Hypermethylation of MST1 in IgG4-related autoimmune pancreatitis and rheumatoid
arthritis
Takataro Fukuhara1*, Takashi Tomiyama1*, Kaneki Yasuda2, Yoshihiro Ueda3, Yoshio
Ozaki4, Yonsu Son4, Shosaku Nomura4, Kazushige Uchida1, Kazuichi Okazaki1, Tatsuo
Kinashi3
1. Division of Gastroenterology and Hepatology, the third Department of Internal Medicine,
JST CREST
2. Department of Urology and Andrology,
3. Department of Molecular Genetics, Institute of Biomedical Science, and JST CREST
4. Department of the first Department of Internal Medicine
Kansai Medical University, 2-5-1 Shin-machi, Hirakata, Osaka, 573-1010, Japan.
* These two authors equally contributed to this study

Address correspondence to Dr. Tatsuo Kinashi, Department of Molecular Genetics, Instituteof Biomedical Science, and JST CREST Kansai Medical University, 2-5-1 Shin-machi,
Hirakata, Osaka, 573-1010, Japan.
E-mail address: kinashi@takii.kmu.ac.jp1Abstract
The serine/threonine kinase Mst1 plays important roles in the control of immune celltrafficking, proliferation, and differentiation. Previously, we reported that Mst1 wasfor thymocyte selection and regulatory T-cell functions, thereby the prevention of
autoimmunity in mice. In humans, MST1 null mutations cause T-cell immunodeficiency
hypergammaglobulinemia with autoantibody production. RASSF5C(RAPL) is an activator
MST1 and it is frequently methylated in some tumors. Herein, we investigated methylation
the promoter regions of MST1 and RASSF5C(RAPL) in leukocytes from patients with
IgG4-related autoimmune pancreatitis (AIP) and rheumatoid arthritis (RA). Increased
number of CpG methylation in the 5’ region of MST1 was detected in AIP patients withextrapancreatic lesions, whereas AIP patients without extrapancreatic lesions were similar
to controls. In RA patients, we detected a slight increased CpG methylation in MST1,
although the overall number of methylation sites was lower than that of AIP patients withextrapancreatic lesions. There were no significant changes of the methylation levels of the
CpG islands in the 5’ region of RASSF5C(RAPL) in leukocytes from AIP and RA patients.
Consistently, we found a significant down-regulated expression of MST1 in regulatory T
cells of AIP patients. Our results suggest that the decreased expression of MST1 in
2

regulatory T cells due to hypermethylation of the promoter contributes to the pathogenesisof IgG4-related AIP.

Keywords,
IgG4-related disease
MST1
DNA methylation
autoimmune disease3

Introduction
Mst1 (also known as Stk4), a ste20-like kinase, regulates a wide variety of cellularfunctions including immune cell trafficking, cell proliferation and apoptosis [1,2]. We
previously demonstrated that MST1 and RASSF5C (RAPL) are effecter proteins of thesmall GTPase Rap1 that regulates integrin activation and cellular morphology. Mst1 forms
a complex with Rap1 and Rassf5c in response to chemokine and antigen receptor
stimulation, leading to conformational changes and clustering of LFA-1 [3,4,5,6]. This
complex also induces polarized lymphocyte morphology. In addition, Rassf5c negatively
regulates lymphocyte proliferation through the G1 cyclin inhibitor p27Kip [7].
Gene-targeting of Mst1 or Rassf5c in mice resulted in hypoplastic lymphoid organs withdefective lymphocyte homing and interstitial migration due to integrin dysfunction [8,9].
Interestingly, mice deficient in either Mst1 or Rassf5c also exhibited autoimmune
phenotypes. The absence of Rassf5c in mice caused lupus-like glomerulonephritis often
accompanied with B-cell lymphoma. On the other hand, Mst1-deficient mice developed
distinct types of autoimmunity, namely an increase of effector/memory lymphocytes with aproduction of organ-specific autoantibodies. Furthermore, leukocyte infiltration wasobserved in multiple organs including the pancreas, liver, stomach, lung, and kidney.
4

