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,
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Dipartimento di Biologia, Università di Trieste, Trieste, Italy;
Istituto di Ricovero e Cura a Carattere Sperimentale, Burlo Garofolo, Trieste, Italy;
Biosciences Division, Los Alamos National Laboratory, Los Alamos, NM 87545; and
Scuola Internazionale Superiore di Studi Avanzati, Trieste, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Phage display of human Ab fragments has proved to be an effective method to investigate in vivo Ab responses in autoimmune disease (8, 9, 10). In this method, a patient’s Ab repertoire is expressed fused to the coat protein of a phage vector that carries the encoded protein gene (11), with each phage carrying a single Ab specificity. In this study, we describe the display of Ab repertoires derived from either intestinal biopsy lymphoctes (IBL) or PBLs from three patients with CD and show that whereas gliadin responses could be selected from all libraries, the tTG response was restricted to the IBL, with most Abs displaying recognition of a single epitope and associated with a highly restricted VH gene family.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DH5aF' (F'/endA1 hsdR17 (rK-
mK+) supE44 thi-1 recA1 gyrA
(Nalr) relA1 D (lacZYA-argF)U169 deoR
(F80dlacD(lacZ)M15)) was used for phage propagation. HB2151
(K12, ara 
(lac-pro), thi/F'
proA+B+,
lacIqZM15) was used to
make soluble single-chain variable fragment (scFv). pDAN5
(12), was used for scFv display. Molecular biology enzymes
were purchased from New England Biolabs (Beverly, MA), Promega
(Madison, WI), or Life Technologies (Rockville, MD), respectively.
Lymphocyte RNA preparation and library construction
Total RNA was prepared as in Ref. 13 from 10 ml of
PBL or IBL from three previously untreated CD adult patients with high
titers of anti-
-gliadin, anti-human tTG (anti-h tTG),
and endomysial Abs. All the patients had HLA-DQ2 histocompatbility Ags.
PBL were purified by Ficoll Hypaque (Pharmacia, Piscataway, NJ), and
IBL were treated directly. cDNA was synthesized by using random
hexamers and SuperScript II reverse transcriptase (Life Technologies).
Ig V regions were amplified by using specific V region primers
(14) and assembled into scFv as reported in Ref.
15 before cloning into pDAN5.
Antigens
Purified -gliadin was prepared as described
(16). Guinea pig tTG (gp tTG), BSA, and lysozyme were
purchased from Sigma (St. Louis, MO). h tTG cDNA was obtained by
amplifying cDNA from an intestinal biopsy with specific primers
(17) and cloning into pTrcHis (Invitrogen, San Diego, CA).
The tTG was extracted as soluble cytoplasm fraction and purified by
nickel nitrilotriacctic acid chromatography (Qiagen, Chatsworth,
CA).
Selection and testing of phage Abs
Rescue of phagemid particles was as described in Ref.
18 . Panning was performed by adding phages diluted in 2%
nonfat milk in PBS (MPBS) to immunotubes (Nunc, Naperville,
IL) coated with purified h tTG or -gliadin (10 µg/ml), washing 20
times with PBS 0.1% Tween 20, and 20 times with PBS, followed by
elution with 1 ml of Escherichia coli cells at 0.5
OD600 for 30 min at 37°C and overnight growth
after addition of ampicillin, helper phage, and kanamycin. The panning
procedure was repeated up to three times. After selection, 48
individual clones from each selection were screened for reactivity to
their respective Ags as well as irrelevant Ags (BSA and lysozme) by
microtiter plate ELISA (18).
