Accepted Article

DR HIRONORI RYOTA (Orcid ID : 0000-0002-5694-7434)

Article type: Original Article

Clinicopathological and immunological features of follicular pancreatitis—a distinct
disease entity characterized by Th17 activation

Hironori Ryota1, Mitsuaki Ishida2, Sohei Satoi1, Hiroaki Yanagimoto1, Tomohisa
Yamamoto1, Hisashi Kosaka1, Satoshi Hirooka1, So Yamaki1, Masaya Kotsuka1, Yoichi
Matsui1, Tsukasa Ikeura3, Kazushige Uchida3, Makoto Takaoka3, Kazuichi Okazaki3,
Koji Tsuta2
1

Department of Surgery, Kansai Medical University, Osaka, Japan2

Department of Pathology and Laboratory Medicine, Kansai Medical University, Osaka,

Japan
3

Department of Internal Medicine 3, Kansai Medical University, Osaka, Japan

Running title: Follicular Pancreatitis: Immune Profile

This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead todifferences between this version and the Version of Record. Please cite this article as doi:
10.1111/his.13802
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Correspondence to: Mitsuaki Ishida, Department of Pathology and Laboratory

Accepted Article

Medicine, Kansai Medical University, 2-5-1, Shinmachi, Hirakata-city, 573-1010 Osaka,
Japan. E-mail: ishidamt@hirakata.kmu.ac.jp

Declaration of conflicts of interest: The authors have declared no conflicts of interest.

Abstract
Aim: Follicular pancreatitis is a recently recognized, distinct clinicopathological entity
characterized by the presence of many intrapancreatic lymphoid follicles with reactive
germinal centres. However, the clinicopathological and immunological features andcauses have not yet been established. We assessed the clinicopathological andimmunological profiles of patients with follicular pancreatitis who underwent surgery.
Methods and Results: This study included three patients with pancreatic masses (agerange: 62-75 years; women:men: 1:2). A histopathological study of the resectedpancreatic masses revealed abundant lymphoid follicles with reactive germinal centres inboth periductal regions and diffusely within the parenchyma. No storiform fibrosis,
obliterative phlebitis, or granulocytic epithelial lesions were observed. The
immunohistochemical examination revealed an IgG4/IgG-positive plasma cell ratio<30% in all patients. Podoplanin (Th17 marker)-expressing lymphocytes were present in
the lymphoid follicles of those with follicular pancreatitis, whereas these were absent innormal lymph nodes and in lymphoid follicles of those with IgG4-related autoimmune
pancreatitis (AIP). An RNA digital counting assay clearly demonstrated that theexpression counts of 20 genes, including dendritic cells and lymphoid follicles markers,
and related cytokines were significantly higher in follicular pancreatitis than in
IgG4-related AIP (p<0.01). The expressions of CCR6 and IL23A, which are genes related
to Th17, were high.

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Conclusions: This study shows that follicular pancreatitis is a histopathologically andAccepted Article

immunologically distinct disease entity of pancreatitis and is characterized byupregulated Th17 expression.

Keywords: Follicular pancreatitis, autoimmune pancreatitis, gene expression analysis,
Th17 lymphocyte

Introduction
Several forms of chronic pancreatitis have been recognized as distinct clinicopathologicalentities, including autoimmune pancreatitis (AIP) and groove pancreatitis. AIP is
subclassified into type 1, an IgG4-related disease, and type 2, which is characterized by
the presence of granulocytic epithelial lesions1-3. Recently, Zen et al. proposed a thirdform of chronic pancreatitis, namely follicular pancreatitis, which is characterizedhistopathologically by the presence of duct-centred, dense lymphoplasmacytic infiltration
with many reactive lymphoid follicles bearing germinal centres4. This condition may beunder-recognized and may have been previously reported as pancreatic pseudolymphomaor reactive lymphoid hyperplasia5-10. To the best of our knowledge, only 13 patients havebeen reported on in the English literature4-11, and only 2 patient series have been
published4, 11. The immunological characteristics of IgG4-related AIP have been
established. The activation of type 2 helper T cells (Th2) and regulatory T cells (Treg) inaffected organs is characteristic of IgG4-related AIP. Tissue mRNA levels of Th2
cytokines, such as IL-4, IL-5, IL-10, and IL-13, are significantly higher, and many
lymphocytes expressing IL-4 or IL10 are present in the affected organs. Moreover,abundant infiltration of CD4+/CD25+ Treg cells is also observed in IgG4-related AIPlesions, and a higher expression of FOXP3 mRNA is also noted12. However, theimmunological characteristics of follicular pancreatitis have not been examined4-11. Thus,

