§
* Department of Cell Biology, School of Medicine and Institute of Biomembranes, Utrecht University, 3584 CX Utrecht, The
Netherlands; and Department of Immunology and § Howard Hughes Medical Institute, University of Washington, School of
Medicine, Seattle, Washington 98105
In most human and mouse antigen-presenting cells, the majority of intracellular major histocompatibility complex (MHC) class II molecules resides in late endocytic MHC class II compartments (MIICs), thought to function in antigen processing and peptide loading. However, in mouse A20 B cells, early endocytic class II-containing vesicles (CIIVs) have been reported to contain most of the intracellular MHC class II molecules and have also been implicated in formation of MHC class II-peptide complexes. To address this discrepancy, we have studied in great detail the endocytic pathways of both a human (6H5.DM) and a mouse (A20.Ab) B cell line. Using quantitative immunoelectron microscopy on cryosections of cells that had been pulse-chased with transferrin-HRP or BSA-gold as endocytic tracers, we have identified up to six endocytic subcompartments including an early MIIC type enriched in invariant chain, suggesting that it serves as an important entrance to the endocytic pathway for newly synthesized MHC class II/invariant chain complexes. In addition, early MIICs represented the earliest endocytic compartment containing MHC class II- peptide complexes, as shown by using an antibody against an abundant endogenous class II-peptide complex. The early MIIC exhibited several though not all of the characteristics reported for the CIIV and was situated just downstream of early endosomes. We have not encountered any special class II-containing endocytic structures besides those normally present in nonantigen-presenting cells. Our results therefore suggest that B cells use conventional endocytic compartments rather than having developed a unique compartment to accomplish MHC class II presentation.
MAJOR histocompatibility complex (MHC)1 class II
molecules expressed at the surface of antigen-presenting cells (APCs) such as B lymphocytes,
dendritic cells, and macrophages present bound peptides
derived from exogenous antigens to CD4-positive T lymphocytes. The antigens are internalized by fluid-phase-
and receptor-mediated endocytosis or by phagocytosis and
are proteolytically cleaved in endocytic compartments to
reveal small fragments that can associate with the binding
groove of MHC class II molecules (for reviews see 9, 14, 62). Peptide binding occurs predominantly to newly synthesized rather than to surface-derived class II molecules (3, 10, 34). Delivery of class II molecules from the biosynthetic to the endocytic pathway requires their association
with invariant chain (I-chain; 9, 56). Class II/I-chain complexes are thought to be transported from the TGN to late
endocytic compartments (10, 32, 34, 36, 37, 60) or (in addition) to earlier endosomes either directly or via the plasma
membrane (6, 8, 26, 48, 58). Upon arrival in endosomes,
the I-chain is rapidly degraded starting from the luminal
COOH terminus (47). A portion of the I-chain, CLIP
(class II-associated I-chain peptides) remains transiently
bound in the class II groove until it is exchanged for antigenic peptide, a process that is facilitated by the class II-
related molecule human leukocyte antigen (HLA)-DM
(H2-M in mice; 11, 45). It is not precisely known at which
site(s) in the endocytic pathway peptide binding takes
place. Peptide-loaded class II molecules are transported to
the plasma membrane via largely unknown pathways (59),
one of which is by exocytosis of MHC class II-enriched
compartments (MIICs; 43, 64). Thus, there is abundant evidence for the crucial role of the endocytic pathway in
class II antigen presentation.
At steady state, APCs harbor a major pool of their intracellular class II molecules in endocytic compartments,
morphologically reminiscent of multivesicular late endosomes and lysosomes and which are collectively called MIICs
(20, 34, 35, 37). Recently, several subtypes of MIICs have
morphologically been distinguished, including those containing internal vesicles and membrane sheets, termed multivesicular and multilaminar MIICs, respectively (38). In a
recent study on human B cells, an MIIC subtype was identified containing abundant I-chain and only a few vesicles.
