Pyroptosis activates conventional type I dendritic cells to mediate the

InkolJM, etal. J Immunother Cancer 2024;12:e006781. doi:10.1136/jitc-2023-006781

Open access

Pyroptosis activates conventional type I

dendritic cells to mediate the priming of

highly functional anticancer T cells

Jordon M Inkol,

Michael J Westerveld,

Shayla G Verburg,

Scott R Walsh,

Jodi Morrison,

Karen L Mossman,

Sarah M Worfolk,

Kaslyn LF Kallio,

Noah J Phippen,

Rebecca Burchett,

Yonghong Wan,

Jonathan Bramson ,

Samuel T Workenhe

To cite: InkolJM,

WesterveldMJ, VerburgSG,

etal. Pyroptosis activates

conventional type I dendritic

cells to mediate the

priming of highly functional

anticancer T cells. Journal for

ImmunoTherapy of Cancer

2024;12:e006781. doi:10.1136/

jitc-2023-006781

► Additional supplemental

material is published online only.

To view, please visit the journal

online (https:// doi. org/ 10. 1136/

jitc- 2023- 006781).

Accepted 13 March 2024

Department of Pathobiology,

University of Guelph, Guelph,

Ontario, Canada

Department of Biomedical

Sciences, University of Guelph,

Guelph, Ontario, Canada

Department of Medicine,

McMaster University, Hamilton,

Ontario, Canada

Pathology and Molecular

Medicine, McMaster University,

Hamilton, Ontario, Canada

Correspondence to

Dr Samuel T Workenhe;

sworkenh@ uoguelph. ca

Original research

employer(s)) 2024. Re- use

permitted under CC BY- NC. No

commercial re- use. See rights

and permissions. Published by

BMJ.

ABSTRACT

Background Initiation of antitumor immunity is reliant on

the stimulation of dendritic cells (DCs) to present tumor

antigens to naïve T cells and generate effector T cells that

can kill cancer cells. Induction of immunogenic cell death

after certain types of cytotoxic anticancer therapies can

stimulate T cell- mediated immunity. However, cytotoxic

therapies simultaneously activate multiple types of cellular

stress and programmed cell death; hence, it remains

unknown what types of cancer cell death confer superior

antitumor immunity.

Methods Murine cancer cells were engineered to

activate apoptotic or pyroptotic cell death after Dox-

induced expression of procell death proteins. Cell- free

supernatants were collected to measure secreted danger

signals, cytokines, and chemokines. Tumors were formed

by transplanting engineered tumor cells to specically

activate apoptosis or pyroptosis in established tumors and

the magnitude of immune response measured by ow

cytometry. Tumor growth was measured using calipers

to estimate end point tumor volumes for Kaplan- Meier

survival analysis.

Results We demonstrated that, unlike apoptosis,

pyroptosis induces an immunostimulatory secretome

signature. In established tumors pyroptosis preferentially

activated CD103

and XCR1

type I conventional DCs

(cDC1) along with a higher magnitude and functionality

of tumor- specic CD8

T cells and reduced number of

regulatory T cells within the tumor. Depletion of cDC1 or

CD4

and CD8

T cells ablated the antitumor response

leaving mice susceptible to a tumor rechallenge.

Conclusion Our study highlights that distinct types of cell

death yield varying immunotherapeutic effect and selective

activation of pyroptosis can be used to potentiate multiple

aspects of the anticancer immunity cycle.

BACKGROUND

Immunotherapy has demonstrated success

in many types of human cancers.

However, a

few human cancers poorly respond to immu-

notherapy due to tumor intrinsic barriers of

T cell priming and trafficking into the tumor

or immunosuppressive microenvironment

preventing cytotoxic CD8

T cells to kill

cancer cells.

Therefore, there is a need for

therapies that can subvert the intratumoral

immunosuppression by reigniting the anti-

cancer immunity cycle to elevate the magni-

tude and functionality of T cells within the

tumor.

Chemoradiotherapies render tumors

to emit danger signals, cytokines, and

chemokines thereby stimulating the anti-

cancer immunity cycle.

3 4

Collectively,

these biomolecules regulate the activity

of antigen presenting cells as well as the

trafficking of cytotoxic T cells into the

tumor.

Indeed, chemoradiotherapies,

photodynamic therapy,

oncolytic viruses

(OVs),

8–16

and many other immunogenic

cell death (ICD) inducers can modify the

tumor microenvironment and often facili-

tate the eradication of established tumors.

However, most cytotoxic anticancer treat-

ments simultaneously activate a mixture

of premortem stress and programmed

cell death. Furthermore, chemotherapies

directly impact the quantity and/or func-

tionality of immune cells, complicating the

WHAT IS ALREADY KNOWN ON THIS TOPIC

⇒ Chemoradiotherapies activate the immune re-

sponse, although the contribution of individual

stress and cell death to anticancer immunity re-

mains unknown.

WHAT THIS STUDY ADDS

⇒ This study shows that pyroptosis, unlike apoptosis,

is an immunostimulatory type of cell death activat-

ing type I conventional dendritic cells to prime dura-

ble T cell- mediated antitumor immunity.

HOW THIS STUDY MIGHT AFFECT RESEARCH,

PRACTICE OR POLICY

⇒ The implications are pyroptosis inducing agents

should be further tested for development as anti-

cancer therapies for patients with cancer.

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interpretation of mechanisms contributing to clinical

effects and outcomes driven by specific types of cancer

cell stress and cell death.

Depending on the type of cytotoxic agent and

cellular pathways engaged, during ICD, cancer cells

can die by apoptosis,

necroptosis,

pyroptosis

and ferroptosis.

