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*
Department of Medicine, Division of Rheumatology, and
Department of Immunology and Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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heterodimer is assembled in the endoplasmic reticulum, where
it associates with invariant chain
(Ii),3 a chaperone
molecule that promotes proper folding of class II, protects the peptide
binding groove for loading in a later compartment, and directs class II
to the endosomal pathway. In the endosomes, Ii is proteolytically
degraded until only the fragment called class II-associated
leupeptin-induced peptide (CLIP) remains associated with class II
(reviewed in Refs. 4 and 5). Exchange of CLIP
for antigenic peptides is then catalyzed by H-2M before peptide:class
II transport to the cell surface for interaction with the TCR. The enzymes involved in Ii degradation have been recently characterized. Reports using protease inhibitors (6) and knockout mice (reviewed in Refs. 4 and 5) have demonstrated a significant role for the lysosomal cysteinal proteases cathepsin L (CL) and cathepsin S (CS) in Ii processing. Deficiency of CL and CS results in a block in generation of mature SDS-stable class II dimers due to an accumulation of Ii intermediates, namely p12, or small leupeptin-induced peptide (SLIP). Within the immune system, CS is found in bone marrow-derived APCs, i.e., macrophages, dendritic cells, and B cells, whereas CL is found in thymic cortical epithelial cells and macrophages (4). Because of this cell type-restricted expression, CL deficiency results in diminished positive selection of CD4+ T cells but does not significantly affect Ag presentation by bone marrow-derived APCs (7). In contrast, CS deficiency impairs Ag presentation by bone marrow-derived APCs but does not affect CD4+ T cell development (8, 9). While CL and CS appear to be the predominant proteases processing Ii, cathepsin F has also been implicated in Ii degradation in macrophages (10).
In contrast to Ii degradation, little is known about Ag processing, i.e., the proteolytic mechanisms that generate particular T cell epitopes. Early studies found that inhibition of lysosomal acidification interferes with proteolysis and Ag presentation, implicating lysosomal proteases (reviewed in Ref. 3). However, subsequent studies have not clearly defined these proteases. One report found an asparagine-specific cysteinal protease to be important for processing tetanus toxoid (11), although asparagine-specific cysteinal protease’s overall importance for the bulk of Ag processing remains to be determined. Using purified lysosomal enzymes and protease inhibitors of relatively broad specificity, cathepsin B (CB) and cathepsin D (CD) were implicated in Ag processing. However, CB and CD knockout mice were not deficient in Ii processing or Ag presentation (12, 13). Recently, CB, and less so CS, were found to be important for degradation of F(ab')2 internalized via FcR, but generation of peptides for presentation on class II was not assessed (14). Lastly, evaluation of CL and CS has focused on their roles in Ii degradation and not in Ag processing (5, 7, 8, 9).
We have chosen to examine the roles of CL and CS in Ag processing because these proteases are already known to have an important immune cell function, the degradation of Ii. To avoid the heterogeneity of ex vivo-derived APC populations and to allow for large-scale peptide analysis, we used in vitro cell lines expressing CL, CS, or neither. Expression of CL and CS mediates efficient Ii processing as expected. Although the peptide repertoire bound to I-Ab, as assessed by mass spectroscopy, is qualitatively similar regardless of CL or CS expression, a distinct subset of peptides is created or destroyed by the presence of CL or CS. Thus, the generation of a specific epitope may be critically regulated by the presence or the absence of a particular protease. This direct effect of CL and CS on Ag processing may have an important impact on immune responses to particular Ags, such as type II collagen in collagen-induced arthritis (8), and on peptide repertoires displayed by different types of APC, i.e., thymic epithelial cells vs dendritic cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CL knockout mice have been previously described
(7). The following Abs were used:
anti-I-Ab mAb Y3P (15) and
M5/114 (16), anti-CLIP:I-Ab mAb
15G4 (C. Beers, P. Wong, and A. Rudensky, manuscript in preparation)
(8), anti-CD22:I-Ab mAb A8 (G.
Barton and A. Rudensky, manuscript in preparation), polyclonal rabbit
Abs specific for lumenal domain of Ii (gift from L. Karrlson, R.
