(Received for publication, August 26, 1994; and in revised form, October 19, 1994)
From the
Major histocompatibility complex (MHC) class I and class II
molecules have been shown to present peptides of different origin to
T cells. Most peptides presented by class I molecules are
derived from endogenously synthesized proteins, whereas most peptides
presented by class II molecules are from exogenous sources. This
functional dichotomy can largely be achieved by the preferential
intracellular association of the invariant chain (Ii) with MHC class II
molecules, which may inhibit binding of endogenous peptides to class II
molecules and direct them to endocytic compartments where
extracellularly derived peptides can be sampled. Here, we show that Ii
also can associate with a subset of MHC class I molecules and direct
them to endocytic compartments. Ii was coprecipitated with class I
molecules after lysis of human lymphocytes in mild detergent such as 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic
acid or digitonin, and the association was more clearly visualized by
the use of dithiobis[succinimidylpropionate], a
homobifunctional chemical cross-linker. The class I
Ii complex was
reconstituted in Ii negative cells by transfection of corresponding
cDNA clones and was found to be transported through the Golgi to acidic
endocytic compartments. These observations may explain how some
exogenous antigens can be presented by MHC class I molecules and how
MHC class II molecules can bind self peptides derived from MHC class I
molecules in endocytic compartments.
Major histocompatibility complex (MHC) ()class I and
class II molecules are integral membrane glycoproteins whose primary
function is to present bound antigenic peptides to T cells expressing
T cell receptor (1) . MHC class I molecules bind
peptides of intracellular origin, such as those derived from
endogenously synthesized viral proteins, and present them to
CD8
T cells, whereas MHC class II molecules bind
exogenous antigens and present them to CD4
T
cells(2, 3) . This functional difference is believed
to be explained by the capability of class II molecules to bind
intracellularly the invariant chain (Ii), which directs them to
endocytic vesicles where they encounter exogenously derived
peptides(4, 5, 6) .
Ii is a nonpolymorphic type II transmembrane glycoprotein that exists in humans in four forms, designated according to their molecular sizes as p31, p33, p41, and p43 (note that p31 and p33 are referred to as p33 and p35, respectively, by some authors)(7, 8) . These different forms of Ii are generated by the use of alternative translation initiation codons and alternative mRNA splicing(9) . Amino acid residues 12-15 (measured from the Ii p31 N-terminal cytoplasmic tail), which are shared among all the four forms of Ii, have been identified as critical residues for endosomal targeting(10) .
Assembly of MHC class
II and
chains with Ii occurs in the endoplasmic reticulum
(ER). Newly synthesized Ii associates as a trimer with
calnexin(11) , an ER resident molecular
chaperone(12, 13, 14) . Newly synthesized
class II
and
chains also bind to calnexin and dissociate
when a complete nonamer complex, containing three
dimers and
an Ii trimer, is formed(11) . Subsequently, the class
II
Ii complex exits the ER and is transported through the Golgi to
a specialized endocytic compartment, distinct from early/late endosomes
or dense lysosomes, where Ii is proteolytically cleaved and class II
molecules acquire antigenic peptides of exogenous
origin(15, 16, 17, 18, 19) .
Following dissociation of Ii and binding of peptides, class II
molecules are transported to the cell surface for recognition by
CD4
T cells(20) .
Newly synthesized class I
heavy chains also bind to calnexin during their biosynthesis and
assembly(21, 22) . We recently demonstrated in human
cells that, following association with m, class I
heavy chains dissociate from calnexin and subsequently bind peptides of
endogenous origin(22) . These peptides are transported from the
cytoplasm by TAP molecules, and their association with nascent class I
chains in the ER might be facilitated by physical association of the
class I heavy chain
m heterodimer with TAP
molecules (23) . Fully assembled class I complexes leave the ER
and are transported through the Golgi to the cell surface without
intersecting the endocytic route(24) . Segregation of class I
and class II takes place in the trans-Golgi reticulum, where class I
molecules traffic to the plasma membrane while class II molecules are
sorted to endocytic compartments(15) . Thus, the different
origin of peptides presented by class I and class II molecules may
reflect different intracellular pathways through which these molecules
traffic in the cell, which apparently result from the ability of Ii to
direct class II molecules away from the default secretory pathway by
virtue of endosomal targeting/retention
signals(25, 26, 27) .
