A conserved epitope on H+,K+-adenosine triphosphatase of parietal cells discerned by a murine gastritogenic T-cell clone

A Conserved Epitope on H+,K+-Adenosine Triphosphatase of Parietal Cells Discerned by a Murine Gastritogenic T-Cell Clone AKIYOSHI NISHIO,*vf MASAMICHI HOSONO,§ MINORU OKUMA,* and TOHRU MASUDA’

YOSHIHIKO

WATANABE,”

MASAHIKO

SAKAI,”

*First Department of Internal Medicine, ‘Department of Immunobiology, Institute for Immunology, Faculty of Medicine, *Department of Immunology, Chest Disease Research Institute, I’Faculty of Pharmaceutical Science, and !Department of Gastroenterological Endoscopy, Faculty ofMedicine, Kyoto University, Kyoto, Japan

Bac&round/Aims: H*,K+-adenosine triphosphatase (H’,K’-ATPase) of parietal cells is an organ-specific enzyme recognized by autoantibodies found in human and murine autoimmune gastritis (AIG). Murine AIG can be induced in BALB/c mice by thymectomy 3 days after birth and is a T cell-mediated disease. This study examined the specificity of T cells that cause AIG and the role of H+,K’-ATPase in this disease. Methods: From an AIG mouse, a gastritogenic T-cell clone (11-6) was established, and its reactivity to synthetic peptides of H+,K’-ATPase was tested. Results: II-6 cells are CD4’, vp14+, and interferon gamma producers. Adoptive transfer of II-6 cells to syngeneic nude mice resulted in AIG without the production of autoantibodies to parietal cells. The II-6 cells were responsive not only to murine but also to human and porcine parietal cells. Their proliferation was also induced by amino acids 891-905 (CX& and 892-906 (a& of the IX subunit of porcine and human H’,K+-ATPase, respectively. Conc/usions: The T-cell response to a single epitope of H+,K’ATPase, the amino acid sequence of which is conserved among at least three mammals tested, is sufficient to cause AIG. Autoantibodies to parietal cells are not detected in these AIG mice.

adenosine

triphosphatase

bulovesicular the effector been

In this the

suggested

study,

primary

Na+,K+-ATPase, despite

high

mer. We isolated mouse

and

using

utoimmune cious

immune

diseases

Hashimoto’s

parietal

including

AIG

cells from

infiltration

rietal

cells.’ The murine of CD4+

diseased

Murine

and production disease

to syngeneic

T cells

mouse.*-’

mice are directed

birth in that

the gastric

it can be transferred

associated

but

not

Moreover, to either

with

perni-

of organ-specific

type I diabetes

3 days after human

cytic

tion

(AIG)

is a paradigm

thyroiditis.*

thymectomy resembles

gastritis

anemia

(3d-Tx) there mucosa

AIG

auto-

mellitus’

and

induced

by

in BALB/c is selective

mice loss of

corre-

H+,K+-ATPase homology

a responsible the

peptides

on a comparison

T-cell

epitope

synthetic

in AIG

with

clone

of and

autoantigenicity

structural

identified

H+,K+-ATPase

lacks

as has

et al.“’

eight

based between

which

having

in tu-

Therefore,

H+,K+-ATPase,

we synthesized

structures

present

cells.‘-”

by Alderuccio

to H+,K+-ATPase

the for-

from an AIG

on the

cx subunit

of

peptides.

Materials and Methods Mice and Thymectomy Male and female mice were maintained at the Experimental oto University. underwent

BALB/cCrSlc,

Animal Facility, as described

Histological Examination

with

conditions

normal

Ky-

BALB/c mice

previously.”

and lmmunohistochemical

examination,

were fixed in 10% formalin, tion, murine

BALB/cCrSlc

Faculty of Medicine,

Three days after birth,

thymectomy,

and stained

nuinu

under specific pathogen-free

For histological

A

of parietal

T cells may recognize

recently

sponding

(H+,K’-ATPase)

membranes

H&E.

