CAR T cell–induced cytokine release syndrome is mediated by macrophages and abated by IL-1 blockade 2018


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, Sjoukje J. C. van der Stegen
, Justin Eyquem
Alessandra Piersigilli
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|
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Pre-CAR
23
42
66
90
95
100
04
50
100
concentration (pg/ml
mCCL
concentration (pg/ml
mG-CSF
concentration (pg/ml)
hGM-CS
concentration (pg/ml
hIL-
concentration (pg/ml
mCXCL9
concentration (pg/ml
50,000
100,000
150,000
200,000
IL-6
concentration (pg/ml
GM-CSF
concentration (pg/ml
IL-3
concentration (pg/ml
hIFN
concentration (pg/ml
2,000
4,000
6,000
8,000
10,000
2,000
4,000
6,000
8,000
500
1,000
1,500
2,000
04
06
0
50
100
200
400
600
5,000
10,000
15,000
2,000
4,000
6,000
8,000
10,000
500
1,000
1,500
2,000
2,000
4,000
6,000
8,000
10,000
5,000
10,000
15,000
20,000
1,000
2,000
3,000
+ CAR
CAR only
Tumor only
No Tumor
No CAR
20
40
60
concentration
0.001
0.001
0.045
0.018
0.001
0.001
0.001
0.005
0.001
0.009
0.017
0.001
0.001
0.010
0.001
0.029
0.012
0.001
0.001
3 weeks
b
e
Fig. 1
A mouse model of CRS recapitulates clinical CAR T cellinduced CRS.
, Schematic of the mouse model. Raji tumor cells were intraperitoneally
injected in mice and allowed to grow for 3 weeks, and they eventually grew into vascularized solid tumor masses. Thirty million CAR T cells were
transferred, and mice were monitored over the days following transfer. Mice were euthanized, and cells were obtained for analysis through peritoneal
lavage and tissue harvesting.
, Weight change of tumor-bearing mice after 1928z CAR T cell transfer. Weight per mouse is normalized to starting weight
before CAR transfer (Tumor only,
12 mice; Tumor
CAR,
18 mice). At least 20 independent experiments monitoring weight under CRS-eliciting
conditions were performed. A two-way ANOVA was used for statistical analysis.
, Percentage of survival of mice after 1928z CAR T cell transfer (Tumor
only,
12 mice, Tumor
CAR,
18 mice). At least 20 independent experiments monitoring survival under CRS-eliciting conditions were performed.
A log-rank MantelCox test was used for statistical analysis.
, Serum levels of mSAA3 at 42h after 1928z CAR T cell transfer as measured using ELISA
No CAR,
5 mice; Tumor only,
5 mice; CAR only,
5 mice; Tumor
CAR,
7 mice). SAA3 levels under CRS-eliciting conditions were
measured in at least in two independent experiments. A two-tailed unpaired two-sample
-test was used for statistical analysis.
, Serum cytokine levels
4.5h before (pre-CAR) and 24h after 1928z CAR T cell transfer. Mice that eventually died from CRS were grouped under Severe CRS, whereas mice that
survived but lost more than 10% of their initial body weight were grouped under CRS (Severe CRS,
5 mice; CRS,
10 mice; pre-CAR,
16 mice).
Stratification of cytokines on the basis of survival was performed in at least three independent experiments. A two-tailed unpaired two-sample
-test was
used for statistical analysis.
, Species of origin of proinflammatory cytokines (human cytokines from
15 mice; mouse cytokines from
15 mice).
, Percentage of survival of tumor-bearing mice treated with 1928z CAR T cells that received anti-mIL-6R blocking antibody or isotype (vehicle) (Vehicle,
6 mice; Anti-mIL-6R,
5 mice). Experiments measuring survival after blocking mIL-6R were performed once. A log-rank MantelCox test was used
for statistical analysis. All data are shown as mean
s.e.m. Numbers on the graphs are
values.
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0.1783
0.1545
0.156
Tumor + CA
IL-6Ra
TNF
G-CSF
M-CSF
IL-12
IL-12
IFN-

IL-6
IL-6Ra
TNF
G-CSF
IL-12

IFN-
IL-6
IL-6Ra
TNF
G-CSF
M-CSF
IL-12
IL-12

IFN-
IL-6
IL-6Ra
TNF
G-CSF
M-CSF
IL-12

IL-12
IFN-
IL-6
IL-6Ra
TNF
G-CSF
M-CSF
IL-12
IL-12

IFN-
IL-6
IL-6Ra
TNF
G-CSF
IL-12

IFN-
IL-6
IL-6Ra
TNF
G-CSF
IL-12
IFN-
IL-6
IL-6Ra
TNF
G-CSF
IL-12
IFN-
10
5
0
5
Fold change
15
10
5
1
2
3
4
5
40
60
15
10
5
1
2
3
4
5
40
60
80
100
1
2
3
4
5
100
150
Peritoneal monocyt
1
2
3
4
5
0
1
0
1
2
4
5
Splenic DCs
10
0
10
15
25
30
DCs
Macrophages
Monocytes
Eosinophils
DCs
Monocytes
Macrophages
Neutrophils ( 10
Eosinophils
DCs
Monocytes
Neutrophils ( 10
Eosinophils
DCs
Macrophages
6 10
4 10
8 10
0.6502
0.0697
Tumor + CAR
0.001
0.001
0.024
0.008
0.001
0.005
0.001
0.013
0.003
0.005
0.001
0.001
0.048
0.005
0.001
0.023
0.014
0.001
0.001
0.8771
0.5414
0.013
0.001
0.001
0.001
d
e
f
b
c
0.001
0.001
0.001
2.5 10
0.001
0.001
10
0
10
Fig. 2
TumorCAR T cell interactions trigger myeloid cell recruitment and activation.
, Immunohistochemical staining for MAC2 in sections from
3-week Raji tumor explants. Mice had not received CAR T cells. Immunohistochemical staining of tumor explants for MAC2 was performed once.
, Absolute counts of myeloid cell populations obtained by peritoneal lavage 68h after 1928z CAR T cell transfer (No tumor
No CAR,
5 mice; CAR
only,
5 mice; Tumor only,
6 mice; Tumor
CAR,
7 mice). Enumeration of myeloid cells under these conditions was performed at least 10 times.
A two-tailed unpaired two-sample
-test was used for statistical analysis.
from peritoneal lavage. Analysis of peritoneal macrophages for levels of F4/80 and Ly6C was performed at least 10 times.
, Absolute counts of myeloid
cell populations obtained from multiple organs 18h after 1928z CAR T cell transfer (Tumor only,
4 mice; Tumor
CAR
4 mice). Enumeration of cells
9 Tumor only mice
9 Tumor
CAR mice. Cells from three mice of the same group were randomly pooled to create one biological replicate group for the purpose of
obtaining three biological replicate groups of Tumor only mice and three biological replicate groups of Tumor
CAR mice. A binomial test was used for
statistical analysis, and
values were adjusted for false discovery rate (FDR). All data are shown as mean
s.e.m. except for that in
, in which data are
shown as mean
s.d. Numbers on the graphs are
values.
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cells in the presence of tumor was toxicity observed (Supplementary
Figs. 1a and 4a), concurrent with a brisk accumulation of peritoneal
neutrophils, eosinophils, dendritic cells (DCs), monocytes (Fig.
and Supplementary Fig. 5a) and F4/80
int-lo
Ly6C
int-hi
macrophages
(hereafter referred to as CRS-associated macrophages), which dif
fer from the typical F4/80
Ly6C
neg-lo
resident peritoneal phenotype
(Fig.
). The rapid elevation of myeloid cell numbers, which was
already noticeable 18
h after CAR T cell administration (Fig.
suggested that recruitment was a major contributor to myeloid
accumulation. RNA-seq analyses at 18
h showed increased expres
sion of genes promoting the cell cycle, suggesting that cell prolifera
tion may play a secondary role in local myeloid cell accumulation.
matched the therapeutic efficacy of control 1928z-LNGFR CAR
T cells (Fig.
and Supplementary Fig. 8c,d). Therefore, we not only
16
41
65
89
113
137
161
85
90
95
100
1928-mCD40L
mG-CSF
concentration (pg/ml)
mCXCL1
concentration (pg/ml)
mIL-6
concentration (pg/ml)
16.5
40
61
90
95
100
fg
h
d
% weight change
0
100
150
50
100
0
100
50
100
20
40
80
100
macrophages
2,000
4,000
6,000
1928z-LNGFR
1928z-mCD40L
Tumor only
1928z-LNGFR
1928z-mCD40L
Tumor only
1928z-LNGFR
1928z-mCD40L
Tumor only
1928z-LNGFR
1928z-mCD40L
Tumor only
1928z-LNGFR
1928z-mCD40L
200
400
600
800
20,000
40,000
60,000
80,000
100,000
2,000
4,000
6,000
0.009
Peritoneal DCs
Splenic macrophages
BM Ly6C
BM Ly6C
20
40
60
80
0.001
0.001
0.001
0.001
0
10
10
Ly6C-AF647
Fig. 3
8 Tumor only mice,
7 1928z-LNGFR
mice and
5 1928z-mCD40L
5 mice. The experiment was performed once.
, Percentage of CD40
total peritoneal macrophages, obtained by
04
06
08
01
04
06
08
0
100
50
100
50
100
0.5 10
0.5 10
Tumor only
0
100
150
concentration (pg/ml
1928z
LNGF
1928z
IL-1Ra
IL-1

IL-1R1
IL-1R2
IL-1RN
IL-1

IL-1
IL-1R1
IL-1R2
IL-1RN
IL-1
IL-1
IL-1R1
IL-1R2
IL-1RN
IL-1
IL-1

IL-1R1
IL-1R2
IL-1RN
IL-1
IL-1
IL-1R1
IL-1R2
IL-1RN
IL-1

IL-1
IL-1R1
IL-1R2
IL-1RN
IL-1
IL-1
IL-1R1
IL-1R2
IL-1RN
IL-1
IL-1

IL-1R1
IL-1R2
IL-1RN
0
5
10
15
c
g
h
de
f
Fold change
10
0
10
20
30
40
50
60
20
10
0
10
10
20
30
40
50
60
2
4
6
8
10
100
150
Peritoneal monocytes
5
10
15
20
25
0
5
10
15
20
25
0
5
10
15
25
Splenic DCs
10
0
10
20
30
40
50
60
Splenic Ly6C
Splenic Ly6C
04
06
08
0
100
50
100
Time (h)
hIL-2
concentration (pg/ml
hIL-3
concentration (pg/ml
50
100
50,000
100,000
150,000
Peritoneal CRS macrophages
10
Anti-mIL-6
Anakinra
Anti-mIL-6 + Anakinra
Tumor only
10
20
40
2,000
4,000
6,000
8,000
10,000
500
1,000
1,500
5,000
10,000
15,000
Fig. 4
IL-1Ra protects from severe CRS without compromising antitumor efficacy.
, Fold change of IL-1 signaling component gene expression in myeloid
9 Tumor only mice and
9 Tumor
CAR mice. Cells from three mice of the same group were randomly pooled to create one biological
replicate group to the end of obtaining three biological replicate groups of Tumor only mice and three biological replicate groups of Tumor
CAR mice. A binomial
test was used for statistical analysis, and
values were adjusted for FDR.
, Percentage of survival of tumor-bearing mice after 1928z CAR T cell transfer receiving
anakinra or vehicle (PBS) (Anakinra,
11 mice; Vehicle,
10 mice). Results were pooled from two independent experiments. A log-rank MantelCox test was
used for statistical analysis.
, Percentage of peritoneal macrophages expressing iNOS at 18h following CAR T cell transfer. Mice were treated with isotype, mIL-6-
blocking antibody, anakinra or mIL-6-blocking antibody
anakinra (Tumor only,
4 mice; Isotype,
3 mice; Anti-mIL-6,
3 mice; Anakinra,
3 mice; Anti-
mIL-6
Anakinra,
4 mice). Measurement of the percentage of iNOS
macrophages with isotype or anti-mIL-6 blockade was performed in two independent
experiments. Measurement of the percentage of iNOS
macrophages with isotype, anakinra or anakinra
anti-mIL-6-blocking antibody was performed once.
A one-way ANOVA was used for statistical analysis.
, Levels of mIL-1Ra in supernatants of 1928z-LNGFR and 1928z mIL1Ra transduced CAR T cells after 48h
IL-6 and iNOS. The aggravation of CRS by CAR T cells express
ing mCD40L demonstrates that CAR T cells and macrophages
can functionally interact within the tumor microenvironment.
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35.
References
Corresponding author(s):Michel Sadelain
ll accepted life
science papers and provides structure for consistency and transparency in reporting. Every life science submission will use thi
s form; some list

Describe any data exclusions.Tumour burden was evaluated by in vivo bioluminescent imaging two days prior to
Tumour burden was evaluated by in vivo bioluminescent imaging two days prior to
CAR T cell transfer. Outlier mice with extreme burdens (either too high or too low
W
values) given as exact values whenever possible and with confidence intervals noted
uartile range)
]](}]}o}P]
(}(Z}vP]vX

availability of computer code
study.
GraphPad Prism v7, FlowJo X, BD FCAP Array v3, Microsoft Excel for Mac 2011,
Living Image v4.4. RNAseq analysis was performed with the following software

availability of materials
Describe the antibodies used and how they were validated
NALM-6 from ATCC, Raji from ATCC.
of commonly misidentified cell lines maintained by
, provide a scientific rationale for their use.
No cell lines from the ICLAC list were used.

studies involving animals
; when reporting animal research, follow the
ARRIVE guidelines
characteristics of the human research participants.
Buffy coats from anonymous healthy donors were purchased from the New York
Blood Center. The researchers were blind to any covariate characteristics.

s of
identical markers).

were immediately injected intraperitoneally with 5ml ice cold PBS/2mM EDTA.
populations within post-sort fractions.

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