PRMT5 regulates T cell interferon response and is a target for acute graft-versus-host disease Katiri Snyder, … , Robert A. Baiocchi, Parvathi Ranganathan JCI Insight. 2020. https://doi.org/10.1172/jci.insight.131099. In-Press Preview Acute Graft-Versus-Host Disease (aGVHD) is a T cell mediated immunological disorder and the leading cause of non- relapse mortality in patients who receive allogeneic hematopoietic cell transplants. Based on recent observations that PRMT5 and arginine methylation is upregulated in activated memory T cells, we hypothesized that PRMT5 is involved in the pathogenesis of aGVHD. Here, we show that PRMT5 expression and enzymatic activity is upregulated in activated T cells in vitro and in T cells from mice developing aGVHD after allogeneic transplant. PRMT5 expression is also upregulated in T cells of patients who developed aGVHD after allogeneic hematopoietic cell transplant compared to those who did not develop aGVHD.PRMT5 inhibition using a selective small-molecule inhibitor (C220) significantly reduces mouse and human allogeneic T cell proliferation and inflammatory IFN-γ and IL-17 cytokine production. Administration of PRMT5 small-molecule inhibitors significantly improves survival, reducing disease incidence and clinical severity in mouse models of aGVHD without adversely affecting engraftment. Importantly, we show that PRMT5 inhibition retains the beneficial graft versus leukemia (GVL) effect by maintaining cytotoxic CD8 T cell responses. Mechanistically, we show that PRMT5 inhibition potently reduces STAT-1 phosphorylation as well as transcription of pro-inflammatory genes including Interferon Stimulated Genes (ISG) and IL-17. Additionally, PRMT5 inhibition deregulates cell-cycle in activated T cells and disrupts signaling by impacting ERK1/2 phosphorylation. Thus, we have identified PRMT5 as […] Research Immunology Transplantation Find the latest version: https://jci.me/131099/pdf
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PRMT5 regulates T cell interferon response and is a target foracute graft-versus-host disease
Katiri Snyder, … , Robert A. Baiocchi, Parvathi Ranganathan
Acute Graft-Versus-Host Disease (aGVHD) is a T cell mediated immunological disorder and the leading cause of non-relapse mortality in patients who receive allogeneic hematopoietic cell transplants. Based on recent observations thatPRMT5 and arginine methylation is upregulated in activated memory T cells, we hypothesized that PRMT5 is involved inthe pathogenesis of aGVHD. Here, we show that PRMT5 expression and enzymatic activity is upregulated in activated Tcells in vitro and in T cells from mice developing aGVHD after allogeneic transplant. PRMT5 expression is alsoupregulated in T cells of patients who developed aGVHD after allogeneic hematopoietic cell transplant compared to thosewho did not develop aGVHD.PRMT5 inhibition using a selective small-molecule inhibitor (C220) significantly reducesmouse and human allogeneic T cell proliferation and inflammatory IFN-γ and IL-17 cytokine production. Administration ofPRMT5 small-molecule inhibitors significantly improves survival, reducing disease incidence and clinical severity inmouse models of aGVHD without adversely affecting engraftment. Importantly, we show that PRMT5 inhibition retains thebeneficial graft versus leukemia (GVL) effect by maintaining cytotoxic CD8 T cell responses. Mechanistically, we showthat PRMT5 inhibition potently reduces STAT-1 phosphorylation as well as transcription of pro-inflammatory genesincluding Interferon Stimulated Genes (ISG) and IL-17. Additionally, PRMT5 inhibition deregulates cell-cycle in activatedT cells and disrupts signaling by impacting ERK1/2 phosphorylation. Thus, we have identified PRMT5 as […]