embedded

stomachs

and

in paraffin,

For immunohistochemical

sera were obtained

from 3d-Tx

ovaries

sectioned, examina-

mice or nu/nu

BALB/c mice that had been injected with T-cell clones. Indirect immunofluorescence staining with murine sera was performed embedded

using

4-pm

in polyester

sections

of murine

stomachs

that

were

wax after being fixed with 10% neutral-

as well as lympho-

of autoantibodies is cell mediated,

to pabecause

nude mice by implantaby autoantibodies autoantibodies

the a or p subunit

of the in

AIG

of H+,K+-

Abbreviations used in this paper: AIG, autoimmune gastritis; ELBA, enzyme-linked immunosorbent assay; FITC, fluorescein is* thiocyanate; H+,K’-ATPase, H+,K+-adenosine triphosphatase; MAb, monoclonal antibody; MHC, major histocompatibility complex; Bd-Tx, thymectomy 3 days after birth. 0 1994 by the American Gastroenterological Association 00165085/94/53.00

November

1994

buffered

T-CELL

formalin.’

goat anti-mouse

Fluorescem

isothiocyanate

immunoglobulin

the second antibody

(Cappel,

(FITC)-labeled

(Ig) antibody

Durham,

2, in 96-well

was used as

NC).

formed

immunosorbent

using membrane

glandular

stomachs

extract

of epithelial

thymidine

cells of murine

Parietal

(kcpm)

prepared

prepared

in the same manner.

acetic

acid (Sigma)

nutes.

The supernatant

0.02%

incubated

and the supernatants three times, at 1200

cells were also

of syngeneic

mice

saline

the remaining

tissue

were collected.

for

This step

and the pooled cell suspension

rpm for 5 minutes. essential

was

The pellets were resus-

medium

and used as kidney

pulsed

with

before

peptides

were added

1 PCi

harvest.

of [‘HI-

The level of

using a liquid scintillation as mean kilocounts

spec-

per minute

anti-mouse

labeled anti-CD4

at 4°C for 30 minutes

Thy-l.2

(GK1.5,

(HO-13-4,

T-cell receptor

ster) or anti-FTCR

(GL3,

or FITC-labeled Francisco, (14-2,

ham-

monoclonal

incubation

with phycoerythrin-

followed

using a flow cytometer

Mountain

View, CA)

TCR was stained

rat), anti-Vp8.1/8.2

(F23.1,

with anti-

rat), or anti-VP14

by FITC-labeled

MAb (Caltag). The percentage

antibodies

Ig MAb (Caltag, South San

CA) for 30 minutes.

rat) MAbs

(HO-

(Becton Dickinson,

goat anti-hamster

VP6 (44-22-1,

anti-CD8

(TCR) (H57-597,

hamster)

by a further

labeled streptoavidin

FITC-

rat) or FITC-labeled

2.2, rat); and anti-t@

with

mouse);

goat anti-rat

Establishment of Parietal Cell-Specific T-Cell Clones Lymphocytes

(5 X lo>) from the regional

at the lesser curvature cultured

in complete

interleukin

of the stomach medium

Ltd., Osaka, Japan).

Syngeneic

source of antigen-presenting as antigens Complete

medium

consisted

node

(FACScan;

Becton

Dickinson).

Unla-

cells (1 X 10’) Seiyaku,

with 10% fetal calf serum (Gibco New Zealand),

100 U/mL

penicillin,

5 X 10pr mol/ and

100 j.lg/mL

streptomycin.

Cells were passaged

every 7 days for 2 weeks

together

with

irradiated

parietal

Limiting

dilution

were screened

to parietal

syngeneic

by the proliferation

cells. Finally,

cells were established with the synthetic

eight

clones

cells.

and growing

assay for reactivity

responding

peptides

a,,,

to the peptide.

eta1 cell-responding

T-cell

, as described

below. Among

in this study because of its

From another clones,

but

cell line, no pariseveral

clones

sponding to syngeneic spleen cells, were obtained. them, III-l, was used as a control T-cell clone.

One

reof

The assay was performed T cells,

supernatants

cells in 2 mL of medium collected

from 5 X 10’ responder

in a 24-well

in triplicate

culture

after 48 hours and assayed for interferon

assay was based on the antiviral by the reduction

stomatitis

virus on L cells, described

expressed

in reference

reference

murine

4 production anti-mouse

in the cytopathic

units

interferon

or recombinant

were

gamma.

The

alp

as

effect of vesicular

elsewhere.”

as calibrated

The titer was

against

(G002-904-5

was assayed by costimulation IgM antibody

T

plate

activity of the supernatants,

measured

the NIH

11). Interleukin using F(ab’ )L goat

(Cappel) and either the supernatants

interleukin

4 (Sigma)

as the standard.

