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Down-Regulates MHC Expression and Antigen Processing in a Human B Cell Line1
Department of Immunology, Windeyer Institute of Medical Sciences, University College London Medical School, London, United Kingdom
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Abstract |
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is a crucial mediator in the induction of cell-mediated
Th1-type responses but is predominantly a negative regulator of B cell
differentiation and proliferation. This cytokine is therefore a key
factor in determining Th1 vs Th2 differentiation. This study
investigates the action of IFN- in modulation of HLA-DR expression
and Ag presentation by EBV-transformed human B cell lines. In contrast
to its action on the monocyte/macrophage, IFN- down-regulates
surface MHC expression on these B cells, and this regulation is
posttranscriptional. In parallel with MHC down-regulation, there is a
reduced capability to process and present exogenous protein and peptide
Ag to T cell hybridomas. IFN- does not change the rates of fluid
phase endocytosis or exocytosis in this model system but correlates
with an up-regulation of the lysosomal enzymes cathepsins B and
D. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is now regarded as the archetypal Th1-type cytokine, playing a role in
multiple facets of the cellular immune response. Of its many functions,
one of the first to be discovered was the transcriptional up-regulation
of MHC expression in macrophages, and in a wide variety of
nonhemopoietic cells, resulting in enhanced display of Ag to
CD4+ and CD8+ T cells. The molecular pathways
underlying this phenomenon have been studied extensively (1, 2). As a
counterpart to its inductive role in cellular immunity, there have been
several reports that IFN- is inhibitory to the humoral response and
in particular inhibits B cell proliferation, differentiation, and
certain forms of isotype switching (3, 4, 5). Its role in MHC regulation
in this cell type has been studied much less extensively, although
there is one report of MHC class II down-regulation in peripheral
blood B cells exposed to IFN- (6), and the induction of
class II MHC by IL-4 is inhibited (7). The mechanism for this effect is
not understood (1).
In this study, we have established a model system that uses a human
EBV-transformed B cell line transfected with the murine
I-Ak molecule, in which the regulation of class II MHC Ag
processing can be studied independently of the transcriptional
regulation of the MHC molecules themselves. EBV-transformed B cells
have been extensively studied as models of B cell Ag processing and
seem to reflect many of the properties of activated B cells in this
regard. We have therefore used this system to examine the influence of
IFN- on B cell Ag processing using the well-defined model Ag hen egg
lysozyme (HEL).3
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß genes of the murine I-Ak
molecule under the control of the CMV promoter. The plasmid also
contained a hygromycin resistance gene. The construct was a gift of Dr.
A. Venkiteraman (8). Cells were transfected by electroporation (200 V,
960 µF) using a Bio-Rad electroporator (Bio-Rad, Hemel Hempstead,
U.K.). After electroporation, cells were selected in hygromycin (500
µg/ml), and resistant cells were cloned by limiting dilution.
Individual clones were screened for surface I-Ak expression
by flow cytometry (see below). Two clones (WMPT3.3 and WMPT3.72)
obtained from separate pools of transfectants, and showing relatively
high levels of transgene expression, were selected for further
analysis. Cells were periodically cultured with hygromycin to prevent
reversion to the wild-type, and expression of the I-Ak
molecule was monitored by flow cytometry. Expression of the transgene
proved to be very stable.
To investigate the effects of cytokine, cells were seeded at between
5 x 104 and 2.5 x 105 cells/ml and
then cultured for 120 h in 20 U/ml recombinant human IFN-
(Genzyme, Kings Hill, Kent, U.K.). Preliminary experiments showed no
effects at shorter time points. In some experiments, anti-IFN-
antiserum (PreProtech, London, U.K.) or control rabbit serum was added
to the IFN- before addition to the culture medium.
T cell hybridomas
Two murine CD4+ T cell hybridomas that recognize specific I-Ak-restricted HEL determinants were used in this study. IC5.1 (a gift from Dr. P. Fairchild, Cambridge University, Cambridge, U.K.) recognizes an epitope encoded by amino acids 46–61 of HEL. AOIT (a gift from Dr. E. E. Sercarz, UCLA, Los Angeles, CA) recognizes HEL74–86. Hybridomas were cultured in RMPI 1640 and 5% FCS as detailed above.
Ag presentation assays
WMPT3.3 (5 x 104) and 5 x 104 of each of the T cell hybridomas were cocultured for 24 h with titrated concentrations of HEL (Boehringer Mannheim, Mannheim, Germany) in 96-well round bottom sterile tissue culture plates at 37°C in 5% CO2. Each culture condition was set up in triplicate. After 24 h, the plates were centrifuged at 1000 rpm for 5 min, and 50 µl of supernatant from each well were removed and transferred to another 96-well round bottom sterile tissue culture plate. Supernatants were then frozen at -70°C until assayed for IL-2 content. IL-2 was assayed using the CTLL cell line as described (9). Results are expressed as mean [3H]thymidine incorporation. SEMs are not shown but are in the range of 10