By
From the * Department of Cell Biology, Yale University School of Medicine,
New Haven, Connecticut 06520-8002; and the Department of Pediatrics,
National Jewish Center for Immunology, Denver, Colorado 80206
B cell receptor (BCR)-mediated antigen processing is a mechanism that allows class II-restricted
presentation of specific antigen by B cells at relatively low antigen concentrations. Although BCR-mediated antigen processing and class II peptide loading may occur within one or more
endocytic compartments, the functions of these compartments and their relationships to endosomes and lysosomes remain uncertain. In murine B cells, at least one population of class II-
containing endocytic vesicles (i.e., CIIV) has been identified and demonstrated to be distinct
both physically and functionally from endosomes and lysosomes. We now demonstrate the delivery of BCR-internalized antigen to CIIV within the time frame during which BCR-mediated antigen processing and formation of peptide-class II complexes occurs. Only a fraction of
the BCR-internalized antigen was delivered to CIIV, with the majority of internalized antigen
being delivered to lysosomes that are largely class II negative. The extensive colocalization of
BCR-internalized antigen and newly synthesized class II molecules in CIIV suggests that CIIV
may represent a specialized subcellular compartment for BCR-mediated antigen processing.
Additionally, we have identified a putative CIIV-marker protein, immunologically related to the
Ig subunit of the BCR, which further illustrates the unique nature of these endocytic vesicles.
The recognition of MHC class II-restricted antigens by
antigen-specific T cells requires the proteolytic processing of protein antigens to immunogenic peptides by
class II-positive antigen-presenting cells (1, 2). The first
step in antigen processing by B cells involves B cell receptor
(BCR)1-mediated internalization of antigen (3). BCR-internalized antigen is then proteolytically processed and
the resultant peptides preferentially loaded onto newly synthesized class II molecules (6) from which the class II-
associated invariant chain has been removed by the concerted action of acid proteases and the protein HLA-DM/
H-2M (9). The resultant peptide-class II complexes are
then transported to the surface of the B cell.
The intracellular compartments where antigen processing occurs have only recently been characterized and there
is considerable variation in the intracellular localization of
class II molecules among different cell types. Many cells,
such as human lymphoblasts and macrophages, sequester
much of their class II in lysosomes or lysosome-like structures referred to as the MHC class II-enriched compartment (MIIC; reference 10). Although delivery of BCR-internalized antigen to MIIC has been demonstrated (11),
the fate of the antigen delivered to these structures (i.e., complete degradation versus processing and binding to class II
molecules) remains unknown.
In other professional antigen-presenting cells such as
many murine B cell lines, there is little accumulation of
class II in lysosomes under normal conditions (12). Instead, class II is found in endosomes and endosome-related
structures, at least one population of which (class II vesicles
[CIIV]) can be purified and physically separated from conventional endocytic and secretory organelles by cell fractionation techniques (14).
Although many or all endocytic, class II-containing vesicle populations may host the loading of peptides onto class
II molecules, there may be important qualitative differences
regarding the subcellular compartments where antigenic
peptides are generated and efficiently loaded onto class II
molecules. Specifically, although BCR-mediated antigen
presentation appears to involve binding of peptide to newly
synthesized class II molecules (6), presentation of fluid phase proteins by B cells appears to be able to occur via
both newly synthesized and recycling class II molecules (7,
8, 15, 16), possibly reflecting differences in the intracellular sites of peptide generation and class II loading.
Additionally, not all receptors are equivalent at mediating
antigen processing and presentation. In murine B cells, antigen internalized via the transferrin receptor (while presented more efficiently than soluble antigen) is presented
10-100 times less efficiently than the same antigen internalized via the BCR (17). This result may reflect the fact that
the transferrin receptor has far more restricted access to intracellular class II compartments in B cells than does the
BCR (11). Even more dramatic is the demonstration that a
single amino acid substitution in the transmembrane region
of the human IgM BCR (huBCR) can completely abolish
the ability of this receptor to mediate efficient antigen processing and presentation without affecting BCR-mediated
antigen endocytosis and bulk antigen degradation (18, 19).
Thus, antigen uptake and degradation is necessary, but not
sufficient, for antigen processing and presentation.
