By
From the * Howard Hughes Medical Institute and the Department of Immunology, University of
Washington School of Medicine, Seattle, Washington 98195; and the § Department of Microbiology
and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
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Abstract |
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Efficient loading of major histocompatibility complex class II molecules with peptides requires
the invariant chain (Ii) and the class II-like molecule H-2M. Recent in vitro biochemical studies suggest that H2-M may function as a chaperone to rescue empty class II dimers. To test this
hypothesis in vivo, we generated mice lacking both Ii and H-2M (Ii/
M
/
). Antigen presenting cells (APCs) from Ii
/
M
/
mice, as compared with APCs from Ii
/
mice, exhibit a
significant reduction in their ability to present self-peptides to a panel of class II I-Ab-restricted
T cells. As a consequence of this defect in the loading of self peptides, CD4+ thymocyte development is profoundly impaired in Ii
/
M
/
mice, resulting in a peripheral CD4+ T cell population with low levels of T cell receptor expression. These findings are consistent with the
idea that H-2M functions as a chaperone in the peptide loading of class II molecules in vivo.
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Introduction |
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Newly synthesized MHC class II /
dimers are assembled in the endoplasmic reticulum by association
with invariant chain (Ii)1 and then transported into endocytic
vesicles (MHC class II peptide-loading compartment MIIC
or CIIV) with lysosomal and endosomal characteristics, where Ii is proteolytically degraded (for review see reference 1). The final products of Ii processing are short fragments originating from the 81-104 region of Ii, termed
CLIP (class II-associated invariant chain peptide), which
remain bound to the groove of class II molecules until they
are exchanged for self or foreign peptides (2). Mice lacking
Ii (Ii
/
) have significantly reduced expression of cell surface class II molecules, and have only small numbers of
CD4+ T cells in the thymus and periphery (3, 4). Ii
/
splenocytes are severely deficient in presentation of the majority of exogenous protein antigens yet are more amenable
to loading of cell surface class II molecules with exogenously supplied peptides. Biochemical experiments have
shown that in Ii
/
cells, class II molecules are associated
with 20-50-kD polypeptides (5). However, endogenous
peptide-class II complexes, discernible by T cells, have been
detected in Ii-negative fibroblasts (6) or in Ii
/
mice carrying a known epitope encoded by a transgene (7).
The class II-like molecule, H-2M (or HLA-DM in humans) uniquely resides in MIIC or CIIV vesicles (8), and
binds to class II molecules at the low pH of these acidic organelles (9, 10). Experiments with purified class II and
HLA-DM molecules have provided clues to potential functions of HLA-DM in vivo (for review see reference 11). In
these experiments, HLA-DM was shown to catalyze antigenic peptide loading by accelerating the dissociation of CLIP, to act as a peptide editor by removing those peptides
with high dissociation rates, and to prevent isolated empty
class II /
dimers from unfolding or aggregating at low
pH (10). The consequences of H-2M or HLA-DM deficiency include the predominant expression of CLIP-class
II complexes and profound defects in presentation of exogenous antigens (16). However, this defect is not absolute in that some peptides derived from endogenous or exogenous antigens have been shown to be displayed by class II molecules in the absence of HLA-DM or H-2M (20-
22). Although the abundance of CLIP-class II complexes
does not alter the level of surface class II expression, mice
lacking H-2M (M
/
) do have moderately reduced numbers of CD4+ T cells in the thymus and periphery, reflecting the altered peptide repertoire displayed by class II molecules.
To investigate the in vivo functions of H-2M other than
facilitating the removal of CLIP, we analyzed mutant mice
lacking both Ii and H-2M (Ii/
M
/
). A recent publication also describing these mice reported that maturation,
transport, and surface expression of class II molecules was
comparable in Ii
/
and Ii
/
M
/
splenocytes (23). Surprisingly enough in view of these data, CD4+ T cell development in Ii
/
M
/
mice was impaired profoundly compared to mice lacking either Ii or H-2M alone, a finding
not explained by the published analyses of the mice (23).
