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Title Detection of Autoreactive Type II NKT Cells: A Pilot Study of Comparison Between Healthy Individuals and Patientswith Vasculitis.

Author(s)Nishioka, Yusuke; Sonoda, Takaomi; Shida, Haruki; Kusunoki, Yoshihiro; Hattanda, Fumihiko; Tanimura, Shun;Uozumi, Ryo; Yamada, Mai; Nishibata, Yuka; Masuda, Sakiko; Nakazawa, Daigo; Tomaru, Utano; Atsumi, Tatsuya;Ishizu, Akihiro

Citation Cytometry. Part A : the journal of the International Society for Analytical Cytology, 93(11), 1157-1164https://doi.org/10.1002/cyto.a.23618

Issue Date 2018-11

Doc URL http://hdl.handle.net/2115/76239

RightsThis is the peer reviewed version of the following article: Cytometry Part A Volume 93, Issue 11, pages 1157‒1164,Nov 2018, which has been published in final form at https://doi.org/10.1002/cyto.a.23618. This article may be used fornon-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.

Type article (author version)

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File Information Nishioka et al-Cytometry A_accepted manuscript.pdf ()

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1

Detection of autoreactive type II NKT cells: a pilot study of comparison

between healthy individuals and patients with vasculitis

Yusuke Nishioka1, Takaomi Sonoda2, Haruki Shida3, Yoshihiro Kusunoki3,

Fumihiko Hattanda3, Shun Tanimura3, Ryo Uozumi1, Mai Yamada1, Yuka

Nishibata4, Sakiko Masuda4, Daigo Nakazawa3, Utano Tomaru5, Tatsuya Atsumi3,

Akihiro Ishizu4,*

1Graduate School of Health Sciences, Hokkaido University, Sapporo, Japan

2Undergraduate School of Health Sciences, Hokkaido University, Sapporo, Japan

3Division of Rheumatology, Endocrinology and Nephrology, Faculty of Medicine and

Graduate School of Medicine, Hokkaido University, Sapporo, Japan

4Faculty of Health Sciences, Hokkaido University, Sapporo, Japan

5Department of Pathology, Faculty of Medicine and Graduate School of Medicine,

Hokkaido University, Sapporo, Japan

*Corresponding author: Akihiro Ishizu, MD, PhD

Faculty of Health Sciences, Hokkaido University, Sapporo 0600812, Japan

Tel. +81 11 706 3385; fax: +81 11 706 4916

E-mail: aishizu@med.hokudai.ac.jp

mailto:aishizu@med.hokudai.ac.jp

2

ABSTRACT

NKT cells are defined as T cells that recognize hydrophobic antigens presented

by class I MHC-like molecules, including CD1d. Among CD1d-restricted NKT cells, type

I and type II subsets have been noted. CD1d-restricted type I NKT cells are regarded as

pro-inflammatory cells in general. On the contrary, accumulated evidence has

demonstrated an anti-inflammatory property of CD1d-restricted type II NKT cells. In our

earlier study using a rat model with vasculitis, we demonstrated pro-inflammatory

function of CD1d-restricted type II NKT cells and identified that one such cell recognized

P518-532 of rat sterol carrier protein 2 (rSCP2518-532), which appeared on vascular

endothelial cells presented by CD1d. Based on this evidence, we attempted to detect

human CD1d-restricted type II NKT cells in peripheral blood using hSCP2518-532, the

human counterpart of rSCP2518-532, together with a CD1d tetramer in flow cytometry. First,

we determined the binding of hSCP2518-532 to CD1d. Next, we detected CD3-positive

hSCP2518-532-loaded CD1d (hSCP2518-532/CD1d) tetramer-binding cells in peripheral

blood of healthy donors. The abundance of TGF-β-producing cells rather than TNF-α-

producing cells in CD3-positive hSCP2518-532/CD1d tetramer-binding cells suggests the

anti-inflammatory property of SCP2-loaded CD1d (SCP2/CD1d) tetramer-binding type

II NKT cells in healthy individuals. Furthermore, we compared cytokine profile between

healthy individuals and patients with vasculitis in a pilot study. Interestingly, the

percentage of TGF-β-producing cells in SCP2/CD1d tetramer-binding type II NKT cells

in vasculitic patients was significantly lower than that in healthy controls despite the

greater number of these cells. Although further studies to clarify the mechanism and

significance of this phenomenon are needed, SCP2/CD1d tetramer-binding type II NKT

cells in peripheral blood should be examined in more detail to understand the

pathophysiology of vasculitides in humans.

