Human Autoantibodies Modulate the T Cell
Epitope Repertoire but Fail to Unmask a
Pathogenic Cryptic Epitope
This information is current as
of June 17, 2017.
Sonia Quaratino, Jean Ruf, Mohamed Osman, Jin Guo,
Sandra McLachlan, Basil Rapoport and Marco Londei
J Immunol 2005; 174:557-563; ;
doi: 10.4049/jimmunol.174.1.557
http://www.jimmunol.org/content/174/1/557
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References
The Journal of Immunology
Human Autoantibodies Modulate the T Cell Epitope
Repertoire but Fail to Unmask a Pathogenic Cryptic Epitope1
Sonia Quaratino,2* Jean Ruf,† Mohamed Osman,‡ Jin Guo,§ Sandra McLachlan,§
Basil Rapoport,§ and Marco Londei‡
T
he interaction between T and B cells has a key role in the
fine-tuning of Ag-specific responses, and in the last few
years, much work has been performed to unravel how this
cross talk happens (1).
T and B cells can communicate by exchanging information via
the release of soluble factors or cognate interactions (2). One of the
key functions of B cells is to present Ags to CD4⫹ Ag-specific T
cells (3). During the evolution of an Ag-specific B cell immune
response, the high-affinity BCR will allow the B cell to compete
and overcome other types of professional APCs in capturing and
presenting Ags to T cells (4). This characteristic is at the basis of
Ab maturation, because B cells with higher affinity receptors will
outcompete other B cells with lower affinity receptors for T cell
help. Only B cells with higher affinity receptors will receive the
optimal maturative signals from Ag-specific T cells (5). B cells can
induce an anti-inflammatory response by presenting Ags, and in
particular self-Ags, to T cells (6). By capturing Ags via their surface Ag receptor, they will change the type of Ag processing, and
ultimately presentation of the T cell epitope repertoire available to
T cells (5, 7). Abs, which are the soluble form of the BCR, can also
influence T cell epitope availability, and several studies have elu-
*Cancer Sciences Division, Southampton General Hospital, University of Southampton, Southampton, United Kingdom; †Unit 38, Institut National de la Santé et de la
Recherche Médicale, and Laboratoire de Biochimie Endocrinienne et Metabolique,
Faculte de Medecine, Marseille, France; ‡Institute of Child Health, University College, London, United Kingdom; and §Autoimmune Disease Unit, Cedars-Sinai Research Institute and School of Medicine, University of California, Los Angeles, CA
90048
Received for publication May 21, 2004. Accepted for publication October 22, 2004.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by the Wellcome Trust (to S.Q.), European Community
Grants BMH4-98-3703 (to S.Q.) and QLK1-CT-1999-00037 (to M.L.), and National
Institutes of Health Grant DK36182 (to B.R.).
2
Address correspondence and reprint requests to Dr. Sonia Quaratino, Cancer Sciences Division, University of Southampton, Tremona Road, Southampton SO16
6YD, U.K. E-mail address: [email protected]
Copyright © 2005 by The American Association of Immunologists, Inc.
cidated some of the molecular mechanisms that control this occurrence (8 –11).
T and B cell interactions play an intricate role in adaptive responses to foreign agents, such as bacteria and viruses, and in
autoimmune diseases. In particular, in organ-specific autoimmune
diseases such as type 1 diabetes and thyroiditis, both T and B
autoantigen-specific responses are observed, and in most cases,
they are specific markers of disease (12, 13). Although it is well
recognized that B and T cells can cross talk, very little is known on
how this might occur in humans, and in particular how high-affinity autoantibodies can influence the nature of epitope(s) recognized by the self-Ag-specific T cells. Although some studies have
been performed in mouse models (14, 15) or in a mouse/human
hybrid system (16), there is no report that addressed this question
in human autoimmune diseases. This is because the appropriate
tools to perform this study, such as a suitable combination of T cell
clones and human monoclonal autoantibodies (mAbs) specific for
the same autoantigen have not been available. The generation of
human mAbs with overlapping functional characteristics of the
autoantibodies detected in vivo has been a problem for these types
of studies. Mouse mAbs, which are easier to generate, do not always have the same characteristics (epitopes or affinities) as human autoantibodies.
This obstacle has been overcome by the Ig gene combinatorial
library approach that permits the generation of human mAbs specific for foreign or self-Ags (17). Human mAbs specific for thyroid
peroxidase (TPO),3 a dominant autoantigen in autoimmune thyroiditis (12) have been obtained by a gene combinatorial library
(18). These Abs have the same characteristics (affinity and epitope
recognition) of the natural polyclonal autoantibodies present in
patients (19). We have previously generated and described a panel
of well-characterized TPO-specific human self-reactive T cell
3
Abbreviations used in this paper: TPO, thyroid peroxidase; SI, stimulation index;
DC, dendritic cell; MVA, modified vaccinia Ankara.
0022-1767/05/$02.00
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Abs can tune the responses of Ag-specific T cells by influencing the nature of the epitope repertoire displayed by APCs. We
explored the interaction between human self-reactive T cells and human monoclonal autoantibodies from combinatorial Ig-gene
libraries derived from autoimmune thyroiditis patients and specific for the main autoantigen thyroid peroxidase (TPO). All human
mAbs extensively influenced the T cell epitope repertoire recognized by different TPO-specific T cell clones. The action of the
human mAbs was complex, because sometimes the same Ab suppressed or enhanced the epitopes recognized by the 10 different
TPO-specific T cell clones. The human mAbs could modulate the epitope repertoire when TPO was added exogenously and when
expressed constitutively on the surface of APCs. However, they could not unmask an immunodominant cryptic TPO epitope. In
this study, we show that human autoantibodies influence the activity of self-reactive T cells and prove their relevance in concealing
or exposing epitopes recognized by self-reactive T cells. However, our results further stress the biological significance of the
immunodominant cryptic epitope we have defined and its potential importance in the evolution of autoimmunity. The Journal
of Immunology, 2005, 174: 557–563.
