Prolonged Antigen Persistence Within
Nonterminal Late Endocytic Compartments
of Antigen-Specific B Lymphocytes
This information is current as
of July 12, 2017.
Timothy A. Gondré-Lewis, Amy E. Moquin and James R.
Drake
J Immunol 2001; 166:6657-6664; ;
doi: 10.4049/jimmunol.166.11.6657
http://www.jimmunol.org/content/166/11/6657
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References
Prolonged Antigen Persistence Within Nonterminal Late
Endocytic Compartments of Antigen-Specific B Lymphocytes1
Timothy A. Gondré-Lewis,2 Amy E. Moquin, and James R. Drake3
I
mmediately upon introduction of Ag into the body, Ag-specific B lymphocytes are able to bind and internalize Ag via
their cell surface B cell receptor (BCR)4 molecules. Moreover, Ag-specific B lymphocytes are capable of expressing complexes of Ag-derived peptides and MHC class II molecules (the
result of BCR-mediated Ag processing) on their surface as little as
1 h after Ag binding (1). However, Ag-specific effector CD4 T
lymphocytes with which these Ag-specific B lymphocytes need to
interact for full development and differentiation into Ab-producing
and/or memory B lymphocytes will not be present in the body until
3–5 days after the initial exposure to Ag (2). Presently, it is unclear
how Ag-specific B lymphocytes maintain sufficiently high levels
of cell surface peptide-class II complexes to allow efficient interaction with Ag-specific effector CD4 T lymphocytes when they
become available.
Trudeau Institute, Saranac Lake, NY 12983
Received for publication October 4, 2000. Accepted for publication March 29, 2001.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported in part by grants (to J.R.D.) from the U.S. Public Health
Service (AI40236) and Trudeau Institute and in part by a United Negro College
Fund-Parke-Davis Postdoctoral Fellowship (to T.A.G.-L.).
2
Current address: Department of Natural Sciences/Biology, York College of the City
University of New York, 94-20 Guy R. Brewer Boulevard, Jamaica, NY 11451.
3
Address correspondence and reprint requests to Dr. James R. Drake, Trudeau Institute, 100 Algonquin Avenue, P.O. Box 59, Saranac Lake, NY 12983. E-mail address: [email protected]
Abbreviations used in this paper: BCR, B cell receptor; B10, B10.Br; ␤-Hex,
␤-hexosaminidase; DAB, 3,3⬘-diaminobenzidine; DM, HLA-DM (human) or
H2-M (murine); DO, HLA-DO (human) or H2-O (murine); DTAF, dichlorotriazinyl amino fluorescein; Nycodenz, 5-(N-2,3-dihydroxy-propylacetamido)-2,4,6triiodo-N,N⬘-bis(2,3-dihydroxypropyl)isophthalamide; EE, early endosome(s); ER,
endoplasmic reticulum; F-P, fluid phase; HEL, hen egg lysozyme; IFM, immunofluorescence microscopy; LDM, low density membranes; L, lysosome; LE, late endosome; MD4, MD4.B10.Br; PC, phosphorylcholine; RE, recycling endosome(s);
LAMP, lysosome-associated membrane protein.
4
Copyright © 2001 by The American Association of Immunologists
One possibility is that Ag-specific B cells may continually internalize Ag via BCR-mediated endocytosis and process this internalized Ag to peptide-class II complexes. However, this strategy
would be ineffective for Ags that are not continually present in the
body. Moreover, this approach would be hampered by Ag-induced
down-regulation of cell surface BCR molecules (making BCRmediated endocytosis of Ag less efficient). A second possibility is
that B cells may continually sample Ags that have been sequestered on the surface of follicular dendritic cells. However, Ag deposition upon follicular dendritic cells requires the presence of circulating Abs (to allow formation of immune complexes) (3), a
situation that may not exist upon the first encounter with an
Ag. Third, Ag-specific B cells may sequester BCR-internalized
cognate Ag within intracellular compartments and use this Ag depot as a source of antigenic peptides to support the continual production and cell surface expression of antigenic peptide-class II
complexes.
Using normal splenic B cells from a BCR transgenic mouse, we
have determined that Ag-specific B lymphocytes are capable of
sequestering a pool of native Ag (still bound to the BCR) within a
nonterminal endocytic compartment for more than 1 day after the
elimination of environmental Ag. Moreover, B lymphocytes harboring persisting intracellular Ag-BCR complexes are capable of
prolonged cell surface expression of antigenic peptide-class II
complexes derived from the processing of this Ag. These results
demonstrate that B lymphocytes possess a mechanism for prolonging the intracellular persistence of internalized Ag-BCR complexes and suggest that B lymphocytes use the Ag within these
complexes as a source of antigenic peptide for the continued cell
surface expression of peptide-class II complexes.
Materials and Methods
Mice
B10.Br (B10) and MD4.B10.Br (MD4) mice (MD4 transgenic mice expressing hen egg lysozyme (HEL)-specific BCR (4) that have been bred
onto the B10 background) were used from 7 to 12 wk of age. Mice were
0022-1767/01/$02.00
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Although Ag-specific B lymphocytes can process Ag and express peptide-class II complexes as little as 1 h after Ag exposure, it
requires 3–5 days for the immune system to develop a population of Ag-specific effector CD4 T lymphocytes to interact with these
complexes. Presently, it is unclear how B cells maintain the expression of cell surface antigenic peptide-class II complexes until
effector CD4 T lymphocytes become available. Therefore, we investigated B cell receptor (BCR)-mediated Ag processing and
presentation by normal B lymphocytes to determine whether these cells have a mechanism to prolong the cell surface expression
of peptide-class II complexes derived from the processing of cognate Ag. Interestingly, after transit of early endocytic compartments, internalized Ag-BCR complexes are delivered to nonterminal late endosomes where they persist for a prolonged period of
time. In contrast, Ags internalized via fluid phase endocytosis are rapidly delivered to terminal lysosomes and degraded. Moreover,
persisting Ag-BCR complexes within nonterminal late endosomes exhibit a higher degree of colocalization with the class II
chaperone HLA-DM/H2-M than with the HLA-DM/H2-M regulator HLA-DO/H2-O. Finally, B cells harboring persistent Ag-BCR
complexes exhibit prolonged cell surface expression of antigenic peptide-class II complexes. These results demonstrate that B
lymphocytes possess a mechanism for prolonging the intracellular persistence of Ag-BCR complexes within nonterminal late
endosomes and suggest that this intracellular Ag persistence allows for the prolonged cell surface expression of peptide-class II
complexes derived from the processing of specific Ag. The Journal of Immunology, 2001, 166: 6657– 6664.
