From the Departments of Medical Microbiology and
§ Oral and Maxillofacial Surgery, Johannes Gutenberg
University, D-55101 Mainz, Germany
Received for publication, July 21, 2000, and in revised form, December 13, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Antigen-presenting cells degrade endocytosed
antigens, e.g. collagen type II, into peptides that are
bound and presented to arthritogenic CD4+ helper T cells by
major histocompatibility complex (MHC) class II molecules. Efficient
loading of many MHC class II alleles with peptides requires the
assistance of H2-M (HLA-DM in humans), a heterodimeric MHC class
II-like molecule that facilitates CLIP removal from MHC class II
molecules and aids to shape the peptide repertoire presented by MHC
class II to CD4+ T cells. In contrast to the HLA-DM region
in humans, the Collagen type II (CII)1
induces a chronic polyarthritis syndrome in H2q mice
that resembles some of the hallmarks of human rheumatoid arthritis.
Both arthritogenic T and B cells are instrumental in initiating and
perpetuating the debilitating disease. The dissociation of the capacity
to induce a strong anti-CII-directed antibody response without
developing arthritis or to induce an immune response ultimately leading
to arthritis indicates that qualitative differences in humoral and/or
cellular immune responses exist (1-8). Thus, efficient degradation,
processing, and presentation of the autoantigen CII may play a role in
whether an "arthritogenic" immune response ensues or not. In
contrast, not only the antigen-presenting cell, but also the B and T
cell repertoire influences the evolution of an autoimmune response.
CD4+ T cells with an arthritogenic potential have been
described and are able to initiate arthritis in DBA/1 mice (9).
However, to activate, expand, and maintain these CII-specific T cells, the antigen CII has to be taken up, processed, and presented by appropriate antigen-presenting cells by I-Aq molecules.
Previous studies have indicated that substantial differences exist
pertaining to the ability of different antigen-presenting cells to
effectively present CII to T cell hybridomas. To stimulate CD4+ T cells, MHC class II molecules must be loaded with
peptides provided by endogenous or exogenous proteins. This step is
governed by the MHC class II-like H2-M molecules, which facilitate
exchange of invariant chain (Ii)-derived MHC class II-associated Ii
peptides (CLIP) from MHC class II for stably bound antigenic peptides
(10, 11). Although dissociation of CLIP for antigenic peptides might spontaneously occur at endosome/lysosome-like pH (12), experiments using either H2-M-deficient mice or human B cell lines
mutated in the HLA-DM (DM) loci demonstrated that
H2-M/DM is required for that final step of peptide loading by many MHC
class II alleles (13-16).
H2-M/DM appears to function as a peptide editor that serves to
positively select peptides that can stably bind to MHC class II
molecules (17, 18). Thus, expression of certain H2-M alleles associated
with susceptibility to develop CII-induced arthritis may critically
affect the peptide repertoire displayed to the T cell compartment
(19).
In contrast to the DM loci in humans, the H2-M
region in the mouse contains one H2-Ma (Ma) gene,
but two H2-Mb (Mb) genes termed
Mb1 and Mb2 (20). It is still unclear whether
Mb1 and Mb2 are equally expressed in
vivo (15, 21), and no systematic study has been
presented in which a comparison of M Animals--
DBA/1 mice (H2q) and New Zealand
White rabbits were purchased from Charles River Laboratories (Sulzfeld, Germany).
Generation of T2 Transfectants and Culture Conditions--
The
human T2 cell line (T cell × B cell hybrid, 721.174 × CEM.T2), a generous gift from Dr. R. Salter (University of Pittsburgh, Pittsburgh, PA), contains a homozygous deletion in chromosome 6p that
removes the DM gene and the entire MHC class II
region (23). T2 cells were maintained in RPMI 1640 medium (Life
Technologies, Inc., Eggenstein, Germany) supplemented with 10%
heat-inactivated fetal bovine serum, 2 mM
L-glutamate, 100 IU/ml penicillin, and 100 µg/ml
streptomycin (all from Life Technologies, Inc.) and 50 µM
2-mercaptoethanol (Sigma, Deisenhofen, Germany), referred to as
complete medium.
Antibodies--
The hybridoma cell line N22 (anti-MHC class II)
was obtained from American Type Culture Collection (Manassas, VA).
Anti-I-Ab,d,q mAb YE2/36HLK was purchased from Serotec
(Wiesbaden, Germany). Anti-MHC class II mAbs KH116
(anti-I-Aq) and KH118 (anti-I-Aq,b) were from
Pharmingen (Hamburg, Germany). FITC-conjugated secondary staining
reagents (goat anti-hamster IgG, rabbit anti-rat IgG, goat anti-mouse
IgG, and goat anti-rabbit IgG) were purchased from Dianova (Hamburg),
and unlabeled isotype-matched control antibodies were from
Coulter-Immunotech (Hamburg). The rabbit antisera R.M Construction of I-Aq, Maq,
Mb1q, and Mb2q cDNA Expression
Vectors--
I-Aaq (Aaq)and
I-Abq (Abq) were amplified from a
DBA/1 splenocyte cDNA library (19) by polymerase chain reaction
using the ExpandTM Long Template PCR system (Roche
Molecular Biochemicals, Mannheim, Germany) and standard PCR conditions.
The primers used for amplification of the cDNA clones were as
follows: Aaq-sense,
5'-ATACCATGGCGCGCAGCAGAGCTC-3'; Aaq-antisense,
5'-ATAGGATCCTCATAAAGGCCCTGG-3';
Abq-sense, 5'-ATACCATGGCTCTGCAGATCCC-3'; and
Abq-antisense, 5'-ATAGGATCCTCACTGACGGAGCCCT-3'.
