortar and pestle followed by incubation with RBC lysis buffer. Lysis was stopped by adding excess PBS,
centrifugation and resuspension of the cell pellet in PBS. Within 6 hours of irradiation, recipient CD45.1 mice received a total of 0.7-
1 3 107 bone marrow cells from WT or Zbtb46DTR via retro-orbital injection in 100 ml. 6 weeks post reconstitution, recipient mice
received 20ng/g per body weight of diphtheria toxin (DTx) intraperitoneally 3 times weekly for 2 weeks. DC depletion in the spleen
and dLN was assessed 1-week post DT injection. These chimeric mice were used for DC injections in Figure S7 and were housed
at MGH animal facility.
Generation of full or mixed bone marrow chimeric mice for immunizations
Four week old CD45.1+ female mice were exposed to whole body irradiation (2 doses of 500 rads per mouse, 2 hours apart). After at
least 4 hours from the last irradiation, mice were reconstituted with 53 106 bone marrow (BM) cells isolated from sex-matched mice
and injected intravenously. Mice were kept in autoclaved cages and were treated with sulfatrim in the drinking water for 2 weeks after
reconstitution. Then, mice were placed in standard cages and allowed to reconstitute for 6 more weeks. To evaluate the percentage
of chimerism, peripheral blood samples were collected at the end of reconstitution and stained for CD45.1 and CD45.2. For these
experiments, all mice were housed at the BCH animal facility.
To deplete conventional dendritic cells (cDCs), we generated full BM chimeras by reconstituting irradiated mice with BM cells iso-
lated from Zbtb46DTR mice. Reconstituted mice were injected intraperitoneally with saline (control mice) or diphtheria toxin (DTx,
400 ng per mouse) 3 times per week starting from 3 days post-tumor injection for a total of 6 DTx doses.
To specifically deplete selected genes in cDCs, we generated mixed BM chimeras by reconstituting irradiated mice with 4 3 106
BM cells isolated from Zbtb46DTR mice mixed with 13 106 BM cells isolated from wild-type, Nlrp3
/
, Casp1/1
/
 or Ccr7 
/
 mice.
Reconstituted mice were then injected intraperitoneally with DTx (400 ng per mouse as first dose, then 200 ng per mouse) 3 times per
week starting from 12 days prior to tumor injection for a total of 9 (immunization experiments) or 12 (tumor challenge experiments)
DTx doses.
Ligand and Chemical Reconstitution
E. coli LPS (Serotype O55:B5-TLR grade) was purchased from Enzo and used at 1 mg/ml in cell culture or 10 mg/mouse for in vivo use.
In some experiments where indicated, LPS was used at 100ng/ml or 10ng/ml. Monophosphoryl Lipid A from S. minnesota R595
(MPLA) was purchased from Invivogen and used at 1 mg/ml in cell culture or 20 mg/mouse for in vivo use. CpG ODN 1826 used at
1 mg/ml in cell culture, was purchased from Invivogen. oxPAPC was purchased from Invivogen, resuspended in pre-warmed
serum-free media andwas used as 100 mg/ml for cell stimulation, or 65 mg/mouse for in vivo use. PGPCwas purchased fromCayman
Chemical. Reconstitution of PGPC was performed as previously described [26]. Briefly, ethanol solvent was evaporated using a
gentle nitrogen gas stream. Pre- warmed serum-free media was then immediately added to the dried lipids to a final concentration
of 1mg/ml. Reconstituted lipids were incubated at 37C for 5-10mins then sonicated for 20 s before adding to cells. PGPCwere used
at 100 mg/ml for cell stimulation or 65 mg/mouse for in vivo use. In some experiments, oxPAPC or PGPC were used at concentrations
ranging from 25 to 100 mg/ml. EndoFit chicken egg ovalbumin protein with endotoxin levels < 1 EU/mg and OVA 257- 264 peptide
were purchased from Invivogen for in vivo use at a concentration of 200 mg/mouse or in vitro use at a concentration ranging from 1000
to 10 mg/ml. Incomplete Freund’s Adjuvant (F5506) was purchased from Sigma and used for in vivo immunizations at a working con-
centration of 1:4 (IFA:antigen emulsion). Alhydrogel referred to as alum was purchased from Invivogen and used for in vivo immuni-
zation at a working concentration of 2mg/mouse. Where indicated, Addavax which is a Squalene-oil-in-water adjuvant was used
instead of IFA at a working concentration of 1:2 (Addavax:antigen).