This so-called early MIIC was proposed to be a precursor compartment of the classical MIICs (17). Cell fractionation and immunoelectron microscopy (IEM) suggested that these
early MIICs represent the main entry site of newly synthesized class II molecules into the endocytic pathway (17,
38). These results were broadly consistent with those of
Castellino and Germain (6), who showed that class II/I-
chain complexes enter early endocytic compartments. Experiments with exogenous tracers have shown that multivesicular MIICs are positioned earlier in the endocytic
pathway than multilaminar MIICs and gradually mature
into the latter type (24). It is not yet clear how MIICs relate to classical endocytic compartments present in non-APCs and whether APCs have developed special endocytic structures dedicated to class II functioning besides
other endosomes (53, 65). A detailed morphological description of the entire endocytic pathway in an APC and
the distribution of class II herein could help answer these
questions.
In non-APCs, at least four types of endocytic structures
have been distinguished on the basis of endocytic kinetics
and differential distribution of marker proteins, i.e., primary endocytic vesicles, early endosomes (EEs), late endosomes (LEs), and lysosomes (for review see 29). However, due to the plasticity of the endocytic system, no clear
separation between EEs, LEs, and lysosomes can be made.
Nevertheless, several markers have proven to be useful to
make operational distinctions. Thus, the presence of the
transferrin (Tf) receptor and internalized Tf is commonly
used to identify EEs, while the cation-independent and
-dependent (CD) mannose-6-phosphate receptors (MPRs),
involved in the delivery of newly synthesized lysosomal
enzymes to the endocytic pathways, are widely used as discriminative markers present in LEs but absent from lysosomes (16, 19, 25). Lysosomes can also be recognized by
the presence of lysosomal enzymes and lysosome-associated membrane proteins (LAMPS; for review see 21).
However, many cell types show hardly any MPRs in endocytic structures by IEM, while LAMPS and lysosomal enzymes are present in typical dense lysosomes as well as
in LEs (16, 25). Both with IEM and cell fractionation, MIICs
do not contain significant amounts of TfRs but are enriched in lysosomal enzymes and LAMPs and therefore
are categorized as late endocytic or (pre)lysosomal structures (37, 41, 57, 60).
In B cells and macrophages, MIICs have been suggested
to represent peptide-loading compartments (20, 31, 41, 51,
60, 65). In accordance with this function, MIICs contain
high levels of HLA-DM molecules (35, 39, 52). However,
several other studies have shown that class II molecules
can already acquire peptide in early endocytic structures
(6, 13, 18). Using free-flow electrophoresis of mouse A20
B lymphoma cells, Amigorena and coworkers (1, 2) and
Pierre et al. (39) have described a distinct class II peptide-loading compartment named class II-containing vesicle
(CIIV), which was thought to be different from classical endosomes. The relation between CIIVs and MIICs is not
yet clear. The present study presents a detailed IEM characterization of the endocytic pathways in a human B cell
line and in the murine A20 cells and addresses the question of whether these APCs contain specialized and/or conventional class II-containing endocytic compartments. Using
quantitative immunogold labeling and a recently improved
cryosectioning technique (28), we studied the progression of internalized tracers through the endocytic pathway and
subdivided the endocytic pathway into up to six subcompartments. Class II molecules distributed to all types of endocytic compartments downstream of the main Tf-containing EEs in both human and mouse B cells. Using an
antibody selectively recognizing an abundant class II-peptide complex (43, 50), we found that early MIICs seen just
downstream of EEs represent the earliest endocytic structures exhibiting class II-peptide complexes. Our observations do not support the existence of a distinct set of endocytic organelles specialized in class II functioning.
Cells
The human 6H5.DM cell line was obtained by transfection of the human
B-LCL cell line Sweig with I-Ab Immunoelectron Microscopy
Cells were prepared for ultrathin cryosectioning and immunogold labeled
as described (15, 54). Briefly, cells were fixed in 2% paraformaldehyde
and washed in PBS and PBS/0.02 M glycine. Cell pellets were embedded
in 7.5% gelatin, which was solidified on ice and cut into small blocks. After infiltration with 2.3 M sucrose for 3 h at 4°C, the blocks were mounted
and frozen in liquid nitrogen. Ultrathin cryosections were embedded in a
mixture of sucrose and methyl cellulose according to a novel procedure allowing a better visualization of membranes (28). Ultrathin cryosections
were single immunolabeled with 10-nm gold particles or double immunolabeled with 5- and 10-nm or 10- and 15-nm protein A-gold particles.