558
Materials and Methods
T cell clones
Human T cell clones specific for different epitopes of the TPO molecules
have been described previously (20, 21). They were established from the
thyroid infiltrate of a patient with autoimmune thyroiditis and maintained
at 1 ⫻ 106/well in RPMI 1640 supplemented with 10% human AB serum,
100 U/ml penicillin, and 50 ␮g/ml streptomycin (complete medium). Cells
were fed at regular intervals with recombinant human IL-2 (20 ng/ml;
kindly donated by Hoffman-La Roche) and stimulated every 3 wk with
irradiated (4500 rad) allogeneic peripheral blood leukocytes and PHA.
T cell proliferation
The reactivity of the TPO-specific human T cells clones was assessed by
coculturing 1 ⫻ 104 T cells from each T cell clone with either 3 ⫻ 104
glutaraldehyde-fixed autologous EBV-transformed B cell line or 1 ⫻ 103/
well live HLA-matched human DC as APC. APC were incubated with TPO
Ag or peptides with or without appropriate Abs for up to 5 h at 37°C before
the addition of the T cells.
Human purified TPO Ag was used at 0.1 ␮g/ml. The peptide TPO536 –547
(DPLIRGLLARPA) is a gift from D. Wraith (University of Bristol, Bristol,
U.K.). All T cell clones were tested at least twice, and all of the experiments showed a similar profile of responsiveness. Stimulation index (SI)
was calculated as follows: (total cpm ⫺ background cpm)/background
cpm. All of the proliferative assays were performed in triplicate for 72 h in
a round-bottom 96-well microtiter plate, and the cells were pulsed with
[3H]thymidine (1 ␮Ci) during the last 8 h of culture.
Human TPO-specific human and mouse Abs
Human TPO autoantibodies were isolated from Ig gene combinatorial libraries constructed from thyroid-infiltrating lymphocytes from patients
with autoimmune thyroid disease (18). These recombinant autoantibodies
were cloned and expressed as Fab molecules and have high affinity for TPO
(Kd ⬃ 10⫺10 M). Four representative Fabs (SP1.4, WR1.7, TR1.8, and
TR1.9) were produced in bacteria, purified by affinity chromatography, and
used at 3.2 ⫻ 10⫺8 M final concentration. These Fabs define overlapping,
conformational epitopes recognized by all patients (18, 26). One of these
autoantibodies (Fab SP1.4) was converted to a full-length human Ig of
different isotypes (IgE, IgG1, and IgG4) of comparable levels of TPO binding (19). They were used at saturating concentrations equivalent to 1/100
for the IgG1, 1/25 for the IgE, and 1/1.5 for the IgE Abs. In addition to the
human monoclonal autoantibodies, 10 mouse mAbs displaying different
affinity for human TPO were generated as previously described (25) and
were tested at 100 ng/ml for their ability to present TPO to T cell clones.
DC preparation
Human dendritic cells (DCs) were generated according to a well-established procedure (24, 27). Briefly, CD14⫹ cells were isolated from 2 to 7 ⫻
107 HLA-matched PBMC using magnetic cell separation system (Miltenyi
Biotec). The CD14⫹ cell fraction was then incubated for 5–7 days in presence of GM-CSF (20 ng/ml) and IL-4 (10 ng/ml) (R&D Systems). In vitrogenerated DC were CD14⫺, CD1b⫹, and expressing high levels of MHC
class II. DCs were further matured using TNF-␣ (10 ng/ml; a kind gift from
the Centre of Molecular and Macromolecular Studies (Lodz, Poland)).
Construction of recombinant modified vaccinia Ankara (rMVA)
virus expressing TPO
Human TPO open reading frame was excised by cutting the plasmid
pBSK-TPO with XbaI, filling the staggered ends in with Klenow DNA
polymerase, and further cutting with NotI. The resulting full-length cDNA
encoding for the human TPO was ligated into the NotI site of pSC11
plasmid. The pSC11 plasmid carrying the human TPO gene was transfected with Perfect Lipid (Invitrogen Life Technologies). BHK21 cells
were infected with MVA at 0.05 PFU per cell (MVA; a kind gift from Prof.
G. Smith (Imperial College School of Medicine, London, U.K.)). Total
virus from the cells and supernatant were harvested 3 days later and used
for the reinfection of BHK21 cultures. The plaques of rMVA were identified by using X-Gal (5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside)
color selection and purified by five rounds of plaque purification (28). Bulk
stocks of the rMVA were grown and purified by centrifugation of cytoplasmic extracts through a 36% (w/v) sucrose cushion in a Beckman SW28
rotor at 13,500 rpm for 80 min.
Expression of TPO in DC and EBV-transformed B cell line
DC or EBV-transformed B cell lines were cultured at a density of 1 ⫻
106/ml in a 24-well plate at 37°C in CO2 and transduced with sonicated
rMVA-TPO or rMVA-␤-galactosidase (as negative control) at a multiplicity of infection of 10 as described (28). We used the MVA as a delivery
vector to induce TPO surface expression, because we can easily infect both
proliferating (like EBV-transformed B cell lines) and nonproliferating (like
DC) cells (28).
TPO-transduced DC or the EBV-transformed B cell line were used as
APC the following day. The analysis of the surface expression of TPO was
performed between 4 h and 2 days postinfection. In some cases, the EBVtransformed B cell line was transfected with TPO encoded by the pREP4
vector as previously described (21, 29). In all cases, surface expression of
TPO in the EBV-transformed B cell line and its APC activity produced
overlapping results regardless of the method used to induce expression of
the TPO.