W. Johnson Research, San Diego, CA), polyclonal rabbit Abs against the
A and A cytoplasmic
tails (A. Rudensky, unpublished observation), anti-Ii-In-1
(17, 18), goat anti-rabbit IgG biotin (The Jackson
Laboratory, Bar Harbor, ME), streptavidin-allophycocyanin (BD
PharMingen, San Diego, CA), streptavidin-CyChrome 5 (The Jackson
Laboratory), and goat anti-rat IgG Texas Red (Caltag Laboratories,
South San Francisco, CA). OVA , keyhole limpet hemocyanin (KLH), IgM,
hen egg lysozyme (HEL), and sperm whale myoglobin were obtained from
Sigma-Aldrich (St. Louis, MO). SA85 peptide (1102) and SA85 Ag (His)
were gifts from Dr. S. Kahn (University of Washington, Seattle, WA).
The culture medium was RPMI 1640 containing 5% FCS, 2-ME,
penicillin/streptomycin, sodium pyruvate, L-glutamine, and
HEPES (all from Life Technologies, Grand Island, NY). Trypsin/EDTA were
used at 1 mg/ml and 53 mM, respectively (Life Technologies).
Generation of fibroblast lines
Day 16 embryos were harvested from CL-deficient mice, mechanically disrupted, and digested by trypsin/EDTA. Immortalized cells were generated by transfection of SV40 large T Ag (SV3-neo) (19) using Fugene 6 (Roche, Indianapolis, IN) and selection for neomycin resistance. Cells were cloned by limiting dilution, and one clone was selected for further use (MEF9). Class II, H-2M, and Ii expressions were induced by cotransfection of human class II trans-activator (CIITA) gene under a CMV promoter (20) and a plasmid with a hygromycin resistance gene. A clone with high level I-Ab expression was selected (MEF9.C2). CL- or CS-expressing cell lines were generated using retroviral infection (21, 22). The CL retroviral vector was made by cloning CL cDNA into BamHI sites in pMI2 (23), which uses an internal ribosome entry site to express a tail-less human CD2 marker (gift from Dr. M. Bevan, University of Washington). The CS retroviral vector was made by cloning CS cDNA into EcoRI sites in MigR1 (24), which uses an internal ribosome entry site to express green fluorescent protein (GFP) marker (gift from Dr. W. Pear, University of Pennsylvania, Philadelphia, PA). Cell lines were cloned before analysis. Expression of class II, human CD2, and GFP remained stable for at least 1 mo. Cell cultures were routinely refreshed from frozen stocks monthly.
Lysosomal cysteine protease active site labeling
Cells were harvested by treatment with trypsin/EDTA and
incubated in culture medium for 2 h at 37°C with 0.25 µM
Cbz-[125I]Tyr-Ala-CN2
(25), which irreversibly binds to the active site cysteine
via a thiol-ester bond. Cells were lysed in 0.5% Nonidet P-40, 0.15 M
NaCl, 5 mM EDTA, and 50 mM Tris in the presence of protease inhibitors
N--p-tosyl-L-lysine chloromethyl
ketone, aprotinin, and PMSF (Sigma-Aldrich). After centrifugation at
10,000 x g for 10 min, lysates were boiled for 5 min
in SDS reducing buffer and run on a 12% w/v polyacrylamide gel.
125I-labeled proteins were visualized by
autoradiography.
Pulse chase
Fibroblasts were cultured in a six-well plate at 6–7.5 x 105/well overnight at 37°C. Cells were then cultured in methionine/cysteine-free RPMI 1640 with 5% dialyzed FCS for 90 min, pulsed with 0.125 mCi/well 35S-methionine (Trans 35S-label; ICN Pharmaceuticals, Costa Mesa, CA) for 40 min, and chased in the presence of 30x unlabeled methionine/cysteine for the indicated time. Cells were lysed on the plate with 1% Nonidet P-40, 0.01 M Tris (pH 7.3), 0.15 M NaCl with protease inhibitors as above, and I-Ab molecules immunoprecipitated with M5/114 and protein G-Sepharose (Amersham Pharmacia Biotech, Piscataway, NJ). Immunoprecipitates were analyzed by PAGE in 7.5–20% gradient SDS polyacrylamide gels under nonreduced/nonboiled, or reduced/boiled conditions, and visualized by autoradiography.
