HLA-DM and invariant chain are expressed by thyroid follicular cells, enabling the expression of compact DR molecules.
ABSTRACT Thyroid follicular cells (TFC) in Graves' disease (GD) hyperexpress HLA class I and express ectopic HLA class II molecules, probably as a consequence of cytokines produced by infiltrating T cells. This finding led us to postulate that TFC could act as antigen-presenting cells, and in this way be responsible for the induction and/or maintenance of the in situ autoimmune T cell response. Invariant chain (li) and HLA-DM molecules are implicated in the antigen processing and presentation by HLA class II molecules. We have investigated the expression of these molecules by TFC from GD glands. The results demonstrate that class II+ TFC from GD patients also express li and HLA-DM, and this expression is increased after IFN-gamma stimulation. The level of HLA-DM expression by TFC was low but sufficient to catalyze peptide loading into the HLA class II molecules and form stable HLA class II-peptide complexes expressed at the surface of TFC. These results have implications for the understanding of the possible role of HLA class II+ TFC in thyroid autoimmune disease.
- SourceAvailable from: Ricardo Pujol Borrell[Show abstract] [Hide abstract]
ABSTRACT: One salient feature of autoimmune thyroid disease is the inappropriate expression of human leukocyte antigen (HLA) class II molecules by thyroid follicular cells. Metallothioneins (MT) are small proteins induced by tissue stress that can contribute to restoring homeostasis of tissue inflammation and have been found to be increased in a transcriptomic analysis of Graves' disease (GD) glands. To assess the role of MT in the pathogenesis of GD, we analyzed MT-I and -II expression and distribution in GD-affected thyroid glands (n = 14) compared with other thyroid diseases (n = 20) and normal thyroid glands (n = 5). Two-color indirect immunofluorescence and semiquantitative morphometry were applied. The relationship between MT and HLA class II expression was analyzed by their degree of colocalization in GD sections, and in vitro induction kinetics and expression of these molecules on the HT93 thyroid cell line were compared by quantitative RT-PCR and flow cytometry using interferon-γ and zinc as stimuli. MT were clearly overexpressed in nine of 14 GD glands. MT expression distribution in GD was almost reciprocal to that of HLA class II. In vitro analysis of MT and HLA class II demonstrated that MT is induced more slowly and at a lower level than HLA. Moreover, the main MT inducer, zinc, reduces interferon-γ-induced class II expression. These findings show that MT and HLA class II play very different roles in the autoimmune process by affecting the thyroid gland, thereby pointing to the possible role of MT as a marker of cell stress and homeostasis restoration in GD.The Journal of clinical endocrinology and metabolism 11/2011; 97(2):446-54. · 6.50 Impact Factor
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ABSTRACT: T-cell tolerance to self-antigens is established in the thymus through the recognition by developing thymocytes of self-peptide-MHC complexes and induced and maintained in the periphery. Efficient negative selection of auto-reactive T cells in the thymus is dependent on the in situ expression of both ubiquitous and tissue-restricted self-antigens and on the presentation of derived peptides. Weak or inadequate intrathymic expression of self-antigens increases the risk to generate an autoimmune-prone T-cell repertoire. Indeed, even small changes of self-antigen expression in the thymus affect negative selection and increase the predisposition to autoimmunity. Together with other mechanisms, tolerance is maintained in the peripheral lymphoid organs via the recognition by mature T cells of a similar set of self-peptides in homeostatic conditions. However, non-lymphoid peripheral tissue, where organ-specific autoimmunity takes place, often have differential functional processes that may lead to the generation of epitopes that are absent or non-presented in the thymus. These putative differences between peptides presented by MHC molecules in the thymus and in peripheral tissues might be a major key to the initiation and maintenance of autoimmune conditions.Frontiers in Immunology 01/2013; 4:442.