We hypothesized that the limited repertoire of class II-
bound self peptides displayed by Ii
/
cells, accounting for
CD4+ T cell development in Ii
/
mice, is dramatically reduced in Ii
/
M
/
mice because an Ii-independent H-2M
function is required for the presentation of the majority of
endogenous peptides. To assess the effect of H-2M on the
loading of high-affinity endogenous self peptides, we generated a novel panel of T cells specific for self peptide-I-Ab
complexes. We found that the presentation of the majority
of these self epitopes was significantly reduced in the doubly mutant Ii
/
M
/
APCs, relative to Ii
/
APCs. These
studies demonstrate that even in the absence of Ii, H-2M
can greatly enhance self peptide-class II complex formation, suggesting that it functions as a general chaperone in
the peptide loading process. In addition, our data suggest
that in the absence of Ii, surface class II molecules that internalize into or traffic through the endocytic pathway have
access to the variety of antigenic peptides that are present in
H-2M-containing early and late endocytic compartments.
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Materials and Methods |
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Animals.
Each of the mutant mice used in this study are of a mixed B6x129 (H-2b) background, which expresses the MHC class II molecule I-Ab. Thus, the phenotypes of IiFACS® Analyses.
Analyses of T cell surface markers and MHC class II were performed as previously described (20). Experiments to assess cell surface binding of exogenous peptides using synthetic ET Cell Assays.
T cell hybrids specific for I-Ab bound toSynthetic Peptides and Peptide Binding Studies.
Peptides were synthesized on an automated peptide synthesizer (Synergy 432; Applied Biosystems, Foster City, CA) using F-moc chemistry. The purity of peptides used was >90%. For binding assays, affinity purified I-Ab (500 pM) was incubated with titrated amounts of inhibitor peptides (300 nM-700 µM) and biotinylated E ![]() |
Results |
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Analyses of mice
lacking Ii have revealed a significant reduction in CD4+
thymocytes, 20-30% of wild-type numbers, suggesting that
efficient CD4+ thymic selection requires high levels of surface MHC class II molecules and/or the diverse array of
endogenous peptides that are displayed by thymic epithelial
cells in the presence of Ii. In contrast, in H-2M-deficient
mice, which display wild-type levels of class II molecules
predominantly occupied by a single peptide, CLIP, the
number of CD4+ thymocytes is significantly higher than in
Ii/
mice, ~30-50% of the number in wild-type mice.
To assess CD4+ T cell development in Ii/
M
/
mice,
thymocytes and splenic T cells were assessed by flow cytometry for expression of CD4, CD8, and
/
TCR. The
total number of thymocytes did not vary significantly in the
mice of various genotypes. Three electronic gates were used
to analyze the presence of CD4
CD8hiTCRhi (Fig. 1, box 1),
CD4+CD8
TCRhi (Fig. 1, box 2), or CD4+CD8loTCRlo
(Fig. 1, box 3) thymocyte populations isolated from mice
of five distinct genotypes (B6x129, Ii
/
, M
/
, Ii
/
M
/
,
and MHC-II
/
), shown as two parameter dot plots of
CD8 versus TCR-
/
in Fig. 1. The CD4+CD8loTCRlo
cells (box 3) represent a minor population in a wild-type
thymus (Fig. 1 A), which is more prominent in an Ii
/
thymus, concomitant with a marked decrease in CD4+
TCRhi SP thymocytes (Fig. 1 B, box 2). In contrast, the
M
/
thymus shows a 50% reduction in CD4+TCRhi single positive thymocytes (box 2) but no change in the intermediate (box 3) population (Fig. 1 D). Notably, the
Ii
/
M
/
thymus shows a dramatic reduction (approximately sixfold relative to the Ii
/
thymus) in the number
of CD4+TCRhi cells (Fig. 1 C), similar to that observed in
mice lacking class II molecules (MHC-II
/
; Fig. 1 E). In
both Ii
/
M
/
and MHC-II
/
mice, virtually all of the
CD4+ thymocytes display low levels of TCR and have incomplete downregulation of CD8 (box 3). Thus, the partial block in positive selection of CD4 SP thymocytes observed in Ii
/
thymi is exacerbated by the additional loss of
H-2M function in doubly mutant Ii
/
M
/
thymi.