Small

B cells used for this assay were separated

from spleen cells of

BALB/c nude mice. Details

elsewhere.”

are described

Disease Transfer Disease transfer was studied II-6 cells (2 X 10’) stimulated or an equal number

by culturing syngeneic

5 X spleen

cells, and various numbers of irradiated parietal cells in a total volume of 200 PL of complete medium without interleukin

by intravenously

injecting

with parietal cells for 48 hours

of III-1 cells stimulated

only with synge-

neic spleen cells, into six BALBic nude mice (two male and four female, respectively) at the age of 6 weeks. Recipient mice were killed 1 month later, and the stomachs, ovaries (from nohistochemically

5 X lo5 irradiated

(Rockville,

T-cell prolifera-

Cytokine Assay

female mice), and sera were examined

Proliferation Assay 10’ responder

Collection

to parietal

from one cell line; three of them reacted

these three, clone II-6 is described high reactivity

and spleen

of two cell lines was performed,

Type Culture

and were

tion.

The culture

Co.

cells (2.5 X 10”) as a

of RPM1 I640 (Nissui

from American

were used in ascitic form for blocking

recombinant

Takeda Pharmaceutical

spleen

Ltd., Auckland,

L 2-mercaptoethanol,

lymph

at 20 Gy and added to the culture.

Tokyo, Japan) supplemented New Zealand

human

cells and parietal

were irradiated

MD),

of an AIG mouse were

containing

2 (2.5 ng/mL, TGP-3;

obtained

Ig

of stamed cells was determined

beled antiCD4, antiCD8, anti-I-A”,“,“,” (25-9-17S), anti _I_Ek,d.r.’ (14-4-48) MAbs, the last two of which

cells.

colonies

concentrations

? SD.

(MAbs) followed

for 15 mi-

in the same enzyme solution

I5 minutes,

in minimum

2-3-month-old

ethylenediaminetetra-

was discarded,

was repeated pended

were

was determined

biotinylated

with 0.2% trypsin (Sigma Chemical

in phosphate-buffered

blocks were further

centrifuged

from parietal

The kidneys

Co., St. Louis, MO) containing

1409

Flow Cytometry and Antibodies

and porcine

were minced and digested

which

various

Cells (10”) were incubated

were

Human

GASTRITIS

plates for 48 hours. When

All data are expressed

as the antigen.”

cells

mice.”

AUTOIMMUNE

for the last 6 hours

trometer.

Cell Preparation BALB/c

microtiter

cultures,

radioactivity

assay (ELISA) was per-

IN MURINE

were used as the antigen, to the

Enzyme-Linked lmmunosorbent Assay Enzyme-linked

EPITOPE

histologically

for AIG and for autoimmune

and immuoophoritis.

Peptide Synthesis Because autoantibodies to parietal cells are directed to Hf,Kf-ATPase molecules, eight positions consisting of 15-

1410

amino acid residues mary structures

each were chosen by comparing

of the TVsubunit

and the p subunit human tural

GASTROENTEROLOGY Vol. 107, No. 5

NISHIO ET AL.

or murine homology

of murine

Peptides

on a peptide

despite

were synthesized

technique

Biosystems

Inc., Foster City, CA) and purified

tides synthesized ATPase

synthesizer

and the p subunit

were as follows (single-letter

VELGPGPSGDMAAKM;

amino

R+APC+PC (1X10’) R+APC+Kid (1X104)

Pep-

0

of murine H+,K+acid code): a,,,

AKMSKKKAGRGGGKR;

a,,,

, LLCVGLRPQWENHHL;

px4, LRPDVYGERGLKISY;

R+APC+PC (SXlm)

of the IX subunit

a,, ,

ctc,x4, DPSELVEALRTHPEN;

H I

a

on a reversecolumn.

A

b

R+APC

of

using

1

R

with

(430A; Applied

liquid chromatographic

on the basis of the structures

of porcine H+,K+-ATPase

either

the absence

solid-phase

phase high-performance

-II-6

which has high struc-

H+K+-ATPase

in AIG.

the pri-

H+,K+-ATPase’*

H+,K+-ATPase’>

Na+,K+-ATPase,‘“.” with

autoantigenicity

of porcine

p, 11, SEKYFFQESFAAPNH;

PQKPRKDTEPLQVEY; and pz73, VTFNNPP *,,, , HDPYEGKVE. Peptides t&92 (LLCVGLRAQWEDHHL) and Na-a,,,

(GNLVGIRLNWDDRTV)

sis of the a subunit subunit

of human

of human

were produced H+,K+ATPase”

Na+,K+-ATPase,

ber of each peptide in the N-terminal

on the baand the a3

respectively.‘”

means the position

The num-

of amino acid residues

side.

