| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Centre d’Immunologie de Marseille-Luminy, 13288 Marseille cedex 9, France
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
- or
ß-chains, we further show that when an Ag is internalized by the BCR,
the cytoplasmic tails of class II molecules differentially control the
presentation of antigenic peptides to specific T cells depending upon
the importance of proteolytic processing in the production of that
peptide. Integrity of the cytoplasmic tail of the I-Ak
ß-chain is required for the presentation of the hen egg lysozyme
determinant (46–61) following BCR internalization, but that dependence
is not seen for the (34–45) determinant derived from the same protein.
The tail of the ß-chain is also of importance for the dissociation of
invariant chain fragments from class II molecules. Our results
demonstrate that Ags internalized through the BCR are targeted to
compartments containing newly synthesized class II molecules and that
the tails of class II ß-chains control the loading of determinants
produced after extensive Ag processing. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The differential sensitivity of class II Ag presentation to protein synthesis inhibitors, Ii, and H2-M expression that was reported for a collection of antigenic determinants suggests the existence of multiple processing pathways 11, 12, 13, 14, 15 . In parallel with a classical pathway requiring newly synthesized class II molecules, an alternative pathway of presentation involves mature class II molecules that are expressed at the cell surface; these mature molecules recycle through endosomes, where they become available for peptide binding 16, 17 . Peptide loading of newly synthesized and mature class II molecules occurs in distinct endocytic compartments with different intracellular pH and proteolytic activities 18, 19 . Within an Ag, buried or cryptic determinants are usually presented by the classical Ii-dependent pathway involving newly synthesized class II molecules, whereas more superficial determinants can be loaded onto class II molecules recycled from the cell surface independently of Ii 18 .
In addition to the differential effects of inhibitors of protein
synthesis, depending upon the Ag, APCs transfected with class II
molecules lacking the cytoplasmic domain of the ß-chain have also
been shown to be defective in the presentation of some but not all
antigenic determinants 20, 21, 22, 23 . Truncation of the class II and ß
cytoplasmic tails interferes with endocytosis of class II molecules and
with the Ii-independent presentation of some protein determinants. This
finding is consistent with the involvement of class II recycling during
the presentation of these peptides 24, 25 . The studies described
above are derived from a diverse group of Ags that are presented by
multiple cell types. In addition, the role of Ii and of class II
cytoplasmic domains in Ag presentation has been analyzed using Ags
internalized via pinocytosis only; no information is available on Ags
targeted through the B cell receptor (BCR). Ag-specific B cells present
determinants derived from cognate ligands to MHC class II-restricted
CD4+ T cells at concentrations far below those required for
nonspecific B cells 26 . BCR-mediated Ag processing and presentation
are highly efficient as a consequence of the linked roles of the BCR
with regard to the delivery of Ag to the class II peptide-loading
compartment and to signaling. Signals transduced through the BCR
induce changes in the intracellular localization of class II molecules
and affect the Ag-presentation pathway 27, 28 . Because Ags targeted
through the BCR follow an intracellular pathway that is distinct from
that of Ags internalized from the fluid phase 29 , it was of interest
to define the effects of inhibitors of protein synthesis and the role
of class II cytoplasmic domains in the presentation of such Ags.
Our results show that when Ag is taken up via the BCR, the resulting peptides, including the so called Ii-independent peptides, are presented by newly synthesized class II molecules. The differential requirement for MHC class II cytoplasmic domains for the presentation of certain Ag-derived peptides also depends upon whether Ags are endocytosed via pinocytosis or via the BCR. The cytoplasmic tail of class II ß-chain is required for the targeting of class II molecules to compartments in which Ii-dependent antigenic determinants are loaded after BCR-mediated Ag uptake.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We used CBA/J mice that were transgenic for rearranged Ig heavy and light chain genes encoding a high-affinity anti-hen egg lysozyme (HEL) Ab 30, 31 . B cells from these transgenic (Tg) mice express this BCR and secrete an Ab specific for HEL. Splenic B cells were purified from these Tg mice or from CBA/J control mice after T depletion using anti-Thy-1, anti-CD4, and anti-CD8 Abs 32 plus complement; afterward, these cells were immediately used in Ag-presentation assays.