Surface biotinylation
Fibroblasts were plated at 4 x 106 in a T75 flask overnight, washed with cold PBS, and incubated with 1 mg/ml NHS-LC-biotin (Pierce, Rockford, IL) for 30 min at 4°C. Cells were washed with PBS followed by 5% FCS/PBS and lysed in 1% Nonidet P-40 with protease inhibitors. Lysates were precipitated with streptavidin-Sepharose (Amersham Pharmacia Biotech) or M5/114 mAb and protein G-Sepharose. Samples were run under boiled/reduced conditions on a 12% Tris/glycine gel, transferred onto nitrocellulose membrane, immunoblotted with anti-Ii-In-1 or rabbit anti-class II Abs followed by anti-rat or anti-rabbit Ig Abs conjugated with HRP, and visualized by chemiluminescence (Amersham Pharmacia Biotech).
T cell hybridoma assay
T cell hybridomas were previously described (7). T cell hybridomas (5 x 104/well) were cocultured in 96-well flat-bottom plates (Costar) with APCs (2.5 x 104/well) and the indicated Ags for 24–30 h. Supernatants were assayed for IL-2 production using the CTLL-2 indicator cell line and Alamar blue colorimetric assay (Trek, Medina, NY). Data are expressed as OD units (OD570–600). In some experiments, APCs were washed with PBS and fixed in 0.4% paraformaldehyde for 7 min at room temperature. Glycine was added at 0.2 M final concentration to stop the reaction, and the cells were washed three times with culture medium.
I-Ab purification and analysis of I-Ab-bound peptides by mass spectroscopy
Cells were harvested by trypsin/EDTA, washed with PBS, and
stored at -70°C until lysis with 1% Nonidet P-40, 25 mM
iodoacetamide, PMSF,
N--p-tosyl-L-lysine chloromethyl
ketone, and aprotinin in PBS (pH 7.4). Peptide isolation was performed
as previously described (26). I-Ab
was purified by affinity chromatography using Y3P, and peptides eluted
from 50–100 µg purified I-Ab bound to
Y3P-Sepharose by 2.5 M acetic acid.
Microcapillary HPLC and automated tandem mass spectrometry were
performed as previously described (26). Briefly,
12-cm-long microcapillary columns were prepared from 100-µm internal
diameter fused silica capillary tubing (Polymicro Technologies,
Phoenix, AZ) and packed with POROS 10R2 reverse phase resin (PerSeptive
Biosystems, Framingham, MA) as previously described (27).
Microcapillary HPLC was performed using Shimadzu LC-10AD pumps
(Shimadzu, Columbia, MD) with flow splitting to achieve a rate of 1
µl/min with a gradient from 2.5% acetonitrile/0.1% acetic acid to
40% acetonitrile/0.1% acetic acid over 40 min. The column was
inserted directly into the electrospray needle (Thermo Finnigan, San
Jose, CA), and eluted peptides were analyzed on a Thermo Finnigan TSQ
7000 tandem mass spectrometer. For experiments comparing peptide
species isolated from different cell lines, scans across the range of
peptide elution were summed and averaged using Thermo Finnigan software
to generate a profile of the top peptide ions from each sample. Most of
these peptide ions are doubly and triply charged, with a few
quadrupally charged species. Each peptide ion was screened for
coelution of alternatively charged species, and the relative peptide
amount was determined by summing the area under the curve for each
charge species. The values presented are normalized within the sample
such that the most abundant peptide equals 100. The level of detection
is 
1% of the most abundant peptide. Sequencing was performed using
Thermo Finnigan ICL procedures, which automatically collect tandem mass
spectra for the three most abundant ions found in two consecutive scans
in m/z range 400-1800. The acquired mass spectral data were analyzed
using Thermo Finnigan ICIS 8.3 software and by searching National
Center for Biotechnology Information databases using SEQUEST
(28).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To study the roles of CL and CS in Ag processing, we used in vitro
cell lines. This facilitated large-scale analysis of class
II-associated peptides, allowed analysis of both CL and CS under the
same conditions, and eliminated the variability found in ex vivo APC
populations. Thus, we generated an
I-Ab-expressing (Fig. 1
A) embryonic fibroblast cell
line (MEF9.C2) from CL knockout mice (see Materials and
Methods). This line is deficient in both CL and CS, as revealed by
active site labeling, although it does express CB as fibroblasts
normally do (Fig. 1B). We then expressed either CL or CS
using retroviral bicistronic vectors with the human CD2 or GFP marker.