We found that the absence of Mst1 caused a breakdown of checkpoint controls of
self-tolerance. Mst1-deficient mice exhibited impaired thymocyte selection and thymic
Treg development due to defective thymocyte migration and self-antigen recognition in the
medulla [10]. In addition, the antigen-specific suppression by Mst1-deficient Tregs wasseverely impaired both in vitro and in vivo. These defects in Treg suppression were due toimpaired antigen-dependent interactions with dendritic cells and immunological synapse
formation [11]. In agreement with the mouse model, human germline mutations of
STK4/MST1 caused T-cell immunodeficiency with recurrent infections as well as
autoimmune manifestations including an increase of effector-memory lymphocytes,
autoantibody production, and hypergammablubulinemia [12,13,14].
MST1 and RASSF5C have CpG islands in their promoter regions [15,16], suggesting
epigenetic regulation of these genes. Therefore, we explored the possibility that epigenetic
abnormalities of these genes could also lead to the phenotypes of autoimmune diseases. For
this purpose, we examined CpG methylation of MST1 and RASSF5C in IgG4-related
autoimmune pancreatitis (AIP). IgG4-related AIP is characterized by a frequent elevation ofserum IgG4 and infiltration of IgG4-positive plasma cells into the pancreas and otherorgans, therefore it is categorized as a pancreatic manifestation of a novel systemic5

IgG4-related disease (IgG4-RD) [17,18]. IgG4-RD includes a wide variety of diseases thatwere formerly diagnosed as Mikulicz's disease, hypophysitis, Riedel thyroiditis, interstitial
pneumonitis, interstitial nephritis, prostatitis, lymphadenopathy retroperitoneal fibrosis,
inflammatory aortic aneurysm, and inflammatory pseudotumor. The detailed pathogenesisof IgG4-RD remains unknown, but some immunological abnormalities have been reported
including innate immunity [19,20], regulatory T cells (Tregs) [21,22,23,24] and aberrantTh1/2 balance [25,26,27] and regulatory B cells [28].
In this study, we sought to elucidate the epigenetic regulation of IgG4-related AIP byexamining the methylation levels of CpG sites in the 5’ promoter region of MST1 andRASSF5C in leukocytes, and also examine the expression levels of MST1 in Treg frompatients with IgG4-related AIP.

6

Materials and Methods
Patients and mice
A total of 20 patients (15 males and 5 females; aged 51–77 years, mean age 67.6) werediagnosed with IgG4-related AIP according to the international consensus diagnostic
criteria for autoimmune pancreatitis guidelines of the International Association of
Pancreatology. AIP patients were further categorized as those with or without
extrapancreatic lesions (lacrimal gland, thyroid gland, and kidney). RA patients (6 males
and 15 females, aged 21–85 years, mean age 58.5) and healthy controls (9 males and 1
female, aged 26–54 years, mean age 37.6) were also studied for comparison. This study
was approved by the Kansai Medical University ethics committee and all patientsprovided informed consent. The demographics of the patients are shown in Table 1.
Mst1-deficient mice was described before [10].

Bisulfite treatment
Blood samples were taken from the patients before drug treatments and centrifuged for 10
minutes at 1000 g to pellet the cells from the plasma. The buffy coat containing leukocytes
was carefully removed and washed with PBS. The cells were suspended in lysis solution7

(10 mM Tris HCl pH 8.0, 0.4 M NaCl, 10 mM EDTA, 1% SDS and 200 μg/ml proteinaseK) and incubated overnight at 50°C. Genomic DNA was isolated using either phenol-CIA(CIA = 24:1 chloroform: isoamyl alcohol) extraction or column extraction (NucleoSpin
Blood XL, Macherey-Nagal). Genomic DNA was digested with EcoRI, Hind III and Sph I,
and then precipitated. Digested DNA (0.5–1.0 μg) in 19 μl of distilled water was treatedwith 1 μl of 6 N NaOH and incubated at 37° C for 15 min. These samples were reacted with
120 μl of freshly prepared bisulfite solution (107 μl of 2.02 M sodium metabisulfite, 7 μl of
10 mM hydroquinone and 6 μl of 6 N NaOH) and overlaid with mineral oil. The bisulfite
reaction was performed with 15 cycles of 30 sec at 95°C for denaturing and 15 min at 50°C
for reaction. The free bisulfite was removed using the Wizard DNA Clean-Up System(Promega). Then, DNA was eluted with 50 µl of TE buffer pH 8.0, followed by a 5 min
incubation with 2.5 µl of 6 N NaOH. Samples were neutralized with 35 µl of 5 M NH4OAc
and precipitated with ethanol and glycogen. The efficiency of bisulfite reaction was greaterthan 97 %.