In most scenarios, cell death takes

place in the background of premortem endoplasmic,

oxidative, and mitochondrial stress.

As a result,

the individual contribution of premortem stress and

cell death pathways to anticancer immunity is largely

unknown.

Anthracycline and oxaliplatin activate

apoptosis along with autophagic stress response to

emit immunostimulatory secretomes.

Expressing

procell death proteins to drive specific types of cell

death is unraveling immunological outcomes.

23 24

Activation of necroptosis by RIPK3 dimerization, but

not apoptosis after caspase 8/9 expression, elicits

potent antitumor immunity.

23 24

In the same study,

necroptosis driven by RIPK3 lacking RIPK1 interac-

tion domain failed to activate NF- kb mediated inflam-

mation and consequently failed to initiate protective

anticancer immunity.

In contrast, another study

reported terminal cell lysis by expressing mixed

lineage kinase domain like pseudokinase (MLKL)

mRNA activated potent anticancer immunity.

These

studies highlight that so much remains unknown

about how distinct types of cancer cell death can

shape the overall intratumoral immune landscape

and anticancer effect.

Pyroptosis is one of the immune- stimulatory cancer

cell death modalities and it can be induced by chemo-

therapy, photodynamic therapy,

18 26

and OVs,

cytotoxic

CD8

T cells

28–31

and NK cells.

Irrespective of the type

of pyroptosis- inducing stimuli and upstream signaling

cascade, the cleavage of the gasdermin family of proteins

by caspases and granzymes is a terminal event during

pyroptosis.

18 33–36

Enforced expression of full- length gasdermin E

protein promotes granzyme mediated pyroptosis initi-

ating T cell- mediated anticancer effect.

However,

detailed mechanisms by which pyroptosis modifies

the tumor microenvironment and how it modulates

antigen presentation to prime antitumor T cells

remains unknown. Using two murine tumor models,

we report that unlike apoptosis, driven by dimeriz-

able (dd) C- terminal caspase- 8 (dd- C

term

- Cas8), pyro-

ptosis, activated by N- terminal gasdermin E (N

term

Gas)

induced a higher amount of calreticulin on the cell

surface, emitted danger signals ATP and HMGB1, as

well as cytokines and chemokines. These biomole-

cules potently stimulated conventional dendritic cell

(cDC1) and associated expansion and intratumoral

recruitment of antigen- specific cytotoxic CD8

cells and lower amounts of regulatory T cells within

the tumor thereby promoting anticancer effect and

extension of the overall survival of tumor- bearing

mice.

RESULTS

Pyroptosis emits danger signals and proinammatory

cytokines and chemokines

To define the immunological events and anticancer

outcomes after induction of apoptosis and pyroptosis,

we developed genetic models of rapid and synchronized

cell death by inducibly expressing procell death proteins

(figure 1A,D). Doxycycline (Dox) treatment activated

the expression of dd- C

term

- Cas8 (figure 1B) and N

term

Gas

(figure 1E) in mouse colon cancer (MC- 38) and lung

cancer cells derived from K- ras

G12D

p53

loxP/loxP

(KP)

mice (KP1.9)

(online supplemental file 2). Expres-

sion of dd- C

term

- Cas8 activated apoptosis, as evidenced

by cleavage of caspase 3 (figure 1B). In addition, expres-

sion of procell death proteins, dd- C

term

- Cas8 and N

term

Gas

showed reduced cellular metabolism used as a proxy to

measure cell viability (figure 1C,F and online supple-

mental file 2). Pyroptotic but not apoptotic cells showed

elevated surface expression of calreticulin (figure 1G) and

higher amounts of danger signals HMGB1 (figure 1H),

and ATP (figure 1I, online supplemental file 2) in cell-

free supernatants. In addition, compared with untreated

controls, pyroptotic cells emitted significantly increased

amounts of proinflammatory cytokines and chemokines

(IL- 1β, IL- 2, IL- 16, IL- 9, CXCL9, CX3CL1, and CCL3)

and decreased of eotaxin (CCL11) and G- CSF (figure 1J

and online supplemental file 3). On the other hand,

apoptotic cells emitted higher amounts of cytokines

and chemokines (IL- 2, IL- 9, IL- 11, IL- 20, IFN-γ, IFN-β,

eotaxin, CCL10, CCL21, CCL22) (figure 1J and online

supplemental file 4). Cytokines and chemokines downreg-

ulated during apoptosis include IL- 1a, IL- 16, LIF, G- CSF,

GMCSF, CCL12, VEGF, CCL5, CCL2, CX3CL1, and KC

(figure 1J and online supplemental file 4). In addition to

the remarkable differences in the types of cytokine and

chemokines emitted, there were notable differences in

the kinetics of their secretion. Pyroptosis- induced cyto-

kines and chemokines increased over time from 2 hours

to 4 hours. In contrast, most of the cytokine and chemo-

kines emitted during apoptosis showed transient increase

at the 2 hour time point and declined by the 4 hour time

point (figure 1J).

Established tumors of MC- 38- dd- C

term

- Cas8 or MC- 38-

term

Gas expressed the procell death proteins after

feeding of mice with a Dox- medicated diet (figure 2A,B).