- 10/2011; , ISBN: 978-953-307-643-0
International Immunology, Vol. 11, No. 2, pp. 269–277© 1999 The Japanese Society for Immunology
HLA-DM and invariant chain are expressed
by thyroid follicular cells, enabling the
expression of compact DR molecules
Marta Cata ´lfamo1, Laurence Serradell2, Carme Roura-Mir1, Edgardo Kolkowski1,
Mireia Sospedra1, Marta Vives-Pi1, Francesca Vargas-Nieto1,
Ricardo Pujol-Borrell1and Dolores Jaraquemada1,2
Unitat d’Immunologia,1Hospital Universitari Germans Trias i Pujol,2Facultat de Medicina, Universitat
Auto `noma de Barcelona, 08193 Bellaterra, Barcelona, Spain
Keywords: class II molecules, peptides, thyroid autoimmunity, thyroid follicular cell
Thyroid follicular cells (TFC) in Graves’ disease (GD) hyperexpress HLA class I and express
ectopic HLA class II molecules, probably as a consequence of cytokines produced by infiltrating
T cells. This finding led us to postulate that TFC could act as antigen-presenting cells, and in this
way be responsible for the induction and/or maintenance of the in situ autoimmune T cell
response. Invariant chain (Ii) and HLA-DM molecules are implicated in the antigen processing and
presentation by HLA class II molecules. We have investigated the expression of these molecules
by TFC from GD glands. The results demonstrate that class II?TFC from GD patients also express
Ii and HLA-DM, and this expression is increased after IFN-γ stimulation. The level of HLA-DM
expression by TFC was low but sufficient to catalyze peptide loading into the HLA class II
molecules and form stable HLA class II-peptide complexes expressed at the surface of TFC. These
results have implications for the understanding of the possible role of HLA class II?TFC in thyroid
Human autoimmune thyroid diseases (AITD), i.e. Hashimoto’s
thyroiditis, Graves’ disease and primary myxedema, are char-
acterized by an intense immune response to well character-
ized thyroid autoantigens such as thyroglobulin (Tg), thyroid
peroxidase (TPO) and the thyrotropin receptor (TSH-R). This
response includes high-affinity antibodies, some of which
are produced in situ (1), against all these antigens and is
maintained for long periods of time. Thlymphocytes specific
for these antigens are therefore expected to direct and
possibly modulate this response. The recognition of thyroid
follicular cells by the thyroid-specific T lymphocytes is consid-
ered central to AITD pathogenesis (2,3).
TFC express class II molecules in glands affected by AITD
(2,4). They also overexpress class I molecules as well as
associated molecules such TAP (5), LMP-2 and LMP-7 (6).
This phenomenon has led us to postulate that presentation
of autoantigens may play a key role in either breaking the
tolerance to peripheral autoantigens or in the perpetuation of
the autoimmune response. Expression of HLA class II by itself
Correspondence to: D. Jaraquemada, Unitat d’Immunologia, Hospital Universitari Germans Trias i Pujol, Universitat Auto `noma de Barcelona,
Carretera de Canyet s/n, 08916 Badalona, Spain
Transmitting editor: M. FeldmannnReceived 10 December 1997, accepted 26 October 1998
is not sufficient for efficient antigen presentation. The nature
and functionality of class II molecules expressed by TFC
ules such as Ii and HLA-DM.
Class II α and β chains assemble in the endoplasmic
reticulum (ER) forming large complexes with the invariant
chain (Ii-α-β)3which are transported to endo-lysosomal com-
partments by signals in the Ii cytoplasmic domain. Ii particip-
ates in the assembly of αβ complexes in the ER, occupying
the peptide binding site of the αβ dimer with a region spanning
residues 81–104 of Ii, which interacts with MHC in the same
way as a peptide, thus preventing the binding of free peptides
in the ER (7,8). This region corresponds to class-II-associated
invariant chain peptide (CLIP) (9). Class II–Ii trimers are
transported to the secretory route and targeted to the endo-
cytic pathway (10) where Ii chain is cleaved, leaving CLIP
bound to the class II molecule peptide-binding site. CLIP
peptides are removed by immunogenic or autologous pep-
tides generated by endosomal processing, thus forming
HLA-DM expression by thyroid follicular cells
mature αβ peptide stable complexes which are transported
to the cell surface. In these endo-lysosomal compartments,
HLA-DM molecules act as the main catalyzer in the release
of CLIP peptides and binding of other peptides to class II
molecules (11). Cells lacking DM express class II molecules in
DM molecules are MHC-encoded membrane glycoprotein
heterodimers formed by two subunits DMα (33–35 kDa) and
DMβ (30–31 kDa), with structural homology with both class I
and class II molecules (13). The cellular distribution of DM is
similar to class II molecules and their expression is also
induced by IFN-γ. DM molecules are expressed at low levels
in professional APC, are not expressed at the cell membrane
and have a relatively long half-life (14).