The primers were designed with synthetic NcoI and
BamHI restriction sites (underlined) to facilitate cloning
of amplified DNA. Following amplification, the 809-base pair
Aaq and 812-base pair Abq PCR
products were cloned into the EcoRV and BamHI
sites of pBSK+ (Stratagene, Heidelberg, Germany) and
sequenced (19). For expression in eukaryotic cells, the
Aaq cDNA was subcloned
(NcoI-BamHI) into the retroviral vector MFG (27),
yielding MFG-Aaq. Subsequently, an IRES-Neo
cassette (28), consisting of an internal ribosomal entry site (IRES)
sequence from the encephalomyocarditis virus and the neomycin
phosphotransferase gene (Neo), was inserted into the
BamHI site at the 3'-end of the Aaq
cDNA in MFG-Aaq, yielding the vector
DFG-Aaq-IRES-Neo. Next, an
IRES-EcoRI-NcoI fragment was fused to the ATG of
the Abq cDNA in pBSK+, yielding the
plasmid pBSK-IRES-Abq+. After that,
pBSK-IRES-Abq+ was digested with BamHI, and the
IRES-Abq fragment obtained was inserted into the
BamHI site at the 3'-end of the Neo cDNA of
the partially digested DFG-Aaq-IRES-Neo, yielding the
vector TFG-Aaq-IRES-Neo-IRES-Abq. This proviral
construct, capable of coordinately expressing Aaq,
Abq, and Neo, was finally termed
TFG-Aq. The isolation of Maq,
Mb1q, and Mb2q full-length
cDNA clones has been reported previously (19). For expression in
eukaryotic cells, the Abq cDNA in
pBSK-IRES-Abq+ was replaced by Maq
(NcoI-NotI). The IRES-Maq
sequence was subcloned into the BamHI site of the eukaryotic expression vector pCEP4 (Invitrogen, Groningen, The
Netherlands). Subsequently, NotI-NheI-digested
Mb1q or Mb2q cDNA was
inserted into the NotI-NheI sites at the 5'-end
of the IRES-Maq sequence, yielding
pCEP4-Mb1q-IRES-Maq and
pCEP4-Mb2q-IRES-Maq, respectively.
Generation of Stable Transfectant Cell Lines--
T2 cells
stably expressing I-Aq molecules from arthritis-susceptible
DBA/1 mice were generated by retroviral transfection as previously
described (33). Briefly, retroviral supernatant was produced by
transfecting the TFG-Aq proviral construct into the Template cDNA Preparation and PCR--
Total RNA isolation
and cDNA synthesis have been described previously (19). PCR
amplification was performed in an amplification mixture adjusted to 50 µl containing 50-100 ng of cDNA, 10 mM Tris-HCl (pH
8.3), 50 mM KCl, 1.5 mM MgCl2, 0.01 (w/v) gelatin, 1 mM each dNTP, 25 pmol each primer, and 2.5 units of AmpliTaq Gold polymerase (PerkinElmer Life
Sciences, Weiterstadt, Germany). The RT-PCR amplification profile
involved an initial denaturation step (94 °C for 10 min), followed
by 35 cycles of 94 °C for 1 min, 62 °C for 1 min, and 72 °C
for 1 min; the last extension was for 10 min at 72 °C. The following
primers were used for PCR: Ma-sense, 5'-AAGGTATGGAGCATGAGCAGAAGT-3';
Ma-antisense, 5'-GATCAGTCACCTGAGCACGGT-3'; panMb1/2-sense,
5'-GGACCATGGCTGCACTCTGGC-3'; and panMb1/2-antisense, 5'-GCATCACGGGCTCCCTTGTGT-3'. PCR products were resolved on ethidium bromide-stained agarose gels and digitized with a Gelprint 2000i densitometer (MWG Biotech, Ebersberg, Germany).
Ratio RT-PCR Analysis--
The ratio RT-PCR assay performed in
this study was based on the simultaneous amplification of
Mb1 and Mb2 transcripts using panMb1/2 primers
(see above) annealing within conserved regions (exons 1 and 3) of both
Mbq mRNA species (19). Equal amplification
efficiency of both Mb transcripts was assured by comparative
cycle kinetic and linear regression analysis (31) using cloned
Mb1 and Mb2 full-length cDNAs (19).
Discrimination between co-amplified Mb transcripts was
performed by restriction endonuclease analysis. We therefore took
advantage of the polymorphism of Mb1q and
Mb2q genes within exon 2 (19) and used the
restriction enzyme HhaI, which specifically cleaves at
nucleotide +321 within the Mb1q sequence and
nucleotide +264 within the Mb2q sequence of the
403-base pair panMb RT-PCR product. PCR was performed using
AmpliTaq Gold polymerase and standard PCR conditions. After 25 cycles, the amplified mixture was diluted 25-fold in a fresh PCR
amplification mixture containing 25 nCi/µl
[ Western Blot Analysis--
Cells (1 × 107/ml)
were lysed in 20 mM Tris-HCl (pH 7.4) containing 1%
Nonidet P-40 (Sigma), 5 mM MgCl2, 5 µg/ml
chymostatin, 2.5 µg/ml leupeptin, 5 µg/ml pepstatin A, and 200 µM phenylmethylsulfonyl fluoride (all protease inhibitors
were from Roche Molecular Biochemicals) for 30 min at 4 °C. Nuclei
and insoluble debris were removed by centrifugation (14,000 rpm) for 30 min, and the protein concentration was determined by the BCA protein
assay (Pierce). Aliquots corresponding to 10 µg of protein were mixed
with Laemmli buffer, boiled for 5 min, separated on SDS-12.5%
polyacrylamide gels, and then transferred onto Immobilon polyvinylidene
difluoride membranes (Millipore, Eschborn, Germany) by semidry blotting
as described (32). Membranes were blocked overnight with blocking
reagent (Roche Molecular Biochemicals). Antibody binding was detected
by incubation with horseradish peroxidase-conjugated goat anti-rabbit
immunoglobulin (Dianova), followed by enhanced chemiluminescence using
Super Signal Ultra (Pierce).