6658
housed under specific-pathogen free conditions and were maintained at
Trudeau Institute’s Animal Breeding Facility (Saranac Lake, NY). Singlecell suspensions of splenocytes were prepared and lymphocytes isolated
using Ficoll-Paque (Amersham Pharmacia Biotech, Piscataway, NJ).
Ag (HEL) endocytosis by splenic B lymphocytes
BCR-mediated endocytosis. MD4 B cells were pulsed with 100 nM HEL
for 30 min on ice and then washed to remove unbound Ag. The cells were
incubated at 37°C for the indicated times to allow endocytosis of cell
surface Ag-BCR complexes.
Fluid phase (F-P) endocytosis. B10 B cells were pulsed with 100 ␮M to
1 mM HEL for 30 min at 37°C to allow F-P endocytosis of detectable
amounts of HEL. The cells were then washed to remove noninternalized
HEL and were incubated at 37°C for the indicated times to allow intracellular trafficking of the internalized HEL.
PROLONGED Ag PERSISTENCE IN Ag-SPECIFIC B CELLS
for the indicated times before analysis of PC-BSA-HRP endocytosis as
previously published (12).
3,3⬘-Diaminobenzidine (DAB) depletion
A20 ␮WT cells were pulsed on ice with HRP-labeled Ag (e.g., PC-BSAHRP) for 30 min on ice and then washed to remove unbound Ag. Cells
were allowed to internalize Ag at 37°C for 0 – 60 min Subsequently, the
cells were resuspended in 2 ml of 0.5 mg/ml DAB (Polysciences, Warrington, PA) in PBS with or without 0.015% H2O2. The cells were incubated for 45 min at 4°C in the dark, then washed with 2 ml of PBS-BSA.
Cells were lysed in PBS with 0.75% Triton X-100 for 1 h on ice. Cellular
debris and aggregated protein polymers were pelleted from the detergent
lysate by centrifugation at 22,000 ⫻ gavg for 15 min at 4°C. The level of
depletion of HLA-DM/H2-M (DM), HLA-DO/H2-O (DO), and calnexin
was determined by SDS-PAGE and Western blotting. The level of ␤-hexosaminidase (␤-Hex) depletion was determined by enzymatic assay (11).
Flow cytometric analysis of HEL46 – 61–I-Ak expression
B lymphocytes were attached to Alcian Blue-coated coverslips, fixed, permeabilized, and stained as previously reported (5). The primary Abs used
were GL2A7 (antilysosome-associated membrane protein (LAMP) 2, 1/20
dilution of tissue culture supernatant) (6), rabbit polyclonal Abs to H2-M
(1/500 dilution), and H2-O (1/250 dilution), Map.DM1 (anti-HLA-DM, 1/5
dilution of tissue culture supernatant) (7), and 2D1 (anti-HEL, 1/10 dilution
of tissue culture supernatant) (8). A20 cells were incubated with BODIPYfluorescein-labeled transferrin conjugate (T-2873; Molecular Probes, Eugene, OR) at 37°C to label early endosomes (EE) and recycling endosomes
(RE). Appropriate secondary Abs were used to detect primary Abs: donkey
anti-mouse Ig-Texas Red (1/100 dilution; Jackson ImmunoResearch Laboratories, West Grove, PA; 715-075-1510), donkey anti-rabbit Ig-dichlorotriazinyl amino fluorescein (DTAF; 1/100 dilution; Jackson Immuno
Research Laboratories; 711-016-152), donkey anti-rabbit Ig Texas Red (1/
100 dilution; Jackson ImmunoResearch Laboratories; 711-076-152),
donkey anti-rat Ig DTAF (1/100; Jackson ImmunoResearch Laboratories;
712-015-153), and donkey anti-rat Ig Texas Red (1/100 dilution; Jackson
ImmunoResearch Laboratories; 712-076-153). The cells were examined
with a Zeiss Axiophot 2 microscope (Carl Zeiss, Thornwood, NY) using
epi-illumination.
Splenic B cells were allowed to process and present HEL internalized by
either BCR-mediated of F-P endocytosis (incubation of MD4 B cells in 10
nM HEL or B10 B cells in 100 ␮M HEL for 18 –20 h at 37°C in complete
medium, respectively). The cells were then washed to remove environmental Ag, returned to culture in complete medium for the indicated times,
collected, and stained with the C4H3 mAb (13) (a 1/10 dilution of hybridoma supernatant), followed by mouse anti-rat IgG2b-FITC (1/500, no.
10054D; PharMingen, San Diego, CA) and anti-mouse CD45R/B220-PE
(1/200, no. 01125A; PharMingen). After treatment with 1 ␮g/ml propidium
iodide, the samples were analyzed with a FACScan flow cytometer (BD
Biosciences, San Jose, CA). For each data point ⱖ10,000 live B cells (i.e.,
B220-positive, propidium iodide-negative lymphocytes) were analyzed,
and the mean fluorescence intensity of the population was determined.