The capacity of DM to release CLIP and form stable
complexes has been reproduced in vitro (15–18). DM kinetics
as an enzyme catalyzing CLIP release from class II molecules
are of three to 12 class II molecules per minute and per
DM molecule using MHC-CLIP complexes as a preferential
substrate for DM rather than stable MHC-peptide complexes
(19). DM molecules also act as a chaperone for α and β
complexes preventing their aggregation at low pH. DM binds
to empty αβ molecules from where CLIP has been removed
and stabilizes them until they bind peptide (20). Specific
signals in the cytoplasmic domain target DM molecules to
compartments of the endocytic pathway including early and
late endosomes as well as the MHC class II compartment
(MIIC) (21,22). Finally, DM also function as a peptide editor,
preventing binding of low-affinity peptides and favoring the
binding of high-affinity peptides, thus helping the formation
of mature complexes (23). Recently, a new molecule, HLA-
DO, also codified within the MHC class II region has been
described associated to DM, which has the capacity to
modulate DM function (24,25).
The presence or absence of Ii and DM in class II-expressing
TFC is very relevant since it will determine the access of
autologous peptides to the class II molecules. In the absence
or suboptimal amounts of Ii, the majority of class II molecules
at the surface would be expected to be unstable and bound
to unselected peptides. If DM was absent, a majority of
peptides would be CLIP variants. On the other hand, if both
DM and Ii are expressed in sufficient amounts, the array of
peptides presented by class II molecules would be varied
and probably from tissue-specific molecules, some of which
could be important in the maintenance of the autoimmunity.
We have studied the Ii and DM expression by class II-
expressing TFC from GD glands.
Thyroid tissue samples were obtained at surgery from seven
patients with GD, i.e. TB212 (73%), TB242 (0%), TB250 (46%),
TB255 (42%), TB258 (40%), TB260 (45%) and TB378 (49%),
expressing different levels of class II molecules (in brackets)
and one with multinodular goitre (MNG) (TB359), class II–.
Clinical diagnosis was made on the basis of the usual thyroid
function tests and were confirmed by histopathology. The
protocol had been approved by the ethical committee of
M1 (26) is a human fibroblast cell line. TEB158 is an Epstein–
Barr virus-transformed lymphoblastoid B cell line (LCL) from
one thyroid donor, which was used as control in all experi-
ments. All cell lines were maintained in standard culture
conditions in culture medium RPMI ? 10% FCS.
Samples of thyroid tissue obtained at surgery were digested
as described (27). Cell suspensions were cultured overnight
in RPMI 1640 culture medium supplemented with 2 mM
L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin and
10% heat-inactivated FCS at 37°C/5% CO2in tissue culture
flasks. Non-adherent mononuclear cells were collected and
cryopreserved. The adherent cell population (TFC) was
harvested, extensively washed and stored frozen or directly
used. For the induction of HLA class II expression in TFC and
M1 cells, cultures were exposed to 500 U/ml IFN-γ. After 24
or 48 h, cells were pelleted for total RNA extraction or stained
and analyzed by flow cytometry. Recombinant human
IFN-γ was a kind gift from G. R. Adolf (Boehringer Institute,
The following mAb were used: MIC-18 (mAb anti-TPO, from
P. Carayon, University of Marseille, France), EDU-1 (specific
for a monomorphic HLA-class II determinant, from R. Vilella,
Hospital Clı ´nic, Barcelona, Spain), DA6.147 (28) (also specific
for a monomorphic HLA-class II determinant, a gift from
E. O. Long, NIAID, Rockville, MD), VIC-Y1 (29) (anti-human Ii
chain cytoplasmic domain, from W. Knapp, Vienna, Austria),
R-DMB/C (30) (a rabbit anti-DMβ serum, from P. Jensen,