Antibodies
For IEM we used rabbit polyclonal antisera specific for human HLA-DR
Internalization of Tf-HRP and BSA-Gold
Tf was coupled to HRP as described (55). Tf-HRP was added to 6H5.DM
or A20.Ab cells at a concentration of 25 µg/ml for 3 or 10 min at 37°C. The
cells were washed in cold medium and fixed as described above. BSA conjugated to 5-nm colloidal gold particles (54) was used in pulse-chase experiments to label endocytic compartments. Cells were either incubated
with BSA-gold for 3 or 10 min at 37°C, washed extensively in cold medium, and fixed (as described above), or incubated for 10 min at 37°C,
washed extensively, chased for 20 or 50 min at 37°C, and then fixed. Ultrathin cryosections of cells incubated with Tf-HRP, were immunolabeled
with anti-HRP antibody and 10-nm gold particles. Of both 3 and 10 min
time points, 20 cell profiles were randomly selected in the cryosections,
and gold particles were assigned to the various types of endocytic compartments (see Results and Tables I and II). For each compartment, the
percentage of the total immunogold was calculated. The same was done
with 5-nm BSA-gold particles internalized after 3- and 10-min pulse, and
10-min pulse plus 20- or 50-min chase. In the same sections, the contribution of each of the types of endocytic compartments to the total number of
endocytic compartments per cell profile was determined. In 6H5.DM and
A20.Ab cells six and five, respectively, different types of endocytic compartments were described morphologically and ordered according to their
position in the endocytic pathway (see Results). The distinctive morphological characteristics of each of these compartments were then used to
study the distribution of markers of interest. Of each immunolabeling, 20 cell profiles were evaluated quantitatively. Gold particles were assigned to
a compartment when lying within a distance of twice the particle size from
the membrane. Gold particles present on other, nonendocytic organelles
were not taken into account. Background labeling using an irrelevant antibody was negligible.
Table I.
Quantitative Analysis of Tf-HRP and BSA-Gold
Uptake in 6H5.DM Cells
Table II.
Quantitative Analysis of Tf-HRP and BSA-Gold
Uptake in A20.Ab Cells
Materials and Methods
and
genes and HLA-DM genes as described before (43). The mouse lymphoblastoid cell line A20 was transfected with the expression constructs containing cDNA encoding I-Ab
- and
-chains driven by the human
actin promotor and neomycin- and
hygromycin-resistance genes. The expression constructs were identical to
those used to obtain 6H5.DM cells. 48 h after electroporation, the cells
were subjected to selection in culture medium containing 3 mg/ml G418
(GIBCO BRL, Gaithersburg, MD) and 1.2 mg/ml hygromycin B (Boehringer Mannheim, Indianapolis, IN). Stable transfectants (A20.Ab) were
cloned by limiting dilution and screening for surface expression of I-Ab
molecules by flow cytometry using mAb Y3P (12). Both cell lines were
maintained in RPMI 1640 supplemented with 5% FCS, 200 mM L-glutamine,
100 U/ml penicillin and streptomycin, 10 mM Hepes, 1 mM sodium pyruvate, and 5 x 10
5 M
-mercaptoethanol (all from GIBCO BRL) in 5%
CO2 atmosphere at 37°C (sRPMI).
-chain (34), human I-chain NH2 and COOH terminus (11), human HLA-DM
(FS2; 52), human LAMP-1 (61), human CD-MPR (MSC1; kind gift
of Dr. K. von Figura, Georg-August University, Göttingen, Germany; 25),
mouse H-2Mb (K553 in biotinylated form; kind gift of Dr. L. Karlsson,
Johnson Pharmaceutical Research Institute, Scripps Research Institute,
La Jolla, CA,), mouse LAMP-1 (7), HRP (p7899; Sigma Chemical Co, St.
Louis, MO), mouse I-chain luminal part (S22; kind gift of Dr. N. Koch,
University of Bonn, Bonn, Germany; 27), and biotin (Rockland, Gilbertsville, PA). In addition, we used a rat mAb specific for the cytoplasmic tail
of mouse I-chain (In-1; 26), mouse mAbs specific for mouse cathepsin D
(generous gift of Dr. K. von Figura), I-Ab/d/I-E (4), and for E
(DR
)52-68 peptide bound to I-Ab called YAe (12).