|
Analyses of peripheral T cells showed that the number of
splenic CD4+ T cells in Ii/
M
/
mice were reduced relative to the number in Ii
/
or M
/
mice (data not shown).
The level of TCR-
/
displayed on the surface of peripheral CD4+ T cells in M
/
mice was identical to that of
wild-type mice (Fig. 1 F). In contrast, Ii
/
CD4+ T cells
existed as two populations which differed in levels of TCR
expression, one of which overlapped with wild-type T
cells. Remarkably, CD4+ T cells with normal TCR expression were not present in spleens of doubly mutant Ii
/
M
/
mice; the low level of TCR exhibited by all these
CD4+ T cells coincided with that of the CD4+TCRlo population in Ii
/
mice (Fig. 1 F). This low level of TCR expression was also observed on all CD4+ T cells present in
MHC-II
/
mice. The level of TCR-
/
displayed on
splenic CD8+ T cells from each of the mutant mice was
not different from that of wild-type T cells (Fig. 1 G). Thus,
Ii
/
M
/
mice have a nearly complete block in normal
CD4+ T cell development, akin to that observed in mice
lacking MHC class II molecules. These data led us to suggest that a diverse repertoire of endogenous peptides is
achieved via an Ii-independent H-2M function and must
be displayed by class II molecules in the thymus in order to
achieve efficient CD4+ T cell development. To test this
hypothesis, we next studied endogenous peptide:class II
complex formation in the double mutant mice.
The majority of I-Ab molecules that reach the cell surface
in the absence of Ii are associated with kinetically unstable
peptides or polypeptides that can be easily displaced by exogenous high-affinity peptides; in contrast, class II-bound
high-affinity peptides in wild-type cells cannot be displaced
(4). The surface expression of class II molecules on Ii/
and Ii
/
M
/
splenocytes was reduced comparably about
fivefold relative to wild-type levels, as assessed by the I-Ab-
specific mAb Y3P (Fig. 2 A). To demonstrate endogenous
peptide occupancy of these surface class II molecules, wild-type and mutant cells were loaded with 30 µM of the
E
52-68 peptide and expression of the resulting E
52-68-
I-Ab complexes was assessed with the complex-specific
mAb YAe (reference 24; Fig. 2, B and C). M
/
cells did
not bind mAb YAe after incubation with E
peptide. Relative to wild-type cells, Ii
/
cells showed a threefold increase in binding of mAb YAe. Significantly, the doubly
mutant Ii
/
M
/
splenocytes showed a further modest increase in mAb YAe binding. Since the fraction of class II
molecules containing high-affinity peptides in Ii
/
splenocytes is already small, resulting in very efficient loading of
surface class II molecules with exogenous E
52-68, it may
be difficult to observe a further large effect of H-2M deficiency on exogenous peptide loading. Nonetheless, these
data suggest that in the absence of both Ii and H-2M, even
fewer self-peptides are stably loaded onto class II molecules.
|
To investigate whether
H-2M acts to facilitate self-peptide binding in the absence
of CLIP, we assessed the ability of Ii/
M
/
splenocytes
to form particular endogenous self-peptide-I-Ab complexes.
