| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Immunobiology Laboratory, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
cells of islets of Langerhans and
consequently results in insulin deficiency. The elimination of
cells is thought to be mediated by autoreactive T cells, a process that
probably results from improper education of CD4+
and CD8+ T cells.
The invariant chain
(Ii)5 functions as a
chaperone in the intracellular transport of MHC class II proteins as
well as in the loading of these proteins with peptides for presentation
to CD4+ T cells (1). The Ii chain
forms a core trimer that associates with three MHC class II 
subunit dimers in the endoplasmic reticulum (ER), resulting in
generation of the nonameric complex (I)3
(2, 3). This complex is incapable of binding antigenic
peptides, which is important because most endogenous peptide fragments
present in the ER are destined for association with MHC class I
molecules (4, 5). The MHC class II-Ii complex undergoes
extensive glycosylation and is transported from the ER to the
endosomal-lysosomal Ag-processing compartments, a process determined by
the targeting motifs of the Ii cytoplasmic domain (6, 7, 8).
Within these compartments, Ii is removed from MHC class II molecules by
a series of highly regulated proteolytic cleavages, with the accessory
protein HLA-DM catalyzing the final release of a remaining Ii fragment
known as class II-associated invariant peptides (CLIP) from the
MHC class II groove, thereby allowing the binding of antigenic
peptides.
The rate of removal of CLIP from MHC class II proteins may be influenced by MHC class II allelic variation (9, 10). The high affinity interaction of MHC class II molecules with antigenic peptides results in the formation of a stable complex, as evidenced by its resistance to dissociation in SDS sample buffer. The cell surface expression of these MHC class II-peptide complexes then contributes to T cell education, selection, and stimulation.
In fresh human lymphocytes and EBV-immortalized B cells, two major
alternative Ii isoforms function in the intracellular routing of MHC
class II molecules to the endosomal-lysosomal compartments. The
predominant Ii chain is the 33-kDa isoform (Ii p33), but an additional
human-specific 35-kDa isoform (Ii p35) is generated as a result of the
use of an alternative upstream translation initiation site in the
shared mRNA (11). These two Ii isoforms are thus
identical, except that Ii p35 contains an additional 16 aa at the
cytoplasmically located NH2-terminus. The ratio
of these Ii isoforms in cells of fixed lineage appears to be highly
regulated, with the p35 isoform representing 
20% of the total Ii
pool of human B cells. Most MHC class II-Ii complexes contain at least
one Ii p35 polypeptide (2, 3, 12). Other minor isoforms of
Ii (p41, p43) are generated as a result of alternative RNA splicing; Ii
p43 with the expression of the additional exon, similar to Ii p35, also
contains the upstream translation initiation site.
The chaperone functions of Ii p35 and p33 in MHC class II assembly and
transport are distinct, with differences apparent in the route taken by
MHC class II molecules to the cell surface and in peptide acquisition
for Ag presentation. The ratio of the two Ii isoforms thus determines
for MHC class II molecules the choice and timing of compartment
localization, the rate of exit from the ER, the extent of
glycosylation, the rate of intracellular peptide loading, and the
degree of endosomal degradation. Indeed, MHC class II molecules
complexed exclusively with Ii p33 are generally transported indirectly
to the endosomal-lysosomal compartments via the plasma membrane,
whereas MHC class II proteins complexed with Ii p35 move directly to
endosomes-lysosomes without first appearing at the cell surface
(13). The transport route of MHC class II complexes
containing Ii trimers with at least one Ii p35 molecule, the
predominant form of the (I)3 complex, is
exclusively intracellular (13, 14, 15, 16). The association of Ii
with MHC class II proteins results in masking of the ER retention motif
in Ii p35, thus allowing the transport of
(I)3 containing this isoform of Ii out of
the ER (17, 18). The transport of MHC class II-Ii nonamers
containing exclusively Ii p33 is to the cell surface before peptide
loading. This delays Ag presentation by these MHC class II molecules
and results in a cell surface phenotype characterized by abundant class
II proteins that are not loaded with antigenic peptides
(13, 14, 15, 16).