Cell lines
A20 murine B cells, A20huDM (A20 cells expressing transfected human
DM␣- and DM␤-chains) (9), and A20 ␮WT (A20 cells expressing a transfected phosphorylcholine (PC)-specific human mIgM BCR) (10) were
grown in ␣MEM, 10% FCS, and 50 ␮M 2-ME. Medium was supplemented
with 500 ␮g/ml of G418 for A20 ␮WT or 500 ␮g/ml of hygromycin B for
A20huDM.
Nycodenz density gradient centrifugation
A20 ␮WT cells were homogenized, and low density membranes (LDM)
were isolated as previously reported (11). LDM were fractionated on topdown continuous Nycodenz (5-(N-2,3-dihydroxy-propylacetamido)-2,4,6triiodo-N,N⬘-bis(2,3-dihydroxypropyl)isophthalamide; D-2158, Sigma, St.
Louis, MO) density gradients as previously described (9). Briefly, a total of
3 ml of LDM in TEAS250 was added to a 12-ml ultracentrifuge tube. This
was sequentially underlain with 3 ml each of 6, 12, and 18% Nycodenz
stock solutions. The tube was tightly sealed, rotated 90°, and incubated on
its side for 1 h at 4°C to form a continuous gradient. The tube was then
slowly brought to an upright position, and the resultant continuous gradient
was centrifuged at 110,000 ⫻ gavg for 2 h in an SW-41Ti rotor at 4°C.
Fractions (0.5 ml) were collected from the top to the bottom of the gradient,
and the refractive index of the odd numbered fractions was determined.
Western blot analysis
Gradient fractions were diluted by addition of 2 volumes of TEAS250, and
vesicles were collected by centrifugation (11). The samples were analyzed
by SDS-PAGE and Western blot as previously described (11). The primary
Abs used to visualize H2-M (1/5000 dilution) and H2-O (1/3000 dilution)
were rabbit polyclonal Abs obtained from Sebastian Amigorena (Institute
Curie, Paris, France), and Lars Karlsson (R. W. Johnson Pharmaceutical
Research Institute, San Diego, CA), respectively. The primary Ab used to
visualize class II (1/25,000 dilution) was a rabbit polyclonal Ab (11).
PC-BSA-HRP endocytosis
A20 ␮WT cells were pulsed with PC-modified BSA that had been labeled
with the enzyme HRP (PC-BSA-HRP; 0.4 ␮M BSA) for 30 min on ice and
then washed to remove unbound Ag. The cells were then incubated at 37°C
Results
Prolonged Ag persistence within Ag-specific B cells
To study the endocytosis and intracellular trafficking of BCRinternalized Ag in normal B lymphocytes, we took advantage of
the MD4 BCR transgenic mouse (4) (referred to hereafter as MD4)
in which all of the B lymphocytes express an HEL-specific BCR.
Incubation of MD4 splenic B cells with 10 –100 nM HEL allows
for the BCR-mediated binding, endocytosis, and presentation of
HEL in the absence of detectable F-P endocytosis or presentation
of HEL (14). To allow for comparison of the intracellular trafficking of BCR-internalized Ag to the intracellular trafficking of the
same Ag internalized via F-P endocytosis, we also used splenic B
cells from non-BCR transgenic B10 mice. Incubation of B10 B
cells with 100 ␮M HEL allows for the F-P endocytosis, processing, and presentation of HEL by these B cells. In addition, we took
advantage of two distinct anti-HEL mAbs (i.e., 2D1, Ref. 8; and
HyHEL10, Ref. 15) to follow the intracellular movements of HEL
internalized by either BCR-mediated or F-P endocytosis. Importantly, the 2D1 mAb recognizes an epitope on HEL that is on the
opposite face of HEL from the epitope recognized by the MD4
BCR (8). Therefore, the 2D1 mAb can recognize both free HEL as
well as HEL bound to the MD4 BCR. Contrastingly, the HyHEL10
mAb recognizes precisely the same epitope as the MD4 BCR (4).
Therefore, the HyHEL10 mAb can only recognize free HEL and
will not recognize HEL bound to the MD4 BCR.
To follow BCR-mediated and F-P endocytosis of HEL, MD4
and B10 B cells were pulsed with Ag (i.e., 100 nM and 100 ␮M
HEL, respectively), and the intracellular distribution of the internalized HEL was analyzed by double-label IFM. The results presented in Fig. 1 (A and B) demonstrate that under these conditions,
sufficient HEL is internalized by both BCR-mediated as well as
F-P endocytosis to allow detection of internalized Ag. Interestingly, when B cells that had internalized Ag via BCR-mediated
endocytosis were washed (to remove environmental Ag) and then
returned to culture for 24 h before analysis, they still contained
detectable HEL (Fig. 1C). Contrastingly, B cells that had internalized HEL via F-P endocytosis contained no detectable intracellular
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Immunofluorescence microscopy (IFM)
The Journal of Immunology
Ag 24 h after removal of environmental Ag (Fig. 1D). Quantitation
of these results is presented in Fig. 2, A and B. The results in Fig.
2A demonstrate that even 24 h after removal of environmental Ag,
essentially every Ag-specific B cells still contained detectable levels of BCR-internalized intracellular Ag. Contrastingly, as little as
8 h after removal of environmental Ag, very few if any B cells that
had internalized HEL by F-P endocytosis contained detectable levels of Ag (Fig. 2B). These results demonstrate that BCR-internalized Ags can persist for a prolonged period of time within B lymphocytes. Furthermore, because the 2D1 mAb recognizes a
conformational epitope on the HEL molecule (8), these results also
suggest that at least a portion of the BCR-internalized HEL molecules are persisting in a relatively native conformation.