3

Key Words: Type II NKT cell, Sterol carrier protein 2, CD1d tetramer, vasculitis,

cytokine profile

4

INTRODUCTION

NKT cells are defined as T cells that recognize hydrophobic antigens presented

by class I MHC-like molecules, including CD1d [1]. Among CD1d-restricted NKT cells,

there are two major subsets, type I and type II [2]. They are classified based on their usage

of TCR and reactivity to marine sponge-derived glycolipid, α-galactocylceramide (α-

GalCer). CD1d-restricted type I NKT cells are defined by the expression of a conserved

TCR α-chain (Vα24-Jα18 in humans and Vα14-Jα18 in rodents) that pairs with a limited

repertoire of β-chains; thus, they are usually called invariant NKT (iNKT) cells. Invariant

NKT cells can bind to α-GalCer [3]. On the contrary, CD1d-restricted type II NKT cells

have a more variable TCR repertoire and do not react with α-GalCer [4]. This type of cell

can recognize sulfatides and hydrophobic peptides.

Difference in function between the subsets of CD1d-restricted NKT cells has

been discussed. CD1d-restricted type I NKT (iNKT) cells can recognize microorganism-

derived glycolipids presented by CD1d and trigger both innate and acquired immune

responses against the microorganism [3]. Therefore, these cells are regarded as pro-

inflammatory cells in general. In contrast, CD1d-restricted NKT cells reactive with

autologous peptides (suggestive of type II phenotype) have been shown to exhibit an anti-

inflammatory property in normal mice [5].

Accumulated evidence has revealed the anti-inflammatory property of CD1d-

restricted type II NKT cells. Halder et al. demonstrated that the activation of sulfatide-

reactive CD1d-restricted type II NKT cells ameliorated the experimental murine hepatitis

[6]. Terabe et al. showed that CD1d-restricted type II NKT cells were essential for the

down-regulation of tumor immunosurveillance in mice [7]. On the other hand, the

association of impaired CD1d-restricted type II NKT cells with inflammatory diseases

has also been reported. Liao et al. demonstrated that the disordered regulation of CD1d-

5

restricted type II NKT cells could cause spontaneous development of colitis in CD1d

transgenic mice [8]. In addition, we have shown that pro-inflammatory autologous

vascular endothelial cell-reactive CD1d-restricted type II NKT cells were involved in the

development of vasculitis in rats [9]. Recently, we identified P518-532 of rat sterol carrier

protein 2 (rSCP2518-532), FFQGKLKIAGNMGLA, as the epitope, which was recognized

by one such CD1d-restricted type II NKT cell [10].

Invariant NKT cells can be detected as CD3-positive α-GalCer-loaded CD1d

(α-GalCer/CD1d) multimer-binding cells in flow cytometry [11-13]. In contrast, CD1d-

restricted type II NKT cells are less well-characterized due to the lack of a suitable antigen

that can substitute for α-GalCer. Hence, we attempted to detect human CD1d-restricted

type II NKT cells in peripheral blood using hSCP2518-532, FFQGPLKITGNMGLA, the

human counterpart of rSCP2518-532, together with a CD1d tetramer in flow cytometry.

SCP2 is an intracellular protein that is expressed widely in organs and cells

involved in lipid metabolism, including the liver and vascular endothelial cells [14]. It

also plays a pivotal role in intracellular lipid transfer [15], which suggests hydrophobicity

of SCP2 molecule. On the other hand, CD1d preferably presents hydrophobic antigens.

Therefore, it is reasonable to consider that CD1d can present SCP2 peptides.

In this study, we first determined the binding of hSCP2518-532 to CD1d. Next,

we detected CD3-positive hSCP2518-532-loaded CD1d (hSCP2518-532/CD1d) tetramer-

binding cells in peripheral blood of healthy donors and demonstrated their anti-

inflammatory property. Furthermore, we compared the prevalence and cytokine profile of

SCP2-loaded CD1d (SCP2/CD1d) tetramer-binding type II NKT cells between healthy

donors and patients with vasculitis in a pilot study.