PRMT5 regulates T cell interferon response and is a target for acute graft-versus-host 1disease. 2 31Katiri J. Snyder, 1Nina C. Zitzer, 1Yandi Gao, 1Hannah K. Choe, 1Natalie E. Sell, 1Lotus 4Neidemire-Colley, 1Anora Ignaci, 1Charuta Kale, 1Raymond D. Devine, 2Maria G. Abad, 3Maciej 5Pietrzak, 4Min Wang, 4Hong Lin, 4Yang W. Zhang, 1Gregory K. Behbehani, 2Jane E. Jackman, 61Ramiro Garzon, 4Kris Vaddi, 1Robert A. Baiocchi and 1Parvathi Ranganathan 7 8 91Division of Hematology, Department of Internal Medicine, Comprehensive Cancer Center, 10
2Department of Chemistry and Biochemistry, 3Department of Biomedical Informatics, The Ohio 11
State University, Columbus, OH. 4Prelude Therapeutics, Wilmington, DE.12
intravenously into F1 recipients on day 0 along with TCD-BM (10x106 cells) and B6 donor 557
splenocytes (20 x 106 cells). Treatment groups included vehicle and PRMT5 inhibitor C220 558
2mg/kg, administered by oral gavage once a week starting day +7 post-transplant. TCD-BM and 559
P815 cells (leukemia alone) served as the control group. P815-induced leukemic death was 560
defined by the occurrence of either macroscopic tumor nodules in liver and/or spleen or hind-leg 561
paralysis. GVHD death was defined by the absence of leukemia and the presence of clinical and 562
histo-pathological signs of GVHD. 563
564
In vivo imaging 565
Xenogen IVIS imaging system (Caliper Life Sciences) was used for live animal imaging. Mice 566
were anesthetized using 1.5% isofluorane (Piramal Healthcare). XenoLight RediJect D-Luciferin 567
Ultra Bioluminescent Substrate (150 mg/kg body weight; 30 mg/mL in PBS; Perkin Elmer) was 568
injected intraperitoneally and IVIS imaging was performed 10 minutes after substrate injection. 569
Whole body bioluminescent signal intensity was determined weekly using IVIS Living Image 570
software v4.3.1 (Caliper Life Sciences), and pseudocolor images overlaid on conventional 571
photographs are shown. Data were analyzed and presented as photon counts per area. 572
573
Degranulation assay 574
CD8 degranulation assay was measured by CD107a mobilization and experiments were set up 575
based on previously published protocols (40, 98). P815 cells were incubated at 37°C overnight. 576
On the next day, the culture supernatant was removed from the wells, and effector cells were 577
added to the wells. Effector T cells: Naive CD8 T cells isolated from BoyJ CD45.1+ splenocytes 578
by negative selection (StemCell Technologies) were used as effector T cells: (a) CD8+ T cells 579
without any treatment (CD8-resting), (b) CD8+ T cells stimulated with allogeneic BMDCs in the 580
presence of DMSO or (c) C220 (100nM) for 5 days. CD8+ T cells were then mixed with P815 581
cells at effector/target (E/T) ratio (5:1), centrifuged at 1000 RPM for 3 minutes, and co-cultured in 582