Preparation and testing of soluble scFv
Phagemids from individual colonies were infected into HB2151,
grown to OD600 0.2, induced with 1 mM,
isopropyl-
-D-thiogalactopyranoside, and further grown
overnight at 28°C. scFv were used directly as supernatants of induced
bacterial cultures. ELISA was performed with soluble scFv essentially
as described for phage particles. Culture supernatants containing scFv
were serially diluted with MPBS, added to microtiter plate wells, and
detected with a mAb recognizing the SV5 tag (19) found at
the scFv C terminus, an HRP-conjugated secondary anti-mouse Ig
antiserum (Dako, Carpinteria, CA), and 3,3',5,5'-tetramethylbenzidine
dihydrochloride as substrates. Competitive ELISA was performed as
conventional ELISA, except that 100 µl of individual scFv diluted
1:10 with MPBS was mixed with equal volumes of serially diluted CD sera
positive for tTG.
Immunofluorescence
Immunofluorescence was performed on histological sections of human umbilical cord prepared according standard techniques. scFvs from bacterial culture supernatant were added to the sections, incubated for 30 min at room temperature in a moist chamber, followed by mAb SV5 and FITC-labeled anti-mouse IgG (Dako). Double labeling was performed by adding CD sera, diluted 1:200, to the same section and revealed with tetramethylrhodamine B isothiocyanate-labeled anti-human Ig antiserum (Dako).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The scFv libraries were constructed from either PBL or IBL from
three untreated adult CD patients with high anti--gliadin and h
tTG Ab titers, the latter determined both by anti-endomysium Abs
assayed on human umbilical cord sections as well as h tTG ELISA
(20). The biopsy materials were obtained from patients
undergoing intestinal biopsy to confirm their diagnoses.
VH and VL chains were amplified from PBL and IBL cDNA by PCR using a set of oligonucleotides that recognize all human V genes (14). For the VH chains, the 3' primer was specific for IgA Abs.
VH and VL amplificates were assembled by PCR (15) and cloned into the phagemid vector pDAN5 (12) to obtain the primary libraries. The six libraries ranged in size from 5 x 106 to 5 x 107, and 20 clones picked at random from each were shown to contain full-length scFv with different BstNI restriction pattern, confirming the diversity and integrity of the libraries. The libraries are reported according to the donor patient, and given reference numbers 2, 3, and 4.
Library selection
Abs were affinity selected on purified cloned h tTG or
-gliadin. According to this method, V genes derived from a
patient’s lymphocytes are used to express a patient’s Ab repertoire
fused to the coat protein of a filamentous phage vector. Each phage
carries a single Ab specificity and can be selected by subjecting the
phage library to recursive rounds of binding, washing, and elution on
the target Ag. After every cycle of selection the eluted phages were
reamplified for the next cycle and tested by ELISA against the Ag used
for the selection. When the polyclonal signal of the eluted phages was
positive by ELISA, the selection was considered concluded, and 48
individual clones were analyzed from each selection. This was adopted
to avoid overgrowth of deleted (21) or well-expressed
positive clones during the cycles of amplification. Clones positive on
the Ag used for the selection (tTG or -gliadin) and negative on
control Ags (BSA and lysozyme) were analyzed by BstNI
fingerprinting and sequencing to determine the number of different
clones, and these results are reported in Table I
. Interestingly, after only one cycle of
selection, all three IBL libraries showed a variable number of positive
clones to h tTG, whereas no positive clones to tTG were obtained after
three cycles of selection with the PBL libraries. To further control
this result, the antigenic quality of the cloned h tTG was assessed by
using a large naive library described in Ref. 12 . This was
prepared from Vµ, V
,
and V
genes with an estimated diversity of
7 x 107 different VH
and VL genes recombined to a final estimated
diversity of 1011. After two cycles of
selections, 12 different clones to tTG were identified, confirming that
the failure of the PBL selections was not attributable to the low
efficiency of the system in isolating rare scFv but rather to the lack
of such Abs in the libraries. In contrast, different Abs against
-gliadin were obtained from all six CD libraries, as well as the
naive library after two rounds of selection. The total number of
positives and different positives clones is reported in Table I.
