539
Characterization of H+,K+-ATPase T cell epitopes in
human autoimmune gastritis
Mathijs P. Bergman1, Amedeo Amedei2, Mario M. D’Elios2, Annalisa Azzurri2, Marisa
Benagiano2, Carlo Tamburini2, Ruurd van der Zee3, Christina M. VandenbrouckeGrauls1, Ben J. Appelmelk1 and Gianfranco Del Prete2
1
Department of Medical Microbiology and Infection Control, VU University Medical Center,
Amsterdam, The Netherlands
Department of Internal Medicine, University of Florence, Florence, Italy
3
Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht
University, Utrecht, The Netherlands
2
Human autoimmune gastritis (AIG) is an organ-specific inflammatory disorder leading to
gastric atrophy and pernicious anemia. Gastric H+,K+-ATPase was identified as the autoantigen in both human disease and experimental murine AIG (EAIG). Studies of EAIG significantly contributed to current knowledge of human AIG, but to what extent EAIG mimics AIG
is still debated, and the autoantigenic epitopes in AIG are yet unknown. This study aimed to
identify the H+,K+-ATPase epitopes recognized by gastric T cell clones from AIG patients, to
define their TCR V g usage and epitope-induced cytokine response. Sixteen H+,K+-ATPasereactive CD4+ gastric T cell clones of four AIG patients were tested for proliferation to overlapping 15-mer peptides spanning the § and g chains of H+,K+-ATPase. We identified 6 epitopes in the § chain and 5 in the g chain; TCR V g usage was not restricted. Four (36%) of the
11 H+,K+-ATPase epitopes recognized in AIG were found to overlap with epitopes that are
relevant in EAIG, including a previously described gastritogenic epitope. Gastric T cell recognition of the peptide epitopes resulted in secretion of Th1 cytokines. Our data suggest a
striking similarity between human AIG and EAIG, at the epitope level, with regard to cytokine
secretion and likely also with regard to pathogenic mechanisms.
Key words: H ,K -ATPase / Synthetic peptides / Human gastric Th1 cells / IFN- + / Murine autoimmune gastritis
+
+
1 Introduction
Chronic autoimmune gastritis (AIG) is an organ-specific
inflammatory disease leading to hypochloridria and gastric atrophy and eventually to pernicious anemia [1]. AIG
is characterized by lymphocytic infiltrates in the mucosa
of the gastric fundus and corpus and by destruction of
parietal and zymogenic cells, resulting in mucosal atrophy [2]. In most AIG patients, either with or without
pernicious anemia, serum anti-parietal cell autoantibodies (PCA) are detectable. The target autoantigen recognized by PCA is the gastric H+,K+-ATPase, the proton
pump, localized in the parietal cell canaliculi [3, 4]. Gastric H+,K+-ATPase is also the major autoantigen in experi-
[I 23569]
The first two authors contributed equally to this publication.
Abbreviations: AIG: Autoimmune gastritis EAIG: Experimental autoimmune gastritis PCA: Parietal cell autoantibody MI: Mitogenic index
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Received
Revised
Accepted
30/9/02
27/11/02
30/12/02
mental autoimmune gastritis (EAIG), an organ-specific
autoimmune disease inducible in mice. EAIG is assumed
to represent a suitable model for the human disease. In
non-thymectomized animals, EAIG can be elicited by
immunization with either gastric mucosal extracts or
purified H+,K+-ATPase [5–7]. Neonatal thymectomy in
susceptible mouse strains also results in EAIG ([8],
reviewed in [9]). Similar to AIG patients, EAIG mice
develop autoantibodies to the catalytic § and the glycoprotein g subunits of parietal cell H+,K+-ATPase [10] and
inflammatory mononuclear cell infiltrates of the gastric
mucosa with subsequent loss of parietal and zymogenic
cells [11]. The gastric infiltrates include both CD4+ and
CD8+ T cells, macrophages and B cells [12], resembling
those observed in human AIG [2]. EAIG is mediated by
H+,K+-ATPase-specific CD4+ T cells, but not by CD8+
T cells, B cells or antibodies [9, 13].