When a similar analysis was performed using the HyHEL10
mAb (which is able to recognize free HEL, but not HEL associated
with the MD4 BCR) an interesting result was obtained. The results
of HyHEL10 staining of B cells that had internalized HEL via F-P
FIGURE 2. Kinetic analysis of Ag persistence after BCR-mediated and
F-P endocytosis. Splenic B cells were allowed to internalized Ag by either
BCR-mediated (A and C) or F-P (B and D) endocytosis, washed, and
chased for various times at 37°C as described in Fig. 1. The cells were then
fixed, permeabilized, and stained for B220 as well as Ag (HEL) using
either the 2D1 mAb (A and B), which recognizes both free and BCR-bound
HEL, or the HyHEL10 mAb (C and D), which recognizes only free HEL.
For each time point, 100 B220-positive cells were analyzed for the presence of Ag (HEL), and the percentage of B220-positive cells containing
detectable Ag was determined. Each line represents the results from an
independent experiment. The results shown in Figs. 1 and 2 were compiled
from five or more independent experiments. Results from concurrent experiments are denoted by the same icon.
endocytosis (Fig. 2D) closely mirrored the results obtained with
the 2D1 mAb. This was expected, because both mAbs should be
able to recognize the non-BCR-associated HEL in these cells.
However, the results obtained with the MD4 B cells were significantly different. As shown in Fig. 2C, HyHEL10 initially did not
detect any free HEL on or within MD4 B cells. However, after 1– 8
h of incubation at 37°C, non-BCR-associated HEL could be detected within intracellular compartments of the vast majority of
MD4 B cells, demonstrating that some of the HEL had dissociated
from the BCR (notably, no HyHEL10 staining of HEL-BCR complexes present at the plasma membrane of HEL-pulsed MD4 B
cells was observed at any time point). Furthermore, after 24 h of
incubation, the HEL that had been released from the BCR was, like
the HEL internalized via F-P endocytosis, degraded, as indicated
by the decrease in HyHEL10 at this time. However, these B cells
still contain HEL-BCR complexes, as indicated by the high level
of staining with the 2D1 mAb (Fig. 2A). These results are in line
with our observation that BCR-mediated processing and presentation of HEL by MD4 B cells exhibits a lag of 2– 4 h before expression of detectable peptide-class II complexes at the cell surface
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FIGURE 1. Prolonged intracellular persistence of BCR-internalized Ag
within nonterminal late endocytic compartments. Splenic B cells were allowed to accumulate Ag (i.e., HEL) for 1 h by either BCR-mediated uptake
or F-P endocytosis (i.e., MD4 B cells pulsed with 10 nM HEL (A, C, E, and
F) or B10 B cells pulsed with 100 ␮M HEL, respectively (B and D)). The
cells were then either fixed immediately (A and B) or washed to remove
noninternalized Ag and returned to culture for an additional 24 h before
fixation (C–F). For A–D, the fixed cells were permeabilized and stained
with anti-B220-FITC (to mark B cells) as well as with the 2D1 anti-HEL
mAb (followed by anti-muIgG1-btn and streptavidin-Texas Red) to localize both free and BCR-bound Ag (i.e., HEL). E, The cells were stained for
persisting Ag (2D1, red) as well as the late endosomal/lysosomal protein
LAMP-2 (green). F, FITC-dextran (fixable; 10 mg/ml) was included during
the Ag pulse of the MD4 B cells. After fixation, the permeabilized B cells
were stained for persisting Ag with the 2D1 mAb, followed by antimuIgG1-btn and streptavidin-Texas Red. Note that the equivalent level of
staining of intracellular vesicles for BCR- and F-P-internalized HEL (A and
B, respectively) suggests that similar levels of HEL protein were internalized via F-P- and BCR-mediated endocytosis.
6659
PROLONGED Ag PERSISTENCE IN Ag-SPECIFIC B CELLS
(J. R. Drake, unpublished observation) and demonstrate that Agspecific B lymphocytes possess a mechanism to allow the prolonged intracellular persistence of cognate Ag-BCR complexes
(compared with Ag internalized via F-P endocytosis).
A20huDM) (9), in which we could use available reagents to analyze the intracellular distribution of DM and DO by double-label
IFM; 2) our extensive experience analyzing the cell biology of Ag
processing within these cells using the techniques of double-label
IFM and subcellular fractionation (5, 9, 11, 12, 21, 22); and 3) the
availability of a transfected A20 cell line (i.e., A20 ␮WT) (10) that
expresses an Ag-specific BCR that can mediate the endocytosis,
processing, and presentation of BCR-bound Ag.
Using A20huDM cells in which we have previously demonstrated a similar intracellular distribution of the endogenous murine DM and transfected human DM molecules (9, 21), we examined the relative distributions of DM and DO by double-label IFM.
As shown in Fig. 4A, the human DM and endogenous murine DO
proteins have notably distinct subcellular distributions within
A20huDM cells. As previously reported (9), the DM protein is
most highly enriched within relatively large intracellular vesicles
located near the periphery of the cell. This pattern of staining is
reminiscent of the distribution of the LE/L marker LAMP-2 (Fig.
4C) (9), which we have previously reported to extensively colocalize with DM in A20 cells (9). Contrastingly, the DO protein is
most highly enriched within a population of intracellular vesicles
whose distribution is reminiscent of the distribution of the Golgi
apparatus within these cells (9, 11, 22). To further characterize the
subcellular distribution of DO in A20 cells, we compared the subcellular distribution of DO to markers of the early and late aspects
of the endocytic pathway (i.e., EE/RE and LE/L, respectively). As
illustrated in Fig. 4B, DO exhibits a moderate degree of overlap
with internalized transferrin present in EE and RE. Although most
of the transferrin-containing vesicles contain DO, there is a population of DO-positive vesicles that does not contain detectable
levels of transferrin. Contrastingly, and as expected from the low
level of colocalization between DM and DO (Fig. 4A), DO exhibits
little if any colocalization with the LE/L marker LAMP-2 (Fig.