Emory University School of Medicine, Atlanta, GA), MaP.DMB/
C (31) (specific for a cytoplasmic tail peptide of DMβ chain)
and CerCLIP.1 (15) (specific for CLIP-associated class II
molecules) (both from P. Cresswell, Yale University School of
Medicine, New Haven, CT). Human serum with high-titer TPO
antibodies was also used for immunofluorescence staining of
Series of 5 µm cryostat sections from a class II?GD gland
(TB212) were stained by double-indirect immunofluorescence
(32) combining a human anti-TPO serum (a patient’s serum
with high-titer TPO antibodies, used at 1:10,000) with either
anti-class II (EDU-1), anti-Ii (VIC-Y1) mAb or a rabbit anti-
HLA-DMβ serum (R-DMB/C), using TRITC-conjugated goat
anti-human Ig and FITC-conjugated goat anti-mouse IgG or
goat anti-rabbit Ig, as second reagents (all from Southern
Biotechnology, Birmingham, AL). For double class II/DM
staining, EDU-1 and R-DMB/C were used with TRITC–goat
anti-mouse IgG and FITC–goat anti-rabbit Ig respectively as
secondary antibodies. To check possible cross-reactions
among the antibodies and non-specific binding to the tissue,
sections were stained following the above protocols but
replacing the primary antibody by normal mouse serum or
replacing the rabbit anti-R-DMB/C antiserum by normal rabbit
serum and by omitting each of the layers in turn. To block
non-specific binding, 1% of BSA was added to the PBS used
HLA-DM expression by thyroid follicular cells
to dilute antibodies. Between incubations (30 min for each
layer), preparations were washed in PBS.
LCL and overnight cultured TFC were fixed and permeabilized
before staining with anti-TPO, anti-class II, anti-Ii and anti-DM
antibodies (33), and the same was done after 48 h culture in
the presence of IFN-γ. Live cells were used for CerCLIP.1
staining, and surface class II and TPO controls. Antibody
binding was detected with GAM–FITC. Samples of 10,000
cells were analyzed on a FACScan using the Lysys II software
(both from Becton Dickinson, San Jose, CA).
Northern blot hybridization
Total RNA was isolated using the guanidine thiocyanate
method (34). Samples (10 µg) were separated by electro-
phoresis on formaldehyde agarose gels and transferred over-
night to a Hybond N?membrane (Amersham, Buckingham-
shire, UK) using 20?SSC as transfer buffer. Hybridization
was performed with 2?106c.p.m./ml of an α-32P-labeled 0.8
kb DMA probe (from P. Cresswell), at high stringency (68°C)
in solution containing 7% SDS, 0.25 M Na2HPO4, 1% BSA
and 1 mM EDTA. After 20 h hybridization, the membrane was
washed in 20 mM phosphate buffer, 1 mM EDTA and 1%
SDS at 68°C, and exposed at –70°C. Hybridization with a 1.2
kb Ii probe and with a 1.3 kb HLA-DRα (both from E.
Long, NIAID, Rockville, MD) was performed using the same
conditions. Membranes were subjected to autoradiography.
Cells at a concentration of 5?107cells/ml were lysed in lysis
buffer (0.01 M Tris, pH 7.4, 0.15 M NaCl, 1% Nonidet P40,
5 mM EDTA, 10 mM iodoacetamide, 1 mM PMSF, 2 µM
pepstatine, 5 µM aprotinin and 5 µM leupeptin), and aliquots
corresponding to 1.5?106and 0.75?106cells were heated
for 4 min at 95°C, loaded onto a 12% acrylamide gel and
electrophoresed in SDS–PAGE. Proteins were transferred to
nitrocellulose. Blots were blocked for 30 min in Blotto (TBS,
0.05% Tween 20 and 5% skimmed milk), incubated for 2 h
with a rabbit anti-DMβ serum (30), in TBSN/milk. After three
(10 min) washes in TBSN, blots were incubated for 1 h with
an anti-mouse IgG conjugated to horseradish peroxidase
in TBSN and the bands developed using the enhanced
chemiluminescence ECL kit (Amersham). The bands were
analyzed by densitometry using a Scanjet IIc scanner (Hewlett
Packard) connected to Power Macintosh 4400/200 and the
Scan Analysis program (Biosoft, Cambridge, UK). Ratios
between densitometry results for DM and DR from 1.5?106
and 7?105cell samples were calculated.