Analysis of 44- plex cytokines/chemokines in pyroptotic

tumor homogenates showed significant increase in

IL- 1a, IL- 6, IFN-β, IL- 16, CXCL2, CCL21, and CCL22

(figure 2C and online supplemental file 5). Furthermore,

pyroptotic tumors had decreased levels of cytokines and

chemokines associated with protumor macrophages, and

myeloid- derived suppressors cells (CCL2, CCL5, M- CSF)

(figure 2C and online supplemental file 5). On the other

hand, apoptotic tumors showed significantly higher levels

of IL- 9, IL- 4 and CXCL2 and significant downregulation

of IL- 16, CCL5, IFN- b, LIF, IL- 17, GMCSF (figure 2C and

online supplemental file 6). Overall pyroptosis induces

a profound immune- stimulating cytokine profile in vivo.

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Figure 1 MC- 38 cells undergoing pyroptosis express calreticulin on the cell surface and emit higher level of HMGB1 and

ATP. (A, D)Schematics of respective apoptosis and pyroptosis effector proteins. (B, E)Immunoblots showing expression of

dd- C

term

Cas8 or N

term

Gas in Dox- treated MC- 38 cells. (C, F)Kinetics of cell viability after inducible expression of N

term

Gas (N=5)

or dd- C

term

Cas8 (N=5). Error bars, SD. P values were determined using two- way ANOVA with Tukey HSD with signicance

indicated (****p<0.0001). (G)ELISA showing HMGB1 in cell- free supernatants of pyroptotic cells (N=3). Error bars, SD. P values

were determined using two- way ANOVA with Tukey HSD with signicance indicated (****p<0.0001, ***p<0.001, **p<0.01).

(H)Extracellular ATP secretion at different time points post apoptosis (N=5) and pyroptosis (N=5). Error bars, SD. P values were

determined using two- way ANOVA with Tukey HSD with signicance indicated (*p<0.083). ((I)Heatmap of Log

fold changes

of cytokines and chemokines secreted 2 and 4 hours after induction of pyroptosis (N=3) and apoptosis (N=3) compared with

baseline at 0 hour for each respective group. Hierarchical clustering distance was dened on Pearson correlation coefcients.

ANOVA, analysis of variance; dd, driven by dimerizable; HSD, honest signicant difference.

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Pyroptosis preferentially activates type I cDCs

To evaluate the immune landscape after cell death, we

established chimeric tumors by transplanting a mixture

of parental MC- 38 along with either MC- 38- dd- C

term

- Cas8

or MC- 38- N

term

Gas cells. Prior to establishing tumors, we

confirmed that parental and N

term

Gas expressing cells

have similar in vitro growth kinetics (online supplemental

file 7), as a result, transplantation faithfully recapitulated

chimeric tumors that activate apoptosis (online supple-

mental file 8) or pyroptosis after feeding of mice with

Dox- medicated diet (online supplemental file 8).

To define the early processes influencing adap-

tive immunity after induction of cell death we quanti-

fied cDC within the tumor and draining lymph nodes

(figure 3 and online supplemental file 9). Within the

tumor, unlike apoptosis, pyroptosis preferentially

induced a 24- hour time peak of cDC1 quantity with a

higher phagocytic capacity as evidenced by uptake of

ZsGreen expressed in tumors (figure 3A). Moreover,

pyroptosis activated cDC1 had expressed SIINFEKL

epitope in the context of MHC (figure 3A). Consistent

with this, pyroptosis exposed tumors showed elevated

cDC1 activation markers including CD40, CD80 and

CD86 (figure 3B). Tumor draining lymph nodes showed

a delayed but similar pattern of pyroptosis- specific

increase in cDC1 quantity, phagocytic activity, antigen

presentation and costimulatory molecule expression at

the 48- hour time point (figure 3C,D). Both apoptosis

Figure 2 Pyroptotic tumors have an inammatory cytokine and chemokine signature. (A, B)Immunoblotting of N

term

Gas (N=3)

or C

term

Cas8 (N=2) in tumor homogenates harvested 6 hours after Dox- medicated diet. (C)Heatmap of log2 fold changes of

cytokines and chemokines in pyroptotic (N=5) and apoptotic (N=5) tumor homogenates 24 hours after induction compared with

untreated control homogenates of each respective group. Hierarchical clustering distance was dened on Pearson correlation

coefcients.

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and pyroptosis increased the total cDC2 count within

the tumor, although pyroptosis had significantly higher

number (figure 3E). Both types of cell death had intra-

tumoral peak of cDC2 mediated phagocytic activity at

the 24- hour time point (figure 3E). However, only pyro-

ptosis exposed cDC2 showed elevated expression of

SIINFEKL epitope (figure 3E). Both types of cell death

stimulated CD40 expression in cDC2, although apop-

tosis induced CD80 and pyroptosis preferentially acti-

vated CD86 (figure 3F). In the tumor draining lymph

node, apoptosis and pyroptosis exposed mice did not

show significant difference in cDC2 quantity, phago-

cytic activity, antigen presentation and costimulatory

molecules expression. However, both apoptosis and

Figure 3 Intratumoral pyroptosis preferentially activates conventional type I dendritic cell (cDC1) in the tumor and draining

lymph node. Quantication of total cDC1 within the tumor (N=4) and TdLN (N=4) as well as phagocytic capacity and antigen

presentation (A, C).Evaluation of costimulatory marker presence on conventional type I DCs (B, D).Quantication of total

cDC2 within the tumor (N=4) and TdLN (N=4) as well as phagocytic capacity and antigen presentation (E, G).Evaluation of

costimulatory marker presence on conventional type I DCs (F, H).Error bars, SD. P values were determined using two- way

ANOVA with Bonferroni correction with signicance indicated. *p <0.05, **p<0.01, ***p<0.001, ****p<0.0001; ANOVA, analysis of

variance; ns, not signicant.

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pyroptosis exposed mice had a higher cDC2 quantity

and phagocytic activity (figure 3G,H).