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we studied the clinicopathological characteristics of three patients with follicularAccepted Article

pancreatitis and assessed the gene expression profile of this disorder. Our aim was todetermine the characteristic gene expression profile of follicular pancreatitis compared tothat of IgG4-related AIP, and to determine whether follicular pancreatitis is a distinct
clinicopathological entity of pancreatitis.

Materials and Methods
Patients
Three patients with pancreatic masses who underwent surgery at the Department ofSurgery, Kansai Medical University (Osaka, Japan) between January 2006 and March2017 and received diagnoses of pseudolymphoma, lymphoid hyperplasia, and follicularpancreatitis were enrolled in this study.
The study was conducted in accordance with the Declaration of Helsinki and the study
protocol was approved by the institutional review board of our hospital (protocol no.
2017001, 1501-2).

Immunohistochemistry and in situ hybridization
Formalin-fixed and paraffin-embedded (FFPE) blocks of the resected specimens were cut
into 4 μm-thick sections. Macrodissections of the lesions were performed using 18-G
needles. Subsequently, the samples were deparaffinized and rehydrated.
Immunohistochemical analyses were performed using an autostainer (Discovery XT
System; Roche Diagnostics, USA, and Autostainer Link 48; Agilent Technology, Santa
Clara, USA) according to the manufacturer’s instructions. The primary antibodies used inthis study are shown in the Supplemental Table 1. In situ hybridizations for kappa andThis article is protected by copyright. All rights reserved.

lambda light chains were also performed using an autostainer (Discovery XT System;

Accepted Article

Roche Diagnostics, USA).
Lymph nodes of a patient with intraductal papillary mucinous adenoma and pancreatic
tissues of patients with IgG4-related AIP were used as controls.
For podoplanin staining, complete circular staining of lymphocytes was considered to bea positive result. IgG4+ and IgG+ cells were counted using printed photographs of thesame microscopic field under a ×40 objective lens in 10 high power fields13.
Immunohistochemical and in situ hybridization examinations were assessedindependently by 2 pathologists who were blinded to the patients’ clinical features.

RNA extraction
For mRNA extraction, 5 μm-thick sections from the FFPE blocks were cut andNucleoSpin® total RNA FFPE kit (Macherey-Nagel, Germany), including on-column
treatment with DNase, was used. A quantitative evaluation of RNA was performed using
the Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE).
RNA quality was evaluated based on a 260/280 nm ratio. We excluded samples in which
the total amount of RNA was less than 50 ng/μL or the 260/280 ratio was less than 1.6.

Digital mRNA counts and analysis
We studied the expression of 770 immune-related genes (Supplemental Table 2) in threepatients with follicular pancreatitis, two patients with IgG4-related AIP, two patients withchronic alcoholic pancreatitis, and one patient with a normal pancreas using the nCounter
PanCancer Immune Profiling Panel (NanoString Technologies, Inc., Seattle, WA. USA).
The nCounter assay was performed according to the manufacturer’s instructions. The

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RNA was hybridized with the probe sets for 16 hours at 67°C, and the samples were thenAccepted Article

processed using an automated nCounter Sample Prep Station (NanoString Technologies,Inc.). Cartridges containing immobilized and aligned reporter complexes were
subsequently imaged on an nCounter Digital Analyzer (NanoString Technologies, Inc.)
that had been set at a data resolution of 555 fields of view. Reporter counts werecollected and normalized using nSolver analysis software version 3.0 (NanoString
Technologies, Inc.).