The cytokines expressed in EAIG lesions are predominantly of the Th1 type, and neutralization of IFN- + prevents disease [12, 14]. The lack of IL-4 and the expression of IFN- + , together with the observation of a prefer0014-2980/03/0202-539$17.50 + .50/0
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H+,K+-ATPase T cell epitopes in human AIG
Eur. J. Immunol. 2003. 33: 539–545
M. P. Bergman et al.
Eur. J. Immunol. 2003. 33: 539–545
ential migration of Th1 cells into the gastric mucosa,
suggest a key role for Th1 cells in gastric pathology
associated with EAIG [15]. In human AIG, most of the
H+,K+-ATPase-specific T cell clones derived from the
gastric mucosa of patients express a polarized Th1 cytokine profile and all clones induce Fas-Fas ligandmediated apoptosis in target cells and perforin-mediated
cytotoxicity against H+,K+-ATPase presenting cells [16].
In EAIG similar mechanisms for tissue destruction have
been proposed [17].
In the present study, we determined the submolecular
epitope specificity of H+,K+-ATPase-reactive T cell
clones derived from the gastric mucosa of four AIG
patients. Recognition of the appropriate autoantigen epitope led to proliferation and cytokine production by the
clonal progeny of autoreactive gastric T cells. This study
also revealed a striking similarity between 4 out of 11
H+,K+-ATPase T cell epitopes identified in human AIG
and those relevant in EAIG, which further proves that
EAIG is an excellent model for human AIG.
2 Results
2.1 TCR V I repertoire of gastric CD4+ T cell
clones reactive to H+,K+-ATPase
A total number of 104 CD4+ and 36 CD8+ T cell clones
were recovered from the biopsy specimens of the gastric
mucosa of patients. All the CD8+ clones and 88 out of the
104 gastric CD4+ T cell clones failed to proliferate in
response to H+,K+-ATPase, whereas 16 CD4+ clones (3
from donor 1; 5 from donor 2; 2 from donor 3; and 6 from
donor 4) showed significant proliferation [mitogenic
index (MI) range: 17.7–414.0] in response to porcine
H+,K+-ATPase, but not in response to porcine albumin.
The analysis of the TCR V g expression by the 16 H ,K ATPase-specific gastric T cell clones showed a wide repertoire of V g usage (Table 1). Two clones from different
donors (2.P02 and 3.A42) expressed V g 8, whereas two
clones from the other two donors (1.A01 and 4.A33)
shared the expression of V g 6.7. Of the five H+,K+ATPase-specific clones from donor 2, one couple
expressed V g 4, and another couple expressed V g 19.
+
+
2.2 Characterization of the submolecular
specificity of T cell clones reactive to H+,K+ATPase
Eleven T cell clones proliferated in response to a single
peptide of the § or the g chain of H+,K+-ATPase. In contrast, five clones showed significant proliferation to cou-
ples of consecutive 15-mer overlapping peptides, which
suggested that the recognized epitope was partially
shared by both peptides of each couple (Table 1). The
peptide epitopes recognized by 9 out of the 16 H+,K+ATPase-specific clones were located in the smaller
g chain, whereas the epitopes recognized by the other 7
clones were in the longer § chain.
Although expressing different TCR V g (V g 6.7, V g 22 and
V g 23), three of the six clones from donor 4 recognized
the 76–90 amino acid sequence of the g chain, whereas
a fourth clone (V g 16+) from the same donor recognized a
nearby epitope, i.e. the g 81–95 amino acid sequence.
Interestingly, the same g 81–95 sequence was also recognized by a V g 6.7+ T cell clone from donor 1. Another
g chain epitope, which was recognized by the couple of
clones from donor 3 (V g 17+ and V g 8+, respectively), was
the g 231–245 amino acid sequence. Only one clone
(2.R17, V g 8+) out of the five derived from donor 2 found
its epitope in the g chain 111–125 sequence, whereas
one (1.C26, V g 13+) out of the three clones from donor 1
was specific for the g chain 166–185 sequence. Four out
of the five gastric clones from donor 2 recognized epitopes in the § chain of H+,K+-ATPase: one clone in the
§ 1–20 sequence, another one in § 151–165, and a couple
(V g 8+ and V g 19+, respectively) proliferated in response to
the § 881–900 sequence, which also comprises an epitope recognized by gastritogenic T cells in EAIG [8, 18].