Sequences of naturally processed self-peptides bound to
I-Ab molecules in B cells or macrophages, corresponding to
portions of both membrane-bound and cytosolic proteins,
were recently identified in our laboratory using tandem
mass spectrometry (Dongre, A., S. Kovats, P. deRoos, and
A.Y. Rudensky, manuscript in preparation). The peptide
epitopes studied in this work include those derived from
membrane-bound proteins IgM (377-392),
2m (48-58),
LDLr (486-501), and cytosolic proteins actin (163-177),
Clp36 (138-153), and AAT (394-410). Synthetic peptides of
identical amino acid sequences to those eluted from class II
molecules were found to have a high affinity for purified
I-Ab in in vitro binding assays (Fig. 3), comparable to that
of the E
52-68 peptide, a known high-affinity binder (24).
These peptides were used to generate antigen-specific T
cell hybrids upon immunization of H-2M
/
mice (LDLr,
actin, AAT, Clp36) P. Dongre, S. Kovats, P. deRoos, and
A.Y. Rudensky, manuscript in preparation), or mice lacking the specific self-protein, e.g., IgM
/
or
2m
/
(20).
These T cell hybrids were specific for the immunizing peptide bound to I-Ab, as demonstrated by experiments in
which exogenous cognate peptide was added to M
/
splenocytes (Fig. 4, bar graphs). Irrelevant peptides added to M
/
APCs did not stimulate the T cell hybrids (data not
shown). The T cell hybrids also recognized endogenously
formed peptide-I-Ab complexes on wild-type splenocytes
(Fig. 4, line graphs).
|
|
A comparison of the ability of wild-type and mutant
APCs to form these endogenous peptide-I-Ab complexes
was obtained by incubation of variable numbers of ex vivo
isolated splenic APCs with a constant number of T cells in the absence of exogenously added antigen (Fig. 4, A-F).
The comparison of Ii/
and Ii
/
M
/
APCs allows direct assessment of the role of H-2M, since the surface class
II expression is equivalent on splenocytes from these two
types of mice (Fig. 2). Each of the six epitopes was efficiently presented by Ii
/
splenocytes (Fig. 4). The responses of T cells specific for IgM, actin, AAT, and Clp36
epitopes were reduced by approximately fivefold, whereas
the responses of T cells specific for
2m and LDLr epitopes were enhanced about twofold, relative to wild-type APCs.
These data indicate that these endogenous peptides can be
efficiently loaded in vivo onto class II molecules that traffic
through cellular compartments via an Ii-independent route.
In contrast, formation of the six distinct peptide-class II
complexes by doubly mutant Ii/
M
/
splenocytes was
much more variable, demonstrating differential requirements for H-2M function in the absence of CLIP. Presentation of the IgM377-392 epitope was profoundly diminished in the absence of both Ii and H-2M, a striking result
since the IgM377-392-I-Ab complex is expressed very well
in Ii
/
APCs (Fig. 4 A). Presentation of the
2m48-58
epitope was also significantly diminished in Ii
/
M
/
APCs,
(reduced ~10-fold relative to Ii
/
APCs; Fig. 4 B). As
previously described (20), these two epitopes were presented at undetectable or very low levels by M
/
splenocytes (Fig. 4, A and B). Presentation of peptides derived from the cytosolic antigens AAT and Clp36 by Ii
/
M
/
splenocytes was diminished 5-10-fold relative to Ii
/
APCs
(Fig. 4, E and F), while the already low level of presentation of the actin epitope by Ii
/
deficient cells was not
profoundly affected by the absence of H-2M (Fig. 4 D). Interestingly, formation of LDLr486-501-I-Ab complexes was
essentially equivalent in Ii
/
and Ii
/
M
/
cells (Fig. 4
C), and slightly enhanced relative to wild-type cells, suggesting that this peptide preferentially binds to class II molecules via a mechanism independent of both Ii and H-2M. As expected, since the T cells specific for LDLr, actin,
AAT, and Clp36 peptides were generated in H-2M-deficient mice, these peptides were not presented by M
/
splenocytes (Fig. 4, C-F).