Flow Cytometry--
Cells (5 × 105/sample)
were washed in PBS supplemented with 1% bovine serum albumin and
incubated on ice with unlabeled primary mAb for 30 min. After washing
in the PBS and bovine serum albumin, the cells were incubated with an
appropriate FITC-conjugated secondary staining reagent for 30 min at
4 °C: goat anti-hamster IgG, goat anti-mouse IgG, rabbit anti-rat
IgG, or goat anti-rabbit IgG. Background fluorescence was evaluated
using irrelevant matched isotypes and FITC-conjugated goat anti-hamster
IgG, goat anti-mouse IgG, rabbit anti-rat IgG, or goat anti-rabbit IgG.
Cell-surface fluorescent labeling was visualized on an
EPICS®-PROFILE II flow cytometer (Coulter-Immunotech), and
data analysis was performed using EPICS®-ELITE Version 3.0 software.
Immunohistochemistry--
Transfected T2 cell lines were washed
in PBS and cytocentrifuged with a Labofuge 400e (Heraeus Instruments,
Hanau, Germany) at 500 × g for 5 min onto
Superfrost slides (Menzel, Hannover, Germany) with 200,000 cells/dot.
The cytospins were air-dried overnight and afterward stored at
Mb1 and Mb2 Are Differentially Expressed in Lymphoid and
Non-lymphoid Organs--
The H2-M region contains
one Ma gene, but two Mb genes termed
Mb1 and Mb2 (20). Transcription of the
Ma gene and both Mb genes has been observed in
splenocytes of different mouse strains, including mice that carry the
arthritis-susceptible H2q haplotype (19, 33). To
define the H2-M gene expression pattern, we examined
Ma and Mb mRNA expression in lymphoid and
non-lymphoid organs or tissues from arthritis-susceptible DBA/1
(H2q) mice by RT-PCR. Constitutive Ma and
Mb mRNA expression could be detected in each organ
sample (data not shown). Next, we addressed the question of whether
Mb1 and Mb2 are differentially expressed by ratio
RT-PCR analysis (Fig. 1). The ratios of
Mb1 to Mb2 mRNA given as percentages of the
total Mb mRNA are listed in Table I. Mb2 represents the major
Mb transcript in lymphoid organs: 60.4% in spleen; 79.5%
in mesenteric lymph nodes; 82.3% in popliteal lymph nodes; and 60.2, 62.4, and 57.8% in thymus of 4-, 8-, and 12-week-old mice,
respectively (Fig. 1A and Table I). Similarly, Mb2 mRNA was preferentially expressed in muscle (62.1%)
and heart (57.7%). In contrast, Mb1 mRNA was found to
be the dominant transcript in testis, brain, lung, liver, kidney,
pancreas, and small and large intestines. With the exception of large
intestine (57.1%), the relative expression of Mb1 mRNA
averaged ~76% of the total Mb mRNA transcripts (Fig.
1B and Table I).
Both M
In view of the well documented observation that absent or reduced
binding of certain mAbs recognizing conformation-dependent epitopes on MHC class II molecules directly correlates with a failure
to exchange CLIP for other peptides on MHC class II molecules in
DM mutant cell lines (13, 26) or H2-M-deficient
mice (14), we analyzed the I-Aq surface phenotype of the
T2.Aq.pCEP4,
T2.Aq.Maq.Mb1q, and
T2.Aq.Maq.Mb2q cell lines by flow
cytometry using a panel of mAbs to monomorphic (Fig. 2 first
and second lane from left) and polymorphic determinants (Fig. 2, third and fourth lane from left).
Staining with mAb KH118, which also recognizes a monomorphic
determinant on I-Aq molecules (34), confirmed the results
obtained with mAb N22, demonstrating that individual transfectants
express I-Aq molecules at equal levels on the cell surface.
In contrast, differential staining was obtained with mAbs KH116 and
YE2/36HLK, both recognizing polymorphic determinants on
I-Aq (34, 35). Both mAbs equally stained
T2.Aq.Maq.Mb1q and
T2.Aq.Maq.Mb2q cells, but stained
T2.Aq.pCEP4 cells with a reduced intensity. These findings
suggest that the I-Aq conformation on T2.Aq
cells expressing either M
To evaluate whether the expression of the KH116 and YE2/36HLK
epitopes in T2.Aq.Maq.Mb1q or
T2.Aq.Maq.Mb2q cells might result
from CLIP exchange for cognate peptides on I-Aq molecules,
cell-surface levels of CLIP were determined by flow cytometry using the
antibody R.hCLIP73.11, which recognizes a peptide derived from
the CLIP region (amino acids 81-104 of the human p33 Ii isoform). As
expected, T2.Aq.pCEP4 cells exhibited high levels of
anti-CLIP staining compared with
T2.Aq.Maq.Mb1q and
T2.Aq.Maq.Mb2q cells (Fig.