Clones were screened for the marker gene, and CL or CS expression was
verified by active site labeling (Fig. 1B). The level of
expression of these enzymes is comparable to that observed in ex vivo
isolated cells (data not shown). Surface I-Ab
expression is slightly enhanced by CL or CS expression (Fig. 1A).
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It is known that in CL- and CS-deficient APCs, there is
significant inhibition of Ii processing and class II maturation
(7, 8, 9). Similarly, we found using biosynthetic labeling
experiments that CL- and CS-deficient MEF9.C2 cells accumulate the SLIP
(p12) fragment of Ii and inefficiently generate SDS-stable
I-Ab complexes (Fig. 2A). The processing of SLIP to
CLIP is reconstituted upon expression of CL or CS (Fig. 2A).
The kinetics of Ii degradation and SDS-stable class II dimer generation
are slightly faster in cells expressing CS than in cells expressing CL.
We have also analyzed these cell lines by confocal microscopy to
localize class II-bound Ii fragments using 15G4, an mAb raised against
the CLIP:I-Ab complex that also recognizes
SLIP:I-Ab (C. Beers, P. Wong, and A. Rudensky,
manuscript in preparation). The overall levels of lysosome-associated
membrane protein (LAMP) class II and Ii are not markedly different
between the cell lines (data not shown). In contrast, 15G4 shows
intense staining of lysosome–associated membrane protein
(LAMP)-positive vesicles in the CL- and CS-deficient MEF9.C2
cells, but only limited punctate staining in the CL- or CS-expressing
cells (Fig. 2B). The latter vesicles show little staining
with anti-LAMP1 Abs, suggesting that a relatively early
LAMP-negative endosomal compartment serves as the site for early stages
of Ii degradation in both CL- and CS-positive cells. H-2M does localize
to the 15G4- and LAMP-positive compartments (data not shown),
consistent with previous observations in CS-deficient dendritic cells
(29). Thus, 15G4 staining reveals the accumulated
SLIP:I-Ab complexes observed by pulse chase (Fig. 2A), which reside in lysosomal/late endosomal vesicles in
CL- and CS-deficient cells.
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We then assessed the ability of these cell lines to present
exogenous Ags to T cell hybridomas (Fig. 3). We examined the responses of
hybridomas specific to OVA (OB4 and OB15), KLH (2BH11), sperm whale
myoglobin, Trypanosoma cruzi SA85 Ag (71.5), IgM (77.1), and
HEL peptide (BO4). Previous work in I-Ab mice
suggested a global inhibition of exogenous protein presentation in
CS-deficient APCs, presumably due to defective Ii processing (8, 9), whereas peptide presentation was not affected
(9). We found that expression of CL or CS enhances the
presentation of five of six Ag-derived epitopes studied (Fig. 3A). However, the effect of CL or CS ranges from a mild, 3-
to 9-fold augmentation of Ag presentation for two OVA epitopes and KLH
to a marked, >80-fold augmentation for myoglobin and SA85.
Interestingly, the expression of CS significantly diminishes the
presentation of one Ag, murine IgM, but not that of the corresponding
peptide (Fig. 3, A and B). As expected, fixed
fibroblast lines present only IgM peptide and not protein (data not
shown), suggesting that the IgM peptide may escape the effects of CS by
directly binding to surface class II. The effects of CL and CS on Ag
presentation may thus be categorized in three patterns: 1) mild
augmentation, 2) marked augmentation or dependence on, and 3)
inhibition. If CL and CS only affect Ag presentation via Ii
degradation, such disparate patterns would not be expected, suggesting
a role for CL and CS in Ag processing.
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). This apparent reduction in peptide
loading of class II molecules on CL- and CS-deficient cells was
directly confirmed by analysis of the binding of exogenous CD22 peptide
to surface class II using a peptide:I-Ab
complex-specific mAb A8 (Fig. 4A). Fixation does not alter
the pattern of A8 staining of these cells, consistent with a defect in
peptide binding at the cell surface (data not shown). Given the
impairment of Ii processing in the absence of CL and CS (Fig. 2), we
asked whether the inhibition of peptide binding was due to the blockade
of cell surface class II by larger Ii fragments. Indeed, surface SLIP,
but not larger Ii fragments, is increased in the MEF9.C2 cells, as
assessed by flow cytometry and surface biotinylation, followed by class
II or streptavidin immunoprecipitation (Fig. 4). This result is
consistent with prior analysis of CS-deficient B cells and dendritic
cells (8, 9). However, the surface staining of 15G4, which
recognizes both CLIP:I-Ab and
SLIP:I-Ab, is enhanced by the expression of CL or
CS, presumably due to the increased surface expression of
CLIP:I-Ab resulting from efficient degradation of
SLIP (Fig. 4A; also see Fig. 6, upper panel;
molecular mass (m) = 1987.9). We hypothesize that
increased SLIP:I-Ab explains inefficient surface
peptide loading on the CL- and CS-deficient MEF9.C2 cells, although
other possibilities cannot be excluded, such as a decreased amount of
"empty" class II due to an altered peptide repertoire.