The I-A-negative B lymphoma cell line M12C3 33 was transfected with
genomic DNA encoding either intact Ak and
Aßk chains or those I-Ak chains with
truncated cytoplasmic domains 21 . The transfectants, which were
generously provided by W. Wade (Department of Microbiology, Dartmouth
Medical School, Lebanon, NH), have the following MHC class II
phenotypes: wt/ßwt (I-Ak), Cyt-12/ßwt (
cytoplasmic domain-truncated I-Ak molecule (T)), and
wt/ßCyt-18 (ß cytoplasmic domain-truncated I-Ak
molecule (ßT)). Truncation of the or ß cytoplasmic domains was
verified by blotting total lysates with Abs specific for MHC class II
or ß cytoplasmic domains (data not shown).
The I-Ak-restricted, HEL-specific T cell hybridomas 3A9 and 3B11 were generously provided by L. Adorini (Roche Milano Ricerche, Milan, Italy). The 3A9 T cells recognize an Ii-dependent determinant present on the 46–61 peptide 34, 35 , and the 3B11 hybridoma recognizes the HEL 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 peptide independent of Ii 14 . The TS12 T cell hybridoma 36 that was also used in Ag-presentation assays is I-Ak-restricted but specific for the RNase A (43–56) peptide. IL-2-dependent CTLL-2 cells were obtained from the American Type Culture Collection (ATCC) (Manassas, VA). Cell lines were cultured in DMEM (Life Technologies, Gaithersburg, MD) supplemented with 10% FCS, 20 µM 2-ME, 1 mM sodium pyruvate, and 1 mM glutamine.
Abs and reagents
The mouse hybridoma 10.2.16 producing an
I-Ak-specific IgG2a mAb and the anti-mouse Fc
RII/III
2.4G2 mAb were obtained from ATCC. The IgG1 mAb HyHEL-10 recognizes an
epitope on intact HEL 37 . The affinity-purified rabbit polyclonal Ab
specific for the cation-independent mannose 6 phosphate receptor (M6PR)
(produced by B. Hoflack, Pasteur Institute, Lille, France) was a
generous gift of S. Meresse (Centre d’Immunologie de
Marseille-Luminy). The rabbit polyclonal anti-Ii or
anti-H-2Mß Abs were raised against synthetic peptides
corresponding to sequences in the cytoplasmic domains of mouse Ii
(Cyt.Ii) or H-2Mß (Cyt.Mß) 27, 38 . Rabbit polyclonal
anti-CLIP serum (CLIP) was produced following injection with a
peptide corresponding to the mouse Ii (80–104) sequence 6 . The
secondary reagents (donkey anti-mouse Igs, anti-rabbit Igs, and
streptavidin) coupled to FITC or Texas Red that were suitable for flow
cytometry and immunofluorescence as well as unlabeled rabbit
anti-mouse IgG (RAMIG) to bind the BCR were purchased from Jackson
ImmunoResearch (West Grove, PA). Human transferrin (Tf) (Sigma,
St. Louis, MO) was conjugated to FITC and purified on a G25
column. HEL and RNase A were obtained from Sigma.
Liposomes
Liposomes (40 µM total lipids) were composed of 65 mol% dimyristoyl phosphatidyl choline (Sigma), 35 mol% cholesterol (Sigma), and 1 mol% phosphatidylethanolamine derivatized with the heterobifunctional cross-linking agent N-succinimidyl-3-(2-pyridyldithio)propionate, as described previously in detail 39 . These liposomes were prepared in a solution containing 10 mM carboxyfluorescein (Molecular Probes, Eugene, OR), a fluorescent marker, and 5 mg/ml HEL or RNase A in PBS. They were frozen and thawed five times and passed through polycarbonate filters (Nucleopore, Pleasanton, CA) of 80-nm pore size using an Extruder (Lipex Biomembranes, Vancouver, Canada). Liposomes were extruded 10 times until a clear solution was obtained. Unencapsulated material was removed by gel filtration over Sepharose 4B columns. Because surface expression of IgG was comparable (data not shown) between the different transfectants, we used IgG as a target for the Ag encapsulated in liposomes. Liposome-protein A (Lip-PA) were covalently coupled to N-succinimidyl-3-(2-pyridyldithio)propionate-modified (10 mol/mol) protein A from Streptococcus aureus (Pharmacia, Uppsala, Sweden). When incubated in the presence of 10 µg/ml of RAMIG, which binds to protein A, the Lip-PA preparation targets the Ag through the BCR. Liposomes were sterilized by filtration through 0.45-µm Acrodisc filters (Gelman Sciences, Ann Arbor, MI). The concentration of HEL or RNase A was determined by the known molar ratios of these proteins to the coencapsulated fluorescent marker. Liposomes of this size are known to be taken up in coated pits in lymphoid cells 32 .