Results
Clinical characteristics
Table 1 summarizes the clinicopathological features of the three patients with follicularpancreatitis in this series (age range: 62-75 years; women:men - 1:2). The lesions werelocated in the tail of the pancreas in three patients and in the body in one patient (onepatient had two lesions, one in the body and one in the tail). All patients were incidentallyfound to have pancreatic tumours and had no clinical symptoms. Pancreatic cancer was
clinically diagnosed preoperatively in all patients, and distal pancreatectomies were
performed. The serum IgG4 level was not elevated in patient 3 (30 mg/dL). The past
medical histories of all patients were negative for autoimmune disorders.
Histopathological characteristics
Macroscopic features
All 3 patients had a yellowish tumorous lesion in the pancreatic body and/or tail. The
boundaries were unclear, and the sizes of the lesions were approximately 20 to 30 mm indiameter. One patient had two lesions in the pancreatic parenchyma and the others each
had one lesion.

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Accepted Article

Microscopic features
Many variable-sized lymphoid follicles with reactive germinal centres and intact mantle
zones were diffusely distributed in the pancreatic parenchyma (Figure 1). Periductal
lymphoid follicle formations were also noted in all patients (Figure 1). Infiltration oflymphocytes into the ductal epithelial cells was not observed, and there was no evidenceof ductal epithelial cell injury. Moreover, granulocytic epithelial lesions were notobserved in all patients. Mild plasma cell infiltrations were noted around the lymphoidfollicles. Mild interlobular fibrosis and parenchymal atrophy were present, but storiform
pattern fibrosis was not observed. Mild eosinophilic infiltration was seen in all threepatients, however, neutrophilic infiltration was inconspicuous. Obliterative phlebitis was
not identified in any of the patients.
The germinal centres were mainly composed of CD20-positive B-cells with scatteredCD3-positive T-cells, had meshworks of CD21-positive follicular dendritic cells, andwere positive for Bcl-6 and CD10 and negative for Bcl-2 (Figure 2). A few IgG4-positive
plasma cells were observed in all patients, and the ratio of IgG4/IgG-positive plasma cells
was less than 30% in all patients (Figure 2) (Table 1). Of note, podoplanin-positive
lymphocytes were present in the germinal centres of the lymphoid follicles in all patientswith follicular pancreatitis (Figure 3-a, b). These were rarely observed in the lymphoidfollicles of patients with IgG4-related AIP or the lymph nodes of a patient withintraductal papillary mucinous adenoma, however, podoplanin-expressing folliculardendritic cells were present in these specimens.
In situ hybridization analyses demonstrated that kappa- and lambda-chain positive cells
were evenly distributed, and monoclonality was not detected in any of the patient
samples.

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Accepted Article

RNA expression
mRNA count data of 770 genes are shown in the Supplemental Table 2 and acluster-classified gene heat map was prepared from the expression levels of the 770 genes(Figure 4). Follicular pancreatitis exhibited a different gene profile than IgG4-related
AIP. The expression counts of 20 genes were significantly higher in patients withfollicular pancreatitis compared to those with IgG4-related AIP (p<0.01) (Table 2). The
expression levels of dendritic cells and lymphoid follicles markers and related cytokines
were significantly higher in those with follicular pancreatitis. Furthermore, the expression
levels of C-C chemokine receptor type 6 (CCR6), which is highly expressed in Th17 cells,
and interleukin 23A (IL23A), which promotes the growth of Th17 cells, were significantly
high.

Discussion
The clinicopathological characteristics and immunological mechanism of IgG4-related
AIP have been established. However, those of follicular pancreatitis have not yet beenclarified since it is a recently recognized entity and the number of patients is limited. This
study clearly demonstrated the following: (1) RNA expression patterns in follicularpancreatitis are significantly different from those in IgG4-related AIP, with highexpression levels of dendritic cells (LAMP3) and lymphoid follicle markers (ICOS andCR2), related cytokines (LTA), and regulatory factors of Th17 (CCR6 and IL23A), and (2)podoplanin (Th17 marker)-expressing lymphocytes are present in the lymphoid follicles
of patients with follicular pancreatitis.
Th17 represents a population of helper T-cells that was first reported for its involvement
in experimental autoimmune encephalomyelitis in 200513. Since then, Th17 has beenreported to contribute to the pathogenesis of several diseases including psoriasis14,

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rheumatoid arthritis15, inflammatory bowel disease16, and autoimmune thyroid disease17.