6

MATERIALS AND METHODS

Peripheral blood samples

Upon receiving the written informed consent, 10 ml of peripheral blood was

obtained from 8 healthy donors and 5 patients with vasculitis, such as microscopic

polyangiitis and Takayasu arteritis (Table 1). All patients with vasculitis were actively

suffering from the disease. At the earliest opportunity (within 30 min) after blood

sampling, PBMCs were isolated using Ficoll-Paque Plus (GE Healthcare, Tokyo, Japan).

RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) supplemented with 10% FBS which

was used as culture medium. The PBMCs were assayed immediately without being frozen

for storage. This study was approved for practice by our institutional ethics committee,

the Ethical Committee of the Faculty of Health Sciences, Hokkaido University

(Permission No. 16-83).

Binding of hSCP2518-532 to CD1d

Human CD1d monomer (molecular mass, 37 kDa) was purchased from Sino

Biological (Beijing, China). Amino acid sequence of hSCP2518-532 is

FFQGPLKITGNMGLA (molecular mass,

7

treated with the blocking solution from a BioLegend ELISA kit (BioLegend, San Diego,

CA) per the manufacturer’s instruction. Thereafter, avidin-conjugated HRP (50 µl) in the

kit was applied to each well. Finally, 3,3',5,5'-tetramethylbenzidine solution (100 µl) was

added to the samples, and the reaction was stopped by 2N H2SO4 (100 µl). Absorbance at

450 nm was measured using MALTISKAN FC (Thermo Scientific, Waltham, MA).

Experiments were repeated 3 times independently. Each experiment was conducted in

triplicate.

Competition assay

We determined the competitive effects of non-biotinylated hSCP2518-532 and α-

GalCer on the binding of hSCP2518-532B to CD1d. CD1d monomer (100 ng) was mixed

with hSCP2518-532B (100 ng) alone and together with hSCP2518-532B (100 ng) and non-

biotinylated hSCP2518-532 (100 ng) in PBS (10 μl). Alternatively, prior to incubation of

hSCP2518-532B with CD1d, CD1d monomer (100 ng) was mixed overnight with 0, 0.1, 0.5,

1.0, or 2.0 μg of α-GalCer (Funakoshi, Tokyo, Japan) in PBS (15 μl) at room temperature.

The next day, hSCP2518-532B (100 ng) was mixed with the α-GalCer-loaded CD1d (100

ng) in PBS (20 μl). After incubation for 5 h at room temperature, the mixture was adjusted

to 2 ml in PBS and centrifuged at 2,600×g for 30 min using Amicon Ultra 10K. The

remaining solution (100 µl) expected to contain bound antigens, including hSCP2518-532B,

was applied to each well of ELISA plates, and color development was conducted as

mentioned above. The relative binding of hSCP2518-532B to CD1d was determined as

follows: {(Sample O.D.) - (Blank O.D.)} / {(No competitor O.D.) - (Blank O.D.)} × 100.

Experiments were repeated 3 times independently. Each experiment was conducted in

triplicate.

Detection of SCP2/CD1d tetramer-binding type II NKT cells in flow cytometry

8

APC-conjugated human CD1d tetramer was purchased from Medical &

Biological Laboratory (Nagoya, Japan). One hundred ng of hSCP2518-532 and a different

peptide hSCP2526-540, TGNMGLAMKLQNLQL (generated by Scrum), as control were

independently mixed overnight with 10 μl of the CD1d tetramer at room temperature as

previously described [5]. The next day, PBMCs (1 × 106) were stained with 20 µl of PE-

conjugated anti-human CD3 antibody (UCHT-1, mouse IgG1κ) (BD Pharmingen,

Franklin Lakes, NJ) for 30 min on ice. After washing with PBS, the cells were stained

with the APC-conjugated CD1d tetramer without antigen-loading or APC-conjugated

SCP2/CD1d tetramer, for 30 min on ice (total volume, 100 μl) according to the instruction

provided from the reagent manufacturer. After washing with PBS, the cells were analyzed

using FACS Canto II (BD Biosciences, Tokyo, Japan) and data was processed by

FACSDiva software ver. 7.0 (BD Biosciences). In some experiments, PBMCs were

stained with PerCP-conjugated anti-TCR Vα24-Jα18 antibody (6B11, mouse IgG1κ)

(Thermo Fisher Scientific, Yokohama, Japan) together with the PE-conjugated anti-CD3

antibody and APC-conjugated hSCP2518-532/CD1d tetramer.