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The V genes from the different anti-tTG scFv clones were
sequenced and the VH and VL
families as well as the gene segments used were assessed by
screening against the VBASE
(http://www.mrc-cpe.cam.ac.uk/imt-doc/restricted/ok.html) database
(22) (Table II, columns 2
and 5). The amino acid sequence of the complementarity-determining
regions (CDR3) are reported in Table II (columns 3 and 6), and the
numbers of silent (S) mutations leading to base substitution or amino
acid replacement (R), for VH and
VL, determined by comparing the V sequence to the
closest germline sequence, are reported in columns 4 and 7. On the
basis of the features reported above, scFvs selected from the three
libraries (2, 3, 4) were grouped according to the
VH family, the CDR3 sequence, and the framework
mutations. Clones with similar CDR3s with a limited number of mutations
in the other parts of the molecules (CDR1, CDR2, and framework regions)
were considered to be derived from the same ancestor and assigned to
the same group, but given different reference numbers (column 1). In
general, VH gene use was restricted to three
(VH5, VH3,
VH1) of the seven human Ab
VH families (for a complete classification, see
VBASE), with many of the VH genes belonging to
the VH5 family (12/29 cases) with a preferential
use of the DP73 segment (10/29 cases). Interestingly, this was the only
VH gene segment selected from all three
libraries, indicating the possible preferential usage of this segment
in the autoimmune response to tTG. This is in contrast to the
VL sequences, which appeared to be completely
random, with V and V
chains belonging to many of the 10 V and 6
V families being found. The overall ratio of
replacement over silent mutations of both VH and
VL domains (Table II, columns 4 and 7) was always
higher than 1, suggesting that the mutations were the consequence of
Ag-driven selections. A big difference between the scFvs containing
DP73 and those containing other VH segments is
that the DP73 segments appeared to be able to pair with any light
chains, whereas the others appeared to pair only to specific light
chains. This is particularly striking in group 3/D where a series of
somatically mutated VI/DPK9 genes are coupled
exclusively to VH1/DP10 segments. As a general
rule, most of the selected scFv of the three libraries show the same
feature. Because during Ab library construction
VH and VL regions are
randomly assembled, it is likely that these coselected
VH/VL pairs represent cases
where heavy and light chain both need to be present for binding to
occur, and hence may mirror the in vivo pairing, whereas those scFv
containing DP73 appear to be able to pair with any light chain and so
presumably have most of the binding activity located in the heavy
chain. These results are in contrast to those obtained when similar
selections were conducted on a naive library made from PBLs from 40
different healthy donors (12), in which of 12 scFvs
selected, only two VH5 gene segments were found,
and both of these were derived from the COS-25 gene. The remaining 10
scFvs had one VH2, four
VH3, and five VH6
VH genes, whereas the VL
genes were randomly distributed among four different families (data not
shown).
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Soluble scFv were derived from each of the IBL clones recognizing
tTG, and reactivity to h tTG by ELISA identical with that shown by
phage Ab was confirmed. The scFv were also tested in immunofluorescence
for their reactivity to human umbilical cord histological sections (the
classic anti-endomysial staining). The results were compared with
the fluorescence pattern obtained with a human serum positive for
endomysium. In almost all cases (Fig. 1A), the scFv gave an
immunofluorescence pattern identical with those of CD patients (Fig. 1C), and in those cases where the scFv did not stain
umbilical cord sections, the ELISA signals tended to be low (data not
shown), suggesting that either affinities or expression levels were
low. In Table III, column 4, the
subjective evaluation of the fluorescence intensity is reported. When a
selected number of scFv were used in double-labeling experiments, the
two images overlapped perfectly (Fig. 1B).