Ag presentation
A total of 5 x 104 APCs were incubated with or without various doses of Ags diluted from a stock solution of free Ag or Lip-PAs containing encapsulated Ag at 5 mg/ml that were targeted with RAMIG or left untargeted. The APCs were cocultured for 24 h at 37°C with 105 T cell hybridomas. Inhibition of the protein synthesis by cycloheximide (CHX) was realized as follows: splenic B cells and I-Ak-expressing B lymphoma cells were incubated with or without 100 µM or 200 µM of CHX, respectively, for 2 h before adding the different preparations of Ags. Cells pulsed with the Ags for 4 h were washed and subsequently fixed for 30 s with 0.05% glutaraldehyde. A total of 5 x 104 fixed APCs were cocultured for 24 h at 37°C with 105 T cells. IL-2 production in culture supernatants was measured using thiazolyl blue (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (Sigma) to evaluate the growth of the IL-2-dependent CTLL-2 cell line 40 .
SDS-PAGE of surface class II complexes
A total of 15 x 106 cells were labeled by lactoperoxidase-catalyzed iodination and lysed in lysis buffer (1% Nonidet P-40, 150 mM NaCl, 1 mM EDTA, and 50 mM Tris-HCl (pH 7.5)) containing protease inhibitors. Lysates were precleared with protein A-Sepharose beads (Pharmacia) and I-Ak immunoprecipitated with 10.2.16 mAb. Immunoprecipitates were extensively washed. Before electrophoresis on an SDS-polyacrylamide (12.5%) gel, the immunoprecipitated material was divided into two aliquots: the first aliquot was fully denatured at 95°C for 5 min (B); the second aliquot was incubated for 1 h at room temperature (NB) in SDS sample buffer containing 5% 2-ME to preserve the peptide-loaded, compact, MHC class II heterodimers (compact forms (CFs)) 27 .
Western blotting
Washed cells in ice-cold PBS were solubilized in 1 ml of lysis
buffer and incubated for 2 h with 10.2.16 mAb bound to the protein
A-Sepharose beads. Next, immunoprecipitates were washed and pellets
were suspended in reducing sample buffer. Immunoprecipitates were
boiled in SDS and run on 15% SDS-PAGE. After transfer onto an
Immobilon-P membrane (Millipore, Bedford, MA), proteins were blotted
with Cyt.Ii or CLIP Abs. After washing, membranes were incubated
with anti-rabbit Ab conjugated to horseradish peroxidase (Jackson
ImmunoResearch). Labeled proteins were detected using the enhanced
chemiluminescence immunodetection kit (Amersham).
Flow cytometry and immunofluorescence
Staining for flow cytometry analysis was performed as follows:
1.5 x 105 cells/sample were incubated for 30 min on
ice with fluorescent HEL-Lip-PAs in the presence or absence of RAMIG
(10 µg/ml), or with 10.2.16 mAbs (1 µg/ml) in PBS containing 5%
FCS and 0.02% NaN3 (FACS buffer) with 20% 2.4G2 mAb
supernatant and 5% normal mouse serum to prevent binding to
FcRII/III. After washing in FACS buffer, cells labeled with
nonfluorescent 10.2.16 Ab were incubated for 30 min on ice with the
FITC-coupled secondary Ab. Before analysis, cells were washed
extensively in FACS buffer and fixed with 1% paraformaldehyde in PBS.
Samples were analyzed using a FACScan apparatus (Becton Dickinson,
San Jose, CA).
Internalization of the HEL encapsulated in liposomes and determination of the expression of class II molecules and Ii in different cellular compartments were performed by confocal immunofluorescence microscopy as described previously 27 . To demonstrate HEL internalization, we incubated cells with HEL-Lip-PAs in the presence of RAMIG for 4 h prior to immunofluorescence as described above. To label early endosomal compartments, FITC-coupled Tf was exposed to the cells for 20 min at 37°C before the fixation and labeling of other internal molecules. Confluent cells that had been treated with liposomes or Tf or left untreated and cultured on glass coverslips were fixed with 4% paraformaldehyde, permeabilized with 0.05% saponin in PBS, and incubated for 30 min with primary Abs. Under these conditions, the liposome-associated carboxyfluorescein is released and does not interfere with the detection of fluorescent Abs. After washing, the cells were incubated for 30 min with secondary labeled Abs and then washed. The coverslips were mounted onto glass sides with Mowiol. Confocal microscopy was performed using the TCS 4D confocal laser scanning microscope (Leica Lasertechnik, Heidelberg, Germany) interfaced with an argon/krypton ion laser.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To determine whether the presentation of Ags targeted through the
BCR is sensitive to protein synthesis inhibitors, we compared the
presentation efficiency of HEL by splenic B cells that expressed or did
not express a HEL-specific BCR with several HEL-specific T cell
hybridomas in the presence or absence of CHX. Splenic B cells were
isolated from either CBA/J control mice or Tg mice carrying genes
encoding a high-affinity anti-HEL Ab 30, 31 . The presentation of
the 46–61 determinant derived from extensive proteolysis of soluble
HEL to the I-Ak-restricted 3A9 T cell hybridoma requires
newly synthesized class II molecules and Ii expression 14 . Other
determinants, such as the peripheral HEL 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 epitope, can
associate with mature class II molecules and be presented to the 3B11 T
hybridoma independent of protein synthesis 14 . We observe here that
the presentation of soluble HEL targeted by the BCR in Tg splenic B
cells was improved for the two epitopes studied compared with the
presentation of HEL that was internalized nonspecifically (Fig. 1
, A and C). The
presentation of the synthetic HEL (46–61) peptide to the 3A9 hybridoma
is comparable between Tg and control splenic B cells (Fig. 1B), which is consistent with the equivalent expression
level of I-Ak and costimulatory molecules of normal and Tg
splenic B cells (data not shown).