The patterns of protein glycosylation and phosphorylation for MHC class II-Ii complexes also differs between those containing only Ii p33 and those containing at least one molecule of Ii p35 (12, 17). The ER retention signal of Ii p35 delays the exit of MHC class II assemblies containing this isoform and thereby facilitates complex glycosylation (7). In contrast, association of MHC class II molecules exclusively with Ii p33 results in the rapid development of resistance to endoglycosidase H (EndoH) and simple patterns of N-glycosylation, signs of fast transport out of the ER. In addition to the glycosylation pattern representing a marker of intracellular transport rate and route, it may affect the rate of degradation survival of some proteins in the endosomes-lysosomes (19).
Cells expressing recombinant MHC class II proteins in the absence of either Ii p33 or p35 exhibit rapid delivery of the class II proteins to the cell surface as well as enhanced loading with exogenous peptides as a result of the surface expression of these molecules with empty peptide-binding groves (20). The rate of intracellular transport of MHC class II molecules is thus slowest in cells expressing exclusively the p35 isoform of Ii, is faster in cells expressing exclusively the p33 isoform of Ii, and is faster still in cells lacking both Ii isoforms (18).
The MHC class II region of the human genome, also more commonly known
as the HLA region, contains up to 14 different class II loci that are
clustered into three major subregions: HLA-DR, -DQ, and -DP. Each of
these subregions contains at least one functional locus and one
locus. Although the MHC class II region of the genome confers the
greatest statistical risk for type 1 diabetes as well as for other
autoimmune diseases, the complexity of this genetic association is
revealed by the fact that identical twins exhibit <40% concordance
for type 1 diabetes expression (21), and for other
autoimmune disease the concordance rate is even less. Furthermore,
diverse human and murine functional studies suggest that
CD4+ T cells might contribute to the initiation
of autoimmunity, including type I diabetes. Given that
CD4+ T cell education is determined by Ag
presentation by MHC class II molecules, we have now investigated
biochemically the assembly of MHC class II molecules and their
presentation of antigenic peptides in established EBV-immortalized B
cell lines derived from human identical twin pairs discordant for
autoimmune type 1 diabetes. The study of cells from such twins controls
for the possible confounding influence of the MHC class II haplotype on
disease-specific defects in the intracellular transport or peptide
loading of class II molecules.
We now show that B cells derived from diabetic twins exhibit rapid delivery of poorly glycosylated and peptide-empty MHC class II molecules to the cell surface as well as delayed peptide loading. These Ag processing defects were associated exclusively with disease penetrance and were secondary in part to a reduced abundance of the p35 isoform of Ii. Stable transfection of cells from diabetic twins with a vector encoding Ii p35 corrected these defects in the assembly of and peptide presentation by MHC class II molecules. The reduced production of a chaperone protein (Ii p35) that facilitates MHC class II assembly may thus be an important phenotype tracking with disease expression of type 1 diabetes in individuals with disease-conferring class II haplotypes.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The EBV-transformed B cell lines used in the present study were prepared from four sets of identical twins discordant for type 1 diabetes (for >15 years) and from random control individuals. All paired twin sets were derived on the same day, although different twin sets were derived at totally different times. At the time the cell lines were made for the twin sets presented in this paper, the diabetic twins no longer had autoantibody expression indicative of active autoimmune disease. The nondiabetic twins and controls were autoantibody negative and exhibited both normal insulin secretion and fasting blood glucose concentrations at yearly examinations over a period of >10 years. One of the diabetic twins of a single pair of twins developed autoimmune hypothyroidism 17 years after the diagnosis of type 1 diabetes. Permission for blood drawing was obtained after full institutional review of the protocol. In the case of experiments for which data obtained from only one twin set are presented, similar results were obtained for all four twin pairs unless indicated otherwise.
Additional derivative B cell lines in this study included T1 and T2 lymphoblastoid cells purchased through American Type Culture Collection (Manassas, VA) and were donated by Dr. P. Cresswell (New Haven, CT). T2 cells are a mutant cell line derived from T1 cells; they lack a large segment of chromosome 6 that encodes MHC class II genes, the Lmp2 and Lmp7 proteasome genes, and the ATPase peptide transporters Tap1 and Tap2. T2 cells do not express surface MHC class II due to direct gene deletion or MHC class I due to deletion of the obligatory transporter and proteasome assembly genes.