Thus, it has become important to determine the intracellular compartments to which physiologically important receptors (e.g., the BCR) deliver antigens. In this paper, we
demonstrate that, within the time frame during which the
intracellular events of BCR-mediated antigen processing
are known to occur, BCR molecules and BCR-internalized antigen have access not only to predominantly class II-
negative endosome and lysosomes, but also to a novel population of endocytic vesicles that are highly enriched in
newly synthesized class II molecules (i.e., CIIV). Moreover, CIIV contain a putative marker protein, immunologically related to the Ig Cell Culture and Fractionation.
A20µWT (i.e., A20 cells expressing a transfected, phosphorylcholine (PC)-specific human
mIgM BCR (huBCR); reference 19) were cultured in Distribution of huBCR-internalized Antigen in A20µWT FFE
Fractions.
A20µWT cells (2 × 108 total cells) were incubated at
4 × 107 cells/ml for 30 min at 37°C in media containing 2 µg/ml
PC-modified Fab fragments of rabbit Immuno-electron Microscopy Localization of huBCR-internalized
Antigen and BCR Molecules to Isolated CIIV.
A20µWT cells were
incubated in 400-nM PC-modified ovalbumin (PC-OVA) for 20 min at 37°C, homogenized, and fractionated by FFE. Isolated
CIIV, as well as endosomes-lysosomes, were processed for immuno-electron microscopy (immunoEM) as previously described (14). Cryosections were stained with rabbit IgG specific for either
murine class II (14), murine IgG (315-005-046; Jackson Immunologicals, West Grove, PA), human IgM (309-005-095; Jackson Immunologicals; 309-005-095), or ovalbumin (RaOVALBUMI;
East Acres Biologicals, Southbridge, MA). Antibody to class II
molecules, anti-ovalbumin, and both anti-Ig antibodies were visualized with either 1, 5, or 10 nm protein A-gold (14).
Steady-state Distribution of the PC-specific huBCR in A20µWT
Cells.
A20µWT cells were homogenized and fractionated by
FFE. 200 µl of each FFE fraction, along with 50 µl of homogenization buffer containing 5% Triton X-100 and 0.5 mg/ml BSA,
was added to a PC-BSA-coated 96-well plate and the samples allowed to bind for 6 h at 4°C. The plates were washed and probed
with rabbit anti-human IgM (1:1,000; 309-005-095; Jackson Immunologicals) followed by horseradish peroxidase (HRP)-labeled
goat anti-rabbit IgG (1:1,000; 31462; Pierce Chemical Co.,
Rockford, IL). Bound goat anti-rabbit Ig-HRP was detected by
addition of 200 µl of 0.5 mg/ml O-phenylenediamine and
0.015% H2O2 in borate buffer. After sufficient time, 50 µl of 1N
HCl was added and the absorbance (OD 490 nm) measured. The
absorbance above background is reported (background = ~0.200
OD 490 nm).
Surface Labeling and Endocytosis.
A20µWT cells were collected
by centrifugation and washed two times with PBS. Cells were labeled for 15 min at 108 viable cells/ml in PBS pH 7.5 containing
1 mg/ml sulfosuccinimidyl-6-(biotinamido) hexanoate (NHS-
LC-biotin; Pierce Chemical Company; 21335). The labeling was
quenched by addition of 5-10 vol of 10 mM lysine in PBS. The
labeled cells were pelleted and then washed twice in PBS 0.1%
BSA before incubation at 37°C in complete media containing 1 µM PC-OVA (Cells were >98% viable after labeling, washing,
and incubation).
Detection of Biotin-labeled huBCR Molecules in A20µWT FFE
Fractions.
FFE fractions (200 µl) from biotin-labeled/PC-OVA
pulsed A20µWT cells were added to NeutrAvidin (50 µg/ml;
31000; Pierce)-coated plates along with 50 µl of homogenization
buffer containing 5% Triton X-100 and 0.5 mg/ml BSA and allowed to bind for 6 h at 4°C. The plate was washed and probed
with rabbit anti-human IgM (1:1,000, 309-005-095; Jackson Immunologicals) followed by HRP-labeled goat anti-rabbit Ig
(1:1,000; 31462; Pierce). Bound goat anti-rabbit Ig-HRP was
detected as described above. The absorbance above background is
reported (background = ~0.100 OD 490 nm).