FACS analysis
Human DC were collected in ice-cold PBS (supplemented with 1% FCS
and 0.05% azide) and stained using a panel of mouse anti-human TPO
mAbs (IgG1) and a mouse anti-human IgG1 isotype control (Serotec). The
autologous EBV-transformed B cell line was stained with FITC-conjugated
mouse anti-human CD16 (clone 3G8), CD32 (clone AT10), CD64 (clone
M22), and purified mouse anti-human CD23 (MHM6) (all raised in-house).
The counterstain was a biotinylated rat anti-mouse IgG (Southern Biotechnology), followed by PE-conjugated streptavidin (Southern Biotechnology). Cells were than analyzed on a FACS (FACStarPlus; BD Biosciences).
Results
Human autoantibodies of the appropriate isotype capture the Ag
and allow presentation by B cells
To evaluate whether human autoantibodies produce any modulation of the T cell epitope repertoire, autologous EBV-transformed
B cells were pulsed with human TPO or with TPO complexed to
the human autoantibody SP1.4 either on the IgE or IgG4 backbones. Two hours later, the TPO-specific T cell clones were challenged with the different combinations of EBV, Abs, and Ag. As
illustrated in Fig. 1A, the T cell clone 36 (representative of six
other T cell clones) was not activated by the autologous EBV cell
line pulsed with TPO but only by the TPO-expressing EBV cells
(EBV-TPO). In contrast, a very powerful T cell response was observed when the T cells were stimulated with the EBV cell line
pulsed with soluble TPO in the presence of saturating concentrations of the human mAb SP1.4 with an IgE (but not the IgG4) Fc
region (Fig. 1B). The mAb SP1.4 on an IgG1 backbone tested at
increasing concentrations behaved similarly to the SP1.4-IgE, but
IgG4 always failed to induce a T cell response, even when tested
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clones derived from thyroid autoimmune patients (20). They constituted the right tool to explore the interaction between autoantibodies and self-reactive T cells specific for the same autoantigen.
These T cell clones were also instrumental in defining and characterizing a human cryptic epitope of TPO (21) involved in the
development of autoimmunity (22). This cryptic epitope was presented when TPO was expressed as a surface molecule (21), as
normally occurs in vivo on thyroid epithelial cells (23), but not
when DC or other professional APC were loaded with exogenous
TPO (21, 24).
The availability of these tools allowed us to address some basic
questions on the nature of the interaction between T and B cells in
a human autoimmune condition, but, more importantly, to explore
whether these autoantibodies could unmask a bona fide and pathogenic human cryptic self-epitope. The results that we have generated indicate that human autoantibodies can modify the pattern of
T cell reactivity of all 10 TPO-specific T cell clones. In contrast,
only 1 of the 10 mouse anti-human TPO mAbs studied in parallel
(25) exerts any activity on the epitopes recognized by the T cells
clones. Despite their ability to widely alter the TPO T cell epitope
repertoire, none of the tested human and mouse autoantibodies
could unveil the immunodominant cryptic and pathogenic T cell
epitope.
T CELL EPITOPES AND B CELLS
The Journal of Immunology
559
Human monoclonal autoantibodies modulate the epitope
repertoire presented by APC expressing TPO as
membrane-bound molecule
FIGURE 1. Autoantibodies modify the Ag processing capacity of B
cells. A, The exogenous TPO cannot be uptaken by the autologous EBVtransformed B cells and therefore cannot be processed and presented to the
TPO-specific T cell clone 36. B, The autologous EBV-transformed B cell
line pulsed with human TPO (100 ng/ml) and human SP1.4 IgE mAb
generates a substantial proliferative response in T cell clone 36. In contrast,
the same Ab with the IgG4 Fc region is unable to allow capture and processing of TPO. Representative data of four experiments are shown. C,
Challenge of T cell clone 36 with the autologous EBV-transformed B cell
line pulsed with human TPO (100 ng/ml) in presence of increasing concentrations of either human SP1.4 IgG1 or IgG4 mAbs. Representative data
of two experiments are shown.
Because the human mAbs proved to be so efficient in permitting
epitope availability to TPO-specific T cells (Fig. 1), our aim was to
determine whether Abs could also modulate the loading of class II
MHC-restricted T cell epitopes when the TPO is membrane bound
at high concentrations (Fig. 1C). These observations demonstrate
that the human autoantibody was efficient in binding the human
TPO but only when the appropriate Ig backbone was used. It suggests that capture of the Ag/Ab complex, via the specific FcR,
subsequently permitted Ag presentation by the EBV-transformed
B cell line (30). The epitope recognized by T cell clone 36 is
presented upon endogenous processing of TPO (TPO-EBV), and it
is displayed following the piggyback action of the SP1.4 Ab.
These data demonstrate that autoantibodies can capture a soluble
autoantigen (TPO) and allow presentation of the autoantigen to
self-reactive T cells.
Human, but not mouse, monoclonal autoantibodies can alter
TPO presentation
We next wanted to assess whether a previously characterized panel
of 10 mouse anti-human TPO IgG1 mAbs (25) had any ability to
modulate TPO presentation to human T cell clones.
These 10 mAbs were very efficient in detecting TPO expression
in DCs transduced with the TPO gene delivered by rMVA virus
FIGURE 2. Mouse anti-human TPO mAbs recognize TPO expressed on
the surface of DC. A, DCs were transduced with rMVA-TPO as described
in Materials and Methods. After 4 h of infection, cell surface expression of
TPO can be detected in DCs by FACS analysis. The gray line represents
cells only, the dotted line represents cells stained by the isotype control Ab,
and the thick line represents the mouse anti-human TPO mAbs. B, The
autologous EBV-transformed B cell line was stained for CD16 (dotted
line), CD32 (thick line), and CD64 (dashed line). The gray line represents
cells only, and the dash-dotted line represents the isotype control.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
(Fig. 2A). Similarly, all mouse Abs detected TPO expressed on the
surface of the autologous EBV-transformed B cell line 4 h after
rMVA-TPO transduction (data not shown). There was no specific
TPO staining in cells transduced with the control virus rMVA-␤galactosidase (data not shown).