|
A), whereas the presentation of the isolated HEL
(46–61) synthetic peptide to the same T cell hybridoma by Tg or
control B cells was not affected by CHX (Fig. 1B). However,
presentation of the 34–45 determinant derived from intact HEL to the
3B11 T cells was sensitive to CHX in a manner that depended upon the
pathway of HEL uptake. Despite the effects of CHX on overall protein
synthesis, the fact that the differential effect of this drug was
dependent upon the HEL-derived peptide or on its mode of entry into the
APC indicates that there are no effects of CHX on class II molecules
expressed at the cell surface or on other molecules required for the
recognition of these Ags by the T cells. The effect of CHX on
the presentation of certain epitopes consequently appears to result
essentially from the inhibition of class II synthesis and transport,
although effects on any protein in the Ag-presentation pathway cannot
formally be ruled out. The 34–45 epitope can be presented in
association with mature class II molecules recycled from the cell
surface in a manner independent of protein synthesis, when the Ag is
taken up from the fluid phase. Presentation of the 34–45 epitope
became sensitive to CHX when HEL was targeted via the BCR (Fig. 1C). This result indicates that the targeting of an Ag via
the BCR forces its presentation by newly synthesized class II
molecules. Binding, internalization, and presentation of liposome-encapsulated Ags in B cell lines
We extended our observations to B lymphoma cell lines to evaluate
the presentation of particulate Ags targeted through the BCR by intact
or truncated MHC class II molecules. The cell lines used express
surface IgG, which is not specific for HEL. To evaluate the role of
surface Ig in the presentation of HEL, we encapsulated HEL in liposomes
that were covalently coupled to protein A. Lip-PAs were targeted
to B cell surface IgG in the presence of RAMIG, an Ab which binds both
protein A and surface IgG. In the present study, the Ag itself is not
complexed to an Ab. The use of targeted liposomes in which the Ags are
passively entrapped has the advantage of facilitating the uptake of Ag
without affecting its processing, as for cross-linked Ags and immune
complexes 19, 41, 42 . To analyze the liposome-targeting efficiency,
we initially used FACS analysis to compare the binding of protein
A-bearing, HEL containing liposomes (HEL-Lip-PAs) with B cells in the
presence or absence of RAMIG (Fig. 2A). As expected, B cells were
only able to bind the targeted liposomes. Second, we determined whether
targeted HEL-Lip-PAs are internalized in B cells. The intracellular
localization of HEL internalized by BCR was performed by confocal
microscopy and immunofluorescence labeling of the Ag using an
anti-HEL mAb. The Ag encapsulated in liposomes targeted to the BCR
accumulated in large intracellular structures after 4 h (Fig. 2B). Intact Ag (revealed with the HyHEL-10 mAb) did not
appear to colocalize with the MHC class II-enriched compartment that
was induced in response to B cell activation 27 .
|
C). The HEL encapsulated in Lip-PAs targeted to the BCR via
RAMIG was more efficiently presented than soluble HEL or nontargeted
HEL-Lip-PAs. Incubation with anti-FcR mAb did not influence the
BCR targeting of encapsulated HEL or its presentation, indicating that
liposomes are not internalized via the FcR. Thus, the presentation
of liposome-encapsulated Ags is augmented by BCR-mediated uptake in B
lymphoma cells, as is the uptake of free Ag in B splenocytes expressing
an HEL-specific BCR.