Accumulating evidence has shown that the dichotomy in presentation of antigen from endogenous and exogenous origin is not absolute. Some endogenous antigens can be presented by class II molecules(28, 29, 30) , whereas exogenous antigens are in some cases presented by class I molecules(31, 32, 33, 34) . The exact mechanisms accounting for these observations are not well understood. In this paper, we show that Ii can associate with a subset of class I molecules and direct them into acidic endosomal compartments. This observation suggests a mechanism that could explain how exogenous antigens can be presented by class I molecules and how self class I-derived peptides gain access to endosomal compartments for binding to class II molecules.
Figure 1:
Association of a 33-kDa
protein with MHC class I molecules. T2 cells radiolabeled for 4 h with
[S]methionine were lysed in 0.3% CHAPS in the
absence (lanes1-6) or presence (lanes7-12) of 0.1 mM DSP. Immunoprecipitation
was performed with mAbs P3 (control), AF8 (anti-calnexin), L243 (anti-HLA-DR), BBM.1 (anti-
m), HC10 (anti-class I heavy chain
unassociated with
m) and W6/32 (anti-class I
heavy chain associated with
m) followed by resolution
on a 12% SDS-PAGE gel under reducing conditions. The positions of class
I heavy chain (HC),
m, and 33-kDa protein (arrow) are indicated to the right. Molecular markers
are shown to the left.
mAb PIN.1 is an anti-Ii antibody that was generated by immunization with a synthetic peptide corresponding to a sequence located in the cytoplasmic domain of all forms of Ii (residues 12-28, measured from the Ii p31 N terminus)(42) . Ii p31 is the most abundant form, and it was visualized prominently by immunoprecipitation with mAb PIN.1 (Fig. 2A, lane4). Importantly, the 33-kDa protein associated with class I (Fig. 2A, lanes2 and 3) or calnexin (lane1) comigrated with Ii p31. The identity of this 33-kDa protein with Ii p31 was confirmed by peptide mapping with V8 protease. The peptide map of either class I-associated or calnexin-associated 33-kDa protein was exactly the same as that of Ii p31 (Fig. 2B). Thus, we concluded that Ii p31 was associated with class I molecules in T2 cells.
Figure 2:
Association of Ii p31 with MHC class I
molecules in T2 cells. A, T2 cells radiolabeled for 4 h with
[S]methionine were lysed in 0.3% CHAPS with 0.1
mM DSP. Immunoprecipitation was carried out with mAbs AF8
(anti-calnexin), BBM.1 (anti-
2 m), HC10 (anti-class I heavy chain
unassociated with
2 m), and PIN.1 (anti-Ii), followed by
resolution on a 12% SDS-PAGE gel under reducing conditions. The
position of Ii p31 is indicated with an arrow. B, 33
kDa bands were cut out from each lane in A and subjected to
digestion with V8 protease followed by resolution on a 15% SDS-PAGE
gel.
To rule out the
possibility that this novel association might be achieved only in class
II negative, mutant T2 cells with impaired class I assembly, we
examined wild type T1 cells. Unlike T2 cells, T1 cells have normal
class I assembly, which was manifested by the appearance of abundant
assembled class I heavy chains detected with mAb W6/32 (Fig. 3, lane4) and fewer m-unassociated
heavy chains detected with mAb HC10 (lane3).