Tumor-specic CD8

T cells drive the antitumor response

during pyroptosis

We next examined if the cDC1 response generated

during pyroptosis translated into tumor specific CD8

cell response. For this, we established chimeric tumors

expressing the LCMV glycoprotein gp33 epitope. Seven

days after start of Dox- medicated diet, we quantified T

cell responses in the blood or within the tumor. Pyro-

ptosis generated higher number of tumor infiltrating

CD8

T cells compared with apoptosis that also showed a

moderate T cell infiltrate compared with control tumors

(figure 4A). Both apoptosis and pyroptosis showed equiv-

alent increase in gp- 33 specific CD8

T cells in the circu-

lation (figure 4B, online supplemental file 10); however,

pyroptosis primed antigen experienced CD44

CD8

cells displayed a higher activation pattern as defined by

CD69 expression (figure 4C). Consistent with the propor-

tion of activated T cells, pyroptosis primed cytotoxic T

cells showed a higher killing capacity against cocultured

gp33 expressing MC- 38 cells (figure 4D). Restimulation

of TILs with a cocktail of peptides (gp33, and MC- 38

neoantigens, Adgpk, and Rpl18) followed by intracellular

cytokine staining (ICS) revealed only pyroptosis exposed

tumors had a higher IL- 2

, TNF-α

or concurrent IL- 2

and TNF-α

CD8

T cells suggestive of polyfunctionality

(figure 4E–H, online supplemental file 11). Surprisingly,

pyroptosis- induced antigen specific T cells did not secrete

IFN-γ (figure 4G). Consistent with highly functional cyto-

toxic T cells, pyroptosis exposed tumors had a lower

quantity of T

reg

within the tumor (figure 4I).

Pyroptosis extends survival of tumor-bearing mice in cDC1

and T cell-dependent manner

Given the higher magnitude of activated cDC1 and cyto-

toxic T cell response, we evaluated the antitumor bene-

fits of pyroptosis in prophylactic and therapeutic settings.

MC- 38- N

term

Gas and MC- 38- dd- C

term

Cas8 tumor- bearing

mice were fed with Dox- medicated diet to eradicate

primary tumors within 7 days (figure 5A). Rechallenge

of pyroptosis and apoptosis exposed mice with parental

MC- 38 tumors on the contralateral flank conferred 40%

and 85% long- term survival, respectively (figure 5B,C).

All the naïve mice challenged with MC- 38 tumor cells

showed 100% tumor growth. Apoptosis, but not pyro-

ptosis, exposed mice fully succumb to the second tumor

rechallenge done at 100 days. In addition, 40% of the

pyroptosis exposed mice remained refractory against

subsequent rechallenges done at 200 days (figure 5C).

We next examined if the induction of pyroptosis results

in therapeutic benefit. Induction of pyroptosis slowed

the growth of chimeric tumors (figure 5D) and extended

the median survival of mice (median survival of 29 days,

25% of the mice remained tumor- free) compared with

apoptosis (median survival of 12 days, no tumor- free

mice) (figure 5E). The treatment benefit was consistent

in large chimeric MC- 38 tumors where induction of pyro-

ptosis displayed extension of median survival (online

supplemental file 12, apoptosis having 12 days median

survival vs pyroptosis conferring 20 days median survival).

The anticancer effects of pyroptosis extends to a non- T

cell inflamed KP1.9 tumor model since pyroptosis expo-

sure prolonged the median survival of tumor- bearing

mice (median survival of parental tumors—11 days,

KP1.9+KP1.9- N

term

Gas chimeric tumor without Dox-

medicated diet- 14 days, KP1.9+KP1.9- N

term

Gas chimeric

tumor fed with Dox- medicated diet—19 days) (online

supplemental file 13). Collectively, the findings suggest

that in prophylactic and therapeutic settings pyroptosis

provides better antitumor immunity than apoptosis.

To determine the antitumor contribution of cDC1, we

assessed tumor growth and survival in wildtype versus

Batf3

−/−

mice that lack cDC1. While pyroptosis protected

80% of the wildtype mice on tumor rechallenge, Batf3

−/−

mice lacking cDC1 were fully susceptible to tumor

rechallenge (figure 6A) even after transfer of wildtype

CD8

T cells (figure 6B). Furthermore, the depletion

of CD8

T cells but not CD4

(online supplemental file

14) completely ablated the antitumor immune response

of pyroptosis rendering mice susceptible to the tumor

rechallenge (figure 6C).

Discussion/conclusion

In this study, we elucidated that synchronized and rapid

cancer cell apoptosis and pyroptosis have remarkable

differences in danger signal, cytokine, and chemokine

emission as well as anticancer effect dependent on cDC1

and CD8 T

cells.

Our findings that show pyroptosis stimulates anti-

cancer immunity through the release of danger signals

and proinflammatory cytokines is in line with results

from previous reports.

28 32

Consistent with the role of

cDC1 in pyroptosis, we found higher level of chemokines

CCL21 and CCL22 in pyroptotic tumor homogenates. It

is tempting to speculate these chemokines to be associ-

ated with the cDC1- mediated anticancer immunity.

38–40

A recent scRNA seq identified CCL22

cDC1 population

after CD40 agonist therapy in MC- 38 tumors.

In addi-

tion, CCL21 is a CCR7 ligand, and it is crucial for cDC1

trafficking into the draining lymph node. In our study,

the contribution of cDC1 during pyroptosis- mediated

antitumor immunity was best confirmed using Batf3

−/−

mice where loss of cDC1 completely ablated the anti-

tumor response. Supporting this, pyroptosis exposed

tumors and draining lymph nodes are endowed with

a higher quantity of cDC1 displaying uptake of dying

cancer cells, activation markers and presentation of the

antigens in the context of MHC. Previous studies have

elucidated that intratumorally injected necroptotic fibro-

blasts elicit cDC1 dependent antigen presentation and

T cell activation.