DC stimulation and T cell culture
To induce active DCs, DCs were stimulated in complete IMDM with LPS (1 mg/ml) for 15 or 18 or 24 hours as indicated. To induce
hyperactive or pyroptotic DCs, cells were primed for 3 hours with LPS at a concentration of 1 mg/ml, unless otherwise indicated,
then stimulated with oxPAPC or PGPC (100 mg/ml, unless otherwise indicated) or alum (100 mg/ml) for 12 or 15h or 21h as indicated.
Naive DCs were cultured in the presence of complete media alone for the indicated time. T cells were cultured in RPMI-1640e4 Cell Reports 33, 108381, November 17, 2020
ll
Article OPEN ACCESSsupplemented with 10% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin (Sigma-Aldrich), and 50 mM b-mercaptoethanol (Sigma-
Aldrich). All cells were cultured at 37C in a humidified atmosphere containing 5% CO2.
LDH Assay and ELISA
Fresh supernatants were clarified by centrifugation then assayed for LDH release using the LDH cytotoxicity colorimetric assay kit
(ThermoFisher) following the manufacturer’s protocol. Measurements for absorbance readings were performed on a Tecan plate
reader at wavelengths of 490 nm and 680 nm. To measure secreted cytokines, supernatants were collected, clarified by centrifuga-
tion and stored at
20C. ELISA for IL-1b, TNFa, IFNg, IL-2, were performed using eBioscience Ready-SET- Go! (now ThermoFisher)
ELISA kits according to the manufacturer’s protocol.
Antigen uptake and peptide presentation assay
To examine antigen uptake and the endocytic ability of BMDCs, Alexa Fluor 488 labeled-chicken OVA (AF488-OVA) was used
(ThermoFisher). Briefly, naive, active, pyroptotic or hyperactive FLT3L-derived BMDCs previously cultured with media alone, or
treated for 24 hours with LPS alone or in combination with PGPC or alum, were incubated with AF488-OVA (0.5mg/ml) for 45minutes
at 37C, or at 4C (as a control for surface binding of the antigen). BMDCs were then washed and stained with Live/Dead Fixable
Violet dye (ThermoFisher) to distinguish living cells from dead cells. Cells were then fixed with BD fixation solution and resuspended
in MACS buffer. FITC fluorescence of live cells was measured. Fluorescence values of BMDCs incubated at 37C were reported
asMean fluorescence Intensity (MFI) of OVA-AF488 associated cells as normalized to MFI of OVA-AF488 associated cells incubated
at 4C.
To measure the efficiency of OVA peptide presentation on MHC-I, FLT3L-derived BMDCs were treated as described above and
incubatedwith Endofit-OVA protein (0.5mg/ml) for 1 hour at 37C. Cells were thenwashedwithMACS buffer and stained at 4C for 20
to 30 minutes with APC-conjugated anti-mouse H-2Kb antibody (BioLegend), and a PE-conjugated antibody that binds to H-2Kb
bound to the OVA peptide SIINFEKL (BioLegend). Non-OVA-treated DCs served as a negative control and isotype controls were
used as a staining control. The percentage of total surface H-2Kb, and the percentage of cells associated with the OVA peptide
onMHC-I was calculated. Data were acquired on a Fortessa flow cytometer (Becton-Dickenson) and analyzed with FlowJo software
(Tree Star).