Importantly, mAb W6/32 coimmunoprecipitated a protein comigrating with
Ii p31 (Fig. 3, lane4, shown with an arrow). The identity of this class I-associated protein to Ii
p31 was confirmed by resolution on nonequilibrium pH gradient
electrophoresis/SDS-PAGE two-dimensional gels (not shown). This
association was also observed in a human B cell line, JY (data not
shown). Thus, we concluded that the association of Ii p31 with class I
molecules was not attributable to the abnormal class I assembly in T2
cells but was a more general phenomenon that also occurred in cells
with normal class I assembly and class II expression.
Figure 3:
Association of Ii p31 with MHC class I
molecules in T1 cells. T1 cells radiolabeled for 4 h with
[S]methionine were lysed in 0.3% CHAPS with 0.1
mM DSP. Immunoprecipitation was performed with mAbs UPC10 (control), BBM.1 (anti-
2 m), HC10 (anti-class I heavy chain unassociated with
2 m), W6/32 (anti-class I heavy chain associated with
2 m), and PIN.1 (anti-Ii), followed by resolution on a 12% SDS-PAGE gel under
reducing conditions. The position of Ii p31 is indicated with an arrow.
Figure 4:
Reconstitution of class IIi p31
complex in HeLa cells. Stable transfectant clones of HeLa expressing
either HLA-B27 alone (HeLaB27 mock) or both HLA-B27 and Ii p31
(HeLaB27Iip31) were radiolabeled for 20 min with
[
S]methionine and lysed in 0.3% CHAPS with 0.1
mM DSP. Immunoprecipitation was carried out with mAbs L243 (anti-HLA-DR), BBM.1 (anti-
2 m), HC10 (anti-class I heavy chain unassociated with
2 m), W6/32 (anti-class I heavy chain associated with
2 m), and PIN.1 (anti-Ii), followed by resolution on a 12%
SDS-PAGE gel under reducing conditions. The positions of class I heavy
chain (HC),
m, and Ii p31 (arrow)
are indicated to the right.
Figure 5:
Prolonged association of class I molecules
with Ii p31 in the presence of concanamycin B. HeLaB27Iip31 cells were
pulse labeled for 10 min with [S]methionine and
chased in cold media either in the presence or absence of 10 nM concanamycin B (ConB). A, at each
chase time, radiolabeled cells were lysed in 0.5% Triton X-100 in
Tris-buffered saline without DSP. Ii p31 was immunoprecipitated with
mAb PIN.1, and was subsequently subjected to digestion with endo H
(+) or mock digested(-). The samples were resolved on a 12%
SDS-PAGE gel under reducing conditions. Endo H resistant (R)
and sensitive (S) species are indicated to the right. B, at each chase time, radiolabeled cells were lysed in 0.3%
CHAPS with 0.1 mM DSP. Immunoprecipitation was performed with
mAb W6/32, specific for class I heavy chain associated with
m, followed by resolution on a 12% SDS-PAGE gel under
reducing conditions. The positions of class I heavy chain (HC),
m, and Ii p31 are shown to the right.
Figure 6:
Transport of the class IIi p31
complex to endocytic compartments. A, fixed and permeabilized
HeLaB27Iip31 cells were first stained with the anti-Ii PIN.1 antibody,
followed by rhodamine B-conjugated goat anti-mouse Ig antibodies. After
blocking free antigen binding sites of the goat antibodies, cells were
then incubated with FITC-conjugated ME.1 antibody specific for HLA-B27.
The background staining with mAb P3 (control) was negligible (not
shown). B, after incubation of HeLaB27Iip31 cells with Texas
red-conjugated ovalbumin, cells were fixed and permeabilized for
staining with mAb ME.1 followed by FITC-conjugated goat anti-mouse Ig
antibodies. Texas red was visualized through rhodamine filter
sets.