Peptide synthesis
The peptide hemagglutinin (HA)307–319
(PKYVKQNTLKLAT) was derived from influenza A/Texas/1/77 virus H3 HA and
is HLA-DR restricted. The peptide IgC
37–51
(KVQWKVDNALQSGNS) is derived from the Ig chain and is also HLA-DR
restricted. The peptide SGPLKAEIAQRLEY was eluted from HLA-DQ, matches
the sequence of an uncharacterized human protein, and binds the
products of all HLA-DQ alleles with varying affinity. All peptides
were biotinylated at the NH2 terminus (Quality
Control Biochemicals, Hopkinton, MA). They were purified by HPLC,
lyophilized, and reconstituted in water.
Assay of peptide binding to the cell surface
The binding of exogenous peptide to MHC class II molecules on the surface of B cells was performed basically as previously described (22). EBV-transformed cells (3 x 105 cells/ml) in 50 µl of complete culture medium were incubated for 4 h at 4°C with 50 µM biotinylated peptide, washed, and then incubated for 30 min at 4°C with FITC-conjugated streptavidin (4.22 µg/ml; Life Technologies, Gaithersburg, MD). As an alternative to incubation at 4°C, in some experiments cells were treated with 12 mM sodium azide to prevent endocytosis and were incubated with peptide for 2 h at 37°C. Cell fluorescence was analyzed (5000 cells/sample) with an EPICS Elite flow cytometer (Coulter, Hialeah, FL); background fluorescence was simultaneously quantified and subtracted. For greater sensitivity of detection of peptide binding, after incubation with biotinylated peptide the cells were sequentially exposed at 4°C to FITC-conjugated avidin D, to biotinylated Abs to avidin D, and again to FITC-avidin D (Vector, Burlington, CA).
For evaluation of the stability of MHC class II-peptide complexes at the cell surface, EBV-immortalized B cells were incubated with 50 µM biotinylated peptide for 4 h at 37°C, washed, and then incubated for various times at 37°C in the presence of 500 µM unlabeled peptide. Fluorescence was determined by flow cytometry.
Antibodies
Among the various mAbs to human MHC class II used in the present
study, L243 recognizes predominantly dimers of HLA-DR devoid of
intact Ii (American Type Culture Collection), 9.3F10 (Coulter)
recognizes all HLA class II proteins (DR, DP, and DQ), I-2 (Coulter)
recognizes HLA-DR, 16.23 (provided by R. Wank) recognizes the HLA-DR3
dimer, I-3 (Coulter) recognizes a nonpolymorphic region and therefore
binds to all class II proteins (DR, DP, and DQ), and DA6.147 (provided
by K. Guy) recognizes DR and DR dimers. mAb PIN.1 was generated in
response to a peptide corresponding to aa 12–28 of the p33 isoform of
human Ii, but detects the cytoplasmic
NH2-terminal regions of both Ii p33 and Ii p35.
The mAb CerCLIP.1 recognizes human CLIP in association with MHC class
II. Rabbit polyclonal Abs to Ii p35 (R.Ip35N) and Ii p41 (R.1p41) and
control rabbit polyclonal Abs to HC3 were obtained from Affiniti
Research (Mamhead, U.K.). mAb W6/32 recognizes all human MHC class I
molecules and was purchased through ATCC.
Analysis of surface expression of MHC class II and CLIP
EBV-transformed B cells (1 x 106) in the log phase of growth (>95% viability) were incubated for 30 min at room temperature in a final volume of 500 µl with various mAbs to MHC class II or CLIP (1.5–2.0 µg/ml). For nonfluorescent primary Abs, immune complexes were detected with FITC-conjugated goat Abs to mouse IgG (Coulter).
Development of EndoH resistance and stable complex formation by MHC class II
Cells (8 x107) were labeled with 2 mCi of
[35S]methionine (DuPont Biotechnology Systems,
Boston, MA) in 15 ml of methionine-free RPMI 1640 for 30 and 90 min at
37°C and then were incubated for various times in complete culture
medium containing a 100-fold excess of unlabeled methionine. Cells were
lysed at 4°C in a solution containing 10 mM Tris-HCl (pH 7.4), 150 mM
NaCl, 1% Triton X-100, 0.5 mM PMSF, and 0.1 nM TLCK
(N-P-tosyl-L-lysine
chloromethyl ketone), and lysates were subjected to immunoprecipitation
with mAbs (L243) to MHC class II and protein A-Sepharose beads
(Pharmacia, Piscataway, NJ). For determination of EndoH resistance, the
immunoprecipitates were eluted from the beads in 15 µl of EndoH
digestion buffer (0.1 M sodium phosphate (pH 6.5), 0.5% SDS, and 0.1%
NaN3) by heating at 100°C for 6 min, and the
eluted proteins were then incubated for 16 h at 37°C in the
absence or the presence of 2 µl of EndoH. The reaction mixtures were
then analyzed by SDS-PAGE on a 12.5% gel and autoradiography. For
detection of stable MHC class II complexes, the immunoprecipitates
prepared with mAb 9.3F10 or DA6.147 were dissolved in SDS sample buffer
(100 mM Tris-HCl (pH 6.8), 2% SDS, and 2% glycerol) and divided into
two portions, one of which was heated at 100°C for 5 min, and the
other was maintained at room temperature before analysis by SDS-PAGE
and autoradiography.