Steady-state Distribution of Ig Immunological Relationship Between Ig The murine A20µWT cell line (19) was used as a model
cell for these studies. A20µWT cells express a PC-specific
huBCR as well as an endogenous murine IgG2a BCR
(muBCR) and the ability of these cells to process and
present antigens via both BCRs has been well characterized
(4, 19). Importantly, A20µWT cells localize only a small
fraction (<10%) of their total class II to intracellular membranes (13, 14), with little class II present in high density,
hydrolase-rich lysosomes (14). Owing to the lack of class II
molecules in the lysosomes of these cells, it is easy to distinguish, by FFE, a distinctive population of low density,
novel endocytic vesicles (i.e., CIIV) that are enriched in
newly synthesized class II molecules (14). Moreover, comparative analysis of A20 (14, 23) and A20µWT cells (Figs.
1, 2 A, and 4; data not shown) demonstrates that CIIV isolated from both cells possesses the same morphological,
biophysical, biochemical, and immunological characteristics
(e.g., class II-positive, lgp110-negative,
Table 1.
Quantitative ImmunoEM Analysis of FFE Fractions Isolated from Antigen-pulsed A20 µWT Cells
subunit of the BCR, further illustrating the distinct nature of these endocytic vesicles.
MEM,
5% FBS, 50 µM 2-mercaptoethanol, and 500 µg/ml G418.
A20µWT cells were homogenized, fractionated by free flow
electrophoresis (FFE), and the distribution of plasma membrane, lysosomes, and CIIV was determined as previously reported (14).
globulin labeled with 125I
(PC-RGG-125I [2µCi 125I/µg PC-RGG]), homogenized, fractionated by FFE, and the distribution of the plasma membrane
and lysosomes was determined. The distribution of PC-RGG-125I
was determined by counting each FFE fraction in a
counter. Background counts (<100 cpm) were subtracted from the counts
for each sample and the results normalized to a maximum value
of 1.00.
in A20µWT Cells.
Individual
A20µWT FFE fractions were concentrated by centrifugation and
analyzed by SDS-PAGE and Western blotting (14) with a rabbit
antiserum raised against intact, full length, bacterially expressed
murine Ig
(rabbit anti-Ig
, 1:5,000). Binding of rabbit anti-Ig
was detected with HRP-labeled goat anti-rabbit Ig (1:5,000; 31462; Pierce) and enhanced chemiluminescence (ECL; 34080;
Pierce).
and p50Ig
.
A20µWT low
density membranes (LDM) were separated by preparative SDS-PAGE and electroblotted onto nitrocellulose. The blot was
probed with rabbit anti-Ig
(1:1,000) and then extensively washed. The regions of the blot containing either Iga and p50Ig
were individually excised and bound antibodies eluted with 500 µl of 0.1 M glycine, pH 2.5. The eluted antibody was neutralized with 100 µl, 1.0 M Tris, pH 8.0, and 4.5 ml of blotting buffer. The affinity-purified antibody was used to probe Western blots of
total A20µWT LDM and binding revealed with HRP-labeled
goat anti-rabbit Ig (1:5,000; 31462; Pierce) and ECL (34080;
Pierce).
BCR-mediated Delivery of Antigen to Endosome and CIIV.
-Hexosaminidase-
negative multivesicular membrane structures with a characteristic electrophoretic mobility). Importantly, in A20µWT
cells, BCR-mediated antigen processing and peptide loading of class II molecules can occur exclusively in low density endocytic structures without the involvement of high
density lysosomal structures (13, 14).
Fig. 1.
The intracellular
distribution of BCR-internalized
antigen in A20µWT cells.