Cells (107) were washed twice with cold PBS and labeled
followingthe standard protocol
(Amersham) using lactoperoxidase/H2O2and washed again
3 times in cold PBS. Radiolabeled cells were lysed at 107
cells/ml in lysis buffer containing 2% NP-40, 6 mM CHAPS,
50 mM Tris–HCl, pH 8, 150 mM NaCl, 5 mM EDTA, 1 mM
PMSF, 10 mM iodoacetamide, 5 µM aprotinin, 5 µM leupeptin
and 2 µM pepstatin. After 30 min incubation at 4°C, cell
lysates were cleared of nuclei and debris by centrifugation
at 14,000 g for 5 min at 4°C. Lysates were precleared with
an irrelevant antibody, normal rabbit serum and Protein
A–Sepharose beads (Pharmacia), and mixed with the relevant
antibody overnight at 4°C. Antigen–antibody complexes
were isolated with 10 µl rabbit anti-mouse Ig bound-Protein
A–sepharose beads. After one wash with lysis buffer and four
with buffer containing 6 mM CHAPS, 50 mM Tris, pH 8,
150 mM NaCl and 5 mM EDTA, samples were resuspended
in 100 µl non-reducing sample buffer and left for 1 h at room
temperature in order to establish complex stability. One-half
of the sample, boiled at 100°C for 3 min (B), and the other
half (NB) were electrophoresed on SDS–12% polyacrylamide
gels. Gels were dried and subjected to autoradiography.
TFC from GD glands express Ii and HLA-DM
Expression of HLA-DR, Ii and HLA-DM in TFC was studied
by indirect immunofluorescence in thyroid tissue sections
from GD patients, using anti-TPO antibodies in parallel to
define TFC in the sections (Fig. 1b, d and f, labeled red). As
seen in Fig. 1, all three molecules were expressed with
different intensities by TPO?cells (Fig. 1a, c and e, labeled
green) from a class II?GD thyroid gland, TB212. Class II
staining (Fig. 1a) was visible on the apical pole of TFC lining
the coloid space of the follicles as well as in some infiltrating
cells (possibly B lymphocytes, dendritic cells, macrophages
and activated T cells). Ii was detected in the cytoplasm of
TPO?TFC around follicles and also in infiltrating cells
(Fig. 1c). Ii was distributed all over the cytoplasm of TFC, in
contrast with HLA-DM (Fig. 1e), which was detected in TPO?
TFC showing a distribution around the nuclei and near the
basal pole. DM staining was also observed in infiltrating
mononuclear cells. DM?TFC (Fig. 1g) were also class II?
(Fig. 1h). Three thyroid glands (TB212, TB250 and TB255)
of DR, Ii and DM, and showed very similar patterns in all
samples (not shown). Control staining with normal mouse,
normal rabbit and normal human sera were negative in all
cases. Anti-CD3 mAb only stained interstitial infiltrating cells
(data not shown).
HLA-DM and Ii message are detected in unstimulated GD
TFC by Northern blots and can be induced by stimulation
Total RNA was extracted from different TFC samples, separ-
ated from the infiltrating population by overnight culture and
processed (unstimulated) or cultured for 48 h in the presence
sion of HLA-DR, Ii and HLA-DM was detected by hybridization
with specific probes, and results from one experiment are
shown in Fig. 2. Total RNA from a LCL (TEB158) was used
cell line (M1) were used as a negative control for basal
expression and positive for IFN-γ induction. Basal expression
of Ii and DR was detected in class II?GD TFC (TB378, TB250
and TB260) but not in a class II–multinodular TFC sample
(TB359). Basal expression of HLA-DM was also detectable
in all three samples, although the intensity of the signal was
HLA-DM expression by thyroid follicular cells
Fig. 1. TFC from GD glands express HLA-DM and Ii. Cryostat sections
of double staining of TPO (b, d and f, red), HLA class II (a, green;
h, red), Ii (c, green) and HLA-DM (e and g, green) of class II?
much lower (see Fig. 2) than for HLA-DR and Ii. In all cases,
culture in the presence of IFN-γ greatly increased expression
of DM, Ii and DR message in positive samples, and induced
their expression in negative samples. The induction of DM
and Ii message was as efficient as the induction of DR
Fig. 2. HLA-DM message is detected in unstimulated GD TFC by
Northern blotting and can be induced by stimulation with IFN-γ. Total
RNA samples from HLA class II?(TB378, TB250 and TB260) GD
glands and HLA class II–(TB359) MNG gland TFC were hybridized
with HLA-DRα, Ii and DMA probes.
expression and went parallel to the induction of all three
genes in M1 cells. A total of seven other GD samples (TB212,
228, 242, 255, 258, 269 and 270) were tested in separate
sion of DM in class II?samples, no signal in class II–samples
and a strong increase of message expression upon IFN-γ
stimulation (data not shown).