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Discussion |
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In this report, we demonstrate that H-2M functions in
the in vivo loading of antigenic peptides onto class II molecules in a capacity that is distinguishable from its previously
described role in CLIP removal. Using a panel of T cells
specific for high-affinity endogenous self-peptides bound to
the class II molecule I-Ab, we found that for the majority of
epitopes normally expressed in the absence of Ii, additional
loss of H-2M function significantly reduced the ability of
APCs to assemble these self-peptide-class II complexes. Thus,
even in the absence of Ii, class II molecules traffic through
the endocytic vesicles in which H-2M facilitates the binding of antigenic peptides. As a consequence of this deficient loading of self-peptides, CD4+ thymocyte development is
profoundly impaired in Ii/
M
/
mice, resulting in a
peripheral CD4+ T cell population with uniformly low levels of TCR. Thus, we hypothesize that the normal diversity
of class II-bound endogenous peptides displayed on antigen
presenting cells, made possible by the disparate chaperone
functions of both Ii and H-2M, is required for development of a complete repertoire of CD4+ T cells.
Each of the endogenous epitopes we studied were presented well to T cells in the absence of Ii. With the exception of the response to the LDLr epitope, self-antigen-
specific T cell responses to Ii/
M
/
splenocytes were
reduced significantly relative to Ii
/
APCs, suggesting that
even in the absence of Ii, some endogenous peptide-class II
complexes are formed in H-2M containing lysosomal compartments. There are two ways in which Ii-free class II
molecules may enter the endocytic pathway and potentially
benefit from the activities of H-2M. Cell surface class II
molecules can internalize into early endocytic vesicles via
signals in the cytoplasmic tails of class II
and
molecules,
a pathway that is also functional in Ii-expressing cells (25,
26). Alternatively, a lysosomal targeting signal present at
residues 80-82 of the class II
chain may facilitate entry of
newly synthesized class II molecules into the endocytic
pathway in the absence of Ii (27). In view of this, the self-epitopes analyzed here may be generated efficiently and
bind to class II in early or late endocytic compartments accessed by class II molecules internalized from the cell surface or after diversion from the trans-Golgi network. Although some of the high affinity self-peptides studied here
were presented in the absence of Ii and H-2M, the dramatic reduction of mature CD4+ T cells in the Ii
/
M
/
mice indicates that the function of H-2M is generally required in vivo to promote the binding of most self-peptides
to class II molecules.
H-2M may significantly increase the class II-bound repertoire of endogenous peptides in Ii/
APCs by virtue of
its ability to stabilize empty class II molecules or its capacity
to mediate replacement of a limited set of unstable polypeptides with a larger repertoire of relatively stable peptides. Thus, it is perhaps unexpected that the absence of H-2M
did not decrease the steady-state level of I-Ab expression in
Ii
/
M
/
APCs. However, since the majority of class II
molecules in Ii
/
cells are unstably bound to low-affinity
polypeptides, the minor proportion of class II molecules
stably bound to high-affinity peptides in the presence of
H-2M may readily assemble with available polypeptides in
the absence of H-2M, thus maintaining an apparently similar level of surface class II expression. Alternatively, the absence of this minor population in Ii
/
M
/
APCs would
not significantly affect total surface class II expression as measured by flow cytometry.
Biochemical experiments have shown that empty class II
/
dimers are partially unfolded at pH 4.0-5.5 and tend
to form aggregates (28). HLA-DM is able to protect these
/
dimers from denaturation through a stable interaction
(10, 15). In vivo, such chaperone activity would maintain
more class II molecules in a peptide-receptive state in endocytic vesicles until they contacted antigenic peptides, eventually increasing the number of stable peptide-class II complexes at the cell surface. For the high affinity self-peptides
we studied, it is unlikely that the editing function of H-2M
was required to select for their stable binding. These peptides are able to efficiently displace low stability or affinity
peptides or polypeptides bound to surface class II molecules
on Ii
/
cells at neutral pH without the aid of H-2M (Fig.