A20µWT cells were pulsed with 2 µg/ml antigen (i.e., PC-RGG-
125I) for 30 min at 37°C before
homogenization and fractionation by FFE. The distribution of plasma
membrane (PM, major peak of class II), endosomes and lysosomes (E/L, -Hexosaminidase;
thin solid line), and CIIV (minor,
anodally shifted peak of class II) are indicated above the graph. (The distribution of class II (broken line)
was determined by quantitative Western blot of every FFE fraction [reference 14].) Additionally, density gradient analysis of A20µWT cells has demonstrated that, as in A20 cells (14),
-Hexosaminidase-positive lysosomes are
devoid of class II molecules (data not shown), demonstrating that the
-Hexosaminidase activity present in the CIIV-containing FFE fractions
likely represents contamination by
-Hexosaminidase-positive, class
II-negative lysosomes that were occasionally observed during ImmunoEM analysis of these FFE fractions (Table 1). The distribution of
huBCR-internalized antigen (i.e., PC-RGG-125I; thick solid line) was determined by counting each fraction in a
counter. (A value of 1.00 = 16,000 cpm above background in the experiment shown.) The major peak
of huBCR-internalized antigen comigrated with markers for endosomes
and lysosomes with a small amount present in CIIV-containing FFE fractions. Illustrated are results representative of three independent experiments.
[View Larger Version of this Image (28K GIF file)]
Fig. 2.
ImmunoEM localization of BCR-internalized antigen in isolated CIIV as well as endosomes and lysosomes. Bar: 300 nm. (A) ImmunoEM localization of BCR-internalized antigen in isolated CIIV.
A20µWT cells were pulsed with antigen (i.e., 20 µg/ml PC-OVA) for
30 min at 37°C and then fractionated by FFE. Isolated CIIV were then analyzed by multiple label immunoEM with rabbit anti-class II and 5 nm
protein A-gold followed by rabbit anti-OVA and 10 nm protein A-gold.
Isolated CIIV were found to be doubly positive for both class II and
BCR-internalized antigen (PC-OVA; arrows), indicating that a portion of
the BCR-internalized antigen is delivered to CIIV. The specificity of the
staining for class II molecules and BCR-internalized antigen is demonstrated by the lack of label over areas of the section that do not contain
vesicular structures. (B and C) ImmunoEM localization of BCR-internalized antigen in isolated endosomes and lysosomes. Endosome/lysosome-
containing FFE fractions from PC-OVA pulsed A20µWT cells were
double labeled for class II and BCR-internalized antigen as in A. BCR-internalized antigen (PC-OVA; arrows) could be detected in both class II-
negative (B) and class II-positive (C) vesicles.
[View Larger Version of this Image (53K GIF file)]
Fig. 4.
ImmunoEM localization of huBCR molecules in isolated
CIIV. CIIV were isolated from A20µWT cells by FFE and analyzed by
multiple label immunoEM with rabbit anti-huBCR (anti-human IgM)
and 1 nm protein A-gold followed by rabbit anti-class II and 10 nm protein A-gold. Isolated CIIV stained for class II and huBCR (arrows). Bar:
100 nm.
[View Larger Version of this Image (115K GIF file)]
FFE Fraction
Fraction of total
vesicles that contain
class II molecules
Fraction of class II-positive
vesicles* that also contain
BCR-internalized antigen
Fraction of BCR-internalized
antigen-containing vesicles
that are class II positive*
%
%
%
CIIV
75
71
58
E/L
72
21§
34
CIIV- and endosome/lysosome (E/L)-containing FFE fractions from PC-OVA pulsed A20µWT cells were analyzed by multiple label immunoEM
for the presence of class II molecules and BCR-internalized antigen (i.e., PC-OVA). The percent of vesicles labeled for one or both markers determined by an unbiased sampling technique (26). For CIIV FFE fractions, a total of 531 vesicle profiles (from four independent experiments) were analyzed. Values are reported as percentages.
*
Class II-positive endocytic vesicles represent either CIIV (CIIV FFE fractions) or class II-positive endosomes (endosome/lysosome FFE fractions).
In a parallel set of experiments, the distribution of a ligand (i.e., HRP-labeled goat anti-murine IgG) internalized via the endogenous muBCR of
A20µWT cells was determined in CIIV FFE fractions. Quantitative analysis of these samples (292 total vesicle profiles) revealed that 90% of the vesicles that contained muBCR-internalized ligand were class II-positive structures (i.e., CIIV) and 85% of CIIV contain muBCR-internalize ligand.
§
The nonantigen-containing, class II-positive vesicles in the E/L-containing FFE fractions most likely represent contaminating, class II-containing
Golgi-derived vesicles.