Since no detailed morphological characterization of the endocytic system in B lymphocytes has been described so far, we have first analyzed the endocytic compartments in the human B cell line 6H5.DM and the mouse B cell line A20.Ab. The A20 cell line has been used extensively in biochemical fractionation studies (1, 2, 39) but has not been studied morphologically in great detail. We have used Tf conjugated to HRP as a receptor ligand or 5-nm BSA-gold particles as fluid-phase endocytic tracer to study the progression of the internalized tracers through the endocytic pathway in thawed cryosections of 6H5.DM and A20.Ab cells. We took advantage of the improved cryosection morphology (28), which enabled us to subdivide the endocytic pathway into a series of compartments according to morphological and endocytic characteristics. Next, we studied the distribution of a variety of markers of the class II pathway in these compartments by IEM.
Kinetics of Tracer Uptake in 6H5.DM Cells
Examination of ultrathin cryosections of 6H5.DM cells
pulsed with either Tf-HRP or with 5-nm BSA-gold particles
for 3 or 10 min, or pulsed for 10 min and chased for 20 or
50 min, revealed six types of endocytic compartments. Each
of the compartments displayed distinct morphological features (Figs. 1 and 2; see also the schematic representation
of the various compartments in Fig. 3 and Table I). Fig. 1
shows several compartments in cells that had taken up
Tf-HRP (Fig. 1, A-D) or BSA-gold (Fig. 1, E and F).
Tf-HRP, detected by immunogold labeling, was found primarily in small clathrin- and nonclathrin-coated vesicles
and tubules, which together were termed endocytic compartment type 1 (Fig. 1, A and B; see numbering on the figures). Additional compartments displaying Tf-HRP showed
an extended straight or curved tubular morphology with
occasional internal vesicles (type 2; Fig. 1, C and D) or
were typically irregularly shaped, with a few internal vesicles (type 3; Fig. 1, B and D). Type 2 and 3 compartments
could also easily be recognized by an electron-lucent area
inside the curvature of the compartment (Figs. 1, C and 2, A and B). Tf-HRP was not found in compartments other
than types 1-3. After 3 min of uptake, the majority of
BSA-gold particles was detected in types 1 and 2 (not
shown) and to some extent in type 3 compartments (Fig. 1
E). Type 4, 5, and 6 compartments showed barely any
BSA-gold at 10 min (Fig. 1 F) but were reached by BSA-gold after prolonged time periods of uptake (30-60 min, not shown).
For a quantitative evaluation of endocytic tracers present in each of the six morphologically distinguishable subcompartments, Tf-HRP immunogold labeling and BSA-gold particles were counted in 20 random cell profiles at each uptake time (Table I). After 3 min, 73% of the Tf-HRP was detected in type 1, 22% in type 2, and 5% in type 3 compartments. The distribution of Tf-HRP was not significantly different after 10 min of uptake. This result indicates that Tf-HRP is not transported much farther into the endocytic pathway and marks the steady-state recycling pathway of the Tf receptor. We conclude that type 1 and 2 compartments are the classical EEs in these cells.
Analysis of BSA-gold uptake revealed that immediately after 3 and 10 min about one third of the total internalized tracer was present in type 3 compartments. The labeling further increased after 10 plus 20 min, with a simultaneous decrease in types 1 and 2. This indicates that type 3 compartments were positioned downstream of types 1 and 2. At 20 and 50 min chase, type 4, 5, and 6 compartments were sequentially reached by BSA-gold, indicating that these compartments were situated in this order later in the endocytic pathway. Type 4 compartments contained internal vesicles reminiscent of classical multivesicular endosomes in most cell types, type 6 displayed internal membrane sheets, whereas type 5 had an intermediate morphology. Types 4, 5, and 6 did not contain detectable internalized Tf-HRP at any time, which positioned them downstream of recycling EEs. Type 4, 5, and 6 compartments represented MIICs (see below) as defined originally (37) and together comprise the late endocytic compartments (Table I). Both tracer kinetics and morphology indicated that type 3 compartments represent a transitional stage between the early type 2 and the later type 4 compartments.