The types of cytokines and chemok-

ines emitted during necroptosis

and our tumor pyro-

ptosis are remarkably different and it remains intriguing

how distinct ICD- associated secretomes shape cDC1

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Figure 4 Pyroptosis induces a polyfunctional antitumor antigen agnostic T cell response (A)Quantication of the number

of inltrating CD8

T cells in chimeric tumors 7 days after activation of apoptosis (N=5) or pyroptosis (N=5). Error bars,

SD. P values were determined using one- way ANOVA with Bonferroni correction with signicance indicated (*p<0.0083).

(B)Quantication of the frequency of LCMV- gp33- specic CD8

T cells in circulation after activation of pyroptosis (N=5) or

apoptosis (N=5) in chimeric tumors. Errors, SD. P values were determined using one- way ANOVA with Bonferroni correction

with signicance indicated (*p<0.0083). (C)Pyroptosis elicits signicantly higher frequency of antigen experienced CD44

CD69

CD8

T cells within the spleen (N=4). Error bars, SD. P values were determined using Student’s t test with signicance

indicated (****p<0.0001). (D)Percent specic lysis of MC- 38- gp33 cells after coculture with pyroptosis (N=4) and apoptosis

(N=4) stimulated CD8

T cells. Error bars, SD. P values were determined using two- way ANOVA with Tukey HSD with

signicance indicated (****p<0.0001). (E–H)Analysis of IL- 2, TNF- a, IFN- g and IL- 2

+TNFa

coexpressing CD8

T cells after

restimulation with a peptide cocktail (Adgpk and Rpl18 and gp33) (N=5). Error bars, SD. P values were determined using

one- way ANOVA with Bonferroni correction with signicance indicated (****p<0.0001, ***p<0.0002, **p<0.001). (I)Analysis of

CD3

CD4

FoxP3

regulatory T cells isolated from apoptosis (N=5) and pyroptosis (N=5) exposed chimeric tumors. Error bars,

SD. P values were determined using one- way ANOVA with Bonferroni correction with signicance indicated (***p<0.0002).

ANOVA, analysis of variance; HSD, honest signicant difference.

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heterogeneity and functionality. Hence, this model of

pyroptosis can be exploited as a ligand- free system to

understand numerous aspects of cDC1 biology including

chemotaxis factors that facilitate cDC1 trafficking as well

as transcriptome changes that dictate the role of cDC1 in

antigen uptake and presentation.

Pyroptosis activated a high proportion of circulating

and tumor infiltrating cytotoxic T cells (TILs). ICS

analysis of TILs after restimulation with the neoantigen

peptide cocktail showed secretion of IL- 2 and TNF-α but

not IFN-γ. In addition, pyroptosis primed cytotoxic T

cells isolated from spleen of tumor- bearing mice had a

higher proportion of rapidly reactivated antigen expe-

rienced CD44

CD69

expressing T cells that exerted

robust killing of cocultured tumor cells. Consistent with

these qualities of pyroptosis primed T cells, we observed

higher protection conferred to pyroptosis, unlike apop-

tosis, exposed mice at 100 days after the rechallenge.

Future studies will investigate if the functionality of

pyroptosis primed T cells is mediating the long- term

protection against rechallenge. It also remains to be

seen if pyroptosis primed T cells can infiltrate non- T

cell inflamed tumors and function in highly suppressive

tumor microenvironments. At the least, the finding of

Figure 5 Antitumor immunity generated by pyroptosis confers extended survival of tumor- bearing mice in therapeutic and

prophylactic settings (A)Tumor growth kinetics after feeding Dox- medicated diet to MC- 38- N

term

Gas (N=7) or MC- 38- dd-

term

Cas8 (N=7) tumor bearing mice. (B, C)Overall tumor growth and survival of contralaterally implanted MC38- gp33 tumors

in the presence or absence of a prior tumor apoptotic (N=7) or pyroptotic exposure (N=7). P values were determined using

log- rank (Mantel- Cox) test with Bonferroni correction with signicance set at p<0.016. (D, E)Overall tumor growth and survival

of apoptosis (N=7) and pyroptosis (N=7) exposed chimeric tumors. P values were determined using log- rank (Mantel- Cox) with

Bonferroni correction with signicance set at p<0.016. **p<0.0016.

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reduced T

reg

in pyroptosis exposed tumors is suggestive

of its broader immunostimulatory effects in the tumor

microenvironment.

Our findings highlight that pyroptosis provides supe-

rior systemic and long- lasting antitumor immunity

compared with apoptosis. Hence, approaches to activate

cancer cell pyroptosis via pharmacological or genetic

methods can be translated into patients with cancer to

reignite the anticancer immunity. In fact, some cancers

evade immune attack by downregulating the pyroptosis

effector gasdermin proteins. Hence, targeted delivery

of gasdermin A3 provided potent anticancer effect.

addition, engineered OVs have successfully delivered cell

death payloads such as MLKL to induce necroptosis.

Lastly, engineered OVs expressing gasdermin or those

OVs naturally activating pyroptosis

should be rigorously

evaluated in humanized mouse settings to evaluate their

anticancer effect against patient derived xenografts.