Results

0

2

4

6

6

10

0

2

4

6

6

IO

Ill-1 -

Establishment of Parietal Ceil-Specific T-Cell Clones Eight

clones

were established

from an AIG mouse.

A representative clone, termed 11-6, responded not only to murine but also to human and porcine parietal cells, resulting

in proliferation

(Figure

1). The lack of stimula-

tory activity in syngeneic kidney cells indicates the organ specificity of the response observed. The remaining clones showed substantially the same results as 11-6. Furthermore, all of these clones expressed CD4 and @TCR produced

interferon

gamma,

indicating

and

that these cells

belong to T-helper 1 cells (data not shown). Three clones, including 11-6, were Vp14+. A control clone, III-l, established

from another

cell line, did not respond

to pari-

eta1 cells but to syngeneic spleen cells in vitro (Figure 1). These cells also expressed CD4 and produced interferon gamma (data not shown).

Induction of AIG by II-6 Cells but not Ill-l All of the nude mice (two male and four female) that received II-6 cells developed gastritis characterized by severe infiltration of mononuclear cells coupled with the destruction of parietal cells and hyperplasia of mucous neck cells (Figure 2A). These features are characteristic of AIG in Sd-Tx mice.’ However, autoantibodies to parietal cells were undetectable by both indirect immunofluorescence staining and ELISA. Moreover, autoimmune oophoritis was not induced in female mice. In contrast,

-I

[3H]Thymrdineuptake (kcpm) Figure 1. Proliferative responses of the parietal cell-specific T-cell clone II-6 to increasing numbers of (A) murine, (B) porcine, and (C) human parietal cells and (0) the control T-cell clone Ill-l to murine parietal cells. Responder T cells (R), 5 x 104, were incubated with 5 x lo5 syngeneic spleen cells (antigen-presenting cells [APC]) and various numbers of parietal cells (PC) irradiated at 20 Gy for 2 days at 37°C. The cultures were pulsed with 1 pCi of [3Hjthymidine per well for the last 6 hours before harvest. The results shown are mean counts of triplicate samples ? SD. Controls were responder T cells alone and responders plus syngeneic spleen cells with or without 1 x lo* syngeneic kidney cells (Kid).

transfer of III-1 cells into nude mice did not cause AIG (Figure 2B).

Responsiveness of II-6 Cells to Synthetic Peptides The II-6 cells were tested in an in vitro assay for reactivity to the eight peptides that were synthesized according to the primary structures of the cx subunit of porcine H+,K+-ATPase and the p subunit of murine H+,K+-ATPase, respectively, and compared with those

November 1994

T-CELL

EPITOPE IN MURINE AUTOIMMUNE

GASTRITIS

1411

Figure 2. Parietal cell-specific T-cell clone II-6 but not control T-cell clone Ill-l can transfer gastritis to BALB/c nude mice. Stomach sections of a nude mouse with transfer of (A) II-6 cells or (8) Ill-l cells. Six-week-old nude mice were intravenously injected with 2 x lo7 II-6 cells that were activated by parietal cells for 2 days or with an equal number of Ill-l cells activated only by syngeneic spleen cells, then the mice were killed 1 month later. There was severe lymphocytic infiltration and destruction of the gastric gland in the mouse with transfer of II-6 cells (original magnification 200x).

of human Figure

and murine

Na+,K+-ATPase.

3, two peptides,

proliferation

As shown

CX~~,and CX~~~,stimulated

of II-6 cells. However,

the proliferative

sponses of spleen cells from AIG mice to a,,,

in

also, the primary

the

ATPase

re-

Na+,K+-ATPase.

or ass2 were

negligible (data not shown). The molecular structure of Na+,K+-ATPase is similar to that of H+,K+-ATPase;

possesses

Pig HK-ATPase ((I~~)

-

Human HK-ATPase (us&

+

Human NaK-ATPase (Na-(hll)

about

of the cx subunit 60%

T o confirm

II-6 cells to H+,K+-ATPase, to peptide

Na+,,

homology the antigen

of H+,K+with

that

of

specificity

of

the proliferation

of Na+,K’-ATPase

response

that corresponds

to asp1 and a,,, in position (Figure 4) was tested. As shown in Figure 3, Na+,, did not induce the proliferation of 11-6. The proliferative

-

structure

response

was blocked

by anti-I-Ad,

in addition to anti-CD4, antibody (Figure 5); this was true when parietal cells were used as the antigen (data not shown).