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The correspondence of the scFv to the in vivo Ab repertoire was
further confirmed by competitive ELISA. Patient sera positive for tTG
were serially diluted from 1:50 to 1:400, mixed with scFv diluted 1:10,
added to a microplate coated with h tTG or BSA as negative control, and
tested for scFv binding. Five scFv representative of the different
groups were tested and all showed a reduction in the ELISA signals
because of the competition with patient sera. The extent of the
reduction ranged from 10 to 80%, depending on the CD patient serum and
probably reflecting a difference in the titer of the specific serum Abs
competing for the binding. No inhibition was observed by using a
control serum from a healthy donor. In Fig. 2, a typical response of two Abs with
sera from a CD patient and a healthy donor is reported. The conclusion
was that the patient sera contained Abs recognizing the same antigenic
determinants recognized by the scFv or at least reacting to the same
antigenic area.
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To determine how many different tTG epitopes were recognized by
the different selected scFvs, two approaches were taken. In the first,
cross-reactivity of the different scFvs to gp tTG by ELISA was
determined, and it was found (Table III
, column 2) that only a fraction
of the Abs were able to recognize gp tTG with OD values comparable to
those obtained with h tTG, with negative scFvs giving OD values similar
to negative controls. In the second approach, an inhibition ELISA was
conducted in which the binding of different phage scFvs to h tTG coated
to a plate (detected with a peroxidase-labeled anti-phage
monoclonal) was tested in the presence of different soluble scFvs.
Negative controls were the same phages without competitor scFv, and
positive controls the inhibition of binding by soluble scFv
corresponding to that displayed by the phage. In the first experiment,
a single, well-expressed scFv belonging to group 2/A (see below) was
tested against all phage Abs. When a reduction in OD of at least 50%
with respect to the control was registered, the scFv and the phage were
considered as recognizing the same antigenic region. After the first
experiment, a second scFv (group 2/D, see below) was chosen from among
those clones not inhibited by the first scFv and tested. After this,
almost all clones could be grouped into two main association groups,
termed epitope 1 (Ep1) and epitope 2 (Ep2; Table III, column 3). Only
three (10%) of the clones could not be assigned to either of these
groups and are reported in Table III as "x." Clones recognizing Ep1
could be isolated from all three libraries (groups 2/A, 2/B, 2/C, 3/A,
3/B, 4/A, 4/B, 4/C), and the level of inhibition caused by the 2/A scFv
was almost total, with a drop of the OD value to the level of positive
controls, suggesting that all of these scFvs recognized the same
antigenic determinant. Although scFvs recognizing Ep2 were also
selected from all libraries, the level of inhibition (caused by scFv
2/D) was far more variable, ranging from 50 to 100%, suggesting that a
larger antigenic region is involved, with partial inhibition being
caused more by steric hindrance then competition for the same epitope.
Ep1 was recognized almost exclusively by those scFv belonging to
theVH5 family. The only exception was 4/E, with
two segments (VH3/DP54 and
VIII/DP10) not present in any other scFv. All
scFvs recognizing Ep1 also recognized gp tTG, whereas none of the
others was able to do so, suggesting that Ep1 is common to h tTG and
gp tTG.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The primary goal of our study was to determine the site of
synthesis of the autoantibodies to tTG. Until now, CD has been
immunologically diagnosed by using serum Abs to -gliadin or
endomysium. Only recently has the discovery of tTG as the autoantigen
of CD resulted in a number of immunoassays based on gp tTG or h tTG
(20, 23). It is generally accepted that the IgA response
related to CD (anti--gliadin, anti-reticulin,
anti-endomysium, and anti-tTG) is more specific than that of
IgG (24, 25, 26, 27). It is also thought that this Ab response may
originate in the intestinal epithelium (28, 29) although
experimental evidence, with the exception of the induction of
anti-tTG Abs by intestinal biopsies after gliadin treatment
(7), has not been found. For these reasons and on the
basis of earlier studies (see below), we decided to selectively amplify
the IgA immune response. In the course of our study, we were able to
isolate anti-tTG scFv from all three libraries from CD patient
intestinal lymphocytes but from none of the libraries prepared from
PBLs, even after three cycles of selection. This indicates that the
anti-tTG IgA Abs are primarily synthesized at the intestinal level
and the presence in the serum is probably attributable to the spillover
from this source into the blood compartment. A major criticism to this
approach is based on the fact that the
VH-VL pairings occurring in
vivo are not usually recreated in vitro, as VH
and VL genes are paired at random. However, a
number of findings suggest that phage Ab libraries can act as
surrogates for the humoral response, or at least that part of it
originating in the tissue used to create the library. In particular,
most of the binding energy and specificity is provided by the
VH domain rather than the
VL domain, as shown by a number of
cocrystallographic studies (reviewed in Ref. 30).