We assessed whether CHX inhibits the presentation of Ags encapsulated
in liposomes targeted through the BCR (Fig. 3). As seen for splenic B cells, the
presentation of HEL-Lip-PAs to the 3A9 T cell hybridoma was sensitive
to CHX independent of its targeting by the BCR (Fig. 3A).
The presentation by B lymphoma cells of liposome-encapsulated HEL to
the 3B11 T cell hybridoma (Fig. 3B) was resistant to CHX in
the absence of BCR targeting. The same results were obtained with
soluble Ags (data not shown). However, when liposomes are targeted via
the BCR, the presentation of the HEL 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 epitope (Fig. 3B) became sensitive to CHX. Liposomes are present in excess
relative to the number of binding sites on the cell. The excess
liposomes are not removed in a washing step. Those liposomes that are
not binding are capable of being taken up by fluid phase endocytosis,
albeit with reduced efficiency. This result is consistent with the data
presented for the 3B11 hybridoma in Fig. 1. The same pattern of
sensitivity to CHX depending upon BCR targeting was observed for the
RNase A (43–56) epitope (Fig. 3C). These results indicate
that, as for soluble Ags bound to the BCR, the targeting of particulate
Ags through the same receptor directs their presentation to newly
synthesized class II molecules. The engagement of the BCR by Ag
probably transports the Ag beyond the limits of the recycling
pathway.
|
We further analyzed whether the integrity of class II molecules
affects the presentation of BCR ligands. We used B lymphoma cell lines
expressing either full-length I-Ak class II molecules as
described above or I-Ak class II molecules lacking the
cytoplasmic domains of the -chain (T) or the ß-chain (ßT).
FACS analysis revealed that the binding of IgG targeted-liposomes to
cells expressing intact I-Ak molecules or T and ßT
class II molecules was comparable; confocal microscopy indicated that
HEL-loaded liposomes were internalized in all transfectants (data not
shown). Using HEL-Lip-PAs internalized by pinocytosis in the absence of
RAMIG, we repeatedly observed that B cells bearing intact
I-Ak class II molecules present the 46–61 peptide more
efficiently than transfectants expressing I-Ak lacking
either cytoplasmic domain (Fig. 4A). We also noticed that T
cells are slightly more potent APCs for the HEL (46–61) epitope than
ßT transfectants in the absence of Ag targeting. The presentation of
HEL (46–61) by full-length and T molecules was improved by
targeting the liposomes to the BCR (Fig. 4B). However,
despite the equivalent ability of class II molecules lacking their ß
cytoplasmic domain to present the synthetic HEL (46–61) peptide (Fig. 4C), ßT molecules were unable to present the same
determinant following BCR targeting of the intact Ag, indicating that
truncation of class II ß-chain affects the presentation of an epitope
localized in the protein core.
|
, D and E). As shown
previously for free Ags in murine cells 25 , we confirm here using
particulate Ags that this alternative presentation pathway depends upon
the integrity of the and ß class II cytoplasmic domains. Murine B
cells expressing intact I-Ak class II molecules present
proteins internalized via nonspecific pinocytosis more efficiently than
transfectants expressing I-Ak lacking either of the two
cytoplasmic domains (Fig. 4D). The targeting of encapsulated
HEL via the BCR improved the presentation efficiency of all
transfectants and was able to restore the presentation of the HEL
34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 epitope by cytoplasmic domain truncation mutants of class II
molecules to a level that was comparable with that of intact
I-Ak molecules (Fig. 4E). Therefore,
BCR-targeted Ags can reach intracellular class II molecules that are
not accessible to peptides derived from fluid phase Ags. Defects in peptide loading and intracellular distribution of cytoplasmic truncation mutants of I-Ak molecules
To complement our Ag presentation results with MHC class II
surface expression and peptide-loading ability in the different B cell
transfectants, we performed FACS analysis, immunoprecipitation of
surface-iodinated class II molecules, and Western blotting of the MHC
class II-associated Ii. Comparable surface expression of
I-Ak class II molecules was detected on the different
transfectants (Fig. 5A).
However, a marked reduction in the proportion of endogenous
I-Ak CFs was revealed with surface iodination in T B
cells as was a slight reduction in ßT B cells (Fig. 5B).
These CFs, which are resistant to denaturation in SDS detergent at
25°C, reflect the presence of class II heterodimers loaded with
peptides derived from intrinsic proteins or serum components.