METHODS

Cell culture

Murine colon adenocarcinoma (MC- 38) was maintained

in Dulbecco’s Modified Eagle’s media supplemented with

10% fetal bovine serum (FBS) and 100 U/mL of peni-

cillin/streptomycin (P/S). KP 1.9 is a cloned lung adeno-

carcinoma cell line that arose spontaneously in a K- ras

LA1

p53

R172HΔg

mouse model. KP 1.9 cells were a generous gift

from Prof. Alfred Zippelius (University Hospital Basel,

Switzerland), who originally developed the cell line. KP

1.9 cells were maintained in Iscove Modified Dulbecco

media with 10% FBS and 100 U/mL P/S.

Generation of stable N terminus gasdermin E and caspase 8

expressing cell lines

N- terminal gasdermin E (N

term

Gas) and dimerizable (dd)

C- terminal Caspase 8 (dd- C

term

Cas8) DNA sequences

were cloned into the piggyBac (pB) vector (pB- TET)

downstream of a Dox inducible TRE- tight promoter

Figure 6 Pyroptosis- induced antitumor immunity is reliant on BATF3

DCs and T cells (A)Comparison of survival after tumor

rechallenge in naïve (N=8) and pyroptosis (N=8) exposed BATF3

−/−

or wildtype (N=8) C57bl/6 mice. P values were determined

using log- rank (Mantel- Cox) test with Bonferroni correction with signicance set at p<0.016. (B)Comparison of survival in

naïve and BATF3

−/−

pyroptosis exposed mice after adoptive cell transfer of CD8

cells. P values were determined using log-

rank (Mantel- Cox) test with signicance set at p<0.05. (C)Comparison of survival in naïve or pyroptosis exposed mice with or

without CD4 (N=7), CD8 (n=8), or CD4 and CD8 (N=8) T cell depletion. P values were calculated using log- rank (Mantel- Cox)

test with Bonferroni correction with signicance set at p<0.0033. *** p<0.0001; **** p<0.00001. DC, dendritic cell.

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Open access

as previously described.

MC- 38- gp33 and KP- 1.9 were

transfected using a lipofectamine 3000 (Invitrogen) and

the three vector constructs PB- TET- N

term

Gas or PB- TET-

dd- C

term

Cas8 (response plasmid with cell death protein

of interest), PB- CAG- rtTA- Neo (expresses regulatory

protein, rtTA), and pCyL43 (plasmid that encodes for

transposase) in a ratio of 10:5:2, respectively. Trans-

fected cells were selected using neomycin and clones

were screened for inducible expression of procell death

proteins. Cell death activating MC- 38 cell lines were

used to express ZsGreen- SIINFEKL, a fusion protein of

the fluorescent protein ZsGreen and SIINFEKL epitope.

The expression of ZsGreen- SIINFEKL was normalized

between MC- 38- dd- C

term

Cas8 and MC- 38- N

term

Gas cell

lines by selection of subclones with equivalent geometric

mean fluorescent intensities (online supplemental file

15).

Animal experiments

Female C57/Bl6 mice aged 6–8 weeks old (Charles

Rivers Laboratories) were subcutaneously implanted

with 2 million tumor cells as described previously.

13–15

For therapeutic settings, chimeric tumors were estab-

lished by injecting 1×10

parental MC- 38- gp33 or

KP- 1.9 cells together with 1×10

MC- 38- gp33 or KP- 1.9

cells with an inducible cell death cassette. 12–14 days

after implantation, tumors achieved a treatable tumor

volume (80–150 mm

). Prior to start of treatment mice

were randomized among groups to ensure an equal

tumor volume across treatments and controls. For all

studies requiring inducible expression of procell death

proteins mice were fed 625 mg/kg Dox- medicated diet

(Envigo Teklad, Madison USA). In prophylactic experi-

ments, mice were rechallenged with 2×10

MC- 38- gp33

cells injected subcutaneously on the contralateral flank

after eradication of the primary tumor. Batf3

−/−

mice

were purchased from The Jackson Laboratory. For T cell

depletion studies, mice were injected intraperitoneally

on day 0, 3, and 7 with either isotype or 250 mg anti- CD4

(clone GK1.5, BioXcell) and anti- CD8 (Clone 2.43, BioX-

cell). For all experiment groups, tumors were measured

every 3–4 days with tumor volumes of 1000 mm

classified

as endpoint.

Quantication of cell viability

Intracellular ATP was measured using the CellTiter- Glo

kit (Promega) as a surrogate marker for cell viability.

At stated time point, cells were lysed then following the

manufacturer’s instructions and luminescence reading

measured using the Enspire multimode plate reader

(PerkinElmer).

In vitro quantication of extracellular HMGB1, ATP, cytokines,

and chemokines

Cell- free supernatant from dying and control cells were

harvested after centrifugation of the media at 2000 rpm

for 10 min. All samples were immediately frozen at −80°C.

Extracellular HMGB1 was quantified using an ELISA kit

developed by Shino Test (IBL International). The ELISA

was conducted according to the manufacturer’s instruc-

tions. For extracellular ATP analysis, supernatants were

analyzed using an ENLITEN ATP assay kit (Promega)

according to manufacturer’s instructions. Cell- free super-

natants were shipped to Eve Technologies for 44- Plex

murine cytokine/chemokine analysis.

Quantication of surface calreticulin expression

Expression of surface calreticulin after apoptosis or pyro-

ptosis was quantified using flow cytometry. Briefly, cells

were seeded at a density of 150,000 cells/well in a 6- well

plate and allowed to adhere for 16 hours before treatment

with Dox for stated time points. Cells were then collected

and washed with PBS before staining with Fixable Viability

Stain 510 and anti- calreticulin- PE (EPR3924, 1:5000).

Data were acquired on BD FACS Canto II and analyzed

in FlowJo.