4). These data show that (i)
I-Aq molecules are occupied with CLIP in the absence of
M Cell-surface Expression of H2-M--
T2, T2.Aq.pCEP4,
T2.Aq.Maq.Mb1q, and
T2.Aq.Maq.Mb2q cells were examined
for M H2-M molecules not only facilitate CLIP removal, but also select
high affinity binding peptides for MHC class II (17, 18). Presumably,
low affinity binding peptides would not be loaded onto MHC class II
molecules if the MHC class II·H2-M complex exhibits a higher affinity
compared with a potential MHC class II·peptide complex. However, the
role of different M To this end, the efficacy of M Additionally, we have been able to demonstrate that I-Aq
does not necessarily require H2-M to be transported and expressed on
the cell surface (Fig. 2). These MHC class II cell-surface molecules
may be occupied by CLIP since H2-M is not present in these cells to
exchange CLIP for antigenic peptides. Not mutually exclusive, peptides
may also occupy the MHC class II heterodimer, which may exhibit a high
affinity for I-Aq, leading to CLIP replacement. The
apparent lack or need of certain human or mouse MHC class II alleles to
require H2-M/DM for CLIP removal in vivo, e.g.
the murine alleles H2b and H2d
(14, 15), has been attributed to different affinities of these MHC
class II alleles for CLIP as determined by in vitro binding
studies (43). Although the affinity of I-Aq for CLIP has
yet to be defined, we propose that I-Aq exhibits a high
affinity in T2 transfectants at least for human CLIP since virtually
all I-Aq molecules on T2 cells appeared to be associated
with CLIP in the absence of both H2-M isoforms (Fig. 4). In support of
this hypothesis, the I-Ad allele, which exhibits a high
affinity for both human and mouse CLIP in vitro (43),
requires H2-M/DM for CLIP release and acquisition of peptides in
H2-M/DM-deficient T2/Ltk-transfected cells. In contrast, I-Ak, although showing a low affinity for CLIP
in vitro, is also capable of displacing CLIP without the
assistance of H2-M/DM (15).
The observation that both M The obvious difference pertaining to the role of antigen-presenting
cells (macrophage versus B cell) in activation of MHC class
II-restricted and CII-specific CD4+ T cells (46) may stem
from the fact that B cells utilized for CD4+ T cell
stimulation are derived from arthritis-susceptible
H2g7 mice, but not from mice with the
H2q haplotype (43). Of note, the Mb2
allele, which represents the dominant H2-M transcript in B
cells (37), differs in H2g7 mouse strains (33)
compared with the Mb2 allele in H2q mice
(19). Hence, the different capacity of B cells obtained from
H2g7 and H2q mice to select for
arthritogenic peptides provided by CII may stem not only from
differences in the MHC class II binding cleft, but also from a
different peptide repertoire selected by alternate Mb2 alleles.
It is noteworthy that transgenic expression of either M In summary, the results of this study provide further evidence that the
expression of genes involved in MHC class II antigen processing is
tissue-dependent. Yet, our findings established for the
first time that M-chain locus is duplicated in mice, with the
H2-Mb1 beta-chain distal to H2-Mb2 and
the H2-Ma alpha-chain gene. H2-M alleles appear to
be associated with the development of autoimmune diseases. Recent data
showed that M
1 and M
2 isoforms are differentially expressed in
isolated macrophages and B cells, respectively. The tissue expression
and functional role of these heterodimers in promoting CLIP removal and
peptide selection have not been addressed. We utilized the human T2
cell line, which lacks part of chromosome 6 encompassing the MHC class
II and DM genes, to construct transgenic cell lines
expressing the MHC class II heterodimer I-Aq alone or in
the presence of H2-M
1 or H2-M
2 heterodimers. Both H2-M
isoforms facilitate the exchange of CLIP for cognate peptides on
I-Aq molecules from arthritis-susceptible DBA/1 mice and
induce a conformational change in I-Aq molecules. Moreover,
I-Aq cell-surface expression is not absolutely dependent on
H2-M molecules. These data suggest that I-Aq exhibits a
high affinity for CLIP since virtually all I-Aq molecules
on T2 cells were found to be associated with CLIP in the absence of
both H2-M isoforms.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/M
1 and M
/M
2 heterodimers has been conducted. To address the questions of whether (i) I-Aq molecules require, for cell-surface expression,
the presence of H2-M molecules and (ii) different H2-M isoforms are
similar or distinct in peptide loading of MHC class II molecules, we
took advantage of the T2 cell line, which lost, due a genetic defect, the MHC class II and DM molecules. Stable transfectants of the T2 cell
line expressing the I-Aq allele from
autoimmune-prone DBA/1 mice alone or combined with H2-M
1
(M
1) or H2-M
2 (M
2) provide the means to perform a
detailed analysis of molecules involved in the MHC class II antigen
presentation pathway without the possibility that freshly isolated
antigen-presenting cells (e.g. macrophages) from autoimmune DBA/1 mice might be contaminated with trace amounts of different antigen-presenting cells (e.g. B cells).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-C.69.3,
R.M
1/2-C.71.3, and R.hCLIP73.11 were prepared by immunizing rabbits
with C-terminal peptides from M
(amino acids 238-248) (24) and
M
1/M
2 (amino acids 228-243) (25) and a CLIP peptide (amino acids
81-104 of the human p33 invariant chain isoform) (26), respectively,
coupled with an added amino-terminal cysteine to diphtheria
toxoid (Chiron Mimotopes, Victoria, Australia). The antisera
were affinity-purified using
N-hydroxysuccinimide-activated Fast Flow Sepharose 4 (Amersham Pharmacia Biotech, Freiburg, Germany) cross-linked to the
respective M
, M
, or CLIP peptide. Affinity-purified antisera were
screened for specific antibody titers by enzyme-linked immunosorbent
assay and Western blot analysis using T2.Aq cell lines
transfected with Maq, Mb1q, and
Mb2q. Specificity controls included Western blot
analysis of T2 cells alone or T2 cells expressing I-Aq (but
not H2-M genes) using anti-M
or anti-M
antisera (see
Fig. 4).
CRIP
packaging cell line (29). T2 cells (2-5 × 106) were
infected with 2 ml of TFG-Aq retroviral supernatant in the
presence of Polybrene (8 µg/ml) and subsequently plated in 96-well
flat-bottomed plates on irradiated feeder cell layers. Stable
transfectants were selected in complete medium supplemented with 1 mg/ml Geneticin (Life Technologies, Inc.). T2.Aq clones
were screened for high I-Aq expression levels by flow
cytometry using the I-A conformation-independent mAb N22 (30).