25
the presence of anti-CD107a at 37°C overnight. On the next day, the cells were stained with 583
surface markers CD45.1 and CD8, and analyzed by flow cytometry. 584
585
Statistical analysis 586
Survival data were analyzed using Kaplan-Meier and log-rank test methods. Differences between 587
continuous variables at a single time point were analyzed using two-tailed t-tests. Data represent 588
mean ± SD. One-way analysis of variance (ANOVA) with Holm-Sidak post-hoc test was used for 589
comparisons >2 groups unless indicated otherwise. Differences between two groups over time 590
analyzed with Mann-Whitney U. All analyses were performed using GraphPad Prism 7.0. *, 591
p<0.05; **, p<0.01; ***, p<0.001. A P-value less than 0.05 was considered significant. 592
593
Study Approval 594
All animal studies were conducted in accordance with the rules and regulations of the Institutional 595
Animal Care and Use Committee at The Ohio State University. Peripheral blood samples from 596
healthy donors and allogeneic transplant patients were obtained following written informed 597
consent in accordance with the Declaration of Helsinki. Healthy PBMCs were obtained from 598
Versiti and transplant patient samples were selected from a biorepository study, both approved 599
by the IRB at The Ohio State University. 600
601
26
AUTHOR CONTRIBUTIONS 602
K.J.S, N.C.Z, Y.G., N.E.S., L.N.C. and A.I. performed in-vivo murine aGVHD experiments, ex-603
vivo FACS analyses, serum/supernatant ELISAs, western blots, real-time PCR analyses, in-vitro 604
experiments with mouse cells and human PBMCs, signaling experiments, analyzed the data and 605
interpreted the results. C.K., Y.G., N.E.S., R.D. performed mass cytometry experiments. G.B. was 606
involved in supervision and discussion of results of the mass cytometry experiments. M.G.A and 607
J.E.J. performed the in vitro methylation experiments. M.P. provided the bioinformatics data. M.W. 608
and Y.W.Z. performed C220 biochemical assay, H.L. synthesized C220. H.K.C discussed 609
experimental design, provided data and edited the manuscript. R.A.B. provided data discussion 610
and edited the manuscript. R.G. and K.V. edited the manuscript. P.R. designed the study, 611
supervised research, interpreted the data and wrote the manuscript along with NCZ. 612
613
ACKNOWLEDGEMENTS 614 615This work was supported by a New Investigator Award from the Leukemia Research Foundation, 616
Pelotonia New Investigator Award, K12 Paul Calabresi Award and Division of Hematology start-617
up funds (P.R.). Research reported in this publication was supported by the Ohio State University 618
Comprehensive Cancer Center and the National Institutes of Health under grant number P30 619
CA016058. We thank the Target Validation Shared Resource (TVSR) at the Ohio State University 620
Comprehensive Cancer Center for providing the NSG mice used in the preclinical studies 621
described herein. The content is solely the responsibility of the authors and does not necessarily 622
represent the official views of the National Institute of Health. 623
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FIGURES and FIGURE LEGENDS 870
871