Also a couple of clones from donor 4 recognized their
epitope in the § chain, at § 351–365 and § 616–630,
whereas one clone from donor 1 recognized the § 31–50
sequence. In summary, T cell clones from the AIG
patients in this study recognized five different T cell epitopes in the shorter g chain of H+,K+-ATPase, and six different T cell epitopes in the longer § chain.
2.3 Cytokine production induced by H+,K+ATPase peptides
Upon 48-h stimulation with H+,K+-ATPase in the presence of autologous APC, 14 (87.5%) out of the 16 H+,K+ATPase-specific T cell clones produced IFN- + , but not
IL-4 (Fig. 1), which is consistent with a Th1 profile. Two
clones produced both IFN- + and IL-4 (Th0 profile). Finemapping of the peptide specificity based on proliferative
response was assessed by measurement of IFN- + and
IL-4 production upon stimulation with the relevant peptides. In all gastric T cell clones, the original cytokine
profile induced by the entire H+,K+-ATPase antigen could
be elicited by stimulation with the relevant peptide,
whereas flanking peptides or medium alone failed to
induce cytokine production (Fig. 1). Furthermore,
peptide-induced cytokine profiles could be reproduced
in two selected clones (clone 4.A33 and 4.C31) on a sec-
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540
H+,K+-ATPase T cell epitopes in human AIG
541
Table 1. H+,K+ATPase epitope recognition by H+,K+ATPase-specific CD4+ T cell clones derived from the gastric mucosa of
patients with AIG
ond occasion, after storage of the clones by
cryopreservation, and with another batch of APC.
Hence, the data shown in Fig. 1 represent the mean
(+ SD) of two (clone 3.B46) and four (clone 4.A33 and
4.C31) cultures, respectively.
3 Discussion
The major finding of this study is the demonstration of
common epitopes recognized by H+,K+-ATPase-specific
T cells in both human AIG and murine EAIG (Fig. 2). This
strongly enforces the belief that murine EAIG models
reflect the pathology which occurs spontaneously in
human AIG. Moreover, we provide new insight in the
pathogenic mechanisms which underlie AIG, and for-
mally prove our previous observation that human AIG is
mediated by CD4+ H+,K+-ATPase-reactive cytotoxic
T cells in the gastric mucosa [16]. The possibility that the
H+,K+-ATPase-specific T cell clones merely reflect an IL2-induced selective expansion of a few gastric T cells is
unlikely since various T cell clones expressed a different
V g family in their TCR, and those with similar TCR V g
usage were specific for different peptides. Thus, we conclude that gastric autoimmunity is the result of a
polyclonal inflammatory response involving a variety of
H+,K+-ATPase-specific effector T cells. Autoreactive
CD4+ T cells that infiltrate the gastric mucosa express
a largely predominant Th1 cytokine profile upon stimulation with either the entire H+,K+-ATPase antigen or
the specific peptide epitopes able to elicit T cell
proliferation.
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Eur. J. Immunol. 2003. 33: 539–545
M. P. Bergman et al.
Eur. J. Immunol. 2003. 33: 539–545
support this notion, since none of the CD8+ T cell clones
recovered from the gastric mucosa of AIG patients
reported here, nor those described in a previous study
[16], showed reactivity to gastric H+,K+-ATPase.
The expression of TCR V g by the H+,K+-ATPase-reactive
T cell clones suggest that TCR V g usage in human AIG is
less restricted than in EAIG, where one TCR (V g 14) clonotype has been detected in three out of eight T cell
clones from one EAIG mouse, as well as in 43% of thymectomized BALB/c mice with gastritis [18, 19].
Although the number of T cell clones isolated from each
patient is too small to reflect the clonal diversity within
each individual, V g 4, V g 6.7, V g 8 and V g 19 appear more
commonly expressed by H+,K+-ATPase-specific T cell
clones. We found V g 6.7 and V g 8 expression by clones
from donors with different MHC haplotypes.
Fig. 1. IFN- + and IL-4 production induced by H+,K+-ATPase
or selected peptides of the § and g chains. H+,K+-ATPasespecific gastric T cell clones derived from patients with AIG
were co-cultured for 48 h with irradiated autologous APC in
the presence of medium, entire H+,K+-ATPase molecule, the
H+,K+-ATPase § or g chain peptides that induced significant
T cell clone proliferation, and a couple of flanking control
peptides that had failed to induce proliferation of the T cell
clone under study. Supernatants were assayed for their IL-4
and IFN- + content. Results of 3 representative out of 16 gastric T cell clones tested are shown. Results represent the
mean production of cytokines (± SD) in two (in case of clone
3.B46) or four (4.A33; 4.C31) separate cultures. Ruffled edge
of bars indicates cytokine production below the limit of
detection.