Pig H/K ATPase (a,;891 -905) 0 01

0.1

1

10

Pepbde concentrah9n (wg/ml)

Figure 3. Proliferative responses of II-6 cells to synthetic peptides of H’,K’-ATPase and Na’,K’-ATPase. II-6 cells were cultured with increasing doses of synthetic peptides ccsg2,asgl, and NacL,,,. The [3H]thymidine incorporation was measured in the same manner as described in Figure 1.

Human Na/K ATPase (Na-a&871-885) Synthetic peptide Figure 4. Amino acid sequences of synthetic peptides ass2, a,,,, and NaasT1. Identical or homologous amino acid residues among three peptides are boxed.

1412

GASTROENTEROLOGY Vol. 107, No. 5

NISHIO ET AL.

snmulatol

Anubodin

parietal

and the amino acid sequences

cells,‘-”

of H+,K+-

ATPase are highly conserved in mammals including humans, pigs, and mice.x~‘4~‘s~‘x~“-24 We found that a Tcell clone, 11-6, is responsive to human

and porcine

6 cells recognize -I

various mammals.

io

2i (?iIThymdme

uptake (kcpm)

Discussion Murine AIG induced by 3d-Tx is a typical model of T cell-mediated autoimmune diseases,4m” in which endogenous,

but not exogenous

antigens

tal autoimmune encephalomyelitis,” evoke the disease. Many investigations to the cellular

mechanism

of AIG, but little of the pathogenic

is known

spontaneously have been directed

parietal

observed

that an intrathymic

cell population

specificity

T cells that can induce response of parietal

mice followed by 3d-Tx induces

that

parietal

presumed

identified

cells of various mammals,

with

that of the homologous

recog-

was consistent of II-6

cells to

because peptides

portion

CX,,,

of the cx subunit

of rat H+,K+-ATPase.

Alderuccio

3d-Tx mice transfected

with a gene encoding

unit of H+,K+-ATPase

showed tolerance

leading to an escape from epitopes on the p subunit However,

our observation

multiple

epitopes

H+,K+-ATPase

et al. reported

indicates

on both

that

recognition

of

p subunits

of

stimulated

the epitope

determinant

We

encephalomyelitis2’

Therefore,

of gastritogenic give a clue

it is not

by II-6 cells is

in the activation shown

and

of

in experimental

nonobese

diabetic

T cells reactive with analysis of

parietal cells in autoreactive T cells, thus preventing AIG but not autoimmune oophoritis.20 This suggests that T

the p subunit

cells that respond to parietal cells, eliminated or inactivated in the thymus by the stimulation of autoantigens in parietal cells, play a central role in the pathogenesis

It is notable that the epitope recognized by II-6 cells is located in the lumen of the tubulovesicles in parietal cells.”

of AIG. In the present study, we show that AIG is caused by these T cells, although autoantibodies to parietal cells

exposed to the immune system because Thus, how antigen epitopes are presented

were undetectable. This implies that T-helper 1 cells cause tissue destruction, perhaps by a delayed type hypersensitivity mechanism and that autoantibodies to parietal

T cells is a debatable problem. It is well understood that CD4+ T cells recognize antigen epitopes in the context of major histocompatibility complex (MHC) class II. One possible explanation is that parietal cells omitted from

cells are not necessary for the onset of the disease. The failure to produce anti-parietal cell autoantibodies in mice that received II-6 cells can be explained by the absence of T-helper 2 cells involved in the production of autoantibodies to parietal cells. The specificity and role of T-helper 2 cells in the pathogenesis of AIG remains to be elucidated. Anti-parietal cell autoantibodies in murine AIG are directed to either the TV. or p subunit of H+,K+-ATPase in

will

of AIG.

spleen cells from

recognized

T cells, as recently

mice. I6 Isolation

cells,

the cx and

AIG mice at the age of 3 months.

allergic

to parietal

is not necessary for the induction

CZ,,, only negligibly

yet clear whether

that

the p sub-

AIG.“’ This suggests that are also involved in AIG.

or cryptic

to

of the cx

and a892 are identical in all but one of 15 amino acids. Furthermore, the amino acid sequence of aa,, is identical

autoreactive

tolerance

aa,,

are specifically

by the responses

a dominant

cells

H+,K+-ATPase.