35S pulse-chase labeling indicated that the truncation of
either cytoplasmic domain delayed the appearance of peptide-loaded MHC
class II CFs (data not shown). Whereas the proportion of endogenous
compact class II molecules is higher at the surface of ßT than T B
cells, HEL (46–61) presentation efficiency is higher for T than for
ßT B cells. This discrepancy could be explained by a difference in
the access of truncated class II molecules to peptide loading
compartments or by a difference in the conformational stability of
these molecules. Identification of the intermediate Ii fragments
bound to class II molecules is instrumental to follow class II
transport and peptide loading in B cells 27 . In this study, we
monitored the pattern of the full-length Ii (p31) and Ii (p12)
degradation products associated with class II molecules to follow class
II maturation (Fig. 5C). Class II molecules are not
prominently associated with p12 Ii fragments in B cells expressing
either intact or T class II molecules, which can present BCR ligands
(Fig. 5C). Truncation of the I-Ak ß
cytoplasmic domain strongly increased the level of association of the
CLIP-containing p12 Ii fragments. To demonstrate the specificity of the
different bands observed on the gels corresponding to
I-Ak-associated proteins, we used the 10.2.16 mAb for
immunoprecipitation of the non-I-Ak-expressing parental
M12C3 line as a negative control. None of the above bands were observed
(data not shown). A slightly higher m.w. band to p31, which we have not
identified, coprecipitates with class II molecules on these gels.
|
Cyt.Ii Ab. In cells
expressing full-length I-Ak or T molecules (Fig. 6, I-Ak and T lanes), Ab
to these molecules was mainly localized at the cell surface, whereas Ab
to Ii showed a reticulated intracellular staining consistent with an
endoplasmic reticulum expression pattern. In these cells, MHC class II
molecules and Ii are not detected in endolysosomal compartments at the
steady state (Fig. 7 and data not shown).
In ßT B cells, class II molecules are expressed at the cell surface
but are also concentrated with Ii in intracellular compartments (Fig. 6, ßT lane). To determine in which intracellular compartments class
II and Ii accumulate in ßT cells, we performed double
immunofluorescence with different markers specific for various
endocytic compartments: Tf is internalized in early endosomes, M6PR
accumulates in late endosomes, and H-2M is present in lysosome-related
compartments 43 . With such markers, we observed (Fig. 7) that class
II and Ii molecules, which colocalize only slightly with internalized
Tf, accumulate preferentially in late endosomal compartments labeled
with the anti-M6PR Ab and do not colocalize with H-2M. This
intracellular accumulation of class II/Ii complexes in late endosomes,
which correlated with the accumulation of Ii p12 fragments (Fig. 5) and
with an impairment of HEL (46–61) presentation (Fig. 4), suggests that
the ß cytoplasmic domain of class II molecules is involved in the
transport of I-Ak molecules to compartments in which Ii is
terminally degraded and in which class II molecules can be loaded with
epitopes derived from protein cores. The class II ß cytoplasmic
domain allows class II molecules to quit late endosomes to reach deep
lysosome-related compartments, which also contain H-2M.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). We extended these observations to B lymphoma
cell lines in which surrogate Ag-specific presentation could be
achieved by encapsulating Ag in liposomes and targeting it with Ab
directed at the non-HEL-specific cell surface Ig expressed by the lines
(Figs. 2 and 3). Presentation of the Ags encapsulated in liposomes and
targeted to surface IgG is also markedly improved compared with
nontargeted Ags, but to a lesser extent (50- to 100-fold) than in
splenic B cells. Using this system, we found that the presentation of
peripheral determinants, such as the HEL 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 and RNase A (43–56)
epitopes, also became dependent upon protein synthesis. This
experimental system permitted us to use cells transfected with genes
encoding truncated class II molecules and to analyze the role of class
II and ß cytoplasmic domains in the presentation of different
antigenic determinants following fluid phase or BCR-mediated uptake of
Ag by transfected B lymphoma cells. Role of MHC class II tails in the presentation of fluid phase Ags
Previous studies performed in human fibroblasts and B cells 24
or in transfected rat blood leukemia (RBL) cells 25 showed that Ag
presentation was differentially affected by class II cytoplasmic domain
truncations, depending upon the nature of the Ag 19 or the
determinant presented within a protein when Ags were taken up from the
fluid phase. Class II cytoplasmic domains are essential to promote the
internalization of surface class II molecules in these cells 24 . In
agreement with these results, we observed that the alternative
presentation pathway of the HEL 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 epitope was equally affected
by the truncation of or ß class II cytoplasmic domains in murine
B cells, even when this Ag was encapsulated in nontargeted liposomes
(Fig. 4, D and E).