In murine EAIG, two H+,K+-ATPase-specific T cell lines
reactive to distinct peptides of the H+,K+-ATPase § chain
were derived from the gastric lymph nodes of neonatally
thymectomized mice [8]. Both T cell lines are
CD4+TCR § / g +, I-Ad-restricted, and equally potent in
inducing gastritis in nu/nu recipient animals, but one
T cell line secretes Th1- whereas the other one secretes
Th2-type cytokines. However, a major role has been
attributed to IFN- + [14] and to gastric mucosa-infiltrating
Th1 cells in the onset of EAIG and in its pathology [15].
The concordance between the data on cytokine profiles
obtained in human AIG and murine EAIG strongly suggests that also in humans H+,K+-ATPase-specific T cells
have gastritogenic potential.
The onset of EAIG in neonatally thymectomized mice is
abrogated by administration of anti-CD4 antibodies, but
not anti-CD8 antibodies [13], suggesting that CD8+
T cells are not essential in EAIG. Our data in humans
Since recombinant human H+,K+-ATPase was not available, we used purified pig gastric microsomes [20] containing 80–90% H+,K+-ATPase, to screen gastric T cell
clones in our previous study [16]. The proton pump specificity of thus identified T cell clones was formally proved
by their proliferation to appropriate synthetic H+,K+ATPase peptides in the present study. Recognition of porcine H+,K+-ATPase epitopes by human T cells is possible,
since there is 98% homology between the gastric H+,K+ATPase of the two species. Of the 1,035 amino acids of
the human § chain, 1,015 are shared by the porcine
H+,K+-ATPase § chain [21], and murine T cell clones able
to recognize corresponding peptides from either murine,
pig, or human H+,K+-ATPase have been reported [9, 18].
In mice, multiple immunization with overlapping peptides,
and subsequent epitope mapping, have shown that the
g 261–274 peptide is a dominant gastritogenic g chain
T cell epitope of H+,K+-ATPase, but other immunogenic
peptides of the g chain were also identified [7, 22].
§ Chain T cell epitopes have also been reported in EAIG.
The disease can be transferred by T cell lines reactive to
peptides § 633–641 or § 889–899 of the § chain [8, 18].
Importantly, § chain peptides § 891–905 and § 892–906
of porcine and human H+,K+-ATPase, respectively, but
not their homologous counterpart in ubiquitously
expressed human Na+,K+-ATPase, are recognized by a
single murine T cell clone [18]. This suggests that the
organ-specific nature of (E)AIG is antigen dependent. In
AIG patients, 9 out of the 16 H+,K+-ATPase-reactive
T cell clones recognized a peptide epitope within the
290-amino acid g chain, whereas 7 clones reacted to
peptides of the 1,034-amino acid § chain (Table 1). Quite
surprisingly, the epitope § 881–900, which was recognized by both clones 2.P02 and 2.P14 (expressing different TCR V g ), overlaps the § 889–899 amino acid
sequence that is the critical epitope for the TXA51 gastri-
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542
H+,K+-ATPase T cell epitopes in human AIG
543
Fig. 2. Submolecular positions of H+,K+-ATPase § and g chain T cell epitopes identified so far in human AIG (A, lower panel) and
murine EAIG models (A, upper panel), and detailed alignment of common epitopes of § chain (B) and g chain (C) involved in AIG
and EAIG. T cell epitopes in human AIG (black text on light gray background) are all based on porcine H+,K+-ATPase and identified in this study. T cell epitopes in EAIG (white text on dark gray background) were derived form H+,K+-ATPase of the indicated
origin. Boxes indicate overlapping T cell epitopes and corresponding sequences of human H+,K+-ATPase. References are in
parentheses.
togenic T cell line in BALB/c mice [8]. TXA51 line
(TCR V g 4+) also shows reactivity to pig H+,K+-ATPase,
with § 891–905 as minimally required epitope. In addition,
the § 881–900 human T cell epitope overlaps the epitope
of the BALB/cCrSlc gastritogenic T cell clone II-6
(TCR V g 14+), which recognizes § 891–905 and § 892–906
of porcine and human H+,K+-ATPase § chain, respectively
[18] (Fig. 2). In the present study, five different human T
cell epitopes of the g chain were also identified, three of
which overlap peptides that are relevant in EAIG (Fig. 2).