DC injections for DC migration assay
BMDCs generated using FLT3L were prepared from C57BL/6J mice (on a CD45.2 background), harvested on day 8 and suspended
at a concentration of 1x106 cells/ml in complete IMDM. DCs were either left untreated or treated with LPS alone for 15 hours, or
BMDCs were primed with LPS for 3h then treated with PGPC or oxPAPC or alum for 12h. Alternatively, BMDCs from Nlrp3
/
 or
Ccr7
/
 or ASC-citrine mice were primed with LPS for 3h then treated with PGPC for 12h. DCs were cultured in polypropylene tubes
with gentle rotation using MACSmix Tube Rotator (Miltenyi Biotec) in the incubator. Cells were washed, stained for 30 minutes with
CFSE, and then 1x106 live DCswere injected s.c. on the right flank into ly5.1/CD45.1mice in a total volume of 100 ml. 15 hours post DC
injection, single cell suspension from the skin draining lymph nodes were stained with live-dead violet dye in PBS, then washed and
stained inMACS buffer with anti-CD11c, anti-CD45.1 and anti-CD45.2 antibodies (BioLegend). Uninjectedmice (no DCx) served as a
control. Hyperactive DCs thatmigrated to the dLNwere sorted asCFSE+CD45.2+ CD11c+ live cells, and resident DCswere sorted as
CFSEneg CD45.1+ CD11c+ live cells. Sorted cells were cultured in media alone for 24 hours onto 96-well round bottom plates. Su-
pernatants were used for LDH and IL-1b cytokine release as described above. For ASC microscopy experiments using migrated
DCs in the dLN, WT or Nlrp3
/
 DCs were injected as mentioned above without CFSE staining.
Microscopy Imaging
For DC immunostaining, DCs generated using FLT3L were cultured on coverslips and either left untreated (none) or treated with LPS
alone for 18 hours, or BMDCswere primedwith LPS for 3h then treated with PGPC or Alum for 15h. Cells were washed then fixedwith
4% PFA for 15 minutes at room temperature, and blocked using 1% goat serum for 1 hour. For immunofluorescence staining of hy-
peractive DCs that migrated to the dLN, cells were sorted from the dLN as CD11c+ CD45.2+ CD45.1- CFSE+ live cells and plated onto
glass 96 well plates. DCs were stained with iFluor 488 phalloidin (ThermoFisher) and DAPI. For anti-ASC speck staining, hyperactive
DCs were sorted from the dLN as CD11c+ CD45.2+ CD45.1neg and resident DCs as CD11c+ CD45.2neg CD45.1+ then plated on glass
96 wells flat plate. Cells were left to rest in media for 3-4hours, then fixed using 4% PFA. DCs were stained with Alexa Fluor 647
conjugated Phalloidin (ThermoFisher), rabbit anti-ASC (Adipogen) followed by Alexa Fluor 488-conjugated chicken anti-rabbit IgG
(ThermoFisher). ASC-citrine hyperactive DCs or resident DCs were sorted from the dLN then fixed with 4% PFA for 15 minutes at
room temperature, blocked using 1% goat serum for 1 hour, then stained with Alexa Fluor 647 conjugated Phalloidin and DAPI.
Images were acquired using 20x or 63x oil immersion lenses on Zeiss microscope.
Cell tracking and time-series analysis
8 days old FLT3L-DCs were cultured as described above for 15 hours in media alone or in the presence of LPS, or upon priming with
LPS for 3 hours followed by PGPC treatment for 12 hours. Cells were washed twice with PBS then plated onto glass plates for 3 hours
to rest prior to Image acquisition on BioTek instrument. Images were acquired every 2 minutes for 5 consecutive hours. ImageCell Reports 33, 108381, November 17, 2020 e5
ll
OPEN ACCESS Articleprocessing and cell tracking were performed for 4 independent ROI using the image-processing software Fiji (Schindelin et al., 2012).
The recorded time-series were imported using the Bio-Formats plugin (Linkert et al., 2010) and cell tracks were manually tracked
using the Manual Tracking plugin (Fabrice Cordelieres). Floating and dead cells were not tracked. Positional data was then imported
into R and individual cell statistics were calculated (https://cran.r-project.org/web/packages/data.table/index.html; https://cran.
r-project.org/web/packages/dplyr/index.html). Cell tracks were visualized using the ggplot2 package.