Quantication of cytokines, chemokines, and cell death

proteins in tumor homogenates

Tumors were resected from mice and diced into small

pieces before being homogenized in tissue extraction

solution (50 mM Tris, pH 7.4, 250 mM NaCl, 5 mM EDTA,

2 mM Na

, 1 mM NaF, 20 mM Na

, 1 mM b- glyc-

erophosphate, 1% NP- 40). Tumors were incubated for

30 min on ice and debris was clarified by three sequential

steps of centrifugation at 14,000×rpm for 10 min at 4°C.

Tumor homogenates with equal concentration of protein

were shipped to Eve Technologies (Calgary, Canada) for

44- Plex murine cytokine/chemokine analysis.

Isolation of immune cells

Tumors were resected and weighed before being placed

into RPMI containing Liberase TL Research Grade

(250 µg/mL; Roche) and DNase I (1 mg/mL; Sigma-

Aldrich). Tumors were minced with scissors and incu-

bated at 37°C for 35 min for enzymatic digestion. Tumor

pieces were subsequently crushed against a 70 µm filter

to generate a single cell suspension. Cells were washed

with PBS containing 2% FBS and 1 mM EDTA. Leucocytes

were isolated from each respective single cell suspension

using a EasySep Release Mouse Biotin Positive Selection

Kit (Stem Cell Technologies) in conjunction with an

anti- CD45.2- Biotin (Clone 104) according to the manu-

facturer’s guidelines. Tumor draining lymph nodes were

harvested and pressed against a 70 µm filter and rinsed

with Hanks Balanced Salt Solution. Blood was collected

from the retro- orbital sinus and red blood cells were lysed

using ACK buffer (150 mM NH

Cl, 10 mM KHCO

, and

0.1 mM Na

EDTA, pH 7.3).

Surface and intracellular staining of immune cells

Leucocytes were incubated with Fc block (anti- CD16/anti-

CD32) and stained with fluorescently labeled antibodies

and Fixable Viability Stain 510 (BD Biosciences). The

following antibodies were obtained from BD Biosciences:

anti- CD11c- APC- Cy7 (HL3, 1:200), anti- Ly6C- RB545

(AL- 21, 1:100), anti- CD40- BV711 (3/23, 1:100),

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anti- CD80- BV650 (16–10 A1,1:100), anti- CD86- PE- CF594

(GL1, 1:100), anti- I- A

- PE (AF6- 120, 1:100), anti- CD103-

PE- Cy7 (M290, 1:100), anti- CD64- BV786 (X54- 5/71,

1:100), anti- CD3- FITC (17A2,1:200), anti- CD8- APC- Cy7

(53–6.7, 1:100), anti- CD44- PE (SB/199, 1:100), anti-

CD69- APC (H1.2F3, 1:100), anti- CD4- APC- Cy7 (RM4- 5,

1:200), anti- IFN-γ-APC (XMG1.2, 1:100), anti- IL- 2- PE

(JES6- 5H4, 1:100), and anti- TNF-α-BV421 (MP6- XT22,

1:100). The following antibodies were from eBioscience:

anti- FOXP3- PE (FJK- 16s, 1:100), anti- CD11b- Pacific Blue

(M1/70, 1:100), and OVA257- 264 (SIINFEKL) peptide

bound to H- 2Kb Monoclonal Antibody (eBio25- D1.16

(25- D1.16))- PE. Anti- XCR1- BV421 (ZET, 1:100) was

purchased from Biolegend. For ICS analysis, leuco-

cytes were stimulated with gp33 (KAVYNFATM), Rpl18

(KILTFDRL), and Adgpk (ASMTNMELM) peptides

(Biomer Technologies) (1 µg/mL per peptide) for

4 hours at 37°C. GolgiPlug (BD Biosciences) was added

for the last 3 hours of incubation. Cells were stained as

above in addition fixation and permeabilization using

Cytofix/Cytoperm solution (BD Biosciences). To obtain

absolute counts of cells, CountBright Absolute Counting

Beads (ThermoFisher Scientific) were added to samples

following manufacturer’s instructions. Other reagents

included the Tetramer H- 2D(b)- LCMV- gp33- 41- PE from

the National Institutes of Health Core facility. Data were

acquired on a BD FACSCanto II and a Cytek Northern

Lights with downstream analysis completed using FlowJo

software.

In vitro cytotoxicity assay

MC- 38- gp33 cells were labeled with 3 µM carboxyfluo-

rescein succinimidyl ester (CFSE; BD Biosciences) and

seeded in a 96- well plate at 10,000 cells per well and

cocultured with CD8

T cells from the spleen for 10

hours. CD8

T cells were stained with fixable viability

stain 510 (BD Biosciences) and evaluated by flow cytom-

etry. Per cent specific lysis was defined previously as: %

specific lysis=100×(%specific cell death−% basal cell

death)/100−%basal cell death, where basal cell death

is the viability of MC- 38- gp33 cells without T cells and

specific lysis is the viability of CSFE positive MC- 38- gp33

cells.

Immunoblotting

Cells were lysed using radioimmunoprecipitation buffer

(10 mM phosphate, 137 mM NaCl, 1% NP- 40m 0.5%

sodium deoxycholate and 0.1% sodium dodecyl sulfate)

supplemented with phosphatase inhibitor cocktail

(1.2 mM AEBSF, 13.6 µM bestatin 12.3 µM E- 64 112 µM

leupeptin1.16 µM pepstatin) and protease inhibitor

cocktail (Millipore Sigma, Massachusetts, USA). For

measuring HMGB1 cell- free supernatants were used.