PCR amplification and sequencing of CpG Islands of MST1 and RASSF5C
After bisulfite treatment, the 5’flanking regions of MST1 and RASSF5C were amplified by
8

PCR using Ex Taq DNA polymerase (Takara Bio). The primer sequences were as follows:
5'-TTTTAGTTTGTGAAATGGGATTTAGGATTT-3' (forward) and5'-CCATACCCRTCCTCTTAACCAATAA-3' (reverse) (R represents A and G) for MST1,
and 5'-ATTTTGTAGAGGAAGTGGTTTTAGAATTGT-3' (forward) and5'-ACRTAAACCCCTAACTCTAAACCC-3' (reverse), for RASSF5C. PCR conditions wereas follows: 95°C for 5 min; 35 cycles of 95°C for 30 s, 59°C (MST1) or 57°C (RASSF5C)
for 30 s, and 72°C for 30 s; and a final extension at 72°C for 10 min. PCR products were
purified using Wizard SV Gel and PCR Clean-Up System (Promega), and then cloned into
pGEMT easy plasmids (Promega) using a DNA ligation kit (Takara Bio). At least ten
independent clones were sequenced for each sample using an ABI PRISM 3130xl Genetic
Analyzer.

Intracytoplasmic staining of MST1
Leukocytes were separated from blood of twelve AIP patients (7 males and 5 females;
aged 39–81 years, mean age 64.2) by density gradient centrifugation using Ficoll-Paque
PLUS (GE Healthcare). Cells were permeabilized by CytoPerm/Fix (BD Pharmacies)
according to the manufacture’s protocol, and stained with APC conjugated anti-human9CD4 antibody, anti human FoxP3-PE-Cy7 (eBioscience), rabbit anti-mouse/human MST1
polyclonal antibody (Upstate), followed by anti-rabbit IgG(H+L)-AlexaFlour 488(Lifetechnology). A FACS Canto II (BD Biosciences) was used to collected data, whichwere analyzed with a FlowJo software. To confirm the specificity of the intracytoplasmic
staining, T cells were isolated from wild-type and Mst1-deficient mice using panT
isolation kit according to the manufacture’s protocol, and subjected to immunostaining
and FACS analysis as above.

Statistical analysis
PRISM (GraphPad Software) was used to conduct Mann-Whitney’s U tests for thestatistical analysis of the sequencing results. Corrected p values of <0.05 were considered
statistically significant.

10

Results
CpG sites in MST1 and RASSF5C
MST1 and RASSF5 are located on 20q11.2–q13.2 and 1q32, respectively. CpG plots
identify the CpG islands in the 5’ regions of both MST1 (exon 1) and RASSF5 (exon 1C)(Fig. 1), thus suggesting a potential for epigenetic regulation of these genes by methylation.
To examine the methylation levels of MST1 and RASSF5, genomic DNA was isolated fromthe peripheral leukocytes of healthy controls, IgG4-related AIP and RA patients before drug
treatments. The samples were subjected to bisulfite sequencing to determine the frequencies
and locations of the methylated CpG sites (Fig. 1). The amplified genomic regions of MST1
and RASSF5 contained 43 and 40 CpG sites, respectively. We sequenced a minimum of 10
clones of each sample.

Methylation of CpG sites in MST1
We examined methylation of 43 CpG sites in the amplified genomic region of MST1 inhealthy controls and AIP patients (Fig. 1A, 2). In AIP patients, the frequency of methylated
CpG sites tended to be increased compared to that of healthy controls. although they were
not statistically significant (Fig. 2A). Importantly, the number of the methylation sites in
11

AIP patients were not correlated with age (R2 = 0.0825, Fig. 2B), indicating that theincrease of CpG methylation in AIP patients was not due to aging.
To gain insight into the enhanced methylation in various phenotypes of AIP, AIP patients
further divided into two groups based on the presence or absence of extrapancreatic lesions
and compared to healthy controls. AIP patients without extrapancreatic lesions exhibited
similar CpG methylation of MST1 to healthy controls. In contrast, CpG methylation wasincreased in AIP patients with extrapancreatic lesions (Fig. 2A). The incidence of
methylated CpG sites was significantly increased in AIP patients with extrapancreaticlesions relative to healthy controls (p = 0.0133) and to AIP patients without extrapancreaticlesions (p = 0.0245). Furthermore, a low but significant correlation was observed between
the number of extrapancreatic lesions and that of methylated CpG sites (R2 = 0.2534) (Fig.
2C). Thus, the increase of CpG methylation in AIP patients could reflects pathologicalconditions.

Methylation patterns of MST1 in RA patients
In RA patients, the frequency of methylated CpG sites was increased, which was notstatistically significant, compared to healthy controls (Fig. 3A). The frequency of CpG
12

methylation in RA patients was not correlated with age (Fig. 3B).