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B). This suggests that CL and CS cleave
within the peptide. Consistent with this interpretation, we find that
the SA85 peptide is much more efficiently presented by live, rather
than fixed, APCs, whereas the IgM peptide is presented equally well
(Fig. 3C). The IgM T cell hybridoma 77.1 was generated
against a defined, naturally occurring class II-bound peptide
(26, 30). In contrast, the 71.5 hybridoma was generated
against a protein Ag and the SA85 peptide later identified by screening
overlapping synthetic peptides (31). Thus, it is
reasonable that the SA85 peptide behaves like a protein, requiring
active CL or CS for efficient presentation. Although consensus sites
are not established, cleavage could occur between L-V or E-A in the
SA85 peptide (SHNFTLVASVIIEEA) based on proteolysis of other proteins
by CL and CS (32, 33). Effect of CL and CS on class II peptide repertoire
The observation that CL and CS expression has diverse effects on
Ag presentation suggests a role for these enzymes in Ag processing, in
addition to their role in Ii degradation. To assess the global effect
of CL or CS on epitope generation, we analyzed peptides eluted from
class II by electrospray mass spectrometry. We found that the peptide
repertoire is diverse regardless of CL or CS expression (Fig. 5). A three-dimensional representation of
the mass spectroscopy data with the scan number on the
x-axis reflecting approximate hydrophobicity of the peptide,
mass/charge (m/z) on the y-axis, and signal intensity on the
z-axis shows differences in the spectrum of prominent
peptides presented by each cell line. To analyze the specific effects
of CL and CS, we identified the 18 most abundant peptides from each
line and determined their relative levels of expression in the other
lines (Fig. 6, upper panel).
Surprisingly, the majority of the peptides in each set of 18
were also found within the other sets of top 18 peptides, suggesting
that the qualitative peptide repertoire is not markedly altered by the
expression of CL or CS. After accounting for peptides found in more
than one set of 18, we are left with a pool of 24 unique peptides, 21
of which are expressed at detectable levels in all three cell lines.
However, the effect of CL or CS on the expression of an individual
peptide is quite variable, significantly enhancing or diminishing
relative peptide expression (Fig. 6, lower panel). In fact,
one peptide (m = 2804) appears to be created by CL or
CS, as it is easily found in CL- and CS-expressing cells, but is
undetectable in MEF9.C2 cells. Two peptides appear to be destroyed by
either CL or CS, as they are found in MEF9.C2 cells, but are
undetectable in CL-expressing (m = 2114.4) or
CS-expressing (m = 2052.9) cells. The patterns we see
by mass spectroscopy correlate with the T hybridoma presentation
assays, as presentation of some Ags are augmented and others diminished
(Fig. 3). Therefore, although the bulk of peptides can be generated
without CL or CS, these proteases appear to play an important role in
the processing of a subset of Ags, shaping the repertoire of class
II-associated peptides.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this paper we show that the presence of CL or CS is not required for the generation of most epitopes, although CL or CS can affect the level of presentation, presumably through their effects on Ii processing. However, CL or CS can strongly affect the generation of a subset of antigenic epitopes in both a positive and a negative fashion, suggesting a direct role for these proteases in Ag processing. This would support the idea that Ag processing within the endosomal compartment is the action of multiple proteases with overlapping, but not entirely redundant, specificities.
The use of embryonic fibroblast lines in this study allowed us to
directly compare the effects of CL and CS in the same cell type. If we
had used ex vivo-derived APCs we would have been restricted to
macrophages, as they are the only cell type that expresses both CL and
CS. However, macrophages are a heterogeneous cell population and may
have diverse levels of cathepsin and class II expression. Variability
in cathepsin expression in these cells is also enhanced by exposure to
cytokines (C. Beers and A. Rudensky, manuscript in preparation)
(34). Furthermore, it is likely that IFN-
-induced
macrophages express less mature CL compared with cortical thymic
epithelial cells, as suggested by a defect in Ii processing in
CS-deficient macrophages despite the expression of CL (C. Beers and A.