Figure 1. PRMT5 expression and activity is upregulated in activated mouse and human T 872cells in vitro and in vivo. Murine B6 T cells were stimulated with either CD3/CD28 Dynabeads 873or with allogeneic BALB/c DCs for 4 days. (A, B) PRMT5 mRNA expression in T cells quantified 874by real-time PCR (n=5). Expression relative to unstimulated (US) T cells, b-actin used as a 875normalizer. Results are represented as mean ± S.D. of 2-3 independent experiments. Each 876symbol represents an individual donor. (C) Prmt5 protein and Histone H3R8 symmetric dimethyl 877protein (H3R8me2s) analyzed by western blot. One representative western blot of three 878independent experiments is shown. (D) T cells isolated from healthy human donor (HD) PBMCs 879were stimulated with CD3/CD28 Dynabeads and PRMT5 mRNA expression (n=3 donors) 880quantified by real-time PCR (E) immunoblotted for PRMT5 protein expression and function (n=2 881donors). (F) In vitro PRMT5 methyltransferase activity in nuclear extracts derived from US vs. 882CD3/CD28 stimulated human T cells. Results show specific activity measured in ng dimethylated 883H4-Arg3 produced per 20 µg nuclear extract. Each symbol represents an individual replicate 884measurement, with mean ± S.D. One representative assay of 3 independent experiments is 885shown. (G) Splenic T cells isolated from lethally irradiated B6D2F1 mice that received TCD-BM 886or TCD-BM+ B6 allogeneic splenocytes around 25 days post-transplant. Messenger RNA 887expression (n=9) and (H) and protein expression. 2-3 spleens were pooled from recipient mice to 888make one pooled sample and three pooled samples were analyzed for PRMT5 protein expression 889by western blot. (I) Healthy human PBMCs were collected from healthy donors (HD PBMCs, n=7) 890and from patients after allogeneic BMT with or without GVHD at the time of collection 891(Supplemental Table 2). Patient samples were matched for day of transplant at which GVHD 892occurred. PBMCs were stained and analyzed for PRMT5 expression on T cells using mass 893cytometry; the gating strategy is shown in Supplemental Figure 8. Each symbol represents an 894individual donor. Data represent mean ± S.D. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. 895
D
Human Donor 1 CD3/CD28 (hrs)
US 24 48 72
Human Donor 2 CD3/CD28 (hrs)
US 24 48 72
E
Figure 1
B
Mouse T cells CD3/CD28 (hrs)
US
H3R8me2s
β actin
PRMT5
24 48 72 US Allo stim
Mouse T cells
Human T cells
H3R8me2s
β actin
PRMT5
C
H3R8me2s
TCD-BM + allo splN=3
TCD-BMN=3
β actin
PRMT5
72KDa
20 KDa
42 KDa
Mouse T cellsG H Human T cellsI
Mouse T cells Mouse T cellsA
FHuman T cells
US CD3/CD28 0.10
0.15
0.20
0.25
Met
hyla
ted
H4
(ng)
***
TCD-BM TCD-BM + allo spl
0
1
2
3
4
5 ****
No disease
aGVHD
Fold
cha
nge
Prmt5
mR
NA
US CD3/CD28 0
1
2
3
4**
Fold
cha
nge
PRMT5
mR
NA
US CD3/CD28 0
1
2
3
4
5 **
Fol
d ch
ange
Prmt5
mR
NA
US Alloactivated0.0
0.5
1.0
1.5
2.0
2.5 ***
HD PBMCs non-GVHD GVHD0
10
20
30
40
PRM
T5 e
xpre
ssio
n (R
aw m
edia
n va
lues
)NS
**
33