We then investigated what Fc␥Rs were expressed by the autologous EBV-transformed B cell line by staining for CD16, CD32,
and CD64. Of these, only CD32 (the Fc␥RII) is expressed by the
autologous EBV-transformed B cell line (Fig. 2B). Furthermore,
the Fc⑀RII CD23 was also not expressed (data not shown).
In view of these results, we proceeded to address the effect of
these mouse anti-human TPO Abs on TPO presentation to human
T cell clones.
Although all Abs detected surface expression of TPO in APC,
only 1 (no. 15) of 10 mouse mAbs was able to allow TPO presentation to specific T cell clones (Fig. 3). The human autoantibody SP1.4, either on an IgG1 or IgE backbone, was very efficient
in enhancing TPO Ag presentation by the EBV-transformed B
cells to all of the six tested T cell clones (Fig. 3). Because all
mouse anti-human TPO mAbs detected surface expression of TPO
in both DC and EBV cell lines (Fig. 2A and data not shown), the
failure of all but one of the mAbs to increase Ag presentation
suggested that the epitope specificity of the Abs may play a key
role in the Ag/epitope processing and presentation to T cells. This
indicates that human high-affinity autoantibodies have a vast and
unexpected potential to modulate Ag recognition by self-reactive T
cells.
560
as in thyrocytes (23). For this purpose, we tested four Abs, expressed as Fabs, that represent ⬎80% of the human autoantibody
repertoire (26) to assess whether they protect or footprint specific
domains of the Ag during processing in EBV-TPO, that we have
previously reported to stimulate selected TPO-specific T cell
clones (21, 29).
In this experiment, the EBV-TPO cell line was coincubated for
2 h with the four human autoantibodies of different specificities
expressed as Fabs, to avoid potential interference via FcR binding
and piggyback effect, before adding the T cells.
T cell clone 59 only marginally recognized TPO expressed by
the EBV cell line, but in the presence of the Fab WR1.7, we observed a significant boost in T cell proliferation, suggesting that
this Fab was able to modulate Ag processing operated by the TPOEBV (Fig. 4). In contrast, none of the other tested Fabs seemed to
have any function, even at high concentrations. However, each of
these human autoantibodies was able to alter the pattern of responsiveness of the tested T cell clones (Table I). Indeed, Fab TR1.8,
TR1.9, and SP1.4, used at the saturating concentration of 10⫺8 M,
proved to be very efficient in either boosting or suppressing the T
cell determinants recognized by T cell clones 23 and 58 (Table I).
In some cases, the Fabs produced a dramatic enhancement in the
presentation of the recognized epitope, as observed for Fab TR1.8
and T cell clone 23 or for Fab WR1.7 and T cell clone 59. In other
cases, Fab WR1.7 resulted in a remarkable suppression of the displayed epitope, as shown for T cell clones 23 and 58. In all cases,
the Fabs suppressed or enhanced the recognition of human TPO by
individual T cell clones.
The experiment was repeated with five more TPO-specific T cell
clones analyzed with different TPO epitope recognition (Fig. 5).
Furthermore, T cell clones 12, 27, and 39 recognized the TPO Ag
only when presented by monocytes or DCs (data not shown), but
not when presented upon endogenous processing by the EBVTPO. T cell clones 40, 58, and 60, on the contrary, recognized also
the EBV-TPO cell line. Fig. 5 shows a complex pattern of epitope
FIGURE 4. Fabs interfere with Ag processing and presentation. T cell
clone 59 was challenged with the autologous TPO-EBV with or without
four human autoantibodies expressed as Fabs at increasing concentrations
(from 10⫺12 to 10⫺7 M). These data represented two separate experiments.
modulation operated by the four Fabs (summarized in Table II),
further stressing their ability to alter, by boosting or suppressing
presentation of different T cell determinants, the T cell responsiveness of the human self-reactive T cells.
The human autoantibodies do not allow the presentation of a
cryptic epitope
We have so far described that human mAbs are highly efficient in
modulating the T cell epitope repertoire. Therefore, we addressed
the question whether Abs could unmask the cryptic TPO epitope
recognized by pathogenic and immunodominant T cell clone 37
(21, 22). This TPO epitope is presented in vivo only upon endogenous processing in thyroid epithelial cells and in vitro in TPOtransduced cells such as EBV-transformed B cells (21). We have
demonstrated that T cell clone 37 is not stimulated by fully competent DCs pulsed with TPO, but, on the contrary, it is anergized
by a tolerogenic epitope presented by DCs (24).
We now tested whether the TPO epitope repertoire presented by
DCs could be influenced by the human TPO-specific autoantibodies to display the cryptic epitope to T cell clone 37. As shown in
Fig. 6A, T cell clone 37 failed to recognize the DCs pulsed with
TPO but was only stimulated by its cognate TPO536 –547 peptide,
and none of the human autoantibodies could unmask this cryptic
epitope when DCs presented TPO upon exogenous processing.
Table I. Enhancement and suppression of the TPO epitopes generated
upon endogenous processinga
SI
Antigenic Challenge
EBV-TPO
⫹WR1.7
⫹TR1.8
⫹TR1.9
⫹SP1.4
Clone 23
Clone 58
Clone 59
22
2.5
78
6
13
19
1.8
5.5
11.8
20.9
4.2
46.5
4.5
4.6
4.2
a
Three TPO-specific T cell clones were challenged with the TPO-EBV with and
without the four different human Fabs. Each Fab fragment (16 ng/ml) was coincubated
with the TPO-EBV for 2 h prior to the addition of the T cells. SI was calculated as
described in Materials and Methods. A SI ⬍ 3 is considered no proliferation in
response to the specific trigger.