Determination of the rates of synthesis and surface appearance of MHC class II
Cells (3 x 107) were labeled with
1 mCi of [35S]methionine in 5 ml of
methionine-free RPMI 1640 for 30 min at 37°C and then incubated for
various times at 27°C in complete culture medium containing a
100-fold excess of unlabeled methionine. The cells were suspended in 1
ml of Hanks’ solution containing
N-hydroxysuccinamide-S-S-biotin (NHS-SS-biotin; Pierce,
Rockford, IL) at a concentration of 3 mg/ml, incubated for 3 min on
ice, and then lysed for 30 min on ice in the lysis solution described
above. Lysates were subjected to immunoprecipitation with mAb to HLA-DR
and protein A-Sepharose beads. Proteins were eluted from the beads in
100 µl of PBS containing 2% SDS. A portion (20 µl) of the eluted
proteins was used for total MHC class II synthesis (results multiplied
by 5). A second portion (80 µl) of the eluted proteins was diluted
into 1 ml of PBS containing 1% Triton X-100 and subjected to
precipitation with streptavidin-Sepharose beads (Pierce) for 2 h
at room temperature. The resulting precipitates were washed three times
with precipitation buffer and suspended in SDS sample buffer for cell
surface MHC class II (results multiplied by 0.8).
Cells (3 x 107) were labeled with 10
mCi/ml of [35S]methionine in 5 ml of
methionine-free RPMI 1640 for 15–20 min at 37°C and then incubated
for 15, 30, 45, 60, 90, 120, 180, or 240 min at 27°C in complete
culture medium with a 100-fold excess of unlabeled methionine. After
labeling, using buffers similar to those used for MHC class II,
cells were subjected to immunoprecipitation with mAb to MHC
class I and protein A-Sepharose beads.
Immunoblot analysis
Cells (2 x 107) were lysed in 500 µl of a solution containing 10 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.15 M NaCl, 0.2 mM EDTA, 0.02% NaN3, PMSF (100 µg/ml), aprotinin (1 µg/ml), and leupeptin (1 µg/ml), and equal amounts of lysate protein were subjected to SDS-PAGE on a 12.5% gel. The separated proteins were transferred to a polyvinylidene difluoride membrane, which was then subjected to immunoblot analysis with polyclonal Abs to Ii p35, Ii p41, or mAb HC3. Immune complexes were detected with appropriate secondary Abs labeled with HRP and an ECL kit (Amersham Pharmacia Biotech, Piscataway, NJ).
Stable transfection of B cell lines with an Ii p35 vector
The human Ii p35 cDNA (in which the second translation start
site was mutated) was provided by E. Long (11) and was cut
from the expression vector Sp64 to construct the expression vector
pBabe Ii p35 out of BamHI sites. EBV-transformed B cells (1
x107) were incubated for 5 min on ice in a
volume of 100 µl containing 30 µg of pBabe/Ii p35 plasmid DNA and
were then subjected to electroporation at 220 V and 960 
in a 0.4-cm
cuvette. Transfected cells were cultured for 2 days without selection
and then were maintained in the presence of puromycin (0.25 µg/ml)
for >2 mo. At least three stable and independently derived transfected
cell lines were established from each EBV cell line.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The abilities of APC to stimulate T cells as well as to induce positive or negative T cell selection are dependent on the total number of surface MHC class II-peptide complexes. With the use of flow cytometry with four different mAbs to MHC class II molecules, we therefore initially examined the total surface density of these molecules on EBV-immortalized B cell lines from identical twin pairs discordant for type 1 diabetes as well as on control cell lines. The MHC class II surface density revealed by the three mAbs, L243, I-2, and I-3, did not differ significantly between diabetic twin cell lines and either matched nondiabetic twin cells or non-HLA-matched random control EBV cell lines (Table I). In contrast, the MHC class II surface density revealed by mAb 9.3F10, which recognizes HLA-DR, -DP, and -DQ, was significantly greater for the diabetic cell lines than for the matched twin cells or control cells. Although the exact conformation of MHC class II molecules recognized by mAb 9.3F10 is not known, the Ab detects a subset of APC with an enhanced ability to present exogenous Ag, an indirect indication of its preferential binding to peptide-empty forms of MHC class II (23). These results suggested that the overall surface density of MHC class II molecules did not differ between B cells from diabetic or nondiabetic twins or between twin cells and control cells, but that the expression of peptide-empty MHC class II molecules on the surface of the diabetic B cells might be increased.