Flow cytometry and cell sorting
After FcR blockade, treated FLT3L-BMDCs were washed and stained in PBS with Live Dead Fixable dye (ThermoFisher) for 20 mi-
nutes at 4C. Cells were then washed again and stained for 20 minutes at 4C in MACS buffer (PBS with 1% FCS and 2 mM EDTA)
containing the following fluorescently conjugated antibodies purchased from BioLegend: anti-CD11c, anti-I-A/I-E, anti-H-2Kb, and
anti-CCR7. For cDC1 and cDC2 sorting, FLT3-derived DCs were stained for 20 minutes at 4C with monoclonal anti-SIRP alpha
(eBioscience), anti-mouse CD24 (BioLegend), monoclonal anti-CD11c (ThermoFisher), anti-mouse MHC Class II (VWR), anti-mouse
CD45R (BD), anti-CD64 (BD) andmonoclonal anti-F4/80 antibody (ThermoFisher). To assess DC depletion in the dLN following diph-
teria toxin treatment, single cell suspension forms the dLN were stained with following antibodies (BioLegend): anti-CD45.1, anti-
CD45.2, anti-MHC-II, anti-CD11c, and anti-CD64 (BD), and monoclonal anti-F4/80 antibody (ThermoFisher).
Single cell suspension from the tumor or draining inguinal lymph nodes, or spleen, or skin inguinal adipose tissue or skin biopsies
were stained for 20 minutes in PBS at 4Cwith Live Dead Fixable Violet or green dye (ThermoFisher) to determine the viability of cells
prior to antibodies staining. Cells werewashed then resuspended inMACS buffer (PBSwith 1%FCS and 2mMEDTA) and stained for
20 minutes at 4C with the following fluorescently conjugated antibodies (BioLegend): anti-CD8a, anti-CD4, anti-CD44, anti-CD62L,
anti-CD3, anti- CD103, anti-CD69, anti-CD45. For antigen-specific T cell detection, T cells were stained with OVA-peptide tetramers
at room temperature for 1h. PE-conjugated H2K(b) SIINFEKL and APC conjugated I-A(b) AAHAEINEA (OVA 329-337) were used. I-
A(b) and H2K(b) associated with CLIP peptides were used as isotype controls. Tetramers were purchased for NIH tetramer core fa-
cility. For tetramer staining, FITC anti-CD8.1 was purchased from accurate chemical.
For intracellular cytokine staining, T cells were stimulated with 50ng/ml phorbol 12-myristate 13-acetate (PMA) and 500ng/ml ion-
omycin (BioLegend) in the presence of GolgiStop and brefeldin A for 4-5 h. Cells were then washed twice with PBS, and stained with
LIVE/DEAD Fixable violet Stain Kit (ThermoFisher) in PBS for 20 min at 4C. T cells were then washed with MACS buffer, and stained
for appropriate surface markers as described above. After two washes, cells were fixed and permeabilizated using BD Cytofix/Cy-
toperm kit for 20 min at 4C, then washed with 1X perm wash buffer (BD) per manufacturer’s protocol. Intracellular cytokine staining
was performed in 1X perm buffer for 20- 30 min at 4C using anti-IFN-g (BioLegend). Data were acquired on a BD FACS ARIA or BD
Fortessa. Data were analyzed using FlowJo software.
To determine the absolute number of cells, countBright counting beads (ThermoFisher) were used, following the manufacturer’s
protocol. Appropriate isotype controls were used as a staining control. Data were acquired on a BD FACS ARIA or BD Fortessa (Bec-
ton-Dickenson). Data were analyzed using FlowJo software (Tree Star).
Adoptive DC transfers
For DC transfers, BMDCs generated using FLT3L were harvested on day 8, and suspended at a concentration of 1x106 cells/ml in
complete IMDM. DCs were treated as described above for 18 hours prior to their injection into recipient mice. DCs were cultured in
polypropylene tubes with gentle rotation using MACSmix Tube Rotator (Miltenyi Biotec) in the incubator. 18 hours post-culture, DCs
were washed twice with PBS, counted using trypan blue (GIBCO), then loaded (or not) with antigens such asOVA (serial dilution start-
ing 500 ug/ml) or whole tumor lysates for 1 hour. DCs were washed twice again with PBS and 1x106 cells in 100ul were injected sub-
cutaneously on the right flank of recipient mice. For DC-based immunotherapy experiments, recipient mice received the first DC in-
jection when tumors reached 3-4 mm of size, followed by 2 DC injections every 7 or 10 days.