Rudensky, manuscript in preparation) (7, 8).
We found that CL- and CS-expressing fibroblast cell lines process Ii and present Ag in a manner consistent with previous studies using ex vivo-isolated APCs (8). For example, IgM presentation is diminished by the presence of CS, in agreement with our previous report (8). Analysis of the peptides eluted from class II by mass spectroscopy showed that, as observed in the functional studies with T cell hybridomas, certain peptides are enhanced/generated in the presence of CL or CS, whereas other peptides are diminished/eliminated. Therefore, these data suggest a direct role of CL and CS in the processing of a subset of Ags.
It is possible, however, that CL or CS may not directly affect Ag
processing. Rather, by accelerating SLIP degradation, these proteases
may facilitate class II loading in a different compartment with a
potentially different peptide pool. If this were the case, we would
expect the effects of CL and CS on Ag presentation to be concordant, as
suggested by the similar effects of CL and CS on Ii processing and
class II maturation revealed by both biochemical analysis and
microscopy (Figs. 2 and 3). Thus, both CL and CS should enhance or
diminish the presentation of a particular peptide. In general, this
appears to be true, as the expression of 11 of 24 peptides is affected
concordantly by CL and CS, e.g., within 2-fold of each other (Fig. 6, lower panel). This could therefore be due to the effects of
CL and CS on either Ii or Ag processing. However, 13 peptides are
discordantly affected by CL or CS. For example, the relative levels of
peptides 2434, 1987.9, 2493.4, 1804.8, 1669.5, 1756.5. 2052.9, 2349.2,
and 2332 are significantly diminished by CS expression compared with
CL. In contrast, peptide 2114.4 is rendered undetectable by the
expression of CL but is only mildly diminished by CS. Similarly, other
peptides (m = 1359.8, 1845.4, and 1698.6) are enhanced
by CS disproportionately. This argues that CL and CS play an important
role in the processing of a subset of Ags.
The most straightforward interpretation of the roles of CL and CS in Ag processing is that they directly cleave proteins, for which there is good in vitro evidence (32, 33). However, we cannot formally exclude the possibility that CL or CS affects the processing of protein Ags indirectly, e.g., by activating or inhibiting other proteases. Identification of other proteases involved in Ag processing will be required to evaluate this possibility.
The finding that these cathepsins selectively affect a subset of peptide epitopes suggests that the roles of other cathepsins, e.g., CB and CD, should be reevaluated. Previous testing was based on the presentation of several specific Ags (12, 13). As the nature of the Ags that require processing by individual cathepsins is not known, a more global approach may be more appropriate, e.g., mass spectroscopic analysis of class II-bound peptides from CB- or CD-deficient cells.
Our data indicating a role for CL and CS in Ag processing are consistent with several other observations. For example, H-2q CS-deficient mice are resistant to collagen-induced arthritis. In contrast to I-Ab mice, CS deficiency in I-Aq mice does not result in significant Ii fragment accumulation or delayed class II maturation (8). Nevertheless, CS deficiency inhibits the presentation of collagen II to a T cell hybridoma, suggesting a role for CS in processing collagen II rather than Ii. Secondly, CL x Ii double-knockout mice show a substantial additive defect in positive selection compared with Ii or CL deficiency alone (K. Honey and A. Rudensky, manuscript in preparation). This suggests an Ii-independent effect on positive selection by CL, presumably through diminished generation of MHC class II-bound peptides on thymic epithelial cells.