Quantitative Distribution of Class II, I-Chain, HLA-DM, and YAe in 6H5.DM Cells
Human 6H5.DM cells displayed several useful features for
a study on class II distribution. First, they expressed I-Ab
molecules, which are able to bind peptide derived from the
endogenous DR -chain. These DR
52-68/I-Ab peptide
complexes are recognized by mAb YAe (33), which can be
used in IEM. In addition, the high level of HLA-DM expression caused an increased number of peptide-loaded
class II molecules (42 and M.J. Kleijmeer, unpublished observations). Immunolabeling patterns for the various
markers of the class II pathway in 6H5.DM cells are illustrated in Figs. 1 and 2 and quantitatively represented in
Fig. 3 A. Both HLA-DR and I-Ab molecules were most
prominently present in type 4, 5, and 6 compartments (illustrated for DR in Fig. 2, A, B, and D, and for I-Ab in Fig.
1 F) and to a much lower extent in type 3 (Fig. 2 A). As in
previous studies, types 4, 5, and 6 MIICs contained
LAMP-1, as shown for a type 6 multilaminar MIIC in Fig.
2 A, and were devoid of MPR (Fig. 2 C). DR and I-Ab
were undetectable in type 1 (Fig. 1 A) and type 2 compartments and were present at low levels in type 3 compartments. In contrast, labeling for both the NH2 and COOH
terminus of I-chain was low or undetectable in types 5 and
6 but was high in types 3 and 4 (Figs. 1, D and E and 2 B).
A low but significant amount was also detectable in type 2 compartments (Figs. 1 C and 2 B). Almost 60% of the total COOH terminus labeling was contained in type 3 compartments. Type 3 compartments showed all of the characteristics of early MIICs as defined previously (17), being
small, irregularly shaped multivesicular structures, often
showing an electron lucent area enclosed in the curvature
of the compartment. In type 4, NH2 terminus labeling was
about the same as in type 3, but COOH terminus labeling
was less than half of that in type 3. This may reflect the
degradation of I-chain, which starts at the COOH terminus. As has been observed before (35, 52), DM was
strongly enriched in type 6 multilaminar MIICs (47%). Of
the total peptide-loaded I-Ab molecules (YAe), 11% was
present in type 3 whereas most YAe labeling was found in
type 5, the intermediate MIICs. YAe labeling was absent
from types 1 and 2. Thus, type 3/early MIICs were the earliest endocytic compartments showing YAe epitope (Fig. 2
D) and therefore may represent the earliest loading compartment, situated just downstream of the major Tf-containing EEs (types 1 and 2). Most of the YAe, however,
was found in later type 4, 5, and 6 compartments, which
also contained most of the HLA-DM and class II molecules (Fig. 2 C).
Kinetics of Tracer Uptake in A20.Ab Cells
By morphology, A20.Ab cells showed endocytic compartments very similar to those of 6H5.DM cells (Figs. 4 and
5). However, the typical multilaminar type 6 was not
prominently present, whereas the intermediate type 5 was
absent. In their place, a dense compartment with some internal membranes was found, which we indicated as type
5/6 (Figs. 4 E and 5, A and B). As in 6H5.DM cells, Tf-HRP was mainly found in types 1 and 2 and was absent
from the multivesicular type 4 (Fig. 4 C). Type 1, 2, and 3 compartments were reached by internalized BSA-gold
particles after 3 min of uptake (Fig. 4, A and B). Type 5/6
compartments were not reached by BSA-gold before 10 min of uptake but obtained BSA-gold only after 10-min pulse and 20- or 50-min chase (not shown). As in 6H5.DM
cells, type 2 and 3 often showed characteristic electron lucent areas (Figs. 4, C and D, and 5 C). Notably, A20.Ab
cells contained considerably fewer endocytic compartments
than 6H5.DM cells, i.e., 10 versus 20 per random cell profile, respectively.
Table II shows results of the quantitative evaluation of Tf-HRP and BSA-gold uptake in A20.Ab cells. The transport kinetics of Tf-HRP and BSA-gold were remarkably similar to those in 6H5.DM cells, with the exception that the later types 5/6 were more rapidly reached by BSA-gold, increasing from 10% immediately after 10-min pulse to 56% at 50-min chase. As in 6H5.DM cells, BSA-gold reached type 3 compartments immediately after 3 min of uptake (16%) and had increased to 38% after 10 plus 20 min. These data indicate that also in A20.Ab cells, types 1 through 5/6 were sequentially reached by BSA-gold and that types 1 and 2 represent EEs. Thus, as in the human cells, type 3 compartments displayed an intermediate position between the early types 1, 2, and the later 4 and 5/6 compartments.