4C). These results suggest that in A20 cells, DO is most highly
enriched within the biosynthetic pathway as well as early aspects
of the endocytic pathway. Contrastingly, DM is most highly enriched within LE and terminal L.
To confirm the results obtained by IFM, the steady-state intracellular distribution of DM and DO in A20 ␮WT cells was also
analyzed by subcellular fractionation. Because we have previously
used the technique of Nycodenz density gradient centrifugation to
localize DM to LAMP-2-positive LE and L in A20 cells (9), we
used the same approach to compare the intracellular distribution of
the endogenous murine DM and DO proteins within A20 ␮WT
cells. As previously reported (9) and again illustrated in Fig. 4D,
the endogenous murine DM protein of A20 cells is most highly
enriched in gradient fractions that contain LAMP-2-positive,
␤-Hex-negative LE as well as LAMP-2-positive, ␤-Hex-positive
L. Contrastingly, the endogenous murine DO protein bands to two
distinct regions of the gradient. The population of low buoyant
density DO-containing vesicles near the top of the gradient has
been tentatively labeled EE/RE (see brackets above Western blots)
because of the previously published localization of the EE/RE
marker Rab4 to this region of the Nycodenz gradient (9) as well as
the high level of DO colocalization with internalized transferrin
illustrated in Fig. 4B. The high buoyant density DO-containing
vesicle near the bottom of the gradient comigrates with vesicles
derived from the plasma membrane as well as comigrating vesicles
derived from the early aspects of the biosynthetic pathway (i.e., the
endoplasmic reticulum (ER) and Golgi apparatus). Because DO is
an intracellular protein, the DO molecules within these gradient
fractions must reside in either ER- or Golgi-derived vesicles. Importantly, although the precise identities of the two populations of
DO-containing vesicles remain to be conclusively determined, the
Subcellular localization of persisting Ag-BCR complexes
Having demonstrated that Ag-BCR complexes can persist for a
prolonged period within Ag-specific B lymphocytes, we next
sought to determine the identity of the intracellular compartment(s) in which these complexes were residing. To accomplish
this, the distribution of persisting Ag-BCR complexes was compared with the intracellular distribution of markers for the late
aspects of the endocytic pathway (i.e., LAMP-2, a marker of both
late endosome (LE) and lysosome (L), and FITC-dextran chased
into terminal L). As shown in Fig. 1E, persisting Ag-BCR complexes reside in a subpopulation of LAMP-2-positive vesicles.
However, the persisting Ag-BCR complexes exhibited no colocalization with FITC-dextran, which was introduced into the cells at
the same time as the Ag, but then subsequently chased into terminal L (Fig. 1F). These results demonstrate that persisting Ag-BCR
complexes reside within nonterminal LE. Additionally, the lack of
colocalization between persisting Ag-BCR complexes and FITCdextran (which were initially internalized into the cell at the same
time) suggests that B cells possess a mechanism to prevent the
delivery of Ag-BCR complexes to terminal L.
Trafficking of Ag-BCR complexes through DM- and DOcontaining endocytic compartments
B lymphocytes express two intracellular proteins that are known to
be intimately involved in BCR-mediated Ag processing and class
II peptide loading, DM and DO (16). Therefore, we sought to
compare the distribution of persisting Ag-BCR complexes to the
subcellular distribution of DM and DO in normal B lymphocytes.
As illustrated in Fig. 3 (A and B), upon initial Ag binding, Ag-BCR
complexes are present exclusively at the surface of the B cell,
whereas the DM and DO proteins are restricted to intracellular
compartments. However, after 1– 4 h of incubation at 37°C, a large
fraction of internalized Ag-BCR complexes is localized to endocytic vesicles that contain detectable levels of both DM and DO
(yellow vesicles in Fig. 3, E–H). Nevertheless, at these time points
internalized Ag was also detected in endocytic vesicles that did not
contain detectable levels of DM or DO protein (e.g., red vesicles
indicated by arrows in Fig. 3, F and H). In addition, at these time
points some B cells contained DM- and DO-positive vesicles that
were not accessed by detectable amounts of internalized Ag-BCR
complexes (green vesicles in Fig. 3, E–H). Unexpectedly, when
the distribution of long term (i.e., 24-h) persisting Ag-BCR complexes was compared with the distribution of DM and DO (Fig. 3,
I and J), it was observed that while long-term persisting Ag-BCR
complexes exhibit a high degree of colocalization with DM, they
exhibit a notably lower level of colocalization with DO. These
results demonstrate that some of the endocytic vesicles that contain
persisting Ag-BCR complexes are more highly enriched for the
MHC class II chaperone DM than for the negative regulator of DM
activity, DO. Moreover, these results suggest that DM and DO
have somewhat distinct steady-state distributions within the endocytic pathway of normal B lymphocytes.
Because many laboratories have demonstrated that DM and DO
are tightly associated within B lymphocytes (17–20), we were surprised by this finding. To further investigate the distribution of DM
and DO within the endocytic pathway of the B lymphocyte, we
analyzed the distribution of DM and DO within the A20 murine B
cell line. We chose the A20 model system for three reasons: 1) the
availability of a human DM transfected A20 cell line (i.e.,
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6660
The Journal of Immunology
6661
subcellular fractionation results are completely consistent with the
IFM analysis of the distribution of DM and DO shown in Fig. 4,
A–C, and conclusively demonstrate that in A20 B cells, the majority of the DM and DO molecules have distinct steady-state intracellular distributions.