Taken together, these data suggest a potential new role for CL and CS in Ag presentation. Much of the previous work has focused on the roles of these proteases in Ag presentation and Ii processing in the I-Ab haplotype (7, 8, 9). However, the Ii-degrading function of CL and CS may not be necessary for efficient class II maturation in many other haplotypes, such as I-Ak, I-As, I-Aq, and I-Ag7 (C. Hsieh, unpublished observations) (8, 12, 35), where SLIP can easily dissociate from MHC class II molecules. Thus, CL and CS may be used by the immune system to broaden the array of epitopes generated during Ag processing, with Ii processing being a fortuitous coincidence. Further definition of the specificity of these enzymes may facilitate a greater understanding of their role in Ag processing and the particular immune responses in which they participate.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Alexander Y. Rudensky, Howard Hughes Medical Institute, University of Washington, Room I604J, 1959 NE Pacific Street, Seattle, WA 98195. E-mail address: aruden{at}u.washington.edu
3 Abbreviations used in this paper: Ii, invariant chain; CB, cathepsin B; CD, cathepsin D; CS, cathepsin S; CL, cathepsin L; CIITA, class II trans-activator; CLIP, class II-associated leupeptin-induced peptide; GFP, green fluorescent protein; HEL, hen egg lysozyme; KLH, keyhole limpet hemocyanin; SLIP, small leupeptin-induced peptide; LAMP, lysosome-associated membrane protein; m, molecular mass.
Received for publication October 19, 2001. Accepted for publication January 10, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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receptors. Eur. J. Immunol. 31:1592.[Medline]
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 Y. A. Bulynko, L. C. Hsing, R. W. Mason, D. J. Tremethick, and S. A. Grigoryev Cathepsin L stabilizes the histone modification landscape on the y chromosome and pericentromeric heterochromatin. Mol. Cell. Biol., June 1, 2006; 26(11): 4172 - 4184. [Abstract] [Full Text] [PDF]
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 R. M Nepal, S. Mampe, B. Shaffer, A. H Erickson, and P. Bryant Cathepsin L maturation and activity is impaired in macrophages harboring M. avium and M. tuberculosis Int. Immunol., June 1, 2006; 18(6): 931 - 939. [Abstract] [Full Text] [PDF]
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I-C. Huang, B. J. Bosch, F. Li, W. Li, K. H. Lee, S. Ghiran, N. Vasilieva, T. S. Dermody, S. C. Harrison, P. R. Dormitzer, et al. SARS Coronavirus, but Not Human Coronavirus NL63, Utilizes Cathepsin L to Infect ACE2-expressing Cells J. Biol. Chem., February 10, 2006; 281(6): 3198 - 3203. [Abstract] [Full Text] [PDF]
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Y. Wang, R. M. Baron, G. Zhu, M. Joo, J. W. Christman, E. S. Silverman, M. A. Perrella, R. J. Riese, and M. Cernadas PU.1 Regulates Cathepsin S Expression in Professional APCs J. Immunol., January 1, 2006; 176(1): 275 - 283. [Abstract] [Full Text] [PDF]
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 T. Zavasnik-Bergant, U. Repnik, A. Schweiger, R. Romih, M. Jeras, V. Turk, and J. Kos Differentiation- and maturation-dependent content, localization, and secretion of cystatin C in human dendritic cells J. Leukoc. Biol., July 1, 2005; 78(1): 122 - 134. [Abstract] [Full Text] [PDF]
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 E. J. Beswick, D. A. Bland, G. Suarez, C. A. Barrera, X. Fan, and V. E. Reyes Helicobacter pylori Binds to CD74 on Gastric Epithelial Cells and Stimulates Interleukin-8 Production Infect. Immun., May 1, 2005; 73(5): 2736 - 2743. [Abstract] [Full Text] [PDF]
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B. M. Chain, P. Free, P. Medd, C. Swetman, A. B. Tabor, and N. Terrazzini The Expression and Function of Cathepsin E in Dendritic Cells J. Immunol., February 15, 2005; 174(4): 1791 - 1800. [Abstract] [Full Text] [PDF]
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C. Beers, A. Burich, M. J. Kleijmeer, J. M. Griffith, P. Wong, and A. Y. Rudensky Cathepsin S Controls MHC Class II-Mediated Antigen Presentation by Epithelial Cells In Vivo J. Immunol., February 1, 2005; 174(3): 1205 - 1212. [Abstract] [Full Text] [PDF]
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H. Yang, M. Kala, B. G. Scott, E. Goluszko, H. A. Chapman, and P. Christadoss Cathepsin S Is Required for Murine Autoimmune Myasthenia Gravis Pathogenesis J. Immunol., February 1, 2005; 174(3): 1729 - 1737. [Abstract] [Full Text] [PDF]
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R. Thai, G. Moine, M. Desmadril, D. Servent, J.-L. Tarride, A. Menez, and M. Leonetti Antigen Stability Controls Antigen Presentation J. Biol. Chem., November 26, 2004; 279(48): 50257 - 50266. [Abstract] [Full Text] [PDF]
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