Amino acid sequences g 76–90 and g 81–95 recognized
by human T cell clones are partially overlapping the
murine epitope g 85–109, whereas peptide g 166–185
almost exactly overlaps the murine epitope g 169–193 [7].
We observed predominant Th1-type cytokine production
upon peptide epitope stimulation (Fig. 1) and Fas-Fas
ligand-mediated cytotoxic capacity (not shown) of H+,K+ATPase-specific T cells infiltrating in the gastric mucosa
in AIG, as previously reported [16]. In EAIG, T cell clone
II-6 expresses Fas ligand upon activation, and caused
DNA fragmentation of B cells pulsed with the H+,K+ATPase § 891–905 peptide [17], i.e. the epitope overlapping the peptides recognized by the human T cell clones
2.P02 and 2.P14. Based on recognition of common epitopes and cytokine profiles of autoreactive gastric
T cells, we propose that in murine EAIG and human AIG
similar mechanisms are responsible for target cell
destruction. In strong analogy with EAIG, our results indicate that in human AIG epitopes of gastric parietal cell
H+,K+-ATPase, both on the g and on the § chain, are specific targets for autoreactive T cells, which are responsible for induction and maintenance of the disease.
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Eur. J. Immunol. 2003. 33: 539–545
M. P. Bergman et al.
Eur. J. Immunol. 2003. 33: 539–545
4 Materials and methods
4.4 Generation of H+,K+-ATPase overlapping peptides
4.1 Patients
For the identification of T cell epitopes, soluble overlapping
15-mer peptides, comprising the complete amino acid
sequence of porcine H+,K+-ATPase § and g chains, were
synthesized. To span the 1,034-amino acid § chain and the
270-amino acid g chain, 205 and 56 consecutive peptides
with a 10-amino acid overlap, respectively, were prepared
by automated simultaneous multiple peptide synthesis
(SMPS). The SMPS set-up was developed with a standard
autosampler (Gilson 221) as described previously [23].
Briefly, standard Fmoc chemistry with in-situ PyBop/NMM
activation of the amino acids in a fivefold molar excess with
respect to 2 ? mol/peptide PAL-PEG-PS resin (Perseptive
Biosystems) was used. Peptides were obtained as Cterminal amides after cleavage with 90–95% TFA/scavenger
cocktails. Peptides were analyzed and purified by reversed
phase HPLC and checked via electrospray mass spectrometry on an ion-trap mass spectrometer (LCQ; Thermoquest,
Breda, The Netherlands).
Four women (mean age 44 years, range 28–52 years) with
chronic AIG provided their informed consent for this study
approved by the local Ethical Committee. All patients had
serum PCA, as assessed by indirect immunofluorescence.
None of the patients had intrinsic factor autoantibodies or
hematological abnormalities, and their vitamin B12 serum
levels were within the normal range.
4.2 Generation of gastric T cell clones
Biopsy specimens obtained from the gastric mucosa were
used for confirmation of histology and culture of mucosainfiltrating T lymphocytes. Biopsy specimens of gastric
mucosa were cultured for 7 days in RPMI 1640 medium
supplemented with human IL-2 (50 U/ml; Eurocetus, Milan,
Italy) in order to expand in vivo-activated T cells. Mucosal
specimens were then disrupted, and single T cell blasts
were cloned under limiting dilution (0.3 cells/well) in the
presence of irradiated PBMC as feeder cells and PHA (1%
vol/vol), as described [16]. Each clone was screened for
responsiveness to medium, porcine albumin (5 ? g/ml), and
porcine H+,K+-ATPase (0.5 ? g/ml) by measuring [3H]thymidine uptake after 60-h stimulation in the presence of irradiated autologous APC [16]. Gastric H+,K+-ATPase was purified from pig gastric mucosa as reported [20].