Quantitative Distribution of Class II, I-Chain, and H-2M in A20.Ab Cells
Class II labeling was found in all types of endocytic compartments, except type 1. Fig. 3 B shows the quantitative
immunogold labeling data for class II, H-2M, and I-chain.
More than 50% of the total I-chain labeling was found in
type 3 compartments (illustrated in Fig. 6). I-chain was
also present in type 2 (Fig. 4 B) and type 4 and to a much
lower extent in type 5/6 (not illustrated). Of the total H2-M
labeling only 2% was present in type 2 (Fig. 4 E), 7% in
type 3, almost 30% in type 4, and 60% in type 5/6. Morphology, accessibility to exogenous tracers, and immunolabeling characteristics of type 3 MIICs were similar to those
in 6H5.DM. YAe labeling was too low for demonstration
with IEM. LAMP-1 was mainly present in type 5/6 compartments, similar to cathepsin D (Fig. 5 B). The type 5/6
compartments probably correspond to the late endocytic
MIICs in human B lymphocytes (17, 37, 38). Some LAMP-1
was detected in type 3 (Fig. 5 C). Approximately 60% of the
cathepsin D-positive endocytic compartments contained
class II. MPR labeling was absent from any of the type 1 through 5/6 compartments and was primarily found in adjacent small vesicles and tubules (Fig. 5 A).
In conclusion, both human and mouse B cells displayed multiple types of class II-containing endocytic MIICs with internal membranes, including the early type of MIIC. Early MIICs are located just downstream of the major recycling EEs, contain DM molecules and abundant I-chain, and are the first endocytic compartments displaying class II-peptide complexes as recognized by YAe antibody. In both cell types we have not found any special class II-containing endocytic compartment apparent from the classical ones described above.
This study expands on our previous work describing MIICs in B lymphoblasts, dendritic cells, and macrophages (for review see 24). In all of these cell types we have found late endocytic MIICs with similar morphological and molecular characteristics containing most of the intracellular class II molecules. However, over the years several reports have shown the transient presence of class II molecules in early endocytic compartments (1, 2, 6, 8, 13, 26, 48, 49, 58). The criteria used in the different studies to define early endocytic compartments varied, and their morphological identity was often not revealed. We have now studied for the first time in great detail the endocytic pathway of both mouse A20.Ab and human 6H5.DM B lymphoblasts, using semi-quantitative IEM.
Based on morphology, molecular marker distribution,
and position in the endocytic pathway, six and five types
of endocytic compartments could be distinguished in the
6H5.DM and A20.Ab cells, respectively (Tables I and II;
Fig. 7). Pulse-chase endocytosis of Tf-HRP and BSA-gold
indicated the position of these compartments in the endocytic pathway. Type 1, 2, and 3 compartments were situated early in the endocytic pathway. Types 1 and 2 represented the major Tf-recycling compartments and therefore
most likely were classical EEs in these cells. Type 3 had
the characteristics of a transitional compartment between
the types 1 and 2 and the later type 4 compartments. Abundance of I-chain, the presence of some Tf-HRP, rapid arrival of BSA-gold, irregular shape, and few internal vesicles characterized type 3 as early MIIC (17). As in our previous studies (for review see 24), most of the intracellular class II molecules were detected in type 4, 5, and 6 compartments, which represented LEs (type 4) and lysosomes
(types 5 and 6), because of their marker profiles and because they were reached by internalized BSA-gold in sequence and after types 1, 2, and 3. The morphology of the
multivesicular (type 4), intermediate (type 5), and multilaminar (type 6) as well as their late position in the endocytic pathway matched the characteristics of MIICs as
originally defined (37). Types 5, 5/6, and 6 are probably classical dense lysosomes. We have not encountered any
class II-positive endocytic structure distinct from the types
above. Fig. 7 shows the possible functional connections between the various types of compartments and how the
type numbers may correlate with existing nomenclature.