Accepted Article

Moreover, Th17 plays an important role in the formation of normal lymphoid follicles.
The naive T cells differentiate into Th1 or Th2 upon antigen presentation, and Th17
similarly differentiates. Co-stimulation of TGF-beta and IL-6 is essential fordifferentiation into Th1718, 19. After differentiation from naive T-cells, Th17 is
proliferated by IL23A, which is secreted by dendritic cells. According to a report on theplasticity of Peyer’s patches20, Th17 transforms into follicular helper T-cells and forms
lymphoid follicles.
In this study, expression of Th17 and follicular helper T cell-related markers (CCR6,
IL23A, ICOS, and LTA) was significantly higher in cases of follicular pancreatitis thanthey were in cases of IgG4-related AIP. CCR6 is expressed on Th17 and plays a role in
Th17 migration21. IL-23A is essential for maintaining the survival and proliferation of
Th17 cells22. Moreover, LTA is a chemokine that promotes the formation of lymphoidfollicles. These results suggest that the development of follicular pancreatitis is highly
associated with the activation of Th17. This may be a unique characteristic of this disease
compared to IgG4-related AIP, which is characterized by activation of Th2 and Treg1, 12.
The possible cascade in the development of follicular pancreatitis is shown in Figure 5.
Th17 cell proliferation is aided by IL-23A, which is produced by dendritic cells
expressing LAMP3. Th17 then migrates to the pancreatic parenchyma. Subsequently,
Th17 cells transform into follicular helper T-cells and then aggregate in lymphoidfollicles.
Moreover, to evaluate the localization of Th17 in lesions of follicular pancreatitis, we
examined the immunohistochemical staining for Th17. Podoplanin is specifically
expressed on Th17 among the lymphocytes23. In this study, podoplanin-expressing
lymphocytes were observed in the germinal centres of the lymphoid follicles in all
patients with follicular pancreatitis, but they were scanty in the lymphoid follicles ofpatients with IgG4-related AIP and the lymph nodes of a patient with intraductal

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papillary mucinous adenoma. The presence of podoplanin-expressing lymphocytes may

Accepted Article

be a characteristic of follicular pancreatitis. It has been reported that Th17 may be
involved in the pathogenesis of type 2 AIP, especially in cases leading to the formation of
granulocytic epithelial lesions. However, a comprehensive immune-related gene
expression profile was not analysed in this study24. We performed a comprehensive
analysis of the immune-related gene expression profile of follicular pancreatitis comparedto that of IgG4-related AIP. The findings clearly demonstrated that dendritic cells andlymphoid follicle markers and regulatory factors of Th17 are significantly upregulated incases of follicular pancreatitis. Although Th17 might be involved in the pathogenesis ofboth type 2 AIP and follicular pancreatitis, the significance and role of Th17 may bedifferent in these two conditions. Thus, additional studies are needed to clarify thesignificance of Th17 in AIP.
The clinicopathological summaries of follicular pancreatitis in the previously reported 13
patients4-11 and the three patients in the present study are as follows: (1) this diseaseaffects middle-aged to elderly patients (age range: 41-75 years; mean age: 62 years) witha predilection for male patients (men:women - 10:6), (2) most patients are asymptomatic(10 cases), with pancreatic tumours diagnosed incidentally, (3) there is no common
location (lesions occurred in the pancreatic head in five patients, the body in two, and thetail in five, with dual lesions in the head and body, and body and tail). Of interest, diffuse
dilatation of the pancreatic duct without a tumorous lesion was observed in two patients.
Table 3 summarizes the clinicopathological characteristics of follicular pancreatitis incomparison with AIP. The histopathological characteristics include: (1) many reactive
lymphoid follicles with germinal centres were present in the periductal regions and/or ina diffuse fashion throughout the parenchyma, (2) storiform fibrosis and obliterative
phlebitis, characteristic findings of IgG4-related AIP, were rare or absent, and (3)IgG4/IgG-positive plasma cell ratio was not elevated (<30%). Based on theclinicopathological and immunological characteristics shown in the present study,