Methylation status of RASSF5C
Rassf5c associates with and activates Mst1 kinase [4], and RASSF5C is frequently
methylated in some tumors. Therefore, we used bisulfite sequencing to examine themethylation status of 40 CpG sites in the 5’ region of RASSF5C in AIP and RA patients(Fig. 1B). We found the frequency (Fig. 3C) and location (data not shown) of CpG
methylation sites in the 5’ region of RASSF5C were not significantly changed in AIP
patients with or without extrapancreatic lesions relative to healthy controls as well as RA
patients.

Decreased Mst1 expression in regulatory T cells of AIP patients
Since MST1 CpG methylation was significantly elevated in AIP patients withextrapancreatic lesions, we examined Mst1 expression in T cells from AIP patients. For thispurpose, MST1 expression levels in T cells from additional twelve patients and ten healthy
controls were evaluated with intracytoplasmic staining followed by FACS analysis. We
confirmed the specificity of the immunostaining using wild-type and Mst1-deficient T cells.
13While in wild-type T cells mean fluorescence intensities (MFI) clearly increased compared
to control antibody, Mst1-deficient T cells did not exhibit significant levels of
immunostaining (Fig. 4A). Having confirmed the specificity of the immunostaining for
MST1, The MFI of MST1 was measured in CD4+FoxP3+

regulatory T cells (Treg) andCD4+FoxP3- conventional T cells (Tconv) and expressed as ratios of MST1 levels of Treg
relative to those of Tconv (Fig. 4B). We found that in healthy controls MST1 levels in Treg
were higher than those in Tconv, whereas MST1 levels were comparable in two T cellsubsets of AIP patients. Consequently, the MST1 ratios in Treg of the AIP patients weresignificantly lower than those of the control (Fig. 4C). We also analyzed MST1 expressions
in the subgroups of AIP with or without extrapancreatic lesion. Although MST1 expressions
in both groups were significantly lower than those of healthy controls, there were no further
decrease of MST1 levels in AIP with extrapancreatic lesions. MST1 expressions did notcorrelate to age, as is the case shown in methylation assays (Fig. 4D).

14

Discussion
MST1 has CpG islands in its 5’ promoter region, thus suggesting that MST1 expression
may be regulated by DNA methylation. Understanding that Mst1 deficiency leads toautoimmune-like phenotypes in mice and humans, we explored the possibility that
epigenetic abnormalities of MST1 are an underlying mechanism that contributes to thephenotypes observed in autoimmune diseases. Therefore, we examined CpG methylationlevels in total peripheral blood leukocytes and MST1 expression in Treg from patients thatwere diagnosed with IgG4-related AIP.
IgG4-related AIP is characterized by steroid-sensitive leukocyte infiltration into thepancreas as well as an elevation of serum IgG4 levels. Most IgG4-related AIP patients havelesions in not only pancreas but also other organs. Interestingly, IgG4-related AIP patientswith extrapancreatic lesions exhibited a significant increase in the frequency of CpG
methylation sites in the 5’ region of MST1 in their leukocytes. This was of interest because
in a previous study, we found that in aged mice with Mst1 deficiency was linked to
autoimmune phenotypes associated with leukocyte infiltration in multiple organs andautoantibody production. The increased levels of CpG methylation was modest, ranging
between 5 to 10%, suggesting that only a subset of peripheral blood leukocytes harbored15

hypermethylated CpG sites in MST1.
In line with these findings, MST1 expression levels were significantly decreased in
Foxp3+ Treg from IgG4-RD patients. We previously demonstrated that a deficiency in Mst1
caused the dysfunction of regulatory T cells in the periphery [29]. Those defects likely
contribute to autoimmune pathogenesis of Mst1-deficient mice, as previously shown by the
association of Treg dysfunction to the induction of multi-organ autoimmune diseases
following the disruption of Treg-related genes [30,31,32,33]. Several studies have indicatedalterations in Treg population of IgG4-RD patients [21,22,23,24]. In IgG4-related AIP
patients, the number of CD4+CD25+CD45RA+ naïve Tregs was decreased, whereas thenumber of CD4+CD25+CD45RA- effector/memory Treg was increased [21]. Similar to AIP
patients, Mst1-deficient mice had a decreased number of thymic Tregs whereas thefrequency of CD62L-FoxP3+-induced Tregs were increased in the periphery [10]. In
addition, the antigen-specific suppression by Mst1-deficient Tregs was severely impaired
both in vitro and in vivo. These defects in Treg suppression were due to impaired
antigen-dependent interactions with dendritic cells and immunological synapse formation[29]. The methylation levels of MST1 in PBL was not significantly augmented in IgG4-RD
patients without extrapancreatic lesions, whereas MST1 protein levels in Treg from these16patients were decreased. This might be due to the experimental conditions that genomic
DNA for the methylation assays was derived from total leukocytes, as small numbers ofTreg was not sufficient to perform methylation assays. Alternatively, MST1
down-regulation in Treg could occur without direct DNA methylation of MST1, which were
subsequently stabilized epigenetically, leading to