The level of HLA-DM expression in the cytoplasm of TFC is
Cytoplasmic expression of HLA-DM, Ii and DR molecules was
also studied in overnight cultured and IFN-γ-treated TFC by
flow cytometry. Figure 3A show the cytoplasmic expression
of TPO, class II, Ii and HLA-DM by unstimulated (–) and
stimulated (st) class II?TFC (TB255). HLA-DM expression in
unstimulated cells was very low and there was hardly any
detectable increase after stimulation, whereas cytoplasmic
DR and Ii expression were higher without stimulation, and
their increase after IFN-γ treatment was clearly detectable.
The DM data contrast with the message data shown in Fig. 2,
since the DM message expression was efficiently induced in
all cells tested. The expression of DM in stimulated cultured
TFC was indeed visible by immunofluorescence in the positive
cells (stimulated TB255, ~10% DM?cells), within the cyto-
plasm, in vesicles near the plasma membrane (Fig. 3B). Basal
expression of HLA-DM by unstimulated TB255 cultured cells
was visible but very low (not shown).
DR protein induction by IFN-γ is more efficient than the
induction of HLA-DM protein in TFC
Low expression levels of DM in TFC could be related to the
relatively low level of class II expression by these cells,
compared to LCL. To compare the level of DR and DM
proteins before and after IFN-γ treatment, Western blots were
performed on TFC, stimulated TFC and LCL samples, the
bands analyzed by densitometry, and the relative ratio DM to
DR calculated. Figure 4 show the expression of both proteins
by all samples and their calculated relative ratio for the 1:1
dilution. The data show that HLA-DM is expressed by class
II?TFC (45% DR?TB260) at a lower level relative to DR than
in LCL (DM:DR ratio 0.52 and 0.33 respectively). In addition,
an increase of DM expression after IFN-γ treatment was clearly
visible by Western blot analysis, although the DM:DR ratio in
stimulated cells was lower (0.18). This indicates that DR
induction by IFN-γ is more efficient than DM induction,
explaining the failure to detect a clear increase of DM protein
expression in stimulated TFC by flow cytometry.
HLA-DM expression by thyroid follicular cells
Fig. 3. Expression of HLA-DM in class II?TFC is low but detectable by immunofluorescence. (A) Cytoplasmic staining of HLA-DR, Ii and HLA-
DM in basal (–) and stimulated (st) conditions in GD TFC (TB255) and LCL, detected by flow cytometry. (B) Double-immunofluorescence
staining of HLA-DM (right panels) and TPO (left panels) of IFN-γ-stimulated TFC (TB255).
expressed by TFC demonstrates the expression of ‘compact’
SDS-resistant surface HLA-DR complexes
Mature, peptide-bound HLA class II complexes are mostly
as dimers in SDS–PAGE (‘compact’ forms) (35). Since TFC
express Ii and DM, transport of DR complexes to the endo-
lysosomal compartments would be secured by Ii and loading
of peptides produced in the endo-lysosomes would be facilit-
ated by DM molecules, capable of displacing CLIP peptides.
However, levels of DM were relatively low so, only if these
levels were not suboptimal, DR molecules expressed by TFC
should therefore be mature, SDS-resistant, peptide-bound DR
dimers. To characterize the conformational characteristics of
class II molecules expressed by TFC, surface (125I-labeled)
DR complexes were immunoprecipitated with a DRα-specific
mAb (DA6.147) from a detergent-solubilized total cell extract
of IFN-γ-stimulated TFC (TB258). Control samples were LCL
extracts. Samples were incubated in non-reducing SDS
sample buffer and either boiled or kept at room temperature
prior to their analysis by SDS–PAGE. Results shown in Fig. 5
demonstrate that most DR molecules expressed by TFC are
SDS-resistant ‘compact’ forms (αβ), which dissociate into α
suggests that despite low levels, DM molecules expressed by
TFC are sufficient and fully functional.