Transfection of T2.Aq cells with the pCEP4,
pCEP4-Mb1q-IRES-Maq or
pCEP4-Mb2q-IRES-Maq vector was performed by
electroporation as described (26). To obtain stable
T2.Aq.Maq.Mb1q and
T2.Aq.Maq.Mb2q cell lines,
transfectants were cloned by limiting dilution in complete medium
supplemented with 1 mg/ml Geneticin and 0.45 mg/ml hygromycin (Roche
Molecular Biochemicals). Individual clones were screened for the
presence of Maq, Mb1q, and
Mb2q by RT-PCR. Clones that exhibited high M
1
or M
2 protein expression levels determined by Western blot
analysis were used in subsequent experiments.
-32P]dCTP (ICN, Eschwege, Germany), followed by two
additional amplification cycles. The labeled Mb PCR products
or their respective restriction fragments were separated on 6%
polyacrylamide gels. To quantitate individual Mb fragments,
gels were subjected to autoradiography. Corresponding bands were
excised from the gel, and radioactivity was measured with a
-counter
(LS6000TA, Beckman, München, Germany) using a Cerenkov program.
To calculate the ratio of Mb1 and Mb2 mRNA
expression levels, their respective restriction fragments were
corrected for length and cytosine and guanine (GC content) since dCTP
was exclusively radioactively labeled in this assay.
20 °C. Cells were fixed with 4% (w/v) paraformaldehyde (Sigma, München) for 10 min at room temperature before immunostaining. After permeabilization with 0.2% (v/v) Triton X-100 (Sigma) in PBS and
blocking with serum-free protein block (Dako, Hamburg), cells were
incubated with the affinity-purified rabbit anti-human primary
polyclonal antibody anti-DM
or anti-DM
or the corresponding antisera (1:1000 dilution for each one). Negative controls included cells treated with PBS or normal rabbit serum. A Cy3-labeled
anti-rabbit antibody (diluted 1:600; Dianova, Hamburg, Germany)
was used as the second antibody. Optimal working dilutions of the
antibodies were determined in titration experiments. Nuclei were
counterstained with bisbenzimide (1:5000; Sigma). Slides were mounted
in fluorescent mounting medium (Dako) and examined using a Leitz DM RBE
fluorescence microscope (Leica, Heerbrugg, Switzerland); the red Cy3
fluorescence was detected by an N2.1 filter (wavelengths
515-560 and 590).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (64K):
[in a new window]
Fig. 1.
Determination of
Mb1q and Mb2q
expression patterns in lymphoid and non-lymphoid organs and tissues of
arthritis-susceptible DBA/1 mice (H2q).
PCRs were performed with 100 ng of cDNA from the indicated lymphoid
(A) and non-lymphoid (B) organs and tissues in
the presence of 25 nCi/µl [ -32P]dCTP as described
under "Experimental Procedures." Primers were chosen to co-amplify
Mb1- and Mb1-specific transcripts. Co-amplified
Mb isoforms were discriminated by digestion with the
restriction enzyme HhaI, followed by separation on 6%
polyacrylamide gels. Lanes NC show the undigested
panMb1/2 PCR product; lanes H show the
Mb1- and Mb2-specific fragments obtained after
HhaI digestion. The lengths (in base pairs (bp))
of the undigested panMb1/2 PCR products and the restriction
fragments corresponding to Mb1 or Mb2 are
indicated to the right of A and B.
Mb1 and Mb2 are differentially expressed in lymphoid and non-lymphoid
organs of arthritis-susceptible DBA/1 (H2q) mice
1 and M
2 Can Complement for
Conformation-dependent Epitopes on MHC Class II Molecules
in T2 Cells--
The observation that Mb1 and
Mb2 are differentially expressed in lymphoid and
non-lymphoid organs (Table I) implies that both heterodimers, M
1
and M
2, might be functional in I-Aq/peptide assembly.
To address this question, we generated T2 transfectants stably
expressing either I-Aq alone (T2.Aq.pCEP4) or
in combination with M
1
(T2.Aq.Maq.Mb1q) or M
2
(T2.Aq.Maq.Mb2q) derived from DBA/1
mice (H2q). To ensure comparable levels of
I-Aq surface expression as well as M
1 or M
2
heterodimer expression, transgenic T2 cells were analyzed by flow
cytometry (Fig. 2) and Western blotting
(Fig. 3), respectively. Cell-surface
staining with mAb N22, which recognizes a monomorphic determinant
on Ii- or peptide-associated MHC class II
-dimers with similar
efficiency (30), showed that T2.Aq.pCEP4,
T2.Aq.Maq.Mb1q, and
T2.Aq.Maq.Mb2q transfectants
expressed comparable I-Aq levels on the cell surface (Fig.
3, left panel). Similarly, Western blot analysis of
T2.Aq.Maq.Mb1q and
T2.Aq.Maq.Mb2q cells demonstrated
comparable M
1 and M
2 expression (Fig. 3).
View larger version (28K):
[in a new window]
Fig. 2.
Expression of
M 1 or
M
2 in T2.Aq restores
the expression of the KH116 and YE2/36HLK epitopes. MHC class II
and DM mutant T2 cells expressing I-Aq were
generated by retroviral transfection as described under "Experimental
Procedures." To express M
1 or M
2 heterodimers,
T2.Aq cells were super-transfected with
Maq in combination with Mb1q or
Mb2q. Transfection with the pCEP4 vector alone
served as a control. I-Aq surface expression was measured
by flow cytometry using MHC class II conformation-independent mAbs N22
(43) and KH118 (22) and MHC class II conformation-sensitive mAbs KH116
(22) and YE2/36HLK (48). Data are plotted as fluorescence intensity
(mean fluorescence channel) versus cell number. Closed
histograms show binding of specific antibodies, whereas open
histograms show isotype-matched control antibodies. Antibody names
are listed across the top, and cell lines are indicated to the
right.