and peptide

nized by II-6 cells. The epitope

cells of

that the T

~~~~ of the a subunit

H+,K+-ATPase

to an

has been shown.” injection

H+,K+-ATPase

Peptide

in the pathogenesis

a T cell-mediated

enriched

as in experimen-

about the antigen

autoreactive

the disease. However,

in newborn

involved

of porcine

with

suggest

the disease recognize

we showed that peptide

that II-

in parietal

These observations

of human

but also

cells, indicating antigen

In addition, subunit

Figure 5. Inhibitory effects of anti-CD4 and anti-MHC class II MAbs on the proliferative responses of II-6 cells. The [3Hjthymidine incorpo ration in response to the final concentration of peptide a,,, (1 ug/ mL) and irradiated syngeneic spleen cells was measured in the presence of an optimal concentration of monoclonal anti-CD4 (GK1.5), anti-CD8 (HO-2.2). anti-l-A (25-9-173) and anti-l-E (14-44s) antibodies, as described in Figure 1.

parietal

the common

cells that induce lb

not only to murine

immunodominant

Therefore,

epitopes

to further

on H+,K’-ATPase.

this sequence

would

not be directly of its location. to autoreactive

the gastric gland by cell turnover are processed by professional antigen-presenting cells, thereby activating autoreactive T cells. Alternatively, cytokines released by inflammatory cells infiltrating into the gastric mucosa by local nonspecific inflammation may induce expression of MHC class II molecules on parietal cells, leading to presentation of autoantigen to responsible T cells by parietal cells themselves. Indeed, aberrant expression of MHC

November 1994

T-CELL EPITOPE IN MURINE AUTOIMMUNE GASTRITIS

class II on rat parietal

cells was induced

of interferon

gamma.“’

the affected

gastric

molecules

by administration

Furthermore, it is reported that epithelium expresses MHC class II

in AIG mice.”

Identification

of the causative

antigens

diseases will help the development therapeutic

strategies.

has been recently

9. Jones CM, Callaghan JM, Gleeson PA, Mori Y, Masuda T, Toh BH. The parietal cell autoantibodies recognized in neonatal thymectomy-induced murine gastritis are the a and 8 subunits of the gastric proton pump. Gastroenterology 1991; 101:287294.

10. Alderuccio F, Toh BH. Tan SS, Gleeson PA, van Driel IR. An auto immune disease with multiple molecular targets abrogated by the transgenic expression of a single autoantigen in the thymus. J Exp Med 1993; 178:419-426.

of successful immuno-

Peptide-mediated

proposed

in autoimmune immunotherapy

for experimental

allergic

en-

11. Mori Y, Hosono M, Murakami K, Katoh H, Yoshikawa Y, Kuriba yashi K, Kannagi R, Sakai M, Okuma M, Masuda T. Genetic studies on experimental autoimmune gastritis induced by neonatal thymectomy using recombinant inbred strains between a highincidence strain, BALB/c, and a low-incidence strain, DBA/2. Clin Exp lmmunol 1991;84:145-152.

cephalomyelitis.29330

However, it is necessary to know the and agretope residues of an antigenic peptide. In

epitope

the present

study,

peptide

did not stimulate homology This

with

H+,K+-ATPase

information

epitope

Na-a,,,

of Na+,K+-ATPase

II-6 cells despite provides

and agretope

its high

and

a means

residues

structural

Na+,K+-ATPase.

of determining

on CX~~,. Further

the studies

of this issue are in progress. Taken

together,

murine

tive T cells specific

AIG is induced

for H+,K+-ATPase.

pathogenesis of the murine of the human counterpart,

by autoreacWhereas

the

disease may differ from that our findings in the murine

model suggest that the human disease is also mediated by T cells specific for H+,K+-ATPase. It is necessary to investigate ATPase

the autoreactive

in patients

T-cell

response

to H+,K+-

with this disease.

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Received April 6, 1994. Accepted July 11, 1994. Address requests for reprints to: Tohru Masuda, M.D., Department of Immunobiology, Institute for Immunology, Faculty of Medicine, Kyoto University, Yoshida-konoe, Sakyo, Kyoto, 606, Japan. Fax: (81) 757534347. Supported by a grant-in-aid from the Ministry of Health and Welfare, Japan, and the Shimizu Foundation Research Grant 1993.