If CIIV are involved in BCR-mediated antigen processing, then BCR-internalized antigens should be delivered to CIIV within 30-60 min after BCR-mediated internalization, the time required for BCR-mediated antigen processing in A20µWT cells (18). To determine whether this was the case, A20µWT cells were incubated with PC-RGG-125I for 30 min under conditions where antigen internalization and processing occur exclusively via cell-surface huBCR molecules (18, 19). The antigen-pulsed A20µWT cells were then homogenized, fractionated by FFE, and the distribution of huBCR-internalized PC-RGG-125I, as well as that of markers for plasma membrane (PM), endosomes, lysosomes, and CIIV (14), was determined. As shown in Fig. 1, the vast majority of the huBCR-internalized antigen was found in FFE fractions that contained endosomes and lysosomes (fractions 45-53). However, a small but significant amount of labeled antigen was present in the anodally shifted CIIV-containing FFE fractions (fractions 54-58). Thus, BCR-bound antigen could be found in CIIV-containing FFE fractions within the time frame during which BCR-mediated antigen processing is occurring.
This biochemical analysis, along with our previous observations that CIIV-containing FFE fractions consist almost entirely of class II-positive vesicles, strongly suggested
that BCR-internalized antigen can gain access to CIIV. To
demonstrate this point directly, and rule out the possibility
that the BCR-internalized antigen present in the CIIV-containing FFE fractions was contained exclusively in class
II-negative, -Hexosaminidase-positive lysosomes, CIIV-containing FFE fractions from antigen (i.e., PC-OVA)-
pulsed A20µWT cells were examined by multiple label immunoEM (14). As shown in Fig. 2 A, huBCR-internalized
antigen (arrow) was present in CIIV isolated from antigen-pulsed B cells. Additionally, immunoEM analysis of endosome/lysosome-enriched FFE fractions demonstrated that
BCR-internalized antigen was present both in class II-negative endosomes and lysosomes as well as class II-positive
endosomes (Fig. 2, B and C, respectively). Quantitation of
these immunoEM samples (Table 1) revealed that the vast
majority (71-85%) of CIIV were endocytic (i.e., accessible by BCR-internalized antigen) and that the bulk (58-90%)
of the endocytic vesicles within the CIIV-containing FFE
fractions were class II positive. On the contrary, only 34%
of the endocytic vesicles in the endosome/lysosome-containing FFE fractions were class II-positive endosomes,
with the majority of the antigen-containing vesicles being
class II-negative endosomes and lysosomes.
The presence of BCR-internalized antigen in
CIIV raised the question of whether antigen was delivered
to this compartment while still bound to the BCR or after
dissociation of BCR-antigen complexes. To determine
whether antigen is delivered to CIIV while still bound to
the BCR, we first determined whether BCR molecules could be found in CIIV at steady-state. To this end,
A20µWT cells were homogenized, fractionated by FFE,
and the distribution of PC-binding huBCR molecules was
determined by an antigen-specific anti-human IgM ELISA.
As shown in Fig. 3 A, most of the PC-binding huBCR was
present in PM-containing FFE fractions (fractions 35-44) with lower but significant levels also detected in endosome
and lysosome, as well as CIIV-containing FFE fractions
(fractions 45-54 and 55-64, respectively). A similar steady-state distribution was observed for the endogenous muBCR
as determined by Western blotting (data not shown).
To confirm that the huBCR molecules detected in the CIIV-containing FFE fractions were actually localized to CIIV, we performed multiple label immunoEM analysis of FFE-isolated CIIV for the presence of huBCR molecules. As shown in Fig. 4, CIIV do contain huBCR molecules (arrows) at steady-state. Similar analysis of endosome/lysosome-containing FFE fractions demonstrated that huBCR molecules could be found in both class II-positive as well as class II-negative vesicles in these fractions (data not shown).