896 897Figure 2. Inhibition of PRMT5 blunts mouse and human T cell alloreactive response. Cell 898trace violet (CTV) labeled B6 T cells were stimulated with allogeneic BALB/c BMDCs for 4 days 899in the presence of increasing concentrations of PRMT5 inhibitor C220 (A) Histogram plots of 900one representative donor. (B) Percent alloreactive T cell proliferation normalized to no drug. (C) 901IFN-g cytokine in supernatant analyzed by ELISA. B6 T cells were stimulated with BALB/c 902BMDCs for 4 days, T cells re-isolated and immunoblotted for (D) symmterically dimethylated 903arginine residues (SDMA) and (E) H3R8me2s and H3R8me2a. b-actin was used as loading 904control. One representative western blot of three independent experiments is shown. (F) Cell 905trace violet (CTV) labeled CD3+ human T cells were stimulated with allogeneic T cell depleted 906PBMCs for 4 days in the presence of increasing concentrations of C220. Percent alloreactive T 907cell proliferation normalized to no drug. (G) IFN-g cytokine in supernatant analyzed by ELISA. 908Mean ± SD of biological replicates of 3 mouse/human donors shown. *, p<0.05; **, p<0.01; ***, 909p<0.001; ****, p<0.0001 compared to allo-stimulated T cells in absence of C220. 910 911
Undivided T cellsBMDCs+ T cellsBMDCs+ T cells + C220 (nM)
0 0.1nM 1nM 10nM 50nM 100nM
F G
T cellsAllo BMDCs
+
C220
+++ +
+- --
β actin
D
Figure 2
H3R8me2a
H3R8me2s
β actin
E
16 KDa
30 KDa
60 KDap60
SmD
SmB
20 KDa
42 KDa
20 KDa
42 KDa
0nM 0.1nM 1nM 10nM 50nM 100nM0
50
100
BMDCs + T cells + C220 (nM)
CD
3+ T
cel
l pr
olife
ratio
nno
rmal
ized
to 0
nM
Mouse 1Mouse 2
**
********
Mouse 3
0nM 0.1nM 1nM 10nM 50nM 100nM0
50
100
MLR+C220 (nM)
CD
3+ T
cel
l pr
olife
ratio
nno
rmal
ized
to 0
nM
HD1
HD3
HD2
** *
0nM 50nM 100nM0
200
400
600
800
PBMCs MLR + C220 (nM)
IFNγ (
pg/m
l)
**
0nM 50nM 100nM0
400
800
1200
BMDCs + T cells + C220 (nM)
IFN
-γ (p
g/m
l)
***
34
916
Figure 3. Inhibition of PRMT5 improves survival in mouse models of aGVHD. Lethally 917irradiated B6D2F1 recipients received CD45.1+ B6 T cell depleted bone marrow (TCD-BM, 91810x106 cells) or TCD-BM + CD45.1+ B6 splenocytes (15 x 106). Recipients of allogeneic 919splenocytes were treated with C220 (2mg/kg) or vehicle by oral gavage once weekly starting day 9207 post-transplant. (A) Kaplan-Meier survival curve. Log-rank test was used to compare survival. 921Data pooled from 3 independent transplant experiments, with n=6-8 in TCD-BM+ splenocytes 922group in every transplant experiment, TCD-BM group was included for 2 transplants, with n=4 923each transplant. Total n=8 for TCD-BM group, n= 18-24 for TCD-BM +splenocytes groups. (B) 924Clinical GVHD scores. Mann-Whitney U test was used for comparison between groups. B6 into 925F1 transplant was performed as described above, and separate cohort of mice were euthanized 926at day 22 post-transplant (after receiving 3 doses of vehicle/C220 on days 7, 14 and 21) and used 927for (C-E). (C) Histopathological assessment of target tissues (liver and GI), n=5 per group. (D) 928Splenocytes harvested for immunoblotting for pharmacodynamics marker SmD3. (E) 929Densitometry values normalized to b-actin. Data represent mean ± S.D. Each symbol represents 930an individual donor, n=4-5. (F) NSG mice were irradiated with 50 cGy on day -1. On day 0, 931irradiated NSG mice received either T cell depleted- human PBMNCs (TCD-PBMC, 10*106, n=3) 932or human PBMNCs (15*106). Mice that received T cell replete grafts were treated with either 933C220 (2mg/kg, n=12) three times weekly or vehicle (n=10). Survival curve. Log-rank test was 934used to compare survival. (G) Clinical GVHD scores of xenogeneic GVHD model. Data shown is 935combined from 2 independent xeno-aGVHD experiments. *, p<0.05; ****, p<0.0001. 936 937