Functionality of DM molecules is confirmed by the absence
of CLIP-associated DR molecules on the surface of TFC
mAb CerCLIP.1 recognizes CLIP peptides associated to DR
molecules. Expression of such complexes at the surface of
the cells would imply that peptide exchange in the endosomal
compartments was incomplete. That is the case of LCL, where
the rate of class II expression is extremely high and there are
always some molecules associated with CLIP which are
transported to the cell surface. We have compared surface
staining of class II and CerCLIP.1 in LCL and class II?and
class II—TFC, using surface TPO as a positive control for
TFC. As seen in Fig. 6, CerCLIP.1 was mostly negative in
HLA-DM expression by thyroid follicular cells
Fig. 4. HLA-DM induction by IFN-γ is less efficient than induction of
HLA-DR in TFC. Western blot analysis of HLA-DM and HLA-DRα
corresponding to 1.5?106(1:1) and 0.7?106(1:2) of unstimulated
(TB260) and stimulated (TB260 IFN-γ) GD TFC and LCL as control of
Fig. 5. Immunoprecipitation of
expressed by TFC demonstrates the expression of ‘compact’ SDS-
resistant surface complexes. Peptides bound to these molecules are
heterogeneous. Immunoprecipitation of HLA-DR–peptide complexes
with mAb (DA6.147) and analysis by SDS–PAGE in non-reducing
conditions with (B) or without (NB) treatment at 95°C. The figure
shows αβ complexes (NB) and α and β chains dissociation after
125I-labeled HLA-DR molecules
class II?(TB255 and TB258) and class II—(TB242) TFC
samples compared to the positivity in LCL, showing CLIP-
class II complexes in LCL but no such complexes in class
II?TFC. These data confirm the immunoprecipitation data
showing that most class II molecules expressed by TFC
are compact, i.e. associated to stabilizing peptides, again
suggesting a very high efficiency of DM in the removal of
CLIP peptides from class II molecules in TFC.
TFC in autoimmunity express class II molecules (4,36), pre-
sumably due to the effect of cytokines synthesized in situ by
inflammatory cells and T lymphocytes (5,37). The role of
these class II molecules is not clear, although it has been
demonstrated that they can present exogenous peptides (38)
and it has been postulated that they may be involved in the
induction and/or maintenance of the in situ autoimmune T cell
response(2). However, since
co-stimulatory molecules even upon IFN-γ treatment the func-
tion of these ectopic class II molecules remains unknown.
The data presented in this report demonstrate that they are
structurally capable of binding endogenous peptides and
Fig. 6. CLIP-associated class II molecules are nearly absent from
the surface of TFC, confirming that TFC class II are compact
molecules. CerCLIP.1 mAb-specific HLA class II-CLIP complexes
was used for surface staining of HLA class II?(TB255 and TB258)
and HLA class II-(TB242) GD TFC and LCL. The figure also shows
the surface staining of HLA class II (EDU-1) and TPO (MIC-18).
therefore to present them. We have been able to demonstrate
the expression of Ii and DM molecules by TFC from auto-
immune glands and their induction by IFN-γ treatment in vitro.
Expression of Ii was high enough to ensure efficient transport
of class II molecules to the peptide loading compartment.
The expression of HLA-DM by these cells has important
implications if we accept that autoantigen presentation by
TFC may have a role in the disease process. It is known that
DM interact with class II molecules bound to endogenous
peptides (CLIP or others) incapable of forming high stability
class II–peptide complexes. DM acts as a peptide editor
favoring the binding of peptides and the formation of highly
stable long-life complexes. DM has therefore a strong influ-
ence in the repertoire of peptides ultimately bound to class
II molecules expressed at the cell surface (23).
We have also demonstrated that surface HLA class II
molecules expressed by TFC are compact SDS-stable com-
plexes. Immunoprecipitations were made on IFN-γ stimulated
samples to increase the expression of class II, although the
original cells (TB258) were class II?(40%). That, together
with the lack of expression of CLIP-bound complexes at the
surface, indicates that class II molecules expressed by TFC
are loaded with peptides other than CLIP.
These results are important in relation to the presentation
of autoantigens by class II molecules expressed by TFC.