Because we have previously demonstrated that newly synthesized BCR molecules do not traffic through CIIV before arrival at the cell surface (14), these results strongly suggest that the BCR molecules found in CIIV are derived from the PM by endocytosis and suggest that antigen is delivered to this compartment while bound to these internalized BCR molecules. To demonstrate directly that cell surface BCR molecules, internalized in the presence of polyvalent antigen, are delivered to CIIV, A20µWT were surface labeled with biotin, incubated for various times at 37°C in the presence of polyvalent antigen (i.e., PC-OVA), homogenized, and then fractionated by FFE. The level of biotin-labeled (i.e., internalized) huBCR molecules in each FFE fraction was then determined by a human IgM-specific, avidin-capture ELISA. As shown in Fig. 3 B, after 20 min of incubation, internalized huBCR molecules could be detected in CIIV-containing FFE fractions (fractions 53-60) as well as those enriched in endosomes and lysosomes (fractions 45-52). Surprisingly, even though we had found that, under similar conditions, a vast majority of the BCR-internalized antigen was ultimately delivered to endosomes and lysosomes (see Fig. 1), a significant fraction of the internalized huBCR molecules were found in CIIV-containing FFE fractions. Therefore, we suggest that a portion, and possibly all, of the huBCR molecules detected in CIIV by immunoEM (Fig. 4) were derived from the PM after endocytosis, further supporting the contention that antigen is delivered to CIIV while bound to the BCR. Interestingly, as suggested by the presence of huBCR molecules in CIIV isolated from nonantigen-pulsed A20µWT cells, preliminary analysis of the constitutive endocytosis and trafficking of the huBCR of A20µWT cells suggests that BCR endocytosis and delivery to CIIV can occur in the absence of antigen cross-linking (Drake, J.R., unpublished results).
Subcellular Distribution of BCR Subunits and Identification of a Putative CIIV Marker Protein Immunologically Related to IgAlthough both BCR molecules and BCR-internalized antigen clearly gained access to CIIV, as well as endosomes and class II-negative lysosomes, the extent to which these molecules are, or are not, selectively targeted to CIIV remains unclear. To begin to address whether there is any selective targeting of BCR molecules or antigen-BCR complexes to CIIV, we first attempted to determine whether there is any difference in the subunit composition of the BCR molecules present in PM, endosome and lysosome, or CIIV-containing FFE fractions.
To this end, we examined the steady state distribution of
the Ig subunit of the BCR. As shown in Fig. 5 A, the
majority of the 32-kD Ig
protein (arrow B) is present in
PM-containing FFE fractions with lesser amounts detected
in endosome-lysosome and CIIV-containing fractions. The
same distribution was also found for the Ig
subunit of the
BCR as well as the heavy and light chain subunits of both
the huBCR and muBCR (data not shown), suggesting that
the subunit composition of the BCRs in these compartments is similar. Surprisingly, the anti-Ig
antiserum also
recognized a second, 50-kD protein (Fig. 5 A, arrow A)
that appears to be selectively enriched in CIIV-containing
FFE fractions.
To determine whether this 50-kD putative CIIV-marker
protein was immunologically related to Ig (as opposed
to being recognized by antibodies of a second specificity
present in the rabbit anti-Ig
antiserum), we affinity-purified antibodies to both Ig
and the 50-kD protein on
Western blots of A20µWT LDM, and tested the specificity of these purified antibodies. As shown in Fig. 5 B, the unfractionated rabbit anti-Ig
antiserum recognized both Ig
as well as the 50-kD protein present in unfractionated
A20µWT LDM. Although affinity-purified anti-Ig
antibody failed to recognize the 50-kD protein (occasionally, reactivity of the affinity-purified anti-Ig
toward the 50-kD protein was observed although the results were variable,
possibly owing to removal of low affinity/highly cross-reactive antibodies by the affinity purification protocol), affinity-purified antibody against the 50-kD protein recognized both Ig
as well as the 50-kD protein. Because this
antiserum was originally generated against recombinant
whole murine Ig
, these results demonstrate that at least
some anti-Ig
antibodies specifically recognize the 50-kD putative CIIV-marker protein, demonstrating that these
proteins are immunologically related (i.e., that Ig
and the
50-kD protein minimally share one cross-reactive epitope).
Additionally, an antiserum raised against the cytoplasmic
tail of Ig
(20) also demonstrated cross-reactivity to the 50-kD protein, suggesting an immunological relationship between the cytoplasmic tail of Ig
and some region of the
50-kD protein (data not shown).