Cytokine profiling

PBMCs were adjusted to 2.5 × 106/ml in the culture medium and stimulated

with the PE-conjugated anti-human CD3 antibody (20 μl/1 × 106 cells) and anti-human

CD28 antibody (final concentration, 3.5 μg/ml) (BioLegend) in the presence of GolgiStop

(BD Pharmingen) for 16 h at 37 ºC according to the manufacturer’s instruction. The cells

were then stained with 10 µl of the APC-conjugated hSCP2518-532/CD1d tetramer or the

APC-conjugated α-GalCer/CD1d tetramer (Proimmune, Oxford, UK) and then fixed with

a fixation buffer (BioLegend, Cat. No. 420801). After being washed twice with a

permeabilization buffer (BioLegend, Cat. No. 421002), the cells were stained with 10 µl

of FITC-conjugated anti-human TNF-α antibody (Miltenyi, Bergisch Gladbach,

9

Germany) or 5 μl of FITC-conjugated anti-human TGF-β antibody (BioLegend) for 30

min at room temperature. The corresponding IgG1 isotype control (BD Pharmingen) was

used for evaluation of background staining. After then washing with PBS, the cells were

analyzed using FACS Canto II followed by data processing with FACSDiva software ver.

7.0 or Attune flow cytometer (Applied Biosystems, Foster City, CA). In order to exclude

the artifact caused by dead cells in flow cytometry, we utilized LIVE/DEAD fixable dead

cell stain kit (Thermo Fisher Scientific) in some of these experiments.

Statistics

The Student’s t-test was applied for comparison of the mean values between

the two groups in the CD1d binding assay. The Mann-Whitney U-test was applied for

comparison of the two groups in flow cytometry. A p-value of less than 0.05 was

considered statistically significant.

10

RESULTS

Binding of hSCP2518-532 to CD1d

Initially, we determined the binding of hSCP2518-532 to CD1d using hSCP2518-

532B (Fig. 1a). For this purpose, CD1d monomer (molecular mass, 37 kDa) and hSCP2518-

532B (molecular mass,

11

(Fig. 2a). Since the CD3-positive hSCP2518-532/CD1d tetramer-binding cells did not

express the iNKT-specific TCR Vα24-Jα18 (Fig. S1), the possibility of these cells as

iNKT cells could be ruled out. The percentage of hSCP2518-532/CD1d tetramer-binding

cells (SCP2/CD1d tetramer-binding type II NKT cells) in CD3-positive T cells ranged

from 0.27% to 0.58% in healthy individuals (0.43 ± 0.16%). Interestingly, the percentage

was significantly higher in vasculitic patients (1.57 ± 0.74%) than in the healthy controls

(p

12

tetramer did not exhibit non-specific binding to dead cells (data not shown).

Lastly, we compared the cytokine profile between healthy donors (n=8) and

patients with vasculitis (n=5), such as microscopic polyangiitis and Takayasu arteritis

(Figs. 3b and 3c). All patients with vasculitis were actively suffering from the disease.

The percentage of TGF-β-producing cells in SCP2/CD1d tetramer-binding type II NKT

cells was significantly lower in vasculitic patients than in healthy controls (p

13

DISCUSSION

CD1d-restricted NKT cells are classified into type I and type II subsets [2]. The

type I subset is defined by a conserved expression of TCR α-chain (Vα24-Jα18 in humans

and Vα14-Jα18 in rodents), whereas type II subsets have a more variable TCR repertoire.

The physiological roles of these cells can be assessed using Jα18-deficient mice, which

specifically lack the type I subset, and CD1d-deficient mice, which lack both type I and

type II subsets [16]. The roles of CD1d-restricted type II NKT cells can be speculated by

the difference between Jα18-deficient mice and CD1d-deficient mice. However, a

technically sound method to detect human CD1d-restricted type II NKT cells has not been

established.

Although the optimized multicolor immunofluorescence panels to identify

human NK cells have been published [17-20], an attempt to detect CD1d-restricted type

II NKT cells without sorting NK cells is still challenging. Recent studies have revealed

that these cells could recognize a kind of sphingophospholipid sulfatide, which is

abundant in the myelin sheaths of the central nervous system [21]. Based on this evidence,

sulfatide-loaded CD1d tetramer has been initially used to detect CD1d-restricted type II

NKT cells. However, this approach is limited due to the difficulty in the preparation of

ligand-loaded CD1d tetramers to detect CD1d-restricted type II NKT cells specifically.