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FIGURE 3. TPO-specific human autoantibodies modulate the activation
of self-reactive T cells. A panel of six TPO-specific T cell clones was
challenged with the autologous EBV-transformed B cell line pulsed with
human TPO (100 ng/ml) complexed with human autoantibody SP1.4 of
different isotypes. Complexes of TPO and SP1.4-IgG1 or SP1.4-IgE induced a powerful proliferation in all T cell clones analyzed. Ten mouse
anti-human TPO mAbs (IgG1) were tested simultaneously. Only mAb no.
15 was able to modulate the Ag processing by the EBV cell line used as
APC. For simplicity, only three mouse mAbs are shown, because the others
had no effect on T cells, as nos. 40 and 64.
T CELL EPITOPES AND B CELLS
The Journal of Immunology
561
Table II. Enhancement and suppression of the TPO epitopes generated
upon endogenous processinga
T Cell Clones
SP1.4
TR 1.9
TR 1.8
WR 1.7
a
In the same experiment, DCs presented the cognate TPO epitope
to T cell clone 43 (21), and this epitope was significantly upregulated by SP1.4 IgG1 (unpaired t test, p ⬍ 0.0001) and downregulated by all Fabs ( p ⬍ 0.0001).
DCs can strongly stimulate T cell clone 37 only when TPO is
expressed as a surface molecule and presented by endogenous processing (Fig. 6B), confirming that regardless of the type of APC
(epithelial cells, EBV-transformed B cells, or MVA-TPO-transduced DC), only endogenous processing allows the presentation of
the cryptic self-epitope.
Discussion
In autoimmune diseases, the production of autoantibodies and the
activation and expansion of self-reactive T cells is common. The
close interaction between B and T cells in autoimmunity is demonstrated by the colocalization of CD4⫹ T cells and B cells in the
affected tissue that may enable the B cells to present autoantigens
to T cells and at the same time enable CD4⫹ T cells to provide help
for autoantibody production (31).
In this complex interaction, Ags could be internalized through
the B cell-specific membrane receptors and presented to helper
CD4⫹ T cells at very low Ag concentrations. In this way, B cells
can compete and outperform more professional APCs such as DCs
(4). B cell Ag receptors, mannose receptors, and receptors for the
Ig Fc region, promote both internalization and efficient presentation at low Ag concentrations. Thus, binding to specific membrane
receptors concentrate Ags on APCs and mediates efficient
uptake (30).
This has clear biological implications, because direct talk between T and B cells can influence the overall adaptive immune
response to Ag (3, 32–34). It has been described that B cell Ag
presentation to T cells will control the functional response of the
Ag-specific T cells (35, 36). Recently, it has been also proven that
B cells might release a different repertoire of cytokines and be
subdivided into B effector type 1 or 2 (2). They can further influence the progression of autoimmune diseases by acting as regulatory cells (37, 38). In autoimmune diseases, B cells expressing the
appropriate BCR can more avidly capture and present the Ag, thus
27
39
40
58
60
0
0
1
0
1
1
1
0
0
0
0
1
2
2
1
2
1
2
2
1
0
2
1
0
Summary table for Fig. 5. 1, Increase; 2, decrease; 0, no change.
reducing the opportunity for mature DC cells, which normally dictate a proinflammatory type of response (39).
Another way in which B cells can alter the T cell response is via
the specific modulation of the epitope(s) displayed (8). Biochemical analysis demonstrated that both the suppressed and boosted
epitope fall within an extended domain of Ag stabilized or footprinted by this Ab during proteolysis (8). Obviously, changes in
the T cell epitope repertoire will totally modify the T cell response
to foreign or self-Ags. This latter modification, contrary to the one
in which B cells exert their function via the release of soluble
factors, does not require that the Ag is presented by the same
Ag-specific B cells, and indeed the soluble receptors (Abs) have
been shown to modify the type of peptide presented to T cells (8,
40). It has even been postulated that Abs acting in this way could
allow the unmasking of cryptic epitopes (5), which could have an
extremely dangerous effect in the induction and perpetuation of
autoimmunity (41).
Although cryptic epitopes, potentially involved in autoimmunity, have been described in mouse (42), they have been more
difficult to detect in man. We have described one of these cryptic
epitopes in human TPO, a dominant autoantigen in autoimmune
thyroiditis (21). We defined that the pathway in which TPO is
processed dictates the exposure of different epitopes recognized by
self-reactive T cells (21, 43). Intriguingly, we have also described
that this differential pattern of presentation can allow the display of
tolerogenic peptides (24).
Although it has been suggested that autoantibodies could potentially alter the T cell epitope repertoire, no study in man has yet
been performed, because the collection of appropriate autoantibodies and T cell clones to address this point have not been available.
The results we have described above indicate that human monoclonal autoantibodies can alter the pattern of Ag presentation to
self-reactive T cells. Biologically important conclusions have been
generated by our study. The first is that the isotype of the Ab will
significantly influence the Ag capturing and therefore the presentation of the conjugated Ag. For instance, in our study, we described that only Abs complexed to IgG1 or IgE could be captured
by B cells, whereas the same Ab on an IgG4 backbone could not.
This is because B cells only express Fc␥RII receptors, which can
capture IgG1 (30) or IgE Igs (19). In keeping with these previous
observations, the autologous EBV-transformed B cell line expressed neither CD16 nor CD64 (Fig. 2B) and was also negative
for CD23 (data not shown). Thus, the isotype of the Abs will
influence their action as modulator of Ag presentation. In this context, it has to be mentioned that polymorphisms of the FcRs may
alter the ability to bind with different affinity to Igs, and this has
been shown to have important biological consequences (44). More
interestingly, Fc polymorphisms have been associated, at least in
animal models, to predisposition to autoimmune diseases (45– 47).