940 941Figure 4. PRMT5 inhibition reduces donor Th1 and Th17 responses. B6 into F1 transplant 942was performed as described in methods. Mice were sacrificed around day 25 post-transplant, 943(n=5–7 per group) and spleen harvested. (A) Percentage and (B) Absolute numbers of CD45.1+ 944donor cells. (C) Percentage and (D) Absolute numbers of CD45.1+ CD3+ donor T cells. (E) 945Percent CD45.1+ Ki67+ donor CD4+ and CD8+T cells. (F) Representative histograms showing 946intracellular staining for IFN-g, IL-17 and CD25/Foxp3 on donor CD45.1+CD4+ T cells. (G) 947Percent IFN-g +, IL-17+ and CD25/Foxp3+ donor CD45.1+CD4+ T cells. (H) Serum IFN-g 948analyzed by ELISA. Data shown is combined from two independent transplant experiments. Each 949symbol represents an individual mouse. *, p<0.05; **, p<0.01; ***, p<0.001. 950 951
952
953
954
955
Figure 4
Vehicle C220
CD
25
28.0 15.5
7.3116.4
6.34 9.75
3.1180.8
6.70 8.75
2.9981.6
IFN-g
IL-17
Foxp3
CD
4C
D4
A B C
H
F G
D E
Vehicle C2200
5
10
15
20
% d
onor
Fox
p3+
Treg
s
Tregs
NS
Vehicle C2205
10
15
20
% d
onor
CD
4+ IL
-17+
*IL-17
Vehicle C2200
10
20
30
40
% d
onor
CD
4+ IF
N-γ
+
*
IFN-γ
Vehicle C2200
20
40
60
80
100
%C
D45
.1 d
onor
cel
ls
NS
Vehicle C2200
5
10
15
20
25
# C
D45
.1do
nor c
ells
(*10
^6) NS
Vehicle C2200
10
20
30
40
50
% C
D45
.1+
CD
3+ T
cel
ls
***
Vehicle C2200
2
4
6
8
# C
D45
.1+
CD
3+ T
cel
ls (
*10^
6)
*
CD8 CD40
20
40
60
% K
i67+
don
or T
cel
ls
*
**
Vehicle C220 0
100
200
300
IFN
-γ (p
g/m
l)
Serum IFN-γ
*
36
956
957
958
Figure 5. PRMT5 inhibition reduces donor T cell infiltration into liver and GI tract. 959B6 into F1 transplant was performed as described in methods. Mice were sacrificed around day 96025 post-transplant, (n=5–7 per group) and liver (A-J) and small intestine of GI tract (K) harvested. 961Single cell suspensions were made as described in methods. (A) Percentage and (B) Absolute 962numbers of CD45.1+ CD3+ donor T cells in the liver. (C) Percentage and (D) Absolute numbers 963of Foxp3+ donor CD45.1+CD4+ T cells. (E) Representative histograms showing intracellular 964staining, (F) percentage and (G) absolute numbers of donor IFN-g + CD45.1+CD4+ T cells in the 965liver. (H) Representative histograms showing intracellular staining, (I) percentage and (J) absolute 966numbers of donor IL-17+ CD45.1+CD4+ T cells in the liver. (K) Percentage and absolute 967numbers of CD45.1+ CD3+ donor T cells absolute numbers of donor Foxp3+ Tregs, donor IFN-968g + , IL-17+ CD45.1+CD4+ donor T cells in the lamina proproa of the small intestine. One 969representative experiment of two independent transplant experiments is shown. Each symbol 970represents an individual mouse. *, p<0.05; **, p<0.01; ***, p<0.001. 971 972
973
974
Figure 5
13.5 4.75
28.8 13.8
CD
4
IFN- g
CD
4
IL-17
Live
r
Live
rLi
ver
Vehicle C220
A B C D
H
E F G
JI
K
Lam
ina
Prop
ria
Vehicle C2200
2×107
4×107
6×107
# C
D45
.1+ C
D3+ T
cel
ls
**
Vehicle C2200
1×106
2×106
3×106
# do
nor C
D4+ IL
-17+
*
Vehicle C2200.0
5.0×106
1.0×107
1.5×107
# do
nor F
oxp3
+ Tr
egs NS
Vehicle C220-5.0×106
0.0
5.0×106
1.0×107
1.5×107
# do
nor C
D4+ IF
Nγ+
*
Vehicle C2200
20
40
60
80
100
% C
D45
.1+
CD
3+ T c
ells
*
Vehicle C22050
60
70
80
90
% C
D45
.1 C
D3+ T
cel
ls
*