Hence, the development of a new strategy to detect CD1d-restricted type II NKT is

required.

In our earlier study, we identified rSCP2518-532, FFQGKLKIAGNMGLA, as the

epitope, which was recognized by one of rat CD1d-restricted type II NKT cells [10]. In

this study, we attempted to detect human CD1d-restricted type II NKT cells in peripheral

blood using hSCP2518-532, FFQGPLKITGNMGLA, the human counterpart of rSCP2518-

532, together with a CD1d tetramer in flow cytometry.

14

In the beginning of this study, we determined the binding of hSCP2518-532 to

CD1d. Since there was competitive binding with α-GalCer, we thought that hSCP2518-532

could be presented by CD1d adequately. Even though α-GalCer competed with hSCP2518-

532B more than 10 times, the effect was still limited. This may reflect the difference in the

binding formation of peptide-CD1d and glycolipid-CD1d. X-ray analyses have

demonstrated that peptides can bind into the cleft between the α1 and α2 helixes of CD1d

like the binding to MHC. Whereas glycolipids, such as α-GalCer, deeply penetrate A' and

F' pockets of CD1d [22]. There is still another possibility of the higher affinity of

hSCP2518-532 than α-GalCer to CD1d.

Next, we detected SCP2/CD1d tetramer-binding type II NKT cells in peripheral

blood of healthy donors and noted that its prevalence in vasculitic patients was

significantly higher than in healthy donors. We also noted the anti-inflammatory property

of SCP2/CD1d tetramer-binding type II NKT cells but not α-GalCer/CD1d tetramer-

binding type I NKT (iNKT) cells in healthy donors. The comparison of cell surface

markers between SCP2/CD1d tetramer-binding type II NKT cells and iNKT cells has not

yet been completed. These are critical tasks in our future studies.

Since the antigen-presenting molecule CD1d can present diverse hydrophobic

antigens, there seems to be diversity among CD1d-restricted type II NKT cells. Although

it is expected that these heterogeneous CD1d-restricted type II NKT cells assist in

regulating inflammation, further studies are needed to determine if they can react with

antigens other than SCP2 and also exhibit an anti-inflammatory property.

It remains unclear whether the decrease in rate of TGF-β-producing cells in

SCP2/CD1d tetramer-binding type II NKT cells is a cause or a result of vasculitis.

Concerning this issue, we examined the cytokine profile of SCP2/CD1d tetramer-binding

type II NKT cells obtained from one of the vasculitic patients who started to experience

remission after treatment. Remarkably, in this patient, the rate of TGF-β-producing cells

15

in SCP2/CD1d tetramer-binding type II NKT cells was comparable to the control level

(unpublished results). Although further studies are needed, we believe that the reduced

TGF-β production of SCP2/CD1d tetramer-binding type II NKT cells in patients with

active vasculitis may be a result of the disease and, therefore, reversible.

There are some critical limitations in this study. First, we have to note the

disparity in age between the vasculitic patients and healthy controls. Second, most data

presented in this manuscript were obtained from samples containing dead cells. Although

we have confirmed that there is no significant difference in the results regardless of the

presence of dead cells in samples, using the LIVE/DEAD fixable dead cell stain kit, we

have to improve the protocol in our future studies. Third, the percentage of SCP2/CD1d

tetramer-binding type II NKT cells in peripheral blood is very small. Therefore, we have

to pay particular attention to avoid measuring noise. Especially, we have to realize the

possibility of an overestimation for the rates of TGF-β-expressing cells. Further studies

will be crucial to confirm our findings.

This pilot study demonstrates the practicability of detection and cytokine

profiling of SCP2/CD1d tetramer-binding type II NKT cells in peripheral blood with a

limited sample of patients. Although further studies are needed to clarify the pathogenic

role of SCP2/CD1d tetramer-binding type II NKT cells, these cells in peripheral blood

should be examined in more detail for better understanding of the pathophysiology of

vasculitides in humans. Moreover, recent reviews on type II NKT cells suggest the critical

involvement of the cells in microbial immunity, inflammation, autoimmunity, and cancer

[23, 24]. The method to detect autoreactive type II NKT cells could be applicable to

investigate the pathophysiology of diverse diseases as well as vasculitides.