View larger version (74K):
[in a new window]
Fig. 3.
Comparison of
M 1 and
M
2 expression in
T2.Aq transfectants. MHC class II antigen-processing
mutant T2 cells were stably transfected with I-Aq
- and
-chain genes and then super-transfected with Ma and
Mb1 (T2.Aq.Maq.Mb1q),
Mb2 (T2.Aq.Maq.Mb1q), or
pCEP4 control vector (T2.Aq.pCEP4). Laemmli
buffer-solubilized cell lysates (10 µg) derived from the indicated
cell lines were separated on denaturing 12.5% SDS-polyacrylamide gels
and analyzed on Western immunoblots by staining with affinity-purified
rabbit antisera to M
, or M
1, and M
2 monomers as described
under "Experimental Procedures."
1 or M
2 differs from that on the H2-M-negative T2.Aq.pCEP4 cells. However,
expression of the KH116 and YE2/36HLK mAb epitopes in
T2.Aq cells requires the expression of both
Maq and either Mb1q or
Mb2q since transfectants expressing each gene
individually remained KH116- and
YE2/36HLK-negative.2
1 or M
2 heterodimers; (ii) I-Aq assembly,
transport, and ultimately cell-surface expression do not require H2-M
molecules; and (iii) either M
1 or M
2 heterodimers are
sufficient to exchange CLIP for cognate peptides displayed by the
I-Aq molecules.
View larger version (14K):
[in a new window]
Fig. 4.
CLIP persistence in the absence of
M 1 or
M
2. T2.Aq cells
transfected with the pCEP4 control vector alone and with
Maq and either Mb1q or
Mb2q were stained with R.hCLIP73.11
(anti-CLIP-(81-104)) and analyzed by flow cytometry. Cell line
designations are indicated across the top.
and M
protein expression (Fig.
5). Both transgenic T2 cell lines
expressing M
q/M
1q or
M
q/M
2q heterodimers exhibited a strong
cytoplasmic and cellular membrane staining pattern.
View larger version (50K):
[in a new window]
Fig. 5.
Cell-surface expression of H2-M in transgenic
cell lines. T2 cells (a and e),
T2.Aq cells (b and f),
T2.Aq M 1 transfectants (c and
g), and T2.Aq M
2 transfectants
(d and h) were evaluated for expression of M
(a-d) and M
(e-h). T2 cells and
T2.Aq cells were negative for M
and M
. In contrast,
the transfectants showed strong staining of the cytoplasm and the cell
membrane. Magnification × 1000.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 and M
2 heterodimers in mediating these
functions has not been addressed. Previous studies have shown that
Ma, Mb1, and Mb2 are coexpressed in
splenocytes of mice carrying different haplotypes (19, 33, 36),
indicating that M
1 and M
2 heterodimers might be implemented
in antigen presentation by many MHC class II alleles/isotypes. As shown
in this study, Mb2 is predominantly expressed in lymph nodes
and spleen. In contrast, Mb1 represents the predominant
H2-M transcript in solid organs in DBA/1 mice (Fig. 1 and
Table I). A similar distribution of Mb1/Mb2 expression has
also been observed in Balb/c (H2d) and
C57BL/6 (H2b) mice.2 Additionally, we
have recently been able to demonstrate that Mb1 is
predominantly expressed in cells of epithelial and mesenchymal origin.
Mb genes at almost equal levels are expressed in splenic dendritic cells, whereas Mb2 is expressed in B cells
(37-39). Of note, highly purified peritoneal macrophages
constitutively express Mb2, which can be switched to
Mb1 expression upon interferon-
treatment (37).
Thus, the differential pattern of
Ma/Mb1/Mb2 expression may reflect either
the composition of immune cells present in lymphoid organs (reviewed in
Ref. 40) or the local cytokine milieu, which impacts on
Mb1/Mb2 mRNA expression significantly (37,
38). Thus, M
1/M
2 could potentially exert different functions concerning (i) efficiency in removing CLIP from MHC class II
heterodimers or (ii) the selection of the peptide repertoire loaded
onto MHC class II molecules. Here, we show that both isoforms are able
(i) to effectively remove CLIP from MHC class II molecules (Fig. 4) and
(ii) to promote peptide loading of MHC class II molecules as detected
with antibodies that define conformational epitopes in MHC class II
molecules, indicating MHC class II occupancy with peptides except CLIP
(Figs. 2 and 4).
/M
1 or M
/M
2
heterodimers in CLIP removal has not been compared. Here, we show that
both M
1/M
2 isoforms are able to release CLIP from
Aq molecules. This results in a conformational change of
the Aq molecule, a finding that has been observed in
H2-M
/
mice. In these animals,
MHC class II cell-surface expression appears normal, but the
conformational shape of the class II molecule (H2-Ab)
appears to be altered, probably due to the occupancy by CLIP (14, 41,
42). A similar situation was found to be true for Aq
cell-surface molecules: the class II epitope defined by mAbs KH116 and
YE2/36HLK is not accessible if Aq is occupied by CLIP
(Figs. 2 and 4). Examination of peptides harvested from
T2.Aq cells expressing either the M
1 or M
2
heterodimer will ultimately reveal if the peptide repertoire presented
to CD4+ T cells is qualitatively or quantitatively different.
/M
1 and M
/M
2, although
different in exon 2 (19, 33), are equally effective in CLIP removal is
of particular interest in view of previous observations that either
peritoneal macrophages or unsorted spleen cells are able to present
naive or denatured CII to T cells compared with purified splenic
dendritic cells, Langerhans cells, and primed or unprimed B cells
(expressing exclusively Mb2 (37)), which cannot
present CII appropriately (44, 45).