|
Increased surface density of peptide-empty MHC class II on diabetic B cells
The peptide occupancy of surface MHC class II molecules can be
directly quantitated by measurement of the binding of exogenous labeled
peptides (20, 26). With the use of flow cytometry, we
therefore examined the binding of two biotinylated HLA-DR-specific
antigenic peptides (HA307–319 and
IgC37–51) at saturating concentrations to MHC
class II molecules on the surface of diabetic and nondiabetic twin B
cells and on mutant T2 B cells that lack surface MHC class II proteins.
The binding of the HA307–319 peptide to B cells
derived from diabetic twins was markedly increased compared with that
to B cells from the matched nondiabetic twins (Fig. 1A). This difference was
apparent both for cells incubated at 4°C for 4 h (Fig. 1A) and for cells incubated at room temperature in the
presence of sodium azide for 2 h (data not shown), both of which
procedures were chosen to prevent the turnover of surface MHC class II.
Similar results were obtained with the peptide
IgC37–51 (Fig. 1B), but only
background peptide binding fluorescence was evident on T2 cells,
confirming the specificity of peptide binding to MHC class II. These
results were thus indicative of a defect in intracellular peptide
loading of MHC class II molecules in the B cells derived from diabetic
twins.
|
We next examined the transport of newly synthesized MHC class II-Ii protein complexes in diabetic and nondiabetic B cell lines. Cells were pulse-labeled with [35S]methionine and subjected to immunoprecipitation with Abs to MHC class II at various times thereafter. The rate of exit of MHC class II molecules from the ER was assessed by subjecting portions of the immunoprecipitates to treatment with EndoH, which cleaves carbohydrate chains from proteins that have not yet been processed further in the Golgi apparatus (27). Cleavage of N-linked carbohydrates from a protein by EndoH was revealed by an increase in electrophoretic mobility and indicated that the protein was still located in the ER at the time of sampling.
Pulse-chase data for B cells derived from one discordant twin pair are shown in Fig. 2A (similar results were obtained with cells from three different twin sets). At 0 or 30 min into the chase incubation, the MHC class II-Ii complexes in cells from both the diabetic and nondiabetic twins exhibited EndoH sensitivity, indicating that they had not yet traversed the Golgi complex. However, at time zero of the chase, MHC class II molecular complexes of the diabetic twin, compared with those of the nondiabetic twin, appeared to be associated to a reduced extent with the p35 isoform of Ii (Fig. 2A, arrow). Furthermore, after EndoH treatment at this time point, MHC class II-Ii from the diabetic twin, compared with that from the nondiabetic twin, exhibited a reduced extent of band heterogeneity. This reduced electrophoresis heterogeneity was reflective of the results of published pulse-chase studies of MHC class II in transfected cells lacking Iip35, but expressing Iip33 (7, 18). These data at this early time point of pulse-chase thus suggested that the MHC class II-Ii complexes of the diabetic twin had altered EndoH sensitivity and perhaps a reduced association with Iip35.
|
These results indicate the MHC class II-Ii complexes of diabetic B cells exit the ER more rapidly than do complexes from nondiabetic cells or control cells. This pulse-chase pattern of the diabetic cells was reminiscent of data previously obtained with cells expressing exclusively the p33 isoform of Ii and lacking Ii p35 (7).