16

AUTHOR CONTRIBUTIONS

Designed the study and wrote the manuscript: YN, UT, and AI. Performed the

experiments: YN and TS. Provided the patient samples: HS, YK, FH, ST, RU, DN, and

TA. Analyzed the data: YN, TS, HS, YK, FH, ST, RU, MY, YN, SM, UT, and AI.

FUNDING

This work was supported by a Grant-in-Aid from the Ministry of Education, Culture,

Sports, Science and Technology of Japan with grant number 26293082 (AI).

Conflict of interest statement

The authors declare no competing financial interest.

17

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Table 1. Demographics of vasculitic patients and healthy controls enrolled in this study

Patient No. Age Sex (F/M) Diagnosis

1 67 M Microscopic polyangiitis

2 79 F Microscopic polyangiitis

3 77 M Microscopic polyangiitis

4 50 F Microscopic polyangiitis

5 64 F Takayasu arteritis

Healthy controls (n=8) 22-51 F/M=3/5

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Figure 1. Binding of hSCP2518-532 to CD1d

(a) Binding of hSCP2518-532B to CD1d. (b) The binding of hSCP2518-532B to CD1d was

competed by non-biotinylated hSCP2518-532 and α-GalCer. The relative binding of

hSCP2518-532B to CD1d was determined as follows: {(Sample O.D.) - (Blank O.D.)} /

{(No competitor O.D.) - (Blank O.D.)} × 100. Data are presented as mean ± standard

deviations of 3 independent experiments. *p

22

Figure 2. Detection of SCP2/CD1d tetramer-binding type II NKT cells in flow

cytometry

(a) PBMCs of healthy donors were stained with PE-conjugated anti-CD3 antibody and

APC-conjugated CD1d tetramer loaded with or without SCP2 peptide. At first, cells gated

23

in lymphocytes were selected based on the forward scatter (FSC) and side scatter (SSC).

CD3-positive cells within the lymphocyte gate were assayed for the binding to the CD1d

tetramer loaded with or without SCP2 peptides. A few of the CD3-positive cells bound to

the hSCP2518-532/CD1d tetramer. CD1d tetramer without peptide-loading and with the

SCP2 peptide control, hSCP2526-540, were not strongly bound to CD3-positive T cells. The

representative profiles are shown. (b) The representative FACS profiles of PBMCs of a

vasculitic patient. (c) Percentage of hSCP2518-532/CD1d tetramer-binding cells

(SCP2/CD1d tetramer-binding type II NKT cells) in CD3-positive cells in healthy donors

and vasculitic patients. *p

24

Figure 3. Cytokine profiling of SCP2/CD1d tetramer-binding type II NKT cells

PBMCs stimulated by CD3 and CD28 antibodies were then stained with APC-conjugated

hSCP2518-532/CD1d tetramer followed by fixation. After permeabilization of the plasma

membrane, the cells were stained with the FITC-conjugated anti-human TNF-α antibody

25

or FITC-conjugated anti-human TGF-β antibody. At first, cells gated in lymphocytes were

selected based on the characteristic FSC/SSC profile. Next, CD3-positive cells within the

lymphocyte gate were assayed for the binding to the CD1d tetramer loaded with or

without SCP2 peptides, using the FSC channel as previously described [5]. Subsequently,

hSCP2518-532/CD1d tetramer-binding cells were gated for the assessment of the

intracellular cytokine staining. (a) Representative profiles of CD3-positive cells in

PBMCs, hSCP2518-532/CD1d tetramer-binding cells in the CD3-positive cells, and TNF-

α-producing and TGF-β-producing cells in the SCP2/CD1d tetramer-binding cells in a

healthy donor. (b) The corresponding cytokine profiles of CD3-positive hSCP2518-

532/CD1d tetramer-binding cells in a vasculitic patient. The representative FACS profiles

are shown. (c) Comparison of the percentage of TNF-α-producing and TGF-β-producing

cells in SCP2/CD1d tetramer-binding type II NKT cells between healthy donors (n=8)

and vasculitic patients (n=5). Experiments were carried out using the corresponding IgG1

isotype control (BD Pharmingen) for evaluation of background staining. The data are

shown as the corrected data from which the background was subtracted. *