Because the 50-kD putative CIIV-marker protein does
not exhibit a decrease in apparent molecular weight upon
treatment with either endoglycosidase H or F (Drake, J.R.,
unpublished results), we have foregone the more traditional
gp50 designation in favor of p50Ig (the Ig
superscript indicates the immunological relationship of the protein to the
Ig
subunit of the BCR). Although the structure and function of p50Ig
remains unknown, it is unlikely to represent
a highly modified form of Ig
, since it was also detected in
Ig
-negative J774 macrophage-like cell line (Drake, J.R.,
unpublished results). Moreover, p50Ig
is unlikely to be an
artifact of proteolytic activity because it can be detected in
detergent extracts of whole cells prepared in the presence
of a cocktail of protease inhibitors (Drake, J.R., unpublished results). Additionally, preliminary analysis of J774 cells and the murine B cell hybridoma 2C3E1 (21) suggests
a restricted distribution of p50Ig
to CIIV in these cells
(Drake, J.R., and P. Webster, unpublished results).
Considering the presence of p50Ig in the BCR-negative
J774 macrophage-like cell line, p50Ig
, unlike Ig
, may not
be a component of the BCR protein complex. Correspondingly, preliminary analysis has failed to reveal any physical
association between p50Ig
and the BCR of A20µWT cells
(Drake, J.R., unpublished results). Therefore, a more thorough understanding of the possible function of p50Ig
will
have to await its eventual purification and sequencing or cDNA cloning. Interestingly, previous Southern blot analysis of the murine MB-1 gene (which codes for Ig
) indicated the presence of an additional, Ig
-related gene (22),
possibly that coding for p50Ig
. Most importantly, the identification of p50Ig
as a putative marker for CIIV graphically demonstrates the unique biochemical nature of these
novel class II-containing vesicles and provides us with a
tool to study their origin, fate, and relationship to other intracellular compartments.
Previously, we demonstrated that in murine B cells, class II molecules are restricted to relatively early endocytic compartments (i.e., endosomes and CIIV), with little or no class II found in high density lysosomes (14). Because peptide-class II complexes have been demonstrated to form only in low density (i.e., nonlysosomal) compartments in these cells (13, 14), the presence of BCR-internalized antigen in both class II-positive endosomes as well as CIIV suggests that antigen processing and class II peptide loading may occur at either or both of these sites. Given the predominant role of newly synthesized class II molecules in BCR-mediated antigen processing and presentation (6) and the fact that CIIV are an intermediate in the pathway of transport of newly synthesized class II molecules to the cell surface (14, 23), our results strongly suggest a role for CIIV in BCR-mediated antigen processing and class II peptide loading.
Why might B cells possess a novel endocytic compartment for the processing of BCR-internalized antigens? In contrast with endosomes, lysosomes, and MIIC, which readily accumulate nonselectively internalized fluid phase endocytic tracers (10, 24), CIIV do not readily accumulate proteins internalized by fluid phase endocytosis (14). Within endosomes, lysosomes, and MIIC, the vast excess of nonantigenic peptides (i.e., peptides derived from the proteolytic degradation of fluid phase plasma proteins) may effectively compete with antigenic peptides (i.e., peptides derived from BCR-internalized antigen) for binding to class II molecules, preventing the efficient formation of antigenic peptide-class II complexes within these compartments. The absence of these nonantigenic peptides from CIIV may allow for more efficient formation of antigenic peptide-class II complexes in these vesicles. Additionally, because peptide loading onto class II is a relatively slow process (25), the relatively slow transport of newly synthesized class II molecules through CIIV, with class II molecules residing in CIIV for up to 2 h (14), may provide the necessary time for the loading of these class II molecules with antigen-derived peptides.
Address correspondence to James R. Drake at The Trudeau Institute, 100 Algonquin Avenue, P.O. Box 59, Saranac Lake, New York 12983. Phone: 518-891-3080; FAX: 518-891-5126; E-mail: jdrake{at}northnet.org
Received for publication 11 June 1997 and in revised form 19 August 1997.
The research described in this paper was supported by grants from the Public Health Service.The authors would like to thank L. Schaefer for technical assistance, L. Ryan for photographic darkroom assistance, and L. Chicoine for assistance with sectioning and labeling samples for immunoEM analysis. Additionally, the authors would like to thank R. Mitchell (Harvard University, Cambridge, MA) and M. Nussenzweig (Rockefeller University, New York) who generously provided A20µWT cells and careful guidance as to their use.
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