DC and antigen-specific CD8+ T cell coculture
8weeks old femalemicewere s.c. immunizedwithOVA antigen emulsified in IFA on the right back. 7 days later, mice received a boost
injection with OVA antigen emulsified in IFA on the left back. 14 days after the first immunization, splenic CD8+ T cells were sorted
from immunized mice by magnetic cell enrichment using anti-CD8 beads. Total CD8+ T cells were sorted with a purity above 98%.
The percentage of SIINFEKL+ CD8+ T cells was assessed by flow cytometry using tetramer staining and was estimated around 1%–
2% among total CD8+ T cells. Total CD8+ T cells were seeded in 96-well plates at a concentration of 105 cells per well in the presence
of 1x104 DCs (10:1 ratio). 7 days post culture, supernatants were collected and clarified by centrifugation for short-term storage at

20C and cytokine measurement by ELISA. Cells were then processed as described above for an intracellular staining.
In vivo immunization and T cell re-stimulation
8weeks old femalemicewere immunized subcutaneously (s.c.) on the right flankwith either 200 mg/mouse endotoxin-free OVA alone
or with 10 mg/mouse LPS emulsified in incomplete Freund’s adjuvant. Alternatively, mice were immunized with 200 mg/mouse endo-
toxin-free OVA, plus 65 mg/mouse oxPAPC or PGPC, plus 10 mg/mouse LPS emulsified in incomplete Freund’s adjuvant. In some
experiments, mice were injected s.c. with OVA plus LPS emulsified in alum. 7 or 40 days after immunization, CD8+ T cells were iso-
lated from the skin draining lymph nodes of immunized mice by magnetic cell sorting with anti-CD8 beads. Enriched cells were thene6 Cell Reports 33, 108381, November 17, 2020
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Article OPEN ACCESSsorted as live CD45+CD3+CD8+ cells. Purity post-sorting was > 98%. Sorted cells were then seeded in 96-well plates in the presence
of OVA-preloaded BMDC (at a ratio of 1 DC: 10 T cells). Secretion of IFNgwasmeasured by ELISA 5 days later. In some experiments,
the percentage of antigen-specific T cells expressing IFNg was measured by intracellular staining 5 days post-co-culture.
CD107a degranulation assay
CD8+ T cells from the skin draining lymph nodes of immunized mice were isolated by magnetic cell enrichment with anti-CD8 beads,
then sorted as CD45+CD3+CD8+ cells live cells. Freshly sorted CD8+ T cells were resuspended in complete RPMI at a concentration
of 1x106 cells/ml. anti-CD107a (LAMP-1) antibody (BioLegend) was added to thismedia at a concentration of 1 mg/ml, in the presence
of GolgiStop. T cells were then immediately seeded in 96 wells plate at a concentration of 100,000 cells in the presence of 10,000
B16OVA as target cells. Alternatively, CD8+ T cells were seeded alone and stimulated for 5 hours with 50 ng/ml phorbol 12-myristate
13-acetate (PMA) and 500 ng/ml ionomycin (Sigma-Aldrich). 5 hours post-culture, cells were washed withMACS buffe r, stained with
LIVE/DEAD Fixable Violet Dead Cell Stain Kit (Thermofisher), and anti-CD8 (BioLegend). Cells were then Fixed with BD fixation so-
lution for 20 min at 4C and resuspended in MACS buffer. The percentage of CD107a+ CD8+ T cells was determined by flow cytom-
etry on the Fortessa flow cytometer (BD).