4.3 Analysis of TCR V I repertoire of gastric T cell
clones
The repertoire of the TCR V g of H+,K+-ATPase-specific Th
clones was analyzed with a panel of 20 monoclonal antibodies specific to V g 2, V g 7, V g 9, V g 11, V g 14, V g 16, V g 18,
V g 20, V g 21.3, V g 22, V g 23 (Beckman-Coulter Immunotech,
Marseille, France), and V g 3.1, V g 5.1, V g 5.2, V g 5.3, V g 6.7,
V g 8, V g 12, V g 13, and V g 17 (AMS Biotechnology, Wiesbaden, Germany). Isotype-matched nonspecific Ig were used
as negative control. Data acquisition was performed in a
FACSCalibur flow cytometer with the CellQuest software
program (Becton Dickinson, San Josè, CA). From each T cell
clone mRNA was extracted by mRNA direct isolation kit
(Qiagen, Hilden, Germany). For cDNA synthesis the same
amount of mRNA (50 ng) was used, and cDNA was synthesized by Moloney murine leukemia virus reverse transcriptase (MoMuLV-RT; New England Biolabs, Beverly, MA) and
oligo-(dT) primers according to enzyme supplier’s protocol.
cDNA mix of all samples was amplified under equal conditions by a 30-cycle PCR using V g T cell receptor-typing
amplimer kit for V g 1, V g 4, V g 10, V g 15, and V g 19 (Clontech,
Palo Alto, CA) according to the manufacturer’s instructions.
4.5 Proliferative response to H+,K+-ATPase peptides of
gastric T cell clones
The fine antigen specificity was assessed at the peptide
level in 16 H+,K+-ATPase-specific CD4+ clones. In a first set
of experiments, equal amounts of each of the 205 overlapping peptides for the § chain and the 56 peptides for the
g chain of H+,K+-ATPase were pooled in 20 pools. T cell
blasts (4×104) from each clone were cultured in triplicate for
3 days in RPMI 1640 medium supplemented with 5% heatinactivated AB human serum in 96-microwell plates together
with irradiated autologous PBMC (1.5×105) in the presence
of medium alone, porcine albumin (5 ? g/ml), H+,K+-ATPase
(0.5 ? g/ml), or equal aliquots from each of the 20 peptide
pools in which each peptide component was present at a
10 ? g/ml final concentration. At 16 h before harvesting,
0.5 ? Ci [3H]thymidine (Amersham Pharmacia Biotech, Little
Chalfont, GB) were added and radionuclide uptake was
measured. The MI was calculated as the ratio between cpm
obtained in stimulated cultures and those obtained in the
presence of medium alone. In subsequent experiments,
T cell blasts of each clone were retested for proliferation to
the individual peptide components of the peptide pool that
induced an MI G 5 in previous experiments.
4.6 Cytokine production induced by H+,K+-ATPase
peptides
To assess the production of IFN- + and IL-4 by H+,K+ATPase-specific T cell clones in response to the peptide(s)
they recognized, two separate cultures on 1 day (containing
5×105 T cell blasts) of each clone were cultured for 48 h in
0.5 ml medium together with 5×105 irradiated autologous
APC in the presence of medium, H+,K+-ATPase (0.5 ? g/ml),
the H+,K+-ATPase peptide that induced proliferation (10 ? g/
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544
ml), and two flanking control peptides that had failed to
induce proliferation. Each supernatant was assayed in duplicate for IL-4 and IFN- + content by commercial ELISA assays
(BioSource International, Camarillo, CA) [16]. In addition, for
two selected clones (4.A33 and 4.C31; see Fig. 1), reproducibility of results was further investigated by retesting them as
described above, on a second occasion, after cryopreservation and with a fresh batch of APC.
Acknowledgements: We thank J. J. de Pont (Nijmegen,
The Netherlands) for providing purified pig gastric H+,K+ATPase. This work was supported by grants from the Italian
Ministry of University and Research, the University of Florence and the Associazione Italiana per la Ricerca sul
Cancro.
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Correspondence: Ben J. Appelmelk, Department of Medical Microbiology and Infection Control, VUMC Vrije Universiteit Medical Center, van der Boechorststraat 7, 1081 BT
Amsterdam, The Netherlands
Fax: +31-20-4448318
e-mail: BJ.Appelmelk.mm — med.vu.nl
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Eur. J. Immunol. 2003. 33: 539–545