the inflammatory lesions

innon-pancreatic tissues.
Germline null mutations of MST1 in humans led to primary T-cell immunodeficiency
with recurrent infections as well as autoimmune phenotypes [11,12,13]. To date, there havebeen no reported cases of IgG4-RD with immunodeficiency and genetic transmission. We
speculate that modest suppression of MST1 expressions with age is sufficient to maintainbasal immune functions that are capable of limiting microbe invasion, but may lead to
impaired self-tolerance. Indeed, full MST1 expression is required to mediate stableadhesion during negative selection of thymocytes as well as Treg suppression functions[29]. On the other hand, Mst1 is down-regulated in effector T cells, indicating that Mst1 is
not required for effector T-cell functions. Therefore, the compromised expression of MST1
affects self-tolerance rather than immune defense.
RA is characterized with inflammatory lesions of major joints. The frequency of17methylated CpG sites in 5’ region of MST1 of leukocytes from RA patients was slightly
elevated, but not significantly. The distinct methylation level of AIP and RA could reflect
differences of pathological conditions of the immune disorders.
Rassf5c-deficient mice have lupus-like autoimmune diseases and frequent occurrence of
B-cell lymphoma [7]. Epigenetic silencing of RASSF5C was reported in some humantumors [16,34]. However, methylation of CpG sites in RASSF5C was not increased in AIP
and RA patients. Our findings suggested that RASSF5C was not involved in thepathogenesis of AIP nor RA. Furthermore, this supports the notion that our observed
increase of CpG methylation in MST1 is not secondary to global enhancement ofmethylation activity, but rather it is specifically related to MST1 regulation in the
autoimmune disease.
In summary, AIP patients exhibited decreased expression of MST1 in Tregs. AIP withextrapancreatic lesions also showed a significantly increased frequency of CpG sites in
MST1. This increased frequency of methylated CpG sites was correlated with the number ofaffected organs in AIP patients. These results reveal the putative role of MST1 as apathological mechanism driving IgG4-RD.

18

Acknowledgement
We thank Ms. Rie Hamaguchi for technical assistance.
This study was supported by CREST, Japan Science Technology Agency, and KAKENHI
from the Ministry of Education, Science, Sport, and Culture of Japan (a Grant-in-Aid for
Scientific Research, a Grant-in-Aid on Innovative Areas and a Grant-in-Aid for Young
Scientists) and Research Program of Intractable Disease provided by the Ministry of Health,
Labor, and Welfare of Japan.

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25

Figure legends

Figure 1. CpG islands in the 5’ region of MST1 and RASSF5C
CpG plots of the 5’ region of MST1 (A) and RASSF5C (B). The percentage of GC contents
in the 5’ region of MST1 and RASSF5C were plotted using MethPrimer software.
Nucleotide sequences of the CpG sites of MST1 and RASSF5C (RAPL) are shown. The
CpG sites (underlined) and PCR primers (bold lines) for bisulfite-sequencing are indicated.

Figure 2. Increased methylation of CpG sites in AIP patients with extrapancreaticlesions.
A. Average methylation frequencies for all CpG sites in the 5’ region of MST1 from healthy
controls and IgG4-related AIP patients. AIP patients are divided into two groups withextrapancreatic lesions (AIP w) or without extrapancreatic lesions (AIP w/o) for further
analysis.
B. A correlation between age and the average number of methylation at all CpG sites in
IgG4-related AIP patients.
C. A correlation between the number of methylated CpG sites and extrapancreatic lesions.
26

Figure 3. Methylation of CpG sites of MST1 in RA patients and Methylation of

RASSF5C
A. Average frequencies of methylation at CpG sites in the 5’ region of MST1 from healthy
controls and RA patients.
B. A correlation between age and the average number of all methylated CpG sites in RA
patients.
C. Average frequencies of methylation at CpG sites in the 5’ region of RASSF5C in healthy
controls, IgG4-related AIP patients with or without extrapancreatic lesions, and RA
patients.