In vitro cytotoxicity assay
CD8+ T cells from the spleen, or the skin inguinal adipose tissue of survivormice were isolated using anti-CD8MACS beads. Enriched
T cells were then sorted as live CD45+CD3+CD8+ cells. Purity post-sorting was > 97%. B16OVA, B16F-10 or CT26 tumor cells were
seeded onto 96-well plates (1x104 cells/well) in complete DMEM at least 5 hours prior their co-culture with T cells. 105 CD8+T cells
were seeded onto tumor cells for 12h, then cytotoxicity was assessed by LDH release assay using the LDH cytotoxicity colorimetric
assay kit (ThermoFisher) following the manufacturer’s protocol.
Whole tumor lysates preparation
To prepare whole tumor cell lysates (WTL) for prophylactic immunization, tumor cell lines were cultured for 4- 5 days in complete
DMEM. When cells became confluent, supernatants were collected, and the cells were washed and dissociated using trypsin-
EDTA (GIBCO). Tumor cell lines were then resuspended at 5x106 cells/ml in their collected culture supernatant, then lysed by 3 cycles
of freeze-thawing. For immunotherapy experiments, syngeneic WTL were prepared from tumors explants of unimmunized tumor-
bearingmice. Briefly, tumors from unimmunizedmice bearing a tumor 10-12mmof size weremechanically disaggregated using gen-
tleMACS dissociator (Miltenyi Biotec) and digested using the tumor Dissociation Kit (Miltenyi Biotec) following the manufacturer’s
protocol. Tumors were incubated for 45 minutes at 37 degrees in a tube rotator for complete digestion. After digestion, tumor cell
suspensions were washedwith PBS and passed through 100-mm then 70-mm then 30-mmfilters. Single cell suspension was depleted
of CD45+ cells using anti-CD45 TILs microbeads (Miltenyi Biotec). Tumor cells were then counted and resuspended at 5x106 cells/ml
then lysed by 3-4 cycles of freeze-thawing. All prepared Lysates were centrifuged at 12,000 rpm for 15 minutes, and supernatants
were passed through 70-mm and 30-mm filters then stored in aliquots at 
20C until use. WTL were used for immunotherapy or for
BMDC antigen loading at a concentration equivalent to 2.5x105 tumor cells per mice, or at ratio equivalent to 1:10 (DC:tumor cells)
respectively.
Prophylactic immunization and tumor challenges
For immunizations prior to tumor inoculation, mice were injected subcutaneously (s.c.) into the right flank with PBS (unimmunized),
WTL alone at a concentration equivalent to 2.5x105 tumor cells per mice or with WTL plus 10 mg/mice of LPS, or WTL plus LPS plus
65 mg/mice of oxPAPC or PGPC, all emulsified in incomplete Freud’s adjuvant (IFA). In some experiments, LPS is replaced by MPLA.
15 days post immunization, mice were challenged s.c. on the left flank with 3x105 of viable B16OVA cells, or 5x105 of viable MC38-
OVA cells, as indicated. Tumor-free mice were re-challenged s.c. into the upper back with a lethal dose of 6x105 viable B16OVA or
1x106 of viable MC38-OVA cells, as indicated. When indicated, mice were given 100 mg of LEAF anti-mouse/rat IL-1b antibody
(BioLegend) by intravenous (i.v.) injection for five consecutive days; starting two days before receiving the immunization, then on
day 1, day 2, and day 3 post-immunization to ensure chronic depletion of circulating IL-1b. The size of the tumors was assessed
in a blinded, coded fashion every two days and recorded as tumor area (length 3 width) using a caliper. Mice were sacrificed
when tumors reached 2 cm3 or upon ulceration.