Highly specialized endocrine epithelial cells such as TFC
which are responsible for the synthesis of thyroid hormones
HLA-DM expression by thyroid follicular cells
ulin which is transported to the follicular lumen where it is the
major component of the coloid. Thyroglobulin is the precursor
of and a biological storage system for thyroid hormones. A
specialization of the TFC is their unique capacity of endo-
cytosis by which thyroglobulin–hormone complexes enter the
cells, and are hydrolysed in the lysosomes to release T3 and
T4 hormones. Another thyroid-specific autoantigen is the
enzyme TPO, which localizes mainly on the surface at the
apical pole and also in the cytoplasm of the follicular cells.
Other molecules, such as the TSH receptor, are expressed
at much lower density at the opposite basal pole. TSH-R is a
major target for autoantibodies in GD and these autoantibod-
ies act mostly as TSH-R agonists, activating TFC function
including hormone synthesis, endocytosis and even class II
expression (39,40). Whether from the cytoplasm (41) or by
internalization (42), these molecules should have access to
the endocytic pathway and therefore peptides from their
degradation could to bind to class II molecules on their way
to the surface. Efficient transport of class II to the peptide-
loading compartment and peptide exchange is possible since
TFC express Ii and DM. Peptide exchange by DM in these
cells was efficient enough to almost completely prevent
expression at the surface of CLIP-associated complexes. A
possible explanation of the high efficiency of DM in TFC is
the putative absence of HLA-DO from these cells (24,25),
since expression of DO appears to be limited to some cells
(B cells, thymic epithelium) and is not induced by IFN-γ.
HLA-DO down-regulates DM function reducing its efficiency
as a peptide editor. Absence of DO should facilitate maximum
efficiency for DM action.
The peptide pool bound to these TFC class II compact
peptides and may be capable of interacting with tissue
specific T cells. The lack of expression of conventional
co-stimulatory molecules has questioned a role for these
complexes at least in the induction of the autoimmune
response. Only professional APC express B7 in autoimmune
thyroid infiltrates (43). Class II?TFC could be cooperating
with these APC in the infiltrates in such a way that APC would
stimulate naive cells and TFC maintain their activation. This
is assuming that the needs for co-stimulation in the mainten-
ance of a response are less astringent or that the TFC are
able to stimulate T cells trans-co-stimulated by other APC.
This has been demonstrated in vitro with class II?TFC,
incapable of inducing primary allogeneic responses but cap-
able if co-cultured with a B7.1-expressing transfected cell.
Some clones could respond to TFC in a B7-independent
manner producing IL-4 in contrast with the production of
IL-2 ? IL-4 if the presenting cells were LCL (44). Non-
professional APC could also induce incomplete responses or
induce different functional capacity in the T cells such as
changes in the cytokine profiles in response to antigen
(45). On the other hand, conventional APC present in the
autoimmune tissue are able to uptake cell debris containing
the same tissue-specific antigens as those presumably pre-
sented by class II on TFC. In addition, differential processing
by APC and TFC may generate different epitopes which could
lead to differential recruitment and/or signaling to T cells
depending on the type of the presenting cells (46). In vitro,
TFC can induce proliferation of T cell clones (47–49), can be
targets of cytotoxic T lymphocytes, induce IL-4 production by
T cell clones and be recognized by γδ cells (50 and M.
is likely to be complex and will be progressively unveiled as
knowledge on the regulation of antigen presentation, T cell
activation and differentiation expands.
We thank Drs G. R. Adolf, P. Cresswell, E. Long, S. Kovats, W. Knapp,
P. Jensen and R. Vilella for kindly supplying us with reagents. We
would like to thank Dr M. Martı ´ for critically reading the manuscript.
This work was supported by grants from the Fondo de Investigaciones
Sanitarias of the Spanish Health Ministry (FIS 94/0807), from the
DGES of the Spanish Education Ministry (PM95-0191) and in part by a
grant from the ISCI Program of the European Commission (CI1*CT92-
0071). M. C. is supported by the FIS (94/0807), L. S. by the TMR
program of the European Commission (ERBFMBICT961295), C. R.-
M. by the Generalitat de Catalunya (GRQ 93-2015) and M. S. by the
FPI program of the Spanish Education Ministry.
TSH-R thyrotropin receptor
autoimmune thyroid diseases
class II-associated invariant chain peptide
Epstein–Barr virus-transformed lymphoblastoid B cell line
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