A very important outcome of our study was the finding that the
ability to modulate Ag presentation was restricted to the human
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FIGURE 5. Modulation of Ag processing by bound Abs boost and suppress different T cell epitopes. Six TPO-specific T cell clones were challenged with the autologous EBV-transformed B cell line transduced with
rMVA-TPO, with and without four human autoantibodies expressed as
Fabs. The EBV cell line was infected with the rMVA-TPO supernatant for
4 h and incubated with the four different Fabs (3.2 ⫻ 10⫺8 M) for 2 h
before the addition of the T cells. These data are representative of two
separate experiments.
12
562
T CELL EPITOPES AND B CELLS
mAbs, whereas only one mouse anti-human mAb was able to affect TPO presentation by the autologous EBV-transformed B cell
line. However, all of the mouse Abs recognized the human TPO as
shown in Fig. 2, and their inability to influence Ag presentation
could relate to their binding affinities, which may be lower than the
human autoantibodies. Alternatively, they could recognize different epitopes not normally recognized by naturally occurring highaffinity human autoantibodies. Most of the TPO B cell epitopes are
conformational, and thus the exact location has not yet been identified. The epitopes for mouse mAbs 18, 59, 64, and 15, and the
human Fab SP1.4 and WT1.7 are within the A domain of TPO,
whereas the epitopes for mAbs 9 and 47, and the human TR1.8 and
TR1.9 are located within the B domain (48).
We have also used these mouse mAbs for similar studies with
DC as APC, and still found them to be less efficient than the human
monoclonals (data not shown). This further stresses the importance
of performing these types of studies with the proper material. Our
studies further stress the biological difference between mouse and
human mAbs in the modulation of immune responses, suggesting
that Ab binding specificities determine the effect on Ag processing.
Another important result of our study is that the human mAbs
could interfere with the endogenous presentation of TPO expressed on the surface of cells potentially acting as APC. The
effects of the different Abs on the panel of T cell clones recognizing different epitopes of TPO was diverse, and depending on the
combination of Abs and T cells, we could observe both up-regulation and down-regulation of T cell responses in agreement with
other studies (8, 15). The fact that these autoantibodies can alter
the pattern of endogenous recognition is of great biological significance, because TPO is expressed on the surface of thyroid ep-
ithelial cells (23), and these cells can in some cases act as APC (49,
50) and present TPO to T cells (20, 21).
In our previous studies, we have defined an immunodominant
cryptic epitope generated by endogenous processing (21), recognized by 18% of the T cell clones infiltrating the thyroid (20). The
analysis of the effects of the four human monoclonal autoantibodies clearly indicates that this epitope cannot be unmasked in DCs,
even if the Abs are able to influence the epitope repertoire. However, we proved that DCs expressing TPO on their surface were
able to present the cryptic epitope, excluding the possibility of an
intrinsic inability of DCs to generate such an epitope. These data
therefore reinforce the biological significance of the cryptic
epitope we have defined. Although we cannot exclude that other
human mAbs recognizing TPO might influence this recognition,
this is unlikely. Indeed, the pattern of epitope recognition of human anti-TPO Abs is quite restricted, and the Abs used in this
study appear to cover the whole repertoire of naturally occurring
Abs (26).
In conclusion, we have demonstrated that human autoantibodies
can alter the pattern of reactivity by human self-reactive T cells
and further stress the dominance of the cryptic epitope we have
previously described. The understanding of how autoantibodies
can influence presentation of T cell epitopes and why they do not
influence the cryptic epitope will hold important clues on the
mechanisms controlling autoimmunity.
Acknowledgments
We thank Tim Elliott and Anthony Antoniou for critical reading of the
manuscript and helpful discussion, and Mark Cragg for the kind gift of the
FcR mAbs.
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FIGURE 6. Human autoantibodies cannot unmask the cryptic epitope recognized by T cell clone 37. A, T cell clone 37 specific for TPO536 –547 and T
cell clone 43 specific for TPO540 –552, both restricted by HLA-DQB1*0602/DQA1*0102, were stimulated with HLA-matched DCs loaded with 100 ng/ml
human TPO with and without the human Fabs (16 ng/ml) or the SP1.4-IgG1 and SP1.4-IgG4. Clone 37 proliferates only in response to its cognate
TPO536 –547 peptide (DPLIRGLLARPA) but not to the TPO peptide presented upon exogenous processing by DCs. Fabs do not enhance presentation of
the cryptic TPO536 –547 epitope. In contrast, SP1.4-IgG1 and the Fabs have significantly altered presentation of the cognate TPO epitope recognized by T
cell clone 43. B, HLA-matched DCs transduced with MVA-TPO present the TPO cryptic epitope upon endogenous processing and induce proliferation in
T cell clone 37.
The Journal of Immunology
References
25. Ruf, J., M. E. Toubert, B. Czarnocka, J. M. Durand-Gorde, M. Ferrand, and
P. Carayon. 1989. Relationship between immunological structure and biochemical properties of human thyroid peroxidase. Endocrinology 125:1211.
26. Nishikawa, T., G. Costante, M. F. Prummel, S. M. McLachlan, and B. Rapoport.
1994. Recombinant thyroid peroxidase autoantibodies can be used for epitopic
“fingerprinting” of thyroid peroxidase autoantibodies in the sera of individual
patients. J. Clin. Endocrinol. Metab. 78:944.
27. Sallusto, F., and A. Lanzavecchia. 1994. Efficient presentation of soluble antigen
by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis
factor ␣. J. Exp. Med. 179:1109.