Moreover, the IFM and subcellular fraction studies suggest that
DM and DO have distinct distributions within the endocytic pathway of murine B cells. Therefore, we analyzed the trafficking of
BCR-internalized Ag through DM- and DO-containing endocytic
compartments of A20 ␮WT B cells. For this analysis, we used
FIGURE 3. Localization of Ag-BCR complexes within DM- and DOcontaining endocytic compartments. HEL was bound to cell surface BCR
molecules by incubation of MD4 B cells with 100 nM HEL on ice. The
cells were then washed to remove unbound HEL and incubated at 37°C for
the time (in hours) indicated in the upper right corner of each panel to
allow internalization of HEL-BCR complexes. The B cells were then fixed,
permeabilized, and stained for Ag (using the 2D1 murine IgG1 anti-HEL
mAb followed by anti-muIgG1-btn and streptavidin-Texas Red) and either
DM (A, C, E, G, and I) or DO (B, D, F, H, and J) using rabbit anti-DM or
rabbit anti-DO followed by donkey anti-rabbit IgG-DTAF. Shown are representative results from one of four independent experiments. In all four
experiments the majority of the observed B cells displayed a staining pattern for HEL and DM or DO similar to that presented in the figure. For
example, in one representative experiment only 22 of 100 randomly scored
B cells containing 24-h persisting Ag contained one or more intracellular
vesicles that were Ag positive but DM negative. Contrastingly, when the
distribution of persisting Ag and DO was analyzed in the same sample, 76
of 100 randomly scored B cells contained one or more intracellular vesicles
that were positive for persisting Ag, but DO negative.
Downloaded from http://www.jimmunol.org/ by guest on July 12, 2017
FIGURE 4. Differential steady-state localization of DM and DO within
the A20 B cell line. A, A20huDM cells were fixed, permeabilized, and
stained with rabbit anti-murine DO (anti-H2-O) and a murine mAb against
human DM (Map.DM1) (7), followed by donkey anti-rabbit IgG-DTAF
and donkey anti-murine IgG-Texas Red. B, To label E/RE, A20 ␮WT B
cells (expressing only endogenous murine DM and DO molecules) were
allowed to bind and internalize BODIPY-labeled transferrin (Tf) as described in Materials and Methods. The cells were then fixed, permeabilized, and stained with rabbit anti-murine DO, followed by donkey antirabbit IgG-Texas Red. C, A20 ␮WT B cells (expressing only endogenous
murine DM and DO molecules) were fixed, permeabilized, and stained
with rat anti-LAMP-2 (GL2A7) (6) and rabbit anti-murine DO, followed
by donkey anti-rat IgG-DTAF as well as donkey anti-rabbit IgG-Texas
Red. Shown are representative results from one of four (DM vs DO) or five
(DO vs transferrin and LAMP-2) independent experiments. D, A20 ␮WT
B cells were homogenized, and total cellular membranes were fractionated
by Nycodenz density gradient centrifugation as previously reported (9).
The steady-state distributions of the endogenous murine DM, DO, and
class II (II) molecules were determined by SDS-PAGE and Western blotting. The distributions of various subcellular compartments within the gradient are indicated as follows (details in Ref. 9): EE/RE, Rab4-positive
EE/RE; LE, LAMP-2-positive, ␤-Hex-negative MIIC-like LEs; L, LAMP2-positive, ␤-Hex-positive (i.e., ␤-Hex activity ⱖ10 times background)
terminal lysosomes; PM, plasma membrane (major peak of class II); ER,
major peak of calnexin. All of the Western blot results shown in D are from
a single experiment. The results shown are representative of those obtained
in six (DM), five (DO), or seven (class II) independent experiments.
PROLONGED Ag PERSISTENCE IN Ag-SPECIFIC B CELLS
HRP-labeled Ag and took advantage of the observation that delivery of HRP-labeled ligands to various endocytic compartments
allows the HRP-catalyzed DAB-mediated cross-linking of the constituent proteins of those vesicles (23–25). The results presented in
Fig. 5A demonstrate that HRP-labeled Ag is rapidly and efficiently
internalized after binding to the BCR of A20 ␮WT cells. After 15
min of incubation at 37°C, the Ag has attained a steady-state distribution in which approximately half the total amount of cellassociated Ag remains at the cell surface while the other half resides within early endocytic compartments. Importantly, after an
additional 45 min of incubation at 37°C, the same relative distribution is maintained. However, after this time BCR-internalized
Ag would have gained access to latter aspects of the endocytic
pathway.
To assess the kinetics of delivery of BCR-internalized Ag to
DM- and DO-containing endocytic compartments, we analyzed the
extent of HRP-catalyzed DAB-mediated cross-linking of various
intracellular proteins at different times after initiation of BCR-mediated endocytosis of PC-BSA-HRP. As illustrated in Fig. 5B, as
soon as 15 min after initiation of endocytosis, Ag-HRP-mediated
depletion of DO attained a maximal level, consistent with the hypothesis that the DO protein in A20 ␮WT cells is most highly
enriched within the early aspects of the endocytic pathway. The
fact that Ag-HRP-mediated DAB cross-linking of DO never exceeded ⬃30% of the total cellular pool of DO suggests that a
significant portion of the DO protein within the cells resides within
the biosynthetic pathway of the cell (i.e., ER and Golgi apparatus)
and is therefore, like the ER-resident protein calnexin, not accessible to BCR-internalized Ag. This would be consistent with the
partial colocalization between DO and internalized transferrin in
A20 cells as presented in Fig. 4B as well as the partial overlap
between DO and BCR-internalized Ag observed in normal splenic
B cells (Fig. 3E). Contrastingly, Ag-HRP-mediated depletion of
both DM and the terminal L marker ␤-Hex was greatest after 60
min of incubation at 37°C, consistent with the hypothesis that the
majority of the DM protein within the endocytic pathway of A20
B cells is restricted to LE and L. The incomplete depletion of DM
and ␤-Hex suggests that either BCR-internalized Ag is unable to
access every LE and L of the cell or that insufficient time was
allowed to attain maximal DM or ␤-Hex depletion.