However, we observed that the presentation of the 46–61 epitope
derived from intact HEL was also sensitive to the truncation of class
II cytoplasmic tails of - and ß-chains, with a stronger inhibition
observed in truncated ß-chain transfectants (Fig. 4A). Our
results suggest that the ßT cytoplasmic domain is important in
directing class II molecules to compartments involved in the processing
of core epitopes such as HEL (46–61) peptide. In contrast to this
observation, presentation of the HEL (46–61) epitope to the same T
cell hybridoma used here was not affected by truncation of the
cytoplasmic tail of the MHC class II ß-chain transfected with Ii into
RBL cells 25 . This difference could have several explanations. First,
Ag taken up by fluid phase endocytosis by cells transfected with
wild-type class II molecules was presented at least an order of
magnitude more efficiently by the mouse lymphoma used here than by the
rat RBL cells used in that study. Second, RBL cells retain wild-type
class II molecules in intracellular compartments, whereas our murine B
lymphoma transfectants express wild-type class II molecules mostly at
the cell surface (Fig. 6). In APCs in which newly synthesized class II
molecules accumulate intracellularly before being exported to the
surface, truncation of the ß cytoplasmic tail could be of minor
importance. Such class II molecules may reside long enough in late
endosome-related compartments to meet core epitopes. In murine B cells,
truncation of the ß cytoplasmic domain induced an intracellular
accumulation of class II and Ii molecules in compartments in which the
M6PR colocalized (Fig. 7). These cells had a reduced ability to present
the 46–61 peptide derived from HEL, suggesting that the access of ßT
class II molecules to compartments in which Ii is degraded and core
epitopes are produced is regulated differentially in RBL cells and in B
lymphoma cells. In addition, the nature of the compartments in which
class II molecules accumulate constitutively or as a consequence of
ß-chain truncation could be different in both cells.
Influence of BCR targeting on Ag presentation by truncated class II molecules
Our understanding of the role of class II cytoplasmic domains in
different Ag-presentation pathways is based essentially on studies
performed with Ags internalized from the fluid phase. However, the
capture of Ags by B cells is mostly mediated through BCR recognition.
When HEL was targeted by the BCR, we observed a strong augmentation in
the presentation efficiency of the HEL-derived (46–61) epitope,
compared with HEL internalized via the fluid phase (Fig. 4). Ag
targeting to the BCR rescued the presentation of HEL by T class II
molecules to a level that was equivalent to that seen for cells
expressing intact I-Ak class II molecules for Ag taken up
by fluid phase endocytosis. Nevertheless, improved presentation of HEL
(46–61) epitope by ßT class II molecules was not observed under the
same BCR-targeting conditions. The data obtained using the same Ag,
APC, and T cell hybridoma recognizing the HEL-derived peptide 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45
indicate that before the molecules pass into a compartment that is
dependent upon the I-Ak ß-chain, they have access to
compartments which do not require the presence of the I-A ß
cytoplasmic domain.
Whereas the cytoplasmic domain of the class II ß-chain is involved in
the transport of newly synthesized class II molecules into deep
endosomal compartments, the cytoplasmic domain is not necessary for
intracellular class II transport. This finding is supported by
immunofluorescence analysis of class II distribution in the T cells,
which is similar to that seen in the wild-type molecule. The
cytoplasmic domain does not influence the Ii-p12 release from class II
molecules, because an equal quantity of p12 remains associated with
T and full-length I-Ak molecules. The reduced Ag
presentation seen in T cells with Ags internalized via the fluid
phase could result from a conformational instability of these truncated
T molecules to bind peptides; this instability is partially
overcome by a concentration of Ags following BCR internalization in
specific compartments, although the precise cause of the reduced
presentation remains unknown. BCR-mediated uptake directs Ags for
presentation in a manner that is dependent upon protein synthesis, as
shown by the sensitivity to CHX of HEL presentation to T hybridomas,
regardless of the peptide presented (Figs. 1 and 3). As well as acting
on routes of Ag internalization, BCR engagement could also control Ag
presentation by acting on the maturation pathway of newly synthesized
class II molecules 27 .