As a control protein assembled in the ER, the rate of intracellular transport of MHC class I to the Golgi was estimated by the rate of sialylation of MHC class I lysates from two sets of discordant identical twins. Exit of MHC class I exit to the Golgi results in an increase in molecular mass when analyzed by SDS-PAGE. Published data show a reduced rate of MHC class I exit from the splenocytes isolated from spontaneously diabetic nonobese diabetic mice, but not unaffected murine cohorts (28). Diabetic B cells from two diabetic twins showed a 15- to 30-min slowing in the delivery of class I molecules into the trans-Golgi, as evidenced by a delay in achieving a higher molecular mass (data not shown). Nondiabetic twin B cell lines exhibited normal MHC class I exit times comparable to those of control cell lines. Opposing assembly rates of slowed MHC class I compared with accelerated MHC class II in only diabetic B cell lines confirmed that marked rapid MHC class II exit is a specific immune transport dysfunction.
Rapid transport of MHC class II in diabetic B cells
The stability of MHC class II-peptide complexes on the cell surface is an important determinant of the efficacy of Ag presentation to CD4+ T cells. We next examined the appearance and turnover of MHC class II at the surface of diabetic and nondiabetic twin B cell lines. Cells were pulse-labeled with [35S]methionine, incubated in the absence of [35S]methionine for various times, and, immediately before lysis, biotinylated with a membrane-impermeable reagent (NHS-SS-biotin) to allow detection of only those MHC class II molecules expressed on the cell surface. Cell lysates were then subjected to immunoprecipitation with Abs to MHC class II to reveal total pulse-labeled MHC class II molecules, and a portion of the resulting immunoprecipitates was subjected to further precipitation with streptavidin-agarose to reveal surface MHC class II. For the twin pair analyzed in Fig. 2B, MHC class II molecules began to appear on the surface of the diabetic B cells by 1 h into the chase incubation, with maximal surface expression apparent at 2 h. In contrast, the B cells from the paired nondiabetic twin and control B cells exhibited a minimal number of 35S-labeled MHC class II molecules on the surface at 2 h. The overall rate of MHC class II synthesis in the diabetic cell line appeared similar to that in the matched nondiabetic cell line.
The B cells from the diabetic twin also exhibited rapid turnover of surface MHC class II. At 3 h into the chase incubation, a time at which the surface expression of 35S-labeled MHC class II had not yet peaked in nondiabetic twin or control cells, the amount of newly synthesized MHC class II at the surface of the diabetic cells was already decreasing (Fig. 2B). This pattern of rapid delivery and turnover of MHC class II molecules at the cell surface was consistently apparent in all diabetic cells (compared with matched twin cells and random control cells) examined.
The turnover of MHC class II molecules at the cell surface was further
examined with a different approach (26). The half-life of
surface MHC class II complexes loaded with exogenous peptides was
assessed to determine whether peptide loading of the relatively large
proportion of peptide-empty class II molecules on the surface of
diabetic B cell lines would affect the kinetics of internalization or
alter the durability of internalized MHC class II complexes perhaps
deficient in select chaperone proteins. Previous studies have shown
that at saturating peptide concentrations peptide dissociation is
extremely slow and nonstoichiometric. Monitoring the disappearance of
labeled peptide MHC class II provides a measure of surface
internalization and internal stability of the MHC class II-peptide
complexes, since fluorescence can persist until endosomal degradation.
Cells were exposed for 4 h to 50 µM of a biotinylated
HLA-DQ-specific peptide (SGPLKAEIAQRLEY), after which the cells
were washed and then incubated for various times in the presence of 500
µM unlabeled peptide. At time zero of the chase incubation the
fluorescence intensity of diabetic B cells was greater than that of the
matched nondiabetic B cells or control cells, reflecting a larger
number of MHC class II molecules newly filled with the exogenous
peptide (Fig. 2C). The subsequent rate of disappearance of
biotinylated peptide from the cell surface or internally was markedly
greater for diabetic B cells than for nondiabetic or control B cell
lines. Similar results were obtained with the HLA-DR-specific peptide
IgC37–51 and B cell lines derived from four
different identical twin pairs discordant for type 1 diabetes (data not
shown). The loading of the empty peptide binding grooves of MHC class
II molecules on the surface of diabetic B cells thus did not normalize
the increased rate of surface MHC class II internalization or the
internal stability apparent in these cells.