1 or
M
2 heterodimers results not only in a cytoplasmic staining pattern, but also in cell membrane staining (Fig. 5). Earlier studies
showed that H2-M/DM molecules reside in the endosomal/lysosomal system
(MHC class II compartments, MIICs) (25, 47, 48), but
recent studies suggested that H2-M/DM may also be present at the plasma
membrane (21, 22). The physiology of H2-M membrane expression, either
alone or in association with MHC class II molecules, has to be
addressed in future studies.
1 and M
2 can select for MHC class II/peptide assembly utilizing the I-Aq heterodimer from
autoimmune-prone mice. Identification of functional differences between
H2-M isoforms appears to be of particular interest for a better
understanding as to why co-adaptation of two alternative M
chains
has been evolved for immune surveillance in mice without apparently
being required in other species analyzed thus far.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Kirsten Freitag and Claudia Neukirch for excellent technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 311/A16.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 49-6131-393-3645; Fax: 49-6131-393-5580; E-mail: maeurer@mail.uni-mainz.de.
Published, JBC Papers in Press, January 8, 2001, DOI 10.1074/jbc.M006521200
2 W. Walter, unpublished observation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: CII, collagen type II; MHC, major histocompatibility complex; Ii, invariant chain; CLIP, invariant chain-derived MHC class II-associated Ii peptides; mAb, monoclonal antibody; FITC, fluorescein isothiocyanate; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-polymerase chain reaction; IRES, internal ribosomal entry site; PBS, phosphate-buffered saline.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Courtenay, J. S., Dallman, M. J., Dayan, A. D., Martin, A., and Mosedale, B. (1980) Nature 283, 666-668[Medline] [Order article via Infotrieve] |
2. | Wooley, P. H., Luthra, H. S., Stuart, J. M., and David, C. S. (1981) J. Exp. Med. 154, 688-700[Abstract] |
3. | Kerwar, S. S., Englert, M. E., McReynolds, R. A., Landes, M. J., Lloyd, J. M., Oronsky, A. L., and Wilson, F. J. (1983) Arthritis Rheum. 26, 1120-1131[Medline] [Order article via Infotrieve] |
4. | Terato, K., Hasty, K. A., Cremer, M. A., Stuart, J. M., Townes, A. S., and Kang, A. H. (1985) J. Exp. Med. 162, 637-646[Abstract] |
5. | Holmdahl, R., Klareskog, L., Rubin, K., Bjork, J., Smedegard, G., Jonsson, R., and Andersson, M. (1986) Agents Actions 19, 295-305[Medline] [Order article via Infotrieve] |
6. |
Seki, N.,
Sudo, Y.,
Yoshioka, T.,
Sugihara, S.,
Fujitsu, T.,
Sakuma, S.,
Ogawa, T.,
Hamaoka, T.,
Senoh, H.,
and Fujiwara, H.
(1988)
J. Immunol.
140,
1477-1484 |
7. |
Myers, L. K.,
Terato, K.,
Seyer, J. M.,
Stuart, J. M.,
and Kang, A. H.
(1992)
J. Immunol.
149,
1439-1443 |
8. | Chiocchia, G., Boissier, M. C., Manoury, B., and Fournier, C. (1993) Eur. J. Immunol. 23, 327-332[Medline] [Order article via Infotrieve] |
9. | Osman, G. E., Cheunsuk, S., Allen, S. E., Chi, E., Liggitt, H. D., Hood, L. E., and Ladiges, W. C. (1998) Int. Immunol. 10, 1613-1622[Abstract] |
10. | Germain, R. N., Castellino, F., Han, R., Reis e Sousa, C., Romanoli, P., Sadegh-Nasseri, S., and Zhong, G. M. (1996) Immunol. Rev. 151, 5-30[Medline] [Order article via Infotrieve] |
11. | Wolf, P. R., and Ploegh, H. L. (1995) Annu. Rev. Cell Dev. Biol. 11, 267-306[CrossRef][Medline] [Order article via Infotrieve] |
12. | Urban, R. G., Chicz, R. M., and Strominger, J. L. (1994) J. Exp. Med. 180, 751-755[Abstract] |
13. | Morris, P., Shaman, J., Attaya, M., Amaya, M., Goodman, S., Bergman, C., Monaco, J. J., and Mellins, E. (1994) Nature 368, 551-554[CrossRef][Medline] [Order article via Infotrieve] |
14. | Fung-Leung, W. P., Surh, C. D., Liljedahl, M., Pang, J., Leturcq, D., Peterson, P. A., Webb, S. R., and Karlsson, L. (1996) Science 271, 1278-1281[Abstract] |
15. | Stebbins, C. C., Peterson, M. E., Suh, W. M., and Sant, A. J. (1996) J. Immunol. 157, 4892-4898[Abstract] |
16. | Wolf, P. R., Tourne, S., Miyazaki, T., Benoist, C., Mathis, D., and Ploegh, H. L. (1998) Eur. J. Immunol. 28, 2605-2618[CrossRef][Medline] [Order article via Infotrieve] |
17. | van Ham, S. M., Gruneberg, U., Malcherek, G., Broker, I., Melms, A., and Trowsdale, J. (1996) J. Exp. Med. 184, 2019-2024[Abstract] |
18. |
Weber, D. A.,
Evavold, B. D.,
and Jensen, P. E.