Immunotherapeutic immunization and tumor challenges
For immunizations in the context of an immunotherapeutic approach, C57BL/6J were injected on the left flank with 3x105 of viable
B16OVA cells, or 3x105 of B16-F10 cells, or 5x105 of viable MC38-OVA cells, or 3x105 of LLC1 cells. Alternatively, BALB/cmice were
injected on the left flank with 3x105 of viable CT26 cells. When tumors reached 3-4mm of size, mice were either left untreated (un-
immunized) or injected intraperitoneally (i.p.) with 100 mg of anti-PD-1 every two days for 5 consecutive injections. Alternatively, mice
were immunized with WTL at a concentration equivalent to 2.5x105 tumor cells per mice plus 10 mg/mice of LPS and 65 mg/mice of
PGPC emulsified in incomplete Freud’s adjuvant (IFA). Immunizations were followed by two boost injections every 7-10 days. Immu-
nized mice were divided blindly into several groups. Somemice were injected i.p. with 100 mg of Ultra-LEAF anti-CD4, or CD8a start-
ing the day of immunization or the day of boost injection, followed by 3 consecutive injections every 2 days. Other mice were givenCell Reports 33, 108381, November 17, 2020 e7
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OPEN ACCESS Article100 mg of LEAF anti-mouse/rat IL-1b antibody or IL-1 receptor antagonist (IL1RA) by i.v. injection every day for five consecutive days;
starting two days before receiving the immunization, then on day 1, day 2, and day 3 post-immunization to ensure chronic depletion of
circulating IL-1b. The injection of anti-IL-1b antibody or IL1RAwere repeated for every boost injection. Control mice received isotype-
matched rat IgG. All antibodies were purchased from BioLegend, and IL-1RA was purchased for Cayman Chemical. The size of the
tumors was assessed in a blinded, coded fashion every two days and recorded as tumor area (length 3 width) using a caliper. Mice
were sacrificed when tumors reached 2 cm3 or upon ulceration.
Tumor infiltrating T cells in the tumor microenvironment
To assess the frequency of tumor-infiltrating lymphocytes (TILs) in the tumormicroenvironment (TME), mice were dissected and peri-
tumoral tissue was discarded. Tumors were harvested then dissociated using the tumor Dissociation Kit and the gentleMACS dis-
sociator (Miltenyii Biotec), following the manufacturer’s protocol. After digestion, tumors were washed with PBS and passed through
70-mm and 30-mm filters. CD45+ cells were positively selected using anti-CD45 TILs microbeads (Miltenyi Biotec). In prophylactic
immunization experiments, the frequency of CD4+ T cells and CD8+ T cells was calculated among CD3+ CD45+ single live cells.
In LLC1 tumor model experiments, CD8+ T cell infiltration and T resident memory CD8+ T cells in the TME defined as
CD103+CD69+ CD8+ T cells were calculated among CD45+ CD8+CD11bneg CD19neg single cells. Tumor infiltrating CD45+ cells
were cultured for 24 hours with dynabeads mouse T-Activator CD3/CD28 (ThermoFisher) for T cell activation and IFN-g
measurement.
In vivo immunization and B16-F10 pulmonary colonization
To induce experimental lung colonization, 3x105 B16-F10 tumor cells were injected intravenously (i.v.) via tail vein in a volume of
100 ul. 2 days before tumor inoculation, mice were left untreated (unimmunized) or were immunized subcutaneously (s.c.) on the right
flank with WTL alone or with LPS, or with WTL plus LPS and PGPC, all emulsified in Addavax. Mice received a boost injection 5 days
post tumor inoculation. Mice were then sacrificed on day 18 after tumor cell injection, and lung tissues were isolated and fixed. Lung
metastatic nodules present on the surface of the lungs per mouse were enumerated.
Skin biopsies and skin adipose tissue dissociation
Skin punch biopsies were performed at the site of immunization and tumor injection site of survivor mice. Skin was incubated in Dis-
pase solution (Roche, 2.5 mg/ml) for 90 minutes and the epidermis separated from the dermis. The dermis was chopped finely and
incubated in collagenase type III (Sigma, 3 mg/ml) and the epidermis placed in trypsin/EDTA (Sigma) and incubated at 37C for
30 min. For adipose tissue dissociation, tissues were chopped using scissors and dissociated using gentleMACS dissociator (Mil-
tenyi Biotec) in the presence of collagenase A (Sigma, 3 mg/ml), then incubated in a tube rotator for 30 minutes 37C. Cells were
passed through 70-mmand 30-mmfilters, and centrifuged for 10minutes at 1300rpm. The lipid layer was aspirated, and supernatants
discarded. Cells were then treated with ACK lysis solution for 5 minutes at 4C. Single cell suspension from the skin tissue or skin
adipose tissue were suspended in MACS buffer for staining.