Relationships between MHC class II molecule transport and Ag-processing compartments
In ßT B cells, the degradation pattern of Ii shows that Ii-p12
fragments are associated with accumulated intracellular class II
molecules (Fig. 5). ß-chain truncation consequently blocks the access
of class II molecules to compartments in which class II/p12 fragment
complexes are processed into Ii-p10 fragments and class II/CLIP
complexes and in which the HEL (46–61) peptide is generated and/or
loaded onto class II molecules. The di-leucine motifs present in the
cytoplasmic domain of the class II ß-chain have been shown to
regulate Ag presentation by acting on the intracellular transport of
class II molecules 25 ; these motifs act in conjunction with the two
di-leucine motifs present in the cytoplasmic tail of Ii, which are
essential for the transport of class II-Ii complexes to endosomal
vesicles 44 . Moreover, the di-leucine motif of the ß-chain is
required for Ii-independent presentation following fluid phase Ag
uptake 25 . Our results show that the cytoplasmic domain of class II
ß-chains controls the access of newly synthesized class II molecules
to compartments, probably lysosome-related, in which class II molecules
achieve their maturation and in which the Ag internalized by the BCR is
terminally processed.
Our results are consistent with the model illustrated in Fig. 8. Mature class II molecules recycling
through endosomal compartments (compartment 3) in a protein
synthesis-independent but I-A- and ß-chain-dependent manner can
bind peptides such as HEL 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 when Ags are internalized via
pinocytosis. This peripheral compartment seems inaccessible to Ags
endocytosed through the BCR. BCR ligands either are directly targeted
to specialized endosomal compartments (compartment 1) or pass through
early endosomal compartments (compartment 3) and are only released from
immune complexes in late endosomal compartments (compartment 1). These
late endosomal compartments contain newly synthesized class II
molecules that are delivered in a manner not requiring the integrity of
their and ß cytoplasmic domains. These compartments permit mild
Ag degradation or are in continuity with compartments having this
capacity 45 . Class II ß cytoplasmic tails control access to deeper
lysosome-related compartments (compartment 2) and the presentation of
peptides requiring extensive Ag degradation. In addition to the effect
of BCR engagement on the intracellular transport of newly synthesized
class II molecules 27 , our results show that BCR engagement also
targets Ags into compartments that are only accessible to these newly
formed class II molecules. The dual role of BCR in Ag internalization
and in cell signaling thus conveys cognate BCR ligands to newly
synthesized class II molecules to transform B cells into very efficient
APCs.
|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Frédérique Forquet, Centre d’Immunologie de Marseille-Luminy, Parc Scientifique de Luminy, Case 906, 13 288 Marseille, France. E-mail address:
3 Abbreviations used in this paper: Ii, invariant chain; BCR, B cell receptor; CLIP, class II-associated Ii peptide; HEL, hen egg lysozyme; RAMIG, rabbit anti-mouse IgG; T, cytoplasmic domain-truncated I-Ak molecule; ßT, ß cytoplasmic domain-truncated I-Ak molecule; Tg, transgenic; M6PR, mannose 6 phosphate receptor; CHX, cycloheximide; Lip-PA, liposome-protein A; Tf, transferrin; CF, compact form; RBL, rat blood leukemia.
Received for publication August 12, 1998. Accepted for publication December 14, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
ß dimers and facilitates peptide loading. Cell 82:155.[Medline]
ß cytoplasmic domains reveal signaling-independent defects in antigen presentation. Int. Immunol. 7:665.This article has been cited by other articles:
|
|
 |
|
  C. Chalouni, J. Banchereau, A. B. Vogt, V. Pascual, and J. Davoust Human germinal center B cells differ from naive and memory B cells by their aggregated MHC class II-rich compartments lacking HLA-DO Int. Immunol., April 1, 2003; 15(4): 457 - 466. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. M. Snyder, X. Zhang, and L. J. Wysocki Negligible Class II MHC Presentation of B Cell Receptor-Derived Peptides by High Density Resting B Cells J. Immunol., April 15, 2002; 168(8): 3865 - 3873. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Gugasyan, C. Velazquez, I. Vidavsky, B. M. Deck, K. van der Drift, M. L. Gross, and E. R. Unanue Independent Selection by I-Ak Molecules of Two Epitopes Found in Tandem in an Extended Polypeptide Antigen J. Immunol., September 15, 2000; 165(6): 3206 - 3213. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) or targeted to the BCR via
RAMIG (•). As a control for Fc
). The presentation efficiency of soluble HEL is shown for
comparison (
). Ag-presentation assays were performed on numerous
occasions with irrelevant Abs in the presence of protein A-bearing
liposomes. The behavior in the presence of control Abs is not different
from uptake by the fluid phase (data not shown).
) B
cells were used to stimulate the 3A9 (A,
B, and C) or the 3B11 (D
and E) T cell hybridomas with serial dilutions of
nontargeted HEL-Lip-PAs (A and D),
HEL-Lip-PAs targeted through the BCR via RAMIG (B and
E), or HEL (46–61) peptide (C). Results
are representative of five experiments for 3A9 and three experiments
for 3B11.