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follicular pancreatitis should be considered a clinicopathologically distinct disease entity

Accepted Article

and must be considered as a form of autoimmune pancreatitis.
Similar to this study, in most of the previous reports, pancreatic cancer or neuroendocrine
tumours were suspected preoperatively4-11. Therefore, surgical resection was performed
in almost all reported patients. Of interest, one patient with follicular pancreatitis showed
a 50% reduction in tumour size (on imaging) with steroid therapy11. Therefore, diagnostic
and therapeutic strategies for follicular pancreatitis must be established to avoid
unnecessary surgery, given the high complication rate associated withpancreaticoduodenectomy (~60%)25. In addition, clinical applications of Th17 inhibitors
are gaining acceptance in Th17-related diseases26-28, indicating their potential astherapeutic candidates for follicular pancreatitis.
In conclusion, to the best of our knowledge, this is the first report on the pathogenesis offollicular pancreatitis. Although this study is limited by the small number of patients, wehave shown that the development of follicular pancreatitis is highly associated with theactivation of Th17. Follicular pancreatitis should be considered as a clinicopathologicallydistinct disease entity and must be considered a form of autoimmune pancreatitis.

Acknowledgments
We thank all the patients, their families, and investigators who participated in the study.
This study was partially funded by KONICA MINOLTA.
Author contributions statement:
HR, MI, and KT contributed to experimental design, pathological diagnosis, andmanuscript preparation. HR performed molecular experiments. HR and MI performed
immunostaining. SS, HY, TY, HK, SH, SY, MK, YM, TI, KU, MT, and KO contributed
to patient data collection. All authors approved the final version of the manuscript.

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Accepted Article

Figure legends
Figure. 1 Histopathological features of follicular pancreatitis.
a) Case 1: Lymphoid follicles around main pancreatic duct (H&E × 40).
b) Case 2: Diffuse lymphoid follicle formation in the pancreatic parenchyma (H&E × 40).
c) Case 3: Diffuse lymphoid follicle formation in the pancreatic parenchyma (H&E × 40).

Figure 2. Immunohistochemical characteristics of follicular pancreatitis.
Germinal centres are negative for Bcl-2 (× 100). CD21-positive follicular dendritic cells
are present in the lymphoid follicles (x 100). Only a few IgG4-positive plasma cells areobserved (× 100).

Figure 3. Immunostainings for podoplanin in lymphoid follicles.
(a) Podoplanin-positive lymphocytes are present in all cases of follicular pancreatitis.
Podoplanin expression is noted on the cell surface of the lymphocytes(arrows)(×200).
(b) Podoplanin is expressed only in the dendritic cells but not in the lymphocytes in thelymphoid follicles of the lymph node from intraductal papillary mucinous adenoma case(control) and IgG4-related autoimmune pancreatitis (× 200).
Figure 4. Cluster classification and heatmap of gene expression level. Heatmap of thenormalized data, scaled to give all genes equal variance, generated via unsupervised
clustering. Red indicates high expression; green indicates low expression. This plot ismeant to provide a high level exploratory view of the data. The enlarged view of theexpression level, where characteristic changes are present in patients with follicularpancreatitis (right).

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Figure 5. The possible cascade of lymphoid follicles formation in follicular pancreatitis.

Accepted Article

Th17 is proliferated by IL-23A, which is produced by dendritic cells expressing LAMP3,
and migrates to pancreatic parenchyma. Then, Th17 transforms to follicular helper T
cells, followed by formation of lymphoid follicles.
DC: dendritic cell, Th: helper T cell, T: T cell area, B: B cell area, Tfh: follicular helper T
cell, LAMP3: lysosomal associated membrane protein 3, CCR6: chemokine (C-C motif)
receptor 6, ICOS: inducible T cell co-stimulator, LTA: lymphotoxin alphaThis article is protected by copyright. All rights reserved.