2 and data not shown). Rather, H-2M may be primarily
enhancing their presentation by virtue of its ability to bind
to and prolong the half-life of empty class II
/
dimers at
acidic pH in vivo, as suggested by the in vitro studies.
The ability of a particular peptide to bind productively to class II molecules in the absence of H-2M may depend on its unique complement of amino acid side chains, or the location and stability of the antigenic fragment within the various endocytic vesicles. Thus, H-2M-independent peptide binding would occur if the on-rate of the binding reaction is very high, or if the half-life of an antigenic peptide is greater than the half-lives of empty class II molecules, a condition that might exist in the less proteolytic early endocytic compartments that contain little or no H-2M (29). In this regard, it is of interest to note that the LDL receptor protein contains a targeting signal for early endosomes, which may be the site where the H-2M- and Ii-independent epitope of LDLr is generated (32).
Observations with singly mutant Ii/
or M
/
mice
have indicated that efficient positive selection of CD4+ T
cells requires high levels of surface class II molecules and/or a diverse array of class II-bound endogenous peptides (3, 4, 17). Since the majority of surface class II molecules in
Ii
/
APCs are apparently associated with unstably bound
peptides or larger polypeptides, the profound impairment
in CD4+ T cell development in Ii
/
mice suggests that
this majority of class II molecules does not mediate positive
selection. Rather, CD4+ T cells in Ii
/
mice are likely to
be selected on the small minority of stable endogenous
peptide-class II complexes that do reach the cell surface, albeit with a contracted repertoire of high-affinity endogenous peptides. The absence of H-2M function in Ii-deficient cells is likely to further decrease the diversity of the
class II-bound repertoire and/or the number of functional
endogenous peptides, which may be replaced with unstably
bound polypeptides. In both of these scenarios, steady state
surface class II expression would be similar to that of Ii
/
cells. Although distinguishing between these two possibilities is key to understanding the requirements for positive
selection in these mice, we currently lack the means to quantitate the proportion of class II molecules occupied with
high affinity peptides in Ii
/
cells. Nevertheless, these reductions in the number or diversity of functional peptides
apparently puts their expression below a threshold level required for CD4+ positive selection in Ii
/
M
/
mice. In
view of our data, the impairment of CD4+ thymic selection in M
/
mice may be due to the dramatic reduction
in diversity of endogenous peptides bound to wild-type
levels of class II molecules, although this impairment may
be less marked than in Ii
/
mice due to the high density
of the single CLIP peptide-class II complex.
In summary, our findings are consistent with the idea
that H-2M evolved as the primary chaperone assisting in
the loading of antigenic peptides onto MHC class II molecules in the late endocytic compartment, presumably by promoting the stability of empty class II /
dimers. The facilitated dissociation of CLIP, and of other peptides that bind
to class II molecules with a high dissociation rate, may be
more specific manifestations of the general chaperone function of H-2M.
![]() |
Footnotes |
---|
Address correspondence to Dr. Alexander Rudensky, Howard Hughes Medical Institute, University of Washington, Box 357370, Seattle, WA 98195. Phone: 206-685-7644; Fax: 206-685-3612; E-mail: sasha{at}nucleus.immunol.washington.edu
Received for publication 18 September 1997 and in revised form 5 November 1997.
1 Abbreviations used in this paper: AAT, aspartate aminotransferase; CLIP, Class II associated invariant chain peptide; Ii, invariant chain; LDLr, LDL receptor; M, H-2M.We thank Deb Wilson for excellent animal care, and our colleagues for valuable discussions. We also thank
E. Bikoff for Ii/
mice.
This work was supported by National Institutes of Health grants to S.K. (NRSA AI-08903), C.E. Grubin (Predoctoral training grant), and A.Y. Rudensky (AI-34206). A.Y. Rudensky and L. Van Kaer are investigators of the Howard Hughes Medical Institute.
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