Adoptive T cell transfer
For T cell transfer, T circulating CD8+ T cells from the spleen, or T resident memory cells from the skin inguinal adipose tissue of sur-
vivor mice were isolated using anti-CD8MACSbeads (Miltenyi Biotec). Enriched cells were then sorted as live CD45+CD3+CD8+ cells
using FACS ARIA. Purity post-sorting was > 97%. Sorted T cells were then stimulated for 24 h in 24-well plates (23 106 cells/well)
coated with anti-CD3 (4 mg/ml) and anti-CD28 (4 mg/ml) in the presence of IL-2 (Peprotech, 50ng/ml). 5.x105 of activated splenic or
skin inguinal adipose CD8+T cells were transferred by i.v. or intra dermal (i.d.) injection respectively on the right flank into naı̈ve recip-
ient mice 7 days prior tumor challenge. Some mice received both T cell subsets 7 days prior tumor challenge.
RNA sequencing
RNA was extracted using RNeasy Plus Micro kit (QIAGEN) following the manufacturer’s protocol. cDNA was produced from isolated
RNA with the Smart-Seq2 reverse-transcription protocol as described by Picelli et al. (2014) with the following modifications: 1) Con-
centrations of input RNA was normalized to2,000 cells-worth of input RNA per reaction. 2) The Superscript II reverse-transcription
enzyme was replaced with Superscript III (ThermoFisher, #18080-085) which was applied according to manufacturer’s instructions.
Paired-end sequencing libraries were generated from cDNA with the Nextera XT DNA sample Prep Kit (Illumina, #FC-131) according
to the manufacturer’s instructions. Libraries were pooled at an equimolar ratio and sequenced on a NextSeq500/550 sequencer (Il-
lumina) using a 75 cycle v2.5 sequencing kit with a paired end read structure. Following sequencing, runs were demultiplexed using
bcl2fastq v2.2 then aligned with HISAT2 (Kim et al., 2015) against GRCm38 and quantified with RSEM (Li and Dewey, 2011) gener-
ating a gene by sample count matrix further analyzed with Seuratv3 (Stuart et al., 2019) and DESeq2 (Love et al., 2014). Treatment
based pairwise differential expression was calculated with DESeq20s negative binomial expression tests grouped by FLT3L-cDC1
and FLT3L-cDC2 populations. The significance threshold was set at 0.05 andmultiple expression tests were accounted for with Bon-
ferroni p value correction. Gene set module scores are calculated from the average expression levels of eachmodule on a per sample
basis and subtracted by the baseline expression of randomly selected gene sets of same size using Seurat’s AddModuleScore func-
tion. One gene set (Curated Cell Migration Module) was curated based genes found to be significantly differentially expressed withine8 Cell Reports 33, 108381, November 17, 2020
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Article OPEN ACCESSLPS plus PGPC versus LPS treatment in FLT3L cDC1 and FLT3L cDC2 populations. Other gene sets were selected from the Gene
Ontology database (http://www.informatics.jax.org/vocab/gene_ontology/). All genes used within each module are provided within
Table S1.
QUANTIFICATION AND STATISTICAL ANALYSIS
In in vivo studies, n refers to the number of animals per condition from at least 2 independent experiments. Statistical differences
were calculated by using unpaired two-tailed Student’s t test, or one-way ANOVA with Tukey post- test. Dependent
samples were analyzed with paired t tests. Statistical significance for experiments with more than two groups was tested
with two-way ANOVA with Tukey multiple comparison test correction. All experiments were analyzed using Prism 7 (GraphPad Soft-
ware). Graphical data was shown asmean values with error bars indicating the SD or SEM. P values of < 0.05 (*), < 0.01 (**) or < 0.001
(***) ; % 0.0001 (****) indicated significant differences between groupsCell Reports 33, 108381, November 17, 2020 e9