28. Osman, M., M. Tortorella, M. Londei, and S. Quaratino. 2002. MMP and TIMP
expression define the migratory characteristics of human monocyte-derived dendritic cells. Immunology 105:73.
29. Mullins, R. J., Y. Chernajovsky, C. Dayan, M. Londei, and M. Feldmann. 1994.
Transfection of thyroid autoantigens into EBV-transformed B cell lines: recognition by Graves’ disease thyroid T cells. J. Immunol. 152:5572.
30. Amigorena, S., and C. Bonnerot. 1999. Fc receptors for IgG and antigen presentation on MHC class I and class II molecules. Semin. Immunol. 11:385.
31. Yu, S., B. Medling, H. Yagita, and H. Braley-Mullen. 2001. Characteristics of
inflammatory cells in spontaneous autoimmune thyroiditis of NOD.H-2h4 mice.
J. Autoimmun. 16:37.
32. Manca, F., A. Kunkl, D. Fenoglio, A. Fowler, E. Sercarz, and F. Celada. 1985.
Constraints in T-B cooperation related to epitope topology on E. coli ␤-galactosidase. I. The fine specificity of T cells dictates the fine specificity of antibodies
directed to conformation-dependent determinants. Eur. J. Immunol. 15:345.
33. Ozaki, S., and J. A. Berzofsky. 1987. Antibody conjugates mimic specific B cell
presentation of antigen: relationship between T and B cell specificity. J. Immunol.
138:4133.
34. Celada, F., and E. E. Sercarz. 1988. Preferential pairing of T-B specificities in the
same antigen: the concept of directional help. Vaccine 6:94.
35. Stockinger, B., T. Zal, A. Zal, and D. Gray. 1996. B cells solicit their own help
from T cells. J. Exp. Med. 183:891.
36. Macaulay, A. E., R. H. DeKruyff, C. C. Goodnow, and D. T. Umetsu. 1997.
Antigen-specific B cells preferentially induce CD4⫹ T cells to produce IL-4.
J. Immunol. 158:4171.
37. Fillatreau, S., C. H. Sweenie, M. J. McGeachy, D. Gray, and S. M. Anderton.
2002. B cells regulate autoimmunity by provision of IL-10. Nat. Immunol. 3:944.
38. Mauri, C., D. Gray, N. Mushtaq, and M. Londei. 2003. Prevention of arthritis by
interleukin 10-producing B cells. J. Exp. Med. 197:489.
39. Lanzavecchia, A., and F. Sallusto. 2001. Regulation of T cell immunity by dendritic cells. Cell 106:263.
40. Watts, C., and A. Lanzavecchia. 1993. Suppressive effect of antibody on processing of T cell epitopes. J. Exp. Med. 178:1459.
41. Lanzavecchia, A. 1995. How can cryptic epitopes trigger autoimmunity? J. Exp.
Med. 181:1945.
42. Sercarz, E. E., P. V. Lehmann, A. Ametani, G. Benichou, A. Miller, and
K. Moudgil. 1993. Dominance and crypticity of T cell antigenic determinants.
Annu. Rev. Immunol. 11:729.
43. Quaratino, S., C. J. Thorpe, P. J. Travers, and M. Londei. 1995. Similar antigenic
surfaces, rather than sequence homology, dictate T-cell epitope molecular mimicry. Proc. Natl. Acad. Sci. USA 92:10398.
44. Van Wauwe, J. P., J. G. Goossens, and P. C. Beverley. 1984. Human T lymphocyte activation by monoclonal antibodies; OKT3, but not UCHT1, triggers mitogenesis via an interleukin 2-dependent mechanism. J. Immunol. 133:129.
45. Prins, J. B., J. A. Todd, N. R. Rodrigues, S. Ghosh, P. M. Hogarth, L. S. Wicker,
E. Gaffney, P. L. Podolin, P. A. Fischer, A. Sirotina, et al. 1993. Linkage on
chromosome 3 of autoimmune diabetes and defective Fc receptor for IgG in NOD
mice. Science 260:695.
46. Jiang, Y., S. Hirose, M. Abe, R. Sanokawa-Akakura, M. Ohtsuji, X. Mi, N. Li,
Y. Xiu, D. Zhang, J. Shirai, et al. 2000. Polymorphisms in IgG Fc receptor IIB
regulatory regions associated with autoimmune susceptibility. Immunogenetics
51:429.
47. Li, X., J. Wu, R. H. Carter, J. C. Edberg, K. Su, G. S. Cooper, and R. P. Kimberly.
2003. A novel polymorphism in the Fc␥ receptor IIB (CD32B) transmembrane
region alters receptor signaling. Arthritis Rheum. 48:3242.
48. Guo, J., R. S. McIntosh, B. Czarnocka, A. P. Weetman, B. Rapoport, and
S. M. McLachlan. 1998. Relationship between autoantibody epitopic recognition
and immunoglobulin gene usage. Clin. Exp. Immunol. 111:408.
49. Londei, M., J. R. Lamb, G. F. Bottazzo, and M. Feldmann. 1984. Epithelial cells
expressing aberrant MHC class II determinants can present antigen to cloned
human T cells. Nature 312:639.
50. Londei, M., G. F. Bottazzo, and M. Feldmann. 1985. Human T-cell clones from
autoimmune thyroid glands: specific recognition of autologous thyroid cells. Science 228:85.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
1. Clark, E. A., and J. A. Ledbetter. 1994. How B and T cells talk to each other.
Nature 367:425.
2. Harris, D. P., L. Haynes, P. C. Sayles, D. K. Duso, S. M. Eaton, N. M. Lepak,
L. L. Johnson, S. L. Swain, and F. E. Lund. 2000. Reciprocal regulation of
polarized cytokine production by effector B and T cells. Nat. Immunol. 1:475.