Taken together, the results presented in Figs. 3–5 demonstrate
that DM and DO have different steady-state distributions within
the endocytic pathway of both normal murine B cells as well as the
A20 B cell line. In A20 ␮WT B cells, DO appears to be most
highly enriched within the biosynthetic pathway and early aspects
of the endocytic pathway, whereas DM is present at highest levels
within LE and L. Moreover, internalized Ag-BCR complexes are
first delivered to EE, which have a low DM to DO ratio. Subsequently, Ag-BCR complexes are transferred to nonterminal LE,
which have a high DM to DO ratio, where these Ag-BCR complexes may persist for a prolonged period of time.
FIGURE 5. Trafficking of BCR-internalized Ag through DM- and DOcontaining endocytic compartments of A20 ␮WT B cells. A, The BCRmediated endocytosis of Ag (i.e., PC-BSA-HRP) by A20 ␮WT B cells was
analyzed as previously reported (12). Shown are the levels of total cellassociated (F), cell surface (f), and internal (Œ) Ag. The open arrows
above A indicate the time points at which the HRP-mediated DAB crosslinking of intracellular proteins was determined (B). Shown are representative results from one of four independent experiments. B, The HRPcatalyzed DAB-mediated cross-linking of intracellular B cell proteins after
various times of BCR-mediated Ag-HRP internalization. The percentage of
total detectable protein that was depleted upon addition of DAB and H2O2
is shown. Depletion of the ER resident protein calnexin never exceeded
0.5% of the total amount of calnexin. The results presented are from one of
four independent experiments. The error bars indicate the range of depletion values observed at 15 and 60 min for all four independent experiments.
Prolonged expression of antigenic peptide-class II complexes by
B cells harboring persisting Ag-BCR complexes
Although the results presented above demonstrate the prolonged
intracellular persistence of native Ag-BCR complexes within nonterminal late endocytic compartments of Ag-specific B lymphocytes, the immunological impact of this finding remains to be determined. Therefore, because BCR-mediated Ag internalization is
the first step in the pathway of Ag processing and presentation, we
analyzed the level of Ag presentation in B cells harboring persistent Ag-BCR complexes. Specifically, we used a flow cytometric
assay for Ag processing (based on the binding of the peptide-class
II-specific mAb C4H3 that specifically recognizes HEL46 – 61-I-Ak
peptide-class II complexes) (13) to analyze the duration of expression of antigenic peptide-class II complexes by Ag-specific B cells
after removal of environmental Ag.
Accordingly, normal splenic B lymphocytes were allowed to
generate HEL46 – 61-I-Ak peptide-class II complexes via either
BCR-mediated or F-P Ag processing (i.e., MD4 B cells plus 10 nM
HEL or B10 B cells plus 100 ␮M HEL, respectively). The B cells
were then washed to remove environmental Ag and were returned
to culture for various periods of time before quantitation of cell
surface antigenic peptide-class II complexes by flow cytometry. As
illustrated in Fig. 6A, removal of environmental Ag from B cells
that are generating peptide-class II complexes via F-P Ag processing results in a rapid decrease in the level of cell surface peptideclass II complexes such that 24 h after removal of Ag the level of
cell surface peptide-class II complexes has dropped by 40 – 60%.
Contrastingly, removal of environmental Ag from B cells that are
generating cell surface peptide-class II complexes via BCR-mediated Ag processing (and possess persisting intracellular antigenic
peptide-class II complexes) fails to significantly effect the level of
cell surface peptide-class II complexes such that 24 h after removal
of Ag there is essentially no decrease in the level of peptide-class
II complexes expressed by the cells (Fig. 6B). Importantly, the
prolonged peptide-class II expression by the Ag-specific B cells is
not simply a consequence of physiological changes elicited by Ag
binding to the BCR, because Ab-mediated ligation of the BCR on
B10 B cells that are generating peptide-class II complexes via F-P
Ag processing does not change the observed level of peptide-class
II cell surface persistence on these cells (Fig. 6A). Therefore, these
results demonstrate that Ag-specific B lymphocytes that are harboring persistent intracellular Ag-BCR complexes are capable of
prolonged cell surface expression of antigenic peptide-class II
complexes that are the direct result of the processing and presentation of BCR-internalized Ag.
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The Journal of Immunology
Discussion
The results presented in this report demonstrate that after internalization, Ag-BCR complexes persist for a prolonged period within
nonterminal late endocytic compartments of B lymphocytes.
Moreover, the ability of a conformation-specific mAb to recognize
the Ag within these Ag BCR complexes demonstrates that at least
a portion of the Ag within these complexes is in a native conformation. In addition, B cells that harbor persisting Ag-BCR complexes are also capable of prolonged cell surface expression of
antigenic peptide-class II complexes, which are the direct result of
the processing and presentation of BCR-internalized Ag. These
results suggest that the reason for the prolonged peptide-class II
expression by these B cells is the continued processing of the Ag
within the persisting Ag-BCR complexes and transport of newly
formed peptide-class II complexes to the cell surface. This possibility is consistent with our preliminary observation that LEs containing persisting Ag-BCR complexes also contain peptide-class II
complexes derived from the processing of this Ag (as detected
with the HEL46 – 61-I-Ak complex-specific mAb C4H3) (13) (T. A.
Gondré-Lewis and J. R. Drake, unpublished observation). Importantly, it is unlikely that the reason for the prolonged expression of
specific peptide-class II complexes on the surface of the Ag-specific B cells is due to a change in the physiology of the B cell
because of Ag-induced BCR signaling, because signaling of B
cells undergoing F-P Ag processing (with anti-BCR Abs) fails to
extend the duration of peptide-class II complex expression by
these B cells.