Total protein extracted from cells or tumor homogenates

and cell- free supernatants were resolved using 7.5%–

15% sodium dodecyl sulfate polyacrylamide gel electro-

phoresis and transferred to nitrocellulose membranes.

Membranes were blocked in Intercept

(TBS) Blocking

Buffer (Li- Cor Biosciences) followed by incubation

with primary antibodies. The following antibodies were

purchased from Cell Signalling Technologies: anti-

Gasdermin E (Cat#40618, 1:3000), anti-β-actin (Cat#4967,

1:6000), and anti- Cleaved- Caspase- 3 (Cat#9661, 1:3000).

The following antibodies were purchased from Abcam:

anti- Caspase- 8 (Cat#ab25901, 1:3000). Membranes were

then probed with secondary antibody conjugated to an

infrared dye (IRDye 800CW Donkey anti- Rabbit IgG,

1:6000) and analyzed using an Odyssey DLx scanner (Li-

Cor Biosciences).

Immunohistochemistry

Treated and control tumors were excised from eutha-

nized mice and fixed in 10% formalin for 48 hours and

then transferred to 70% ethanol before being embedded

into paraffin blocks. Five µm thick tumor sections were

deparaffinized prior to incubation in citrate buffer at

95°C for 15 min. Tumor sections were blocked with Inter-

cept

(TBS) Blocking buffer (Li- Cor Biosciences) before

addition of primary antibody. Anti- Gasdermin E (Cell

Signalling Technologies, 40618) was diluted in Inter-

cept Blocking Buffer with 0.2% Tween- 20 and used at a

working dilution of 1:500. Primary antibody was incubated

overnight in a humidified chamber at 4°C in the dark.

Signal amplification was completed using an anti- rabbit

secondary antibody conjugated to Alexa- 488 (Abcam,

ab150077, 1:10,000). Secondary antibody incubation was

performed at room temperature in a humidified chamber

in the dark for 2 hours. Sections were counterstained with

diluted DAPI (Abcam) for 5 min at room temperature

in the dark. Slides were rinsed with MiliQ water before

imaging with the Leica DMLB fluorescent.

Statistics and reproducibility

For each statistical test used, normality of the distribution

and equality of variance between groups was evaluated

beforehand. For differences in means, one- way analysis

of variance (ANOVA), two- way ANOVA, Student’s two-

tailed unpaired t- test, and non- parametric Kruskal- Wallis

test were used. The Holm Šidák method was used to

correct multiple comparisons. For analyzing differences

between experimental groups in Kaplan- Meier survival,

the log- rank (Mantel- Cox) test was used with 525 mm

endpoints. Bar graphs are shown as mean±SD. The null

hypothesis was rejected for p<0.05. All statistical tests and

analyses were carried out using GraphPad Prism V.9.

X Jordon M Inkol @always_jord, Michael J Westerveld @MikeWesterveld1 and

Samuel T Workenhe @samworkenhe

Acknowledgements We thank the University of Guelph Centralized and Isolation

Animal Facility staff for help with animal studies.

Contributors JMI and STW conducted most of the experiments presented in

the paper together with MJW, SGV, SRW, SW, KK, NP and JM. MJW and SRW

helped with in vivo experiments involving immune cell analysis. SGV, SW, KK and

NP conducted ATP, HMGB1 and calreticulin quantication and immunoblotting

experiments. JM helped with the tissue section and staining studies. KM nancially

supported the preliminary in vitro experiments of this study when STW was a

postdoctoral fellow in her lab. RB, YW and JB provided p14 splenocytes. JMI and

on August 2, 2024 by guest. Protected by copyright.http://jitc.bmj.com/J Immunother Cancer: first published as 10.1136/jitc-2023-006781 on 4 April 2024. Downloaded from

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STW prepared the gures and wrote the manuscript. STW conceived, supervised,

and is the guarantor for the study.

Funding STW is funded by Canadian Institute of Health Research (PJT 185868 and

PJT 190071), SickKids New Investigator award (NI23- 1064R), Cancer Research

Society (ID# 942502 and 1056603), Ontario Institute for Cancer Research funding

from The Joseph and Wolf Lebovic Cancer Genomics and Immunity Program.

Competing interests None declared.

Patient consent for publication Not applicable.

Ethics approval Mice were maintained at the University of Guelph Isolation Facility

and all procedures were completed in compliance with the Canadian Council on

Animal Care and approved by the University of Guelph Animal Care Committee

(Animal Utilization Protocol (AUP) # 4487).

Provenance and peer review Not commissioned; externally peer reviewed.

Data availability statement Data sharing not applicable as no datasets generated

and/or analyzed for this study.

Supplemental material This content has been supplied by the author(s). It has

not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been

peer- reviewed. Any opinions or recommendations discussed are solely those

of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and

responsibility arising from any reliance placed on the content. Where the content

includes any translated material, BMJ does not warrant the accuracy and reliability

of the translations (including but not limited to local regulations, clinical guidelines,

terminology, drug names and drug dosages), and is not responsible for any error

and/or omissions arising from translation and adaptation or otherwise.

Open access This is an open access article distributed in accordance with the

Creative Commons Attribution Non Commercial (CC BY- NC 4.0) license, which

permits others to distribute, remix, adapt, build upon this work non- commercially,

and license their derivative works on different terms, provided the original work is

properly cited, appropriate credit is given, any changes made indicated, and the use

is non- commercial. See http://creativecommons.org/licenses/by-nc/4.0/.

ORCID iDs

JonathanBramson http://orcid.org/0000-0003-2874-6886

Samuel TWorkenhe http://orcid.org/0000-0001-9521-3903

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