Accepted Article

Table.1 Clinical and histopathological features of three cases of follicular pancreatitis

Case 1Case 2

Case 3

Age/Sex

62/Male63/Male75/Female

Symptoms

Incidental

Incidental

Incidental

Location

Tail

Body and tail

Tail

a

Size

CECT : 29×25mm

CECT : 20×18mm

CECTa: 25×21mm

EUS-FNA b Findings

Aciner cells and benign
lympocytes

Not done (cytology using
ENPDc juice was benign )Not done

Histology

Lymphoid follicles with
Bcl-2 negative germinal
centers in parenchyma

Lymphoid follicles with
Bcl-2 negative germinal
centers in parenchyma

Lymphoid follicles with
Bcl-2 negative germinal
centers in parenchyma

+

+

+

Number of CD21
lymphoid follicles

28

34

17

Plasma cell infiltration

Mild

Mild

Mild

Eosinophilic infiltration

Mild

Mild

Mild

Neutrophilic infiltration

Few

Few

Few

Fibrosis

Mild

Mild

Mild

IgG4/IgG ratio(%)

20

30

15

Serum IgG4
level(mg/dL)d

Not doneNot done30

Alcohol/cigarettes

Socially /none

Duct-centered follicles+

2-3day in a week/
None/none
10 cigarettes for 15 years
Hyperlipidemia

Medical history

Hypertension
Cholelothiasis

a_Contrast-enhanced computed tomographyb_EUS guided fine needle aspirationca_ Endoscopic nasobiliary drainaged_Normal serum IgG4 level = 4.8-105mg/dL

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Benign tumor (Thyroid
and parotid gland)Accepted Article

Table.2 mRNA counts with significantly higher in follicular pancreatitis compared with IgG4-related
autoimmune pancreatitis

numbername

Log2 foldchangeStd error

Lower
confidence
limit

Upper
confidence
limit

p value

1

LAMP3

0.946

0.08480.78

1.11

0.000368

2

IL32

1.3

0.158

0.995

1.61

0.00117

3

ICOS

1.41

0.173

1.07

1.75

0.00123

4

LTA

2.43

0.323

1.8

3.07

0.00167

5

TLR10

2.68

0.375

1.94

3.41

0.00202

6

CR2

5.18

0.757

3.7

6.67

0.00239

7

CCR6

2.14

0.331

1.5

2.79

0.00291

8

BIRC5

1.84

0.289

1.27

2.41

0.00311

9

CDK1

2.22

0.356

1.52

2.91

0.00341

10

CD1C

1.69

0.284

1.13

2.25

0.00401

11

CD40

2.01

0.343

1.34

2.69

0.00422

12

FCER2

4.55

0.781

3.02

6.08

0.00432

13

TNFRSF9

1.88

0.327

1.23

2.52

0.00457

14

ADA

1.42

0.251

0.932

1.92

0.00476

15

PAX5

4.00

0.71

2.61

5.39

0.00489

16

TNFSF8

1.17

0.217

0.746

1.59

0.00568

17

PIK3CD

0.857

0.166

0.53

1.18

0.00675

18

AICDA

3.68

0.763

2.18

5.17

0.00855

19

CCL22

1.61

0.343

0.933

2.28

0.00947

20

IL23A

2.12

0.459

1.22

3.01

0.00995

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Accepted Article

Table.3 Comparison of pathological and clinical features in follicular pancreatitis and autoimmune
pancreatitis

Lymphoid follicles withgerminal centers
Presence of Th17 cells
Storiform fibrosis
Obliterative phlebitis
Significant difference of
IgG4/IgG ratioa
Response to steroids

Follicular pancreatitis
Mainly periductal and diffuse
throughout the parenchyma
Mainly in germinal centers
AbsentRare to absent
AbsentAutoimmune pancreatitis
Rarely(type1)

Potentialb

Gooda: the presence of IgG4 to IgG ratio of >40%b: there was partial response8

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AbsentPresent(type1)
Present(type1)
Present(type1)

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Accepted Article
This article is protected by copyright. All rights reserved.

Accepted Article
This article is protected by copyright. All rights reserved.

Accepted Article
This article is protected by copyright. All rights reserved.

Accepted Article
This article is protected by copyright. All rights reserved.