Furthermore, in selection from very large naive phage Ab libraries
(31), different Ags always selected different
VH genes, whereas some VL
genes were found in scFvs binding to different Ags. Also, in other
libraries made from patients with autoimmune thyroid disease
(32), myasthenia gravis (9), systemic lupus
erythematosis (33), Sjogren’s syndrome (34),
and paraneoplastic encephalomyelitis (35), the Ag
specificities of selected Abs reflect the specificity of the patient’s
humoral response.
Our results with the anti-tTG Abs mirror these findings: selected
Abs give identical staining patterns to patients’ sera on umbilical
cord sections, indicating that the constructed library represents at
least a fraction of the original response to tTG, and the selected
scFvs mirror the serum anti-tTG response very closely. In a
previous study (36), we analyzed the Ig response to tTG of
a library constructed by using PCR primers recognizing all Ig classes
from PBL and we were unable to isolate scFv to tTG, although Abs
against -gliadin could be selected with relative ease. This suggests
that the anti-tTG responses of other Ig isotypes are also likely to be
mediated at the intestinal level. Although the libraries used were
small, this did not prevent selection of anti-gliadin Abs from
all six libraries, indicating that it is unlikely that library size was
the reason for failure to select anti-tTG Abs from the PBL
libraries.
It has recently been shown that although naive B cells may make up the greatest number of B cells, the largest proportion of mRNA is derived from activated B cells (37). If, as is expected, this finding is reflected in intestinal B cells, the very high proportion of anti-tTG scFvs present in the library (as indicated by the high numbers of positive clones after a single round of selection) suggests that the V genes making up these scFvs are derived from activated intestinal B cells.
V family usage and epitopes recognized
The anti-tTG V gene sequences identified showed high levels of
mutation, with the CDR3 of the VH5/DP73 Abs in
groups 2/A and 2/C and the V1/DPK9
VL genes coupled to the 3/C and 3/D
VH genes showing differences clearly indicating
an origin from the same original V gene. This suggests that these V
genes are derived from an ongoing immune response where mutation is
driven by Ag stimulation. Human VH5 is not the
most commonly used variable region by the immune system to build Abs in
healthy donors, although it is over-represented when compared with the
corresponding genome complexity (38, 39). In our study,
the selective usage of this family was found in three different
patients and makes us believe it may be a common feature to the
anti-tTG response of all CD patients, in contrast to those
anti-tTG VH genes selected from the naive
library. scFv from CD patients containing VH5
genes all recognized the same epitope, which, interestingly, is shared
by gp tTG (Ep1). This raises the question of whether the selective use
of this family might be related to the genetic predisposition shown by
CD patients. In fact, even though the CD is associated to specific
HLAs, the genetic linkage is not clear and other factors are required
to account for the familial predisposition (5). Although
the idea that V gene alleles may be responsible for such genetic
predisposition is attractive, no common somatic mutation which could
suggest the existence of a new allele potentially associated to CD, by
virtue of perhaps an intrinsic recognition of tTG, could be identified.
Thus we cannot exclude an epigenetic phenomenon involving a natural
structural affinity of some elements of the VH5
family for Ep1 of tTG, which may play a role in the onset of the
disease. As far as the other Abs are concerned, they seem to be
directed to the second, less well-defined epitope, Ep2, characterized
by its exclusive presence on h tTG, notwithstanding the high homology
between h tTG and rodent tTG (17). The role of these two
epitopes in the disease remains to be determined.