Delayed formation of stable MHC class II complexes in some diabetic B cells
The binding of antigenic peptides by MHC class II molecules
confers stability to the class II dimer, as revealed by failure
of SDS to induce its dissociation at room temperature
(29). The formation of SDS-resistant MHC class II
complexes, a phenomenon called dimer formation, in B cell lines from
diabetic and unaffected twins was investigated by labeling the cells
for 90 min with 0.5 mCi of [35S]methionine,
chasing them in cold medium for 4 and 15 h, and then subjecting
cell lysates to immunoprecipitation with Abs to MHC class II. In the
twin sets the resulting precipitates were dissolved in SDS sample
buffer and either boiled or not before analysis by electrophoresis and
autoradiography. For one twin set the abundance of MHC class II
complexes resistant to SDS at 4 h was markedly greater in the
nondiabetic B cells than in diabetic B cells (Fig. 3A). Although virtually all
MHC class II - and -chains in nondiabetic cells were present in
stable complexes at 15 h, the diabetic cells at this time still
contained substantial amounts of MHC class II not in the form of stable
complexes. In this assay of MHC class II stability there was
variability in the discordant diabetic twin sets. As presented, one
twin set was markedly discordant in dimer formation, two other twin
sets were mildly discordant, and one twin set showed no differences
between the paired twin sample sets, thus suggesting that this assay
less reliability tracks a phenotype of interrupted MHC class II
assembly and disease expression.
|
The altered transport kinetics and peptide loading of MHC class II molecules detected in diabetic B cell lines were suggestive of a defect in the expression or function of the p35 isoform of Ii (7, 13). We therefore subjected B cells derived from matched sets of diabetic and nondiabetic twins to immunoblot analysis with Abs to Ii p35. The amount of Ii p35 in each diabetic cell line was reduced by 30–60% compared with that in the cell line derived from the corresponding nondiabetic twin (Fig. 3B). In contrast, the abundance of the p41 and p33 isoforms of Ii in B cell lines did not differ between diabetic and nondiabetic twins. Furthermore, as a control protein, the amounts of the proteasome subunit HC3 were similar in the diabetic and nondiabetic twin cells. Given that both p35 and p33 isoforms of Ii are produced from the same mRNA, these data suggested that translation of the p35 isoform is specifically reduced in diabetic B cells; alternatively, the degradation of Ii p35 may be increased in diabetic B cells. The unlikely possibility that the Ii gene of the diabetic twins contained a somatic mutation was eliminated by sequencing of the promoter, early introns, and coding regions of this gene from all four twin pairs studied and from one random control individual. The gene sequences were identical for all individuals (data not shown) and were in complete agreement with the previously determined sequence.
Correction of MHC class II defects in diabetic B cells by restoration of Ii p35 expression
To define the possible role of the functionally reduced expression of Ii p35 in the MHC class II defects of diabetic B cells, we subjected B cells derived from matched diabetic and nondiabetic twins to stable transfection with an Ii p35 vector. Transfection restored the abundance of Ii p35 in diabetic B cells to that apparent in untransfected nondiabetic cells (Fig. 4A); transfection of the nondiabetic cells had little effect on the overall abundance of Ii p35.
|
The effects of restoration of Ii p35 expression in diabetic cells on the increased rates of appearance and subsequent turnover of newly synthesized MHC class II at the cell surface were also evaluated. The time course of the appearance of MHC class II molecules at the cell surface and their subsequent internalization in stably transfected Ii p35 diabetic B cells did not differ substantially from that observed in the corresponding untransfected or transfected nondiabetic twin cells (Fig. 4C). These results support the critical functional role of deficient Ii p35 in diabetic cells in conferring altered MHC class II assembly and correction of these intracellular defects with restored protein expression.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
A reduced surface density of MHC class II molecules loaded with antigenic peptides and increased rate of turnover of these molecules at the cell surface of diabetic cells might be expected to impair interaction of APC with CD4+ T cells and thereby influence T cell education. It remains to be determined whether the abnormal MHC class II processing apparent in human diabetic cells is a direct cause of disease or whether it represents a phenotype potentially useful for tracking disease expression. Each paired twin set of affected and nonaffected twin blood cells was transformed to EBV cell lines on the same day, and many sets of these paired samples were prepared multiple times over the past 10 years. Therefore, it is unlikely that a random increase in the number of activated B cells at the time of transformation could account for the consistent diabetic twin EBV cell line errors repeatedly observed.