The labeling patterns for I-chain and YAe suggest that
type 3/early MIICs are of particular interest with respect to
class II function. First, early MIICs contained 40 and 55%
of the I-chain labeling in the human and mouse cells, respectively. In types 4, 5, and 6, I-chain labeling progressively decreased, most likely due to progressive proteolysis. The abundance of I-chain COOH terminus epitope in
early MIICs (17, 38, and this study) probably reflects both
class II-bound and -detached I-chain. The sequential COOH- to NH2-terminal degradation of I-chain likely accounts for the earlier disappearance of COOH terminus labeling than of NH2 terminus labeling. An attractive interpretation of the I-chain labeling pattern is that early MIICs
represent an important entry point of class II/I-chain molecules coming from the TGN. This would be in accordance
with our previous observations (17, 38) and with those of
Cresswell (8) and Castellino and Germain (6). However, in
both the 6H5.DM and A20.Ab cells a small but significant
fraction of I-chain was found in type 2 compartments. It is
therefore likely that class II/I-chain complexes can enter
the endocytic pathway even earlier, either directly from
the TGN or via the plasma membrane (48, 49, 58, 63). This
latter pathway is probably a minor one in human B cells,
since major amounts of biosynthetically labeled class II
molecules cotransport with pro-cathepsin D directly from
the TGN to endosomes within the same vesicular intermediates (17). The fact that we found only low class II and
abundant I-chain labeling in early MIICs is possibly due to
differences in specificities and immunolabeling efficiencies
of the antibodies used. In addition, epitopes recognized by
the polyclonal antibody against human class II -chain may
partly be masked when associated with I-chain. Class II/I-chain complexes present in type 2 EEs may therefore have
been underscored. Several recent subcellular fractionation studies on B cells have shown direct transport of
/I-chain complexes from the TGN to late MIIC-like compartments (32, 41, 57, 60). Our present morphological observations are broadly consistent with those of Castellino
and Germain (6) who used a refractionation and density
shift protocol on murine splenic B cells and found that
most newly synthesized class II/I-chain complexes first enter EEs and then migrate farther down the endocytic system (see also 49). In their study, EEs were marked by labeling with HRP during 10 min of fluid phase endocytosis.
We have defined EEs by their Tf content after 3 min of receptor-mediated uptake. After 10 min of BSA-gold fluid
phase uptake, tracer had already accessed early MIICs significantly (17, this study). It therefore seems likely that the
EE subcellular fraction in the study of Castellino and Germain (6) included early MIICs. Mellman et al. (30) proposed a model in which class II/I-chain complexes are first
transported to EEs for removal of I-chain, whereupon
class II molecules are included in CIIVs that are formed
from EEs by a distinct budding process.
A second important aspect of early MIICs is their possible involvement in peptide loading. Although the majority
of the DR52-68/I-Ab complexes, recognized by the YAe
antibody, was found in type 4, 5, and 6 compartments (31),
we now demonstrate the presence of the YAe epitope already in early MIICs. Early MIICs contain lysosomal enzymes so that degradation of I-chain and of endocytosed antigen may occur simultaneously here (17). Since early
MIICs also harbor HLA-DM molecules, important requirements for antigen processing and peptide loading of class
II molecules are met in this compartment. Appearance of
antigen-presenting capacity after antigen uptake and acquisition of class II SDS stability, as indications of peptide
loading, have led to the notion that late MIICs are the primary sites of peptide loading in macrophages and B cell lines
(6, 21, 41, 60, 65). In human B lymphoblasts, West et al. (60)
detected peptide loading activity first in a compartment distinct from early (Tf+) and late (MPR+) endosomes and
with a morphology similar to MIICs. Recently it was shown
that in addition, EEs can be involved in peptide loading (13, 18). Such an activity was dependent on the type of antigen used and, more interestingly, varied between different
epitopes present in one particular antigen. According to
Castellino and Germain (6), SDS-stable complexes present
in EEs may also represent recycling class II-peptide complexes (40, 44, 46). Our quantitative IEM shows that at
steady state a little more than 10% of an abundant endogenous class II-peptide complex is present in early MIICs but is undetectable in EEs. However, most of the complexes were found in late MIICs, which may have accumulated there after traversing the endocytic pathway and/or
represent complexes formed de novo in later compartments. We have not found YAe labeling in any other type
of vesicle, distinct from early and late MIICs, which argues against the possible existence of a unique compartment
serving as the exclusive assembly site of class II-peptide
complexes.