An additional interesting property of the persisting intracellular
Ag-BCR complexes is that they exhibit a higher degree of colocalization with the MHC class II peptide exchange catalyst DM
than with the DM modulator DO. DO is widely thought to be an
inhibitor of the peptide exchange activity of DM (18, 20), and it
has been proposed that the function of DO is to focus Ag processing in B cell onto BCR-internalized Ag (16, 26). The colocalization results presented in this report suggest that DO may achieve
this focusing by restricting DM-mediated class II peptide loading
to intracellular compartments where Ag-BCR complexes persist
for a prolonged period of time, allowing a greater time for the
processing of BCR-internalized Ag and increasing the efficiency
by which antigenic peptides are loaded onto MHC class II molecules. However, this observation raises the question of how DM
and DO (two proteins that are known to associate within the ER of
the B cell) (19) establish different steady-state intracellular distributions within the endocytic pathway of these cells.
Although the limited number of published reports on the intracellular distribution of DM and DO in nontransfected B lymphocytes suggests that DM and DO have similar intracellular distributions (19, 26), the precise distributions of these two proteins
within the endocytic pathway of normal B cells and the ability of
the BCR to deliver Ag to these subcellular compartments remain to
be extensively studied. Although double-label IFM analysis of murine splenic B cells suggests that DM and DO have an overlapping
intracellular distribution (Fig. 1 in Ref. 19), the relative intensity of
staining of individual vesicles for DM and DO was variable, suggesting that in some cases there may be differences in the intracellular distributions of these two proteins. Moreover, it is known
that B cells produce DM in excess over DO (leading to the presence of both DM-DO complexes as well as free DM molecules)
(17, 18). Therefore, it is possible that (as already suggested by
Alfonso and Karlsson; Ref. 16) “free DM and DM-DO complexes
may be sorted to different compartments of the endosomal/lysosomal system.” Furthermore, DM and DO molecules may access
the endocytic pathway as DM-DO complexes, which subsequently
dissociate, allowing the active intracellular targeting motifs in the
cytoplasmic tails of DM (27) and DO (28) to establish distinct
steady-state distributions of the two dissociated proteins. Therefore, although the results presented in this report do not distinguish
between these or other possibilities, they do demonstrate that
BCR-internalized Ags access DO- and DM-enriched endocytic
compartments with distinct kinetics and show that persistent intracellular Ag-BCR complexes exhibit a higher degree of colocalization with DM than with DO.
Finally, the prolonged intracellular persistence of Ag-BCR complexes within nonterminal LE raises the question of the molecular
mechanism of intracellular trafficking of this receptor-ligand complex. Unlike Ag internalized by F-P endocytosis (which is rapidly
delivered to terminal L for degradation), internalized Ag-BCR
complexes are delivered to a nonterminal LAMP-2-positive LE
where they persist for ⬎24 h. Although we do not know the precise
molecular mechanism behind this phenomenon, it is likely to involve the actin-based cytoskeleton, because cytochalasin B treatment of B cells containing persistent Ag-BCR complexes results in
the colocalization of Ag-BCR complexes and FITC-dextran within
terminal L (without causing complete mixing of LAMP-2 and
FITC-dextran) (T. A. Gondré-Lewis and J. R. Drake, unpublished
observation). Because LE to L trafficking appears to involve LE/L
fusion and formation of a hybrid organelle followed by hybrid
organelle fission to regenerate LE and L (29), it is possible that the
actin-based cytoskeleton is necessary for the sorting of Ag-BCR
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FIGURE 6. Prolonged cell surface expression of antigenic peptide-class
II complexes on Ag-specific B cells harboring persistent Ag-BCR complexes. A, B10 B cells were incubated in 100 ␮M HEL (either without
(open icons) or with (filled icons) 10 nM anti-␮ Ab) overnight to allow for
the F-P endocytosis of HEL and cell surface expression of HEL peptideclass II complexes. The cells were then washed to remove environmental
Ag, returned to culture at 37°C for the indicated chase periods, and removed onto ice, and the level of cell surface HEL46 – 61-I-Ak complexes
was determined by staining with the C4H3 mAb, followed by analysis via
flow cytometry. Shown is the level of detectable cell surface peptide-class
II complexes, where the initial level of expression has been normalized to
a value of 1 to allow direct comparison between experiments. Each line
represents the results obtained from a single independent experiment. B,
MD4 B cells were incubated in 10 nM HEL overnight to allow the BCRmediated endocytosis of HEL and cell surface expression of HEL peptideclass II complexes. The cells were then washed to remove environmental
Ag, returned to culture at 37°C for the indicated chase periods, and removed onto ice, and the level of cell surface HEL46 – 61-I-Ak complexes
was determined by staining with the C4H3 mAb, followed by analysis via
flow cytometry. Shown is the level of detectable cell surface peptide-class
II complexes, where the initial level of expression has been normalized to
a value of 1 to allow direct comparison between experiments. Each line
represents the results obtained from a single independent experiment. Results from concurrent experiments are denoted by the same icon.
6663
6664
complexes into reforming LE and/or exclusion of the Ag-BCR
complexes from reforming L. Presently, we are working to further
examine the molecular mechanism of Ag-BCR persistence within
nonterminal LE.
Acknowledgments
We thank Joseph Diehl for technical assistance, Sebastian Amigorena for
the gift of the rabbit anti-H2-M␤ antiserum, Lisa Denzin for the gift of
rabbit anti-HLA-DM␤ antiserum and Map.DM1 anti-human DM mAb,
Lars Karlsson for the gift of rabbit anti-H2-O␤ antiserum, Ralph Kubo for
the gift of rabbit anti-I-Ad antiserum, Ari Helenius for the rabbit anticalnexin antiserum, Dennis Metzger for the gift of the 2D1 hybridoma, and
Lee Lesserman for the gift of the HyHEL10 hybridoma.
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PROLONGED Ag PERSISTENCE IN Ag-SPECIFIC B CELLS