Vehicle C2200.0
5.0×107
1.0×108
1.5×108
2.0×108
# C
D45
.1+ C
D3+ T
cel
ls
**
Vehicle C2200
5
10
15
% d
onor
CD
4+ IFNγ+
*
Vehicle C2200.0
5.0×106
1.0×107
1.5×107
# do
nor C
D4+ IF
Nγ+
***
Vehicle C2200
10
20
30
40
% d
onor
CD
4+ IL-1
7+
p=0.05
Vehicle C2200
2×106
4×106
6×106
8×106
1×107
# d
onor
CD
4+ IL-1
7+
*
Vehicle C2200
10
20
30
40
50
% d
onor
Fox
p3+
Treg
s
NS
Vehicle C2200
2×106
4×106
6×106
8×106
# do
nor F
oxp3
+ Tr
egs
NS
37
975
Figure 6. PRMT5 inhibition preserves graft versus leukemia effect. 976
Firefly luciferase-transduced P815 cells (10,000 cells) were injected i.v. into lethally irradiated F1 977recipients on day 0 along with TCD-BM and B6 donor splenocytes. Treatment groups included 978PRMT5 inhibitor C220 or vehicle control. TCD-BM and P815 cells (leukemia alone) served as the 979control group. (A) Kaplan-Meier survival curve. Log-rank test was used to compare survival. Data 980pooled from 3 independent transplant experiments. N=22 for TCD-BM +P815 alone group, n=17-98119 for TCD-BM+P815 + B6 splenocytes groups. (B-C) Whole-body bioluminescent signal intensity 982of recipient mice (n= 5 per cohort). Mice were imaged on indicated days. Average radiance 983expressed as mean ± SD. One representative experiment of two is shown. (D) H&E stained liver 984sections at original magnification 200 and 400 of two representative recipients showing leukemic 985infiltration in the liver in the TCD-BM + P815 only group. There were no leukemic cells in either of 986the groups that received B6 allogeneic splenocytes. There was lymphocytic infiltration in mice 987receiving B6 allogeneic splenocytes (triangles). (E) Murine CD45.1 BoyJ CD8+ T cells were 988stimulated with allogeneic BALB/c BMDCs ± C220 (100nM) for 5 days. CTL capacity against P815 989tumor cells was assessed by flow cytometric evaluation of intracellular CD107a expression. 990Contour plots of one representative donor. (F) Data expressed as mean ± SD of biological 991duplicates of three independent experiments. *, p<0.05. 992
Day 21 Day 35
All deceased
Figure 6
A B
DC
Day 14
US Allo Stim + DMSO Allo Stim + C220
CD107a
CD
8
86.9 12.3
0.270.53
87.3 11.9
0.260.61
96.5 2.63
0.340.55
E F
TCD-BM+P815
200X
400X
400X
200X
TCD-BM+P815
+ allo. spl. +
Vehicle
TCD-BM+P815
+allo. spl. +
C220
10 20 30 40102
103
104
105
106
107
Days post BMT
Ave
rage
radi
ance
*
NS
TCD-BM+P815
TCD-BM+P815+Allo. spl+C220
TCD-BM+P815 +Allo. spl.+Vehicle
US Allo stim+DMSO
Allo stim+C220
0
5
10
15
20
% C
D8+
CD
107a
+ P8
15 C
TLs
NS
+P815
0 20 40 600
50
100
Days post BMT
Per
cent
sur
viva
l
*
****
38
993
Figure 7. Inhibition PRMT5 deregulates cell-cycle and ERK signaling in mouse and human 994T cells. (A) Murine CD3+T cells stimulated with CD3/CD28 Dynabeads ± C220 for 48hrs. Cell 995cycle analysis based on propidium iodide (PI) incorporation. Representative PI histogram of one 996experiment. (B) Percentage T cells in G0/G1, S and G2/M phases. Mean ± SD of biological 997triplicates of two independent experiments. Significance was determined using two-tailed t-test 998with Holm-Sidak correction for multiple comparisons. (C) Healthy donor T cells were stimulated 999with CD3/CD28 beads for 48hrs ± C220 (100nM). Cells were labeled with IdU for 15 mins at the 1000end before collection for analysis by mass cytometry. Percent Ki67+ Idu+ T cells of 4 individual 1001donors. (D) PBMCS were pre-treated with DMSO or C220 (100nM) for 2hrs and stimulated with 1002PMA/Ionomycin for 5 mins. Phosphorylation of ERK1/2 on CD3+ T cells analyzed by flow 1003cytometry. Histogram of two representative donors. Mean Fluorescence Intensity (MFI) of (E) 1004phospho-ERK1/2 and (F) total ERK1/2 in 7 donors. **, p<0.01; ***, p<0.001 1005 1006