Although the endocytic compartments of 6H5.DM and A20.Ab cells were very similar in most of their characteristics, several differences were noted. 6H5.DM cells contained many multilaminar MIICs (type 6), while A20.Ab cells were mostly devoid of this MIIC type as well as of the intermediate MIICs (type 5). In their place, a dense MIIC (type 5/6) with only few internal membranes was prominently present. Thus, the multilaminar structure may not be the most discriminative morphological feature of MIICs in general. We have previously detected dense instead of multilaminar MIICs in dendritic cells from skin and spleen (22, 23). In 6H5.DM cells, 95% of the 4-6 types of endocytic structures contained class II molecules, in contrast to 60% in A20.Ab cells. Furthermore, sections of 6H5.DM cells showed twice as many profiles of endocytic organelles than A20.Ab cells. A20.Ab cells have been reported to contain <5% of their total class II molecules intracellularly (1) versus up to 36% in human B cells (17). Together these data illustrate that the A20.Ab cells exhibit relatively poorly developed intracellular antigen processing and binding organelles, a feature that may hold for other mouse B cells as well (M.J. Kleijmeer, unpublished observations).
A most puzzling question concerns the relationship between CIIVs in mouse A20 cells (30) and MIICs defined in several human and mouse APCs (24). As far as we could test by IEM, our A20.Ab did not contain a distinct structure with all the characteristics reported for CIIVs. Nevertheless, many CIIV features matched those of early MIICs. Like CIIVs, early MIICs were not a major station in the Tf-recycling pathway, were markedly distinct from LEs and lysosomes, and contained peptide-loaded class II molecules. Furthermore, CIIV-enriched fractions were described to contain multivesicular structures (1) reminiscent of type 3 and 4 compartments in the present study. In addition, HLA-DM occurred only at relatively low levels in both early MIICs (this study) and CIIVs (39). On the other hand, while CIIVs were the predominant location of intracellular class II molecules and appeared to serve as an exclusive site for compact forms of class II (2), we found only 25% of the intracellular class II molecules in early MIICs of A20.Ab cells and most in the later MIICs. A possible explanation for this could be that we have used A20 cells transfected with I-Ab. Since the M5/114 antibody recognizes both I-Ab and I-Ad, overexpression of one may have caused a shift to additional late endocytic compartments in our study. In both 6H5.DM and A20.Ab cells the most striking feature of early MIICs was the enrichment in I-chain. This feature differs markedly from the low I-chain content reported for CIIVs (1, 2). It should be noted, however, that this may partly be caused by the specificity of antibody MKD6 used by Amigorena et al. (1), which does not efficiently recognize class II/I-chain complexes (5). Finally, since CIIVs were isolated by free-flow electrophoresis, they may represent a subset of early endocytic membranes that, because of their charge, show a unique deflection pattern.
In conclusion, our present observations show a major fraction of I-chain present in early MIICs, positioned just downstream of EEs. Most of the class II and HLA-DM molecules distribute to typical late endosomal/lysosomal MIICs (Fig. 7). Thus, MIICs appear to represent a heterogenous set of conventional late and lysosomal compartments, identical to their counterparts in non-APCs. The current, poorly defined connotations of EE, LE, and lysosome are insufficient to describe what we can distinguish in the endocytic pathway of class II by IEM. Since understanding of these compartments and the relationships between them is still incomplete, the term MIIC continues to be a convenient designation. We propose that APCs employ their full endocytic system for antigen processing and loading, which would enable them to process both easily degradable and proteolytically resistant antigens into peptides shortly or late after antigen uptake.
Received for publication 17 February 1997 and in revised form 22 August 1997.
Address all correspondence to Hans J. Geuze, Medical School, Department of Cell Biology, AZU H02.314, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. Tel.: (31) 30-2507652. Fax: (31) 30-2541797. E-mail: h.j.geuze{at}lab.azu.nlWe would like to thank Dr. G. Raposo for helpful discussions, and R. Scriwanek is gratefully acknowledged for his excellent photographical work.
This work was supported by the Dutch Organization of Scientific Research (NWO) grant 901-09-241.
APC, antigen-presenting cell; CD, cation dependent; CIIV, class II-containing vesicle; EE, early endosome; HLA, human leukocyte antigen; I-chain, invariant chain; IEM, immunoelectron microscopy; LAMPS, lysosome-associated membrane proteins; MPR, mannose 6-phosphate receptor; LE, late endosome; MHC, major histocompatibility complex; MIIC, MHC class II-enriched compartment; Tf, transferrin.
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