3. Rock, K. L., B. Benacerraf, and A. K. Abbas. 1984. Antigen presentation by
hapten-specific B lymphocytes. I. Role of surface immunoglobulin receptors.
J. Exp. Med. 160:1102.
4. Lanzavecchia, A. 1985. Antigen-specific interaction between T and B cells. Nature 314:537.
5. Watts, C., A. Antoniou, B. Manoury, E. W. Hewitt, L. M. McKay, L. Grayson,
N. F. Fairweather, P. Emsley, N. Isaacs, and P. D. Simitsek. 1998. Modulation by
epitope-specific antibodies of class II MHC-restricted presentation of the tetanus
toxin antigen. Immunol. Rev. 164:11.
6. Saoudi, A., S. Simmonds, I. Huitinga, and D. Mason. 1995. Prevention of experimental allergic encephalomyelitis in rats by targeting autoantigen to B cells:
evidence that the protective mechanism depends on changes in the cytokine response and migratory properties of the autoantigen-specific T cells. J. Exp. Med.
182:335.
7. Davidson, H. W., and C. Watts. 1989. Epitope-directed processing of specific
antigen by B lymphocytes. J. Cell Biol. 109:85.
8. Simitsek, P. D., D. G. Campbell, A. Lanzavecchia, N. Fairweather, and C. Watts.
1995. Modulation of antigen processing by bound antibodies can boost or suppress class II major histocompatibility complex presentation of different T cell
determinants. J. Exp. Med. 181:1957.
9. Antoniou, A. N., and C. Watts. 2002. Antibody modulation of antigen presentation: positive and negative effects on presentation of the tetanus toxin antigen via
the murine B cell isoform of Fc␥RII. Eur. J. Immunol. 32:530.
10. Stockinger, B. 1992. Capacity of antigen uptake by B cells, fibroblasts or macrophages determines efficiency of presentation of a soluble self antigen (C5) to T
lymphocytes. Eur. J. Immunol. 22:1271.
11. Berg, M., R. Uellner, and J. Langhorne. 1997. Fc␥ receptor II dependency of
enhanced presentation of major histocompatibility complex class II peptides by a
B cell lymphoma. Eur. J. Immunol. 27:1022.
12. Weetman, A. P. 2001. Determinants of autoimmune thyroid disease. Nat. Immunol. 2:769.
13. Wucherpfennig, K. W., and G. S. Eisenbarth. 2001. Type 1 diabetes. Nat. Immunol. 2:767.
14. Falcone, M., J. Lee, G. Patstone, B. Yeung, and N. Sarvetnick. 1998. B lymphocytes are crucial antigen-presenting cells in the pathogenic autoimmune response
to GAD65 antigen in nonobese diabetic mice. J. Immunol. 161:1163.
15. Dai, Y., K. A. Carayanniotis, P. Eliades, P. Lymberi, P. Shepherd, Y. Kong, and
G. Carayanniotis. 1999. Enhancing or suppressive effects of antibodies on processing of a pathogenic T cell epitope in thyroglobulin. J. Immunol. 162:6987.
16. Reijonen, H., T. L. Daniels, A. Lernmark, and G. T. Nepom. 2000. GAD65specific autoantibodies enhance the presentation of an immunodominant T-cell
epitope from GAD65. Diabetes 49:1621.
17. Huse, W. D., L. Sastry, S. A. Iverson, A. S. Kang, M. Alting-Mees, D. R. Burton,
S. J. Benkovic, and R. A. Lerner. 1989. Generation of a large combinatorial
library of the immunoglobulin repertoire in phage lambda. Science 246:1275.
18. Portolano, S., G. D. Chazenbalk, P. Seto, J. S. Hutchison, B. Rapoport, and
S. M. McLachlan. 1992. Recognition by recombinant autoimmune thyroid disease-derived Fab fragments of a dominant conformational epitope on human
thyroid peroxidase. J. Clin. Invest. 90:720.
19. Guo, J., S. Quaratino, J. C. Jaume, G. Costante, M. Londei, S. M. McLachlan, and
B. Rapoport. 1996. Autoantibody-mediated capture and presentation of autoantigen to T cells via the Fc⑀ receptor by a recombinant human autoantibody Fab
converted to IgE. J. Immunol. Methods 195:81.
20. Dayan, C. M., M. Londei, A. E. Corcoran, B. Grubeck-Loebenstein,
R. F. L. James, B. Rapaport, and M. Feldmann. 1991. Autoantigen recognition by
thyroid infiltrating T cells in Graves’ disease. Proc. Natl. Acad. Sci. USA
88:7415.
21. Quaratino, S., M. Feldmann, C. M. Dayan, O. Acuto, and M. Londei. 1996.
Human self-reactive T cell clones expressing identical T cell receptor ␤ chains
differ in their ability to recognize a cryptic self-epitope. J. Exp. Med. 183:349.
22. Quaratino, S., E. Badami, Y. Y. Pang, I. Bartok, J. Dyson, D. Kioussis,
M. Londei, and L. Maiuri. 2004. Degenerate self-reactive human T-cell receptor
causes spontaneous autoimmune disease in mice. Nat. Med. 10:920.
23. Khoury, E. L., L. Hammond, G. F. Bottazzo, and D. Doniach. 1981. Presence of
the organ-specific “microsomal” autoantigen on the surface of human thyroid
cells in culture: its involvement in complement-mediated cytotoxicity. Clin. Exp.
Immunol. 45:316.
24. Quaratino, S., L. P. Duddy, and M. Londei. 2000. Fully competent dendritic cells
as inducers of T cell anergy in autoimmunity. Proc. Natl. Acad. Sci. USA
97:10911.
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