(1996)
Science
274,
618-620 |
19. | Walter, W., Loos, M., and Maeurer, M. J. (1996) Immunogenetics 44, 19-26[CrossRef][Medline] [Order article via Infotrieve] |
20. | Cho, S., Attaya, M., Brown, M. G., and Monaco, J. J. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 5197-5201[Abstract] |
21. | Alfonso, C., and Karlsson, L. (2000) Annu. Rev. Immunol. 18, 113-142[CrossRef][Medline] [Order article via Infotrieve] |
22. | Kim, K.-J., Jung, H. H., and Bae, Y. S. (1996) Mol. Cells 6, 684-691 |
23. | Salter, R. D., Howell, D. N., and Cresswell, P. (1985) Immunogenetics 21, 235-246[Medline] [Order article via Infotrieve] |
24. | Cho, S. G., Attaya, M., and Monaco, J. J. (1991) Nature 353, 573-576[CrossRef][Medline] [Order article via Infotrieve] |
25. | Karlsson, L., Peleraux, A., Lindstedt, R., Liljedahl, M., and Peterson, P. A. (1994) Science 266, 1569-1573[Medline] [Order article via Infotrieve] |
26. | Denzin, L. K., Robbins, N. F., Carboy-Newcomb, C., and Cresswell, P. (1994) Immunity 1, 595-606[Medline] [Order article via Infotrieve] |
27. | Dranoff, G., Jaffee, E., Lazenby, A., Golumbek, P., Levitsky, H., Brose, K., Jackson, V., Hamada, H., Pardoll, D., and Mulligan, R. C. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 3539-3543[Abstract] |
28. |
Maeurer, M. J.,
Gollin, S. M.,
Martin, D.,
Swaney, W.,
Bryant, J.,
Castelli, C.,
Robbins, P.,
Parmiani, G.,
Storkus, W. J.,
and Lotze, M. T.
(1996)
J. Clin. Invest.
98,
1633-1641 |
29. | Danos, O., and Mulligan, R. C. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6460-6464[Abstract] |
30. |
Villadangos, J. A.,
Riese, R. J.,
Peters, C.,
Chapman, H. A.,
and Ploegh, H. L.
(1997)
J. Exp. Med.
186,
549-560 |
31. |
Bouaboula, M.,
Legoux, P.,
Pessegue, B.,
Delpech, B.,
Dumont, X.,
Piechaczyk, M.,
Casellas, P.,
and Shire, D.
(1992)
J. Biol. Chem.
267,
21830-21838 |
32. | Kyhse-Andersen, J. (1984) J. Biochem. Biophys. Methods 10, 203-209[CrossRef][Medline] [Order article via Infotrieve] |
33. | Hermel, E., Yuan, J., and Monaco, J. J. (1995) Immunogenetics 42, 136-142[Medline] [Order article via Infotrieve] |
34. | Hasenkrug, K. J., Cory, J. M., and Stimpfling, J. H. (1987) Immunogenetics 25, 136-139[Medline] [Order article via Infotrieve] |
35. | Brickell, P. M., McConnell, I., Milstein, C., and Wright, B. (1981) Immunology 43, 493-501[Medline] [Order article via Infotrieve] |
36. |
Sloan, J. H.,
Hasegawa, S. L.,
and Boss, J. M.
(1992)
J. Immunol.
148,
2591-2599 |
37. | Walter, W., Loos, M., and Maeurer, M. J. (1999) Mol. Immunol. 36, 733-743[CrossRef][Medline] [Order article via Infotrieve] |
38. | Walter, W., Scheuer, C., Lingnau, K., Reichert, T. E., Schmitt, E., Loos, M., and Maeurer, M. J. (2000) Immunogenetics 51, 794-804[CrossRef][Medline] [Order article via Infotrieve] |
39. | Walter, W., Lingnau, K., Schmitt, E., Loos, M., and Maeurer, M. J. (2000) Br. J. Cancer 83, 1192-1201[CrossRef][Medline] [Order article via Infotrieve] |
40. | Young, A. Y. (1999) Semin. Immunol. 2, 73-83 |
41. | Miyazaki, T., Wolf, P., Tourne, S., Waltzinger, C., Dierich, A., Barois, N., Ploegh, H., Benoist, C., and Mathis, D. (1996) Cell 84, 531-541[Medline] [Order article via Infotrieve] |
42. | Martin, W. D., Hicks, G. G., Mendiratta, S. K., Leva, H. I., Ruley, H. E., and van Kaer, L. (1996) Cell 84, 543-550[Medline] [Order article via Infotrieve] |
43. | Sette, A., Southwood, S., Miller, J., and Appella, E. (1995) J. Exp. Med. 181, 677-683[Abstract] |
44. | Manoury-Schwartz, B., Chiocchia, G., and Fournier, C. (1995) Eur. J. Immunol. 25, 3235-3242[Medline] [Order article via Infotrieve] |
45. | Michaelsson, E., Holmdahl, M., Engstrom, A., Burkhardt, H., Scheynius, A., and Holmdahl, R. (1995) Eur. J. Immunol. 25, 2234-2241[Medline] [Order article via Infotrieve] |
46. | Korganow, A. S., Ji, H., Mangialaio, S., Duchatelle, V., Pelanda, R., Martin, T., Degott, C., Kikutani, H., Rajewsky, K., Pasquali, J. L., Benoist, C., and Mathis, D. (1999) Immunity 10, 451-461[Medline] [Order article via Infotrieve] |
47. | Sanderson, F., Kleijmeer, M. J., Kelly, A., Verwoerd, D., Tulp, A., Neefjes, J. J., Genze, H. I., and Trowsdale, I. (1994) Science 266, 1566-1569[Medline] [Order article via Infotrieve] |
48. | Nijman, H. W., Kleijmeer, M. J., Ossevoort, M. A., Oorschot, V. M., Vierboom, M. P., van de Keur, M., Kenemans, P., Kast, W. M., Genze, H. J., and Melief, C. J. (1995) J. Exp. Med. 182, 163-174[Abstract] |