Role of humoral response to tTG
The role of transglutaminases has been demonstrated in many cellular and extracellular events, mainly because of their cross-linking capabilities. Although transglutaminases are constitutively expressed in many tissues, tTG is primarily found in epithelia. Activation of tTG occurs during apoptosis, where it was originally proposed to be involved in the late phase of apoptosis, during which, by the creation of a net of cross-linked proteins (apoptotic bodies), it is thought to prevent release of the potentially toxic intracellular contents to the extracellular space. An interesting variation on this theme suggests that the role of tTG is to avoid exposure of cryptic intracellular autoantigens to the extracellular space and hence the immune system (40). Although the humoral response at the intestinal level is commonly thought to be less important than the cell-mediated response, total IgA from untreated CD patients have been shown to inhibit intestinal epithelial cell differentiation (41). It will be interesting to see whether scFvs recognizing the two different epitopes identified here have similar effects in this system.
In the course of the immune response to gliadins and the autoimmune
response to tTG, autoantibodies to other autoantigens have been shown
to rise in active disease only to disappear when gluten is withdrawn.
(42, 43). As a direct consequence of prolonged exposure to
gluten, CD patients often develop autoimmune disease such as diabetes,
thyroiditis, alopecia, autoimmune hepatitis, and cerebellar ataxia
(44). On the basis of the responses to tTG in the IBL
libraries, a large proportion of the intestinal Ab production appears
to be involved in the recognition of tTG. The response to -gliadin
itself looks less relevant, especially as it is also found in the PBL
library and may result from leakage of gliadin into the systemic
circulation with a corresponding central response. This result raises
the question as to whether the immune response to tTG is itself a risk
factor for other autoimmune pathologies.
Recently, a series of the scFvs described in this work were used by T.
Halttunen in an in vitro fibroblast assay. In preliminary experiments
(unpublished observations), these recombinant anti-tTG Abs
increased fibroblast proliferation. This is in agreement with the model
proposed by Halttunen and Maki (41) on the possible role
of the anti-tTG IgA component found in the serum of CD patients.
These authors demonstrated that murine mAbs to tTG or unfractionated CD
serum IgA inhibited epithelial cell differentiation in an artificial
epithelium model obtained by cocultivating epithelial cell with
fibroblasts or in presence TGF- (45). Their conclusion
was that anti-tTG Abs interfered with the tTG-mediated maturation
of TGF-, and as a result caused inhibition of epithelial cell
differentiation and increased proliferation. This reproduced the severe
intestinal crypt hyperplasia and flattened villi characteristic of CD.
Confirmation of these preliminary results will suggest that the CD
humoral response to tTG is not an epiphenomenon but an important factor
in intestinal mucosa damage, and hence perhaps one of the major
pathological players. The availability of cloned Abs from CD patients
with defined specificities will allow a detailed investigation of this
point and perhaps help in answering the question: why are such Abs made
in CD patients?
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Acknowledgments |
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Footnotes |
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2 R.M. and D.S. equally contributed to the present work.
3 Address correspondence and reprint requests to Dr. Robert Marzari, Dipartimento di Biologia, via Giorgieri 5, Università di Trieste, 34127 Trieste, Italy.
4 Abbreviations used in this paper: CD, celiac disease; scFv, soluble single-chain variable fragment; tTG, tissue transglutaminase; IBL, intestinal biopsy lymphocyte; gp tTG, guinea pig tTG; h tTG, human tTG; CDR, complementarity-determining region; MPBS, 2% nonfat milk in PBS.
Received for publication May 30, 2000. Accepted for publication January 2, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C Esposito, F Paparo, I Caputo, M Rossi, M Maglio, D Sblattero, T Not, R Porta, S Auricchio, R Marzari, et al. Anti-tissue transglutaminase antibodies from coeliac patients inhibit transglutaminase activity both in vitro and in situ Gut, August 1, 2002; 51(2): 177 - 181. [Abstract] [Full Text] [PDF]
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