Figure 4. Decreased expression of MST1 in Tregs of AIP patients
A. The intensity of the anti-human and murine MST1 antibody in lymphocytes from wild
type and MST1 knockout mice.
B. Representative flow cytometry gates on T cell subsets. Mean fluorescent intensities of
intracytoplasmic MST1 staining were measured in CD4+FoxP3+ Treg and CD4+FoxP3Tconv.
27

C. The ratios of MFI of MST1 in Treg relative to Tconv are shown on healthy controls and
AIP patients. AIP patients are divided into two groups with extrapancreatic lesions (AIP w)
or without extrapancreatic lesions (AIP w/o).
D. A correlation between age and the MST1 expression ratio in Treg of AIP patients.

28

29

A. MST1
GC (%)

100

50

Ch20: 435945150
CpG

350

550

950

750

1150

bp

1
2
3
4
5
6
TCTCAGTCTGTGAAATGGGACCCAGGACCCAGGTAGAGGTGCGTTCTCGGCCTGGGGACCGAGTATTTTGTGCGCTCCGGTAACGCA
13 14
10
9
11
12
8
7
GGAAGACAGCGCCACTGACACTCTAGAGACCAGCGGGCACCGCCTGGAGGCGCCTTCACCACTTGGCGGTTCCGGGTCCGCGCCC
29
15 16
17
18
20
19
21
22
23 24 25 26 27 28
CACCGCGCCACAAGACTCACGCCCGAACCACGTGATCAGGGCCGTGGCTCCGCCCCGCTCCCGCGCCGCGCGCCGCTTCCGGTAG
34 35
36
30
33
37
31
32
GGGCGGAAAGCGGAAGTGTGGGAGGGTCTGCGGGGCGGGCTCAGGAGGTCCGCGGGAGGATGGAGCAGTGAGCGGGTCTGGGCG
3940 41 42 43
38
GCTGCTGGCAGCGCCATGGAGACGGTACAGCTGAGGAACCCGCCGCGCCGGTGAGGGGCCACTGGCTAAGAGGACGGGCATGG

B. RASSF5C
GC (%)

100

50

Ch1. 206,730,493
CpG

593

693

793

893

993

1 2
3 4
5
6
7
ACCTTGCAGAGGAAGTGGCTTCAGAACTGCTTTACGCGAGGGGCAGGAAAGGCGCGGGAGGCGGGGGAGGTGCGGAGATGGCGC
8
9 10
11
12 13
14
15
16
17
18 19
TCTGCACGGCGGCGGAGGGAGGGCGCTGGCGCCGGGGACACGAAACCGCAGAGCCCGGACGAGTCAGGGAGTGAGGCGCGAGC
21
22
20
27
28
29
30
31
32
24 25 26
23
CGGGCGCCCGGGGCTCTGCAGGCGCAGGCGGCGCGGGGACAGGAGCAGGTTACCGGGCCGCCCGAGCGCTCGCACCCCGCTGA
40
33
34
35 36
37
38
39
AAGAAACGCAGGCGGCCCGCCGGCTCTGCCTGGTCCGCTACCCGACCAGCTCCCGGCTCGGGGCTCAGAGCTAGGGGCTTACGC

Fig. 1NS
p=0.0133

A.

R2 = 0.0825

p=0.0245

30

30

20

20

numbernumberp=0.145

B.

10
0

10
0

Control

AIP

AIP w

50

AIP w/o

60

70

80

age

C.

number30

R2 = 0.2534

20

10

0

1
extrapancreatic lesions

2

Fig. 2

A.

B.
p=0.0702

R2 = 0.0859

8
6

numberfrequency (%)

30

4

10

2
0

0
Control

C.

20

20

RA

40

60

80

age

frequency (%)

8
6
4
2
0
Control

AIP

RA

Fig. 3

A.

C.

p=0.0140
p=0.0435

MST1 ratio (Treg/Tconv)

Mst1+/+(solid line)
Mst1-/- (dashed line)

MST1

B.
CD4+ Foxp3+ Treg

p=0.0111

6

NS

4

2

FoxP3

0
Control

AIP

AIP w

AIP w/oD.

CD4
CD4+ Foxp3- Tconv
MST1
background (dashed line)
anti-Mst1 Ab (solid line)

MST1 ratio (Treg/Tconv)

R2 = 0.0695

3
2
1

0

40

60

80

age

Fig. 4