Although not reported in this study, we have also examined EBV cell lines from other discordant twin pairs with differential autoimmune disease expression. Both diseased twin EBV cell lines from a rheumatoid arthritis and multiple sclerosis patient compared with their unaffected twin similarly display accelerated MHC class II transport to the cell surface and reduced peptide-loaded surface MHC class II structures. These data are consistent with possible insufficiencies in Iip35 expression as a contributor to the development of diverse forms of autoimmunity without this gene translation defect determining target organ selection.
The avidity of T cells for MHC class II-peptide complexes is thought to be a determinant of T cell selection (30). MHC class II molecules are usually retained in the ER through the formation of mixed Ii trimers that contain at least one Ii subunit with an ER retention signal. One consequence of such multimerization is that Ii p35, which contains an NH2-terminal ER retention signal (7, 8), exerts an effect that is disproportionate to its relative abundance (18). Studies with transfected human B cells have shown that the ER residence time of Ii p33 is <3 h, whereas that of Ii p35 is 6–9 h (18). Cells expressing only the p33 isoform of Ii also do not exhibit complex patterns of protein glycosylation (7). Furthermore, expression of Ii p35 at normal or increased levels in transfected cells promotes Ag presentation by MHC class II molecules by preventing excessive degradation of these molecules in endosomes-lysosomes and thereby presumably increasing the opportunity for their interaction with antigenic peptides (18).
In summary, these findings describe a new epigenetic influence of altered abundance of an important human-specific chaperone protein, Iip35, that possibly explains the selective influence of at-risk MHC class II genes to confer altered Ag presentation in only diabetic twins with disease.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 G.Y. and L.S. contributed equally to this work.
3 Current address: Endocrinologie, Hopital Jean Minjoz, 25030 Besancon Cedex, France.
4 Address correspondence and reprint requests to Dr. Denise L. Faustman, Immunobiology Laboratory, Massachusetts General Hospital, East, Building 149, 13th Street, CNY-3601, Charlestown, MA 02129. E-mail address: faustman{at}helix.mgh.harvard.edu
5 Abbreviations used in this paper: Ii, invariant chain; CLIP, class II-associatedinvariant peptide; EndoH, endoglycosidase H; ER, endoplasmic reticulum; HA,hemagglutinin; NSH-SS-biotin, N-hydroxysuccinamide-S-S-biotin.
Received for publication June 7, 2002. Accepted for publication October 29, 2002.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
dimers. J. Immunol. 150:499.[Abstract]
-N-acetyl-D-glucosamine-1-phosphodiester N-acetylglucosaminidase (formerly called -N-acetylglucosaminylphosphodiesterase) and lysosomal -N-acetylglucosaminidase. Arch. Biochem. Biophys. 222:145.[Medline]
This article has been cited by other articles:
|
|
 |
|
  P. Alard, J. N. Manirarora, S. A. Parnell, J. L. Hudkins, S. L. Clark, and M. M. Kosiewicz Deficiency in NOD Antigen-Presenting Cell Function May Be Responsible for Suboptimal CD4+CD25+ T-Cell-Mediated Regulation and Type 1 Diabetes Development in NOD Mice. Diabetes, July 1, 2006; 55(7): 2098 - 2105. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Khalil, A. Brunet, I. Saba, R. Terra, R. P. Sekaly, and J. Thibodeau The MHC class II {beta} chain cytoplasmic tail overcomes the invariant chain p35-encoded endoplasmic reticulum retention signal Int. Immunol., October 1, 2003; 15(10): 1249 - 1263. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) and nondiabetic (
) twins. Background fluorescence was
subtracted from the data, which are presented as the mean ± SD of
duplicate samples analyzed on the same day. The same experiment was
performed on three separate occasions with similar results.
) and nondiabetic (
) were incubated for 4
h at 37°C with 50 µM of the biotinylated peptide SGPLKAEIAQRLEY,
after which the cells were washed and incubated for the indicated times
in the presence of 500 µM unlabeled peptide. The cells were then
subjected to flow cytometric analysis for quantitation of surface
biotinylation. Data are the mean ± SD of triplicate
determinations and are expressed as a percentage of the fluorescence
intensity for diabetic cells at time zero.