Using direct biochemical analyses of human lymphocytes (Fig. 1Fig. 2Fig. 3) and reconstitution by
transfection in HeLa cells (Fig. 4), we have demonstrated an
association between class I molecules and Ii. The association of class
II molecules with Ii is fairly strong and is maintained in detergents
such as 1% Triton X-100(11) . In contrast, the association of
class I molecules with Ii was too weak at least in vitro to be
maintained in 0.5% Triton X-100 (data not shown). We observed that a
small amount of Ii was coimmunoprecipitated with class I molecules in
milder detergents such as 0.3% CHAPS (Fig. 1) and 1% digitonin
(not shown), and we succeeded in visualizing the association more
clearly in a reproducible way by the use of low concentrations of the
chemical cross-linker DSP (Fig. 1). Previously, mouse class I
heavy chains were expressed artificially in human cells, and an
association with human Ii was reported(53) . Here, we
demonstrate the presence of class IIi complexes under
physiological conditions in untransfected human cell lines. Moreover,
we show that the class I
Ii complex remained assembled in vivo during transport from the ER, through the Golgi, to endocytic
compartments, which was evidenced by pulse-chase experiments (Fig. 5) and immunofluorescence microscopy analysis (Fig. 6). Thus, the association of Ii with class I molecules is
stable in vivo and results in sorting of this pool of class I
molecules out of the direct secretory pathway.
What is the
biological significance of this association? The colocalization of
class IIi complex with endocytosed ovalbumin leads one to
consider the possible involvement of this complex in presentation of
exogenous antigens by class I molecules. Several reports have shown
that exogenous antigens can be presented by class I molecules to T
cells both in vivo and in vitro(31, 32, 33, 34) . Recent
evidence has shown that cells capable of this presentation are
phagocytic macrophages, and indeed phagocytosis by these cells of
exogenous antigens, such as bacteria or ovalbumin linked to beads, and
transport of these antigens to endosomal compartments are critical for
antigen presentation(33, 34) . We speculate that Ii
may transport a subset of class I molecules, as well as class II
molecules, to endocytic compartments, where Ii is cleaved and class I
molecules acquire peptides derived from endocytosed exogenous antigens.
Although a recently identified compartment where peptide loading onto
class II molecules takes place does not contain class I molecules, a
small amount of class I molecules are found in endosomes in a human B
cell line, JY(15) , in which no internalization from the cell
surface is detected for class I molecules(24) . Further studies
are required to determine definitively the extent to which class I
molecules (colocalized with Ii and with class II molecules) are present
in endocytic compartments.
Another possible function of class
IIi complexes is to provide the source of self class I-derived
peptides for binding to class II molecules. Analysis of naturally
processed peptides bound to HLA-DR1 shows that self HLA-A2 derived
peptides as well as those derived from Ii are found in association with
DR1 molecules(54) , and indeed MHC class II molecules have been
shown to present endogenous class I-derived peptides to cytotoxic T
cells in a Brefeldin A-sensitive manner(29) . Given the limited
internalization of class I molecules from the cell surface, Ii-mediated
transport of class I molecules to endocytic compartments might be
crucial to the supply of certain self antigens (such as MHC class I
proteins) for presentation by class II molecules.
Finally, based on
these results and previous studies from our laboratory(22) , we
believe that calnexin may act as a molecular chaperone in the assembly
of class IIi complexes. Calnexin has been shown to play an
important role in the assembly of both class I and class II
molecules(11, 21, 22, 55) . In the
case of class II molecules, calnexin binds newly synthesized
,
and Ii chains and remains associated with assembling intermediate
complexes until the complete nonamer, containing three
dimers and the Ii trimer, is formed (11) . In the case of class
I assembly, we recently showed that newly synthesized class I heavy
chains bind to calnexin and that association with
m
triggers heavy chains to dissociate from calnexin(22) . Since
the interaction between class I heavy chains and Ii was also observed
in
m deficient Daudi cells (data not shown), we
speculate that calnexin may mediate association of a subset of class I
heavy chains with Ii, and that, following association with
m, class I heavy chain
m
Ii
complex may dissociate from calnexin. These findings potentially
indicate stronger parallels between the assembly of class II molecules
and a subset of class I molecules than previously have been recognized.