1007
1008
1009
1010
1011
1012
1013 1014
T cells only CD3/CD28 CD3/CD28 + C220
PI
Cel
l num
ber à
US
STIM
STIM+C220
p-ERK1/2
CD3+ T cells Donor 1 Donor 2
A B C
D E
Figure 7
CD3/CD28 CD3/CD28+C220
0
10
20
30
% K
i67+
IdU
+ C
D3+
T c
ells
**
STIM STIM+C2200
1000
2000
3000
4000
5000
MFI
tota
l ER
K1/
2
NS
STIM STIM+C220
400
600
800
1000
1200
1400
1600
1800
MFI
p-E
RK1
/2
***
G0/G1 S phase G2/M0
20
40
60
80
100
% T
cel
ls
T cell only
CD3/CD28
CD3/CD28+ C220
**
**
**
Cell cycle
39
1015 1016 1017Figure 8. PRMT5 regulates interferon response in T cells 1018Human T cells were isolated from healthy PBMCs (n=4) by negative selection. T cells were 1019stimulated CD3/CD28 Dynabeads for 48hrs and treated with vehicle (DMSO) or C220, RNA 1020isolated and RNA-seq performed. (A) Volcano plot showing the top dysregulated genes. (B) Real-1021time qPCR validation in four additional human donor T cells performed for indicated genes. Fold 1022change compared to DMSO, gene expression normalized to b-actin. (C) PBMCs were stimulated 1023with CD3/CD28 ± C220 for 48hrs and then rested for 4hrs followed by 15min pulse with IFN-a 1024(100ng/ml). STAT-1 phosphorylation on CD3+ T cells was analyzed by flow cytometry. Histogram 1025of 3 representative donors. (D) Mean Fluorescence Intensity (MFI) of p-STAT and (E) MFI Total 1026STAT-1, n=6 donors. (F) B6 into F1 transplant was performed, and recipients treated with C220 1027or vehicle as described in methods. Splenocytes were isolated around day 25 post-transplant and 1028pulsed with IFN-a (100ng/ml) for 40 minutes. MFI of p-STAT-1 and (G) total STAT-1 on CD45.1+ 1029CD3+ donor T cells was evaluated by intracellular flow cytometry in both resting and IFN-a pulsed 1030cells. *, p<0.05; **, p<0.01; ***, p<0.001. 1031 1032
1033
A B
-4 -2 0 2 4
02
46
810
12
log2 FC
-log1
0 p-
valu
e
UTS2
IFI35
PIK3CG
IL17F
IFIT3
SHC2
FADS2
IFI44L
VCAM1
ADCY1
LDHC
ABO
IFIT1
SAXO2
ZFP57
TMEM191CFRG1JP
ZBED6
RPS2P7PSPHP1
C
Figure 8
T cells
UNSTIM
STIM
STIM+C220
No IFN
+IFN
+IFN
Donor 1 Donor 2 Donor 3 D E
F G
Resting IFN-α stim Resting IFN-α stim0
50
100
150
200
Geo
met
ric M
FI p
-STA
T1
Vehicle cohort C220 cohort
**
NS
***
Resting IFN-α stim Resting IFN-α stim0
200
400
600
Geo
met
ric M
FI T
otal
STA
T1
Vehicle cohort C220 cohort
NS
NS
*
DMSO C2200.0
0.5
1.0
Fold
cha
nge
com
apar
ed to
DM
SO
IL-17*
DMSO C2200.0
0.5
1.0
IFIT3**
DMSO C2200.0
0.5
1.0
IFIT1***
DMSO C2200.0
0.5
1.0
IFI35***
STIM STIM+C220
0
500
1000
1500
2000
Geo
met
ric M
FI T
otal
STA
T-1 NS
STIM STIM+C220
100
200
300
400
500
Geo
met
ric M
FI p
-STA
T-1 *
40
Table 1. List of top 20 genes with significantly different expression in PRMT5 inhibitor 1034
treated T cells compared to control (DMSO). 1035
1036 Symbol Entrez Gene Name Expr Log
Ratio FDR Adjusted
P Value 1 LDHC lactate dehydrogenase C -3.967 2.97E-06 3.82E-10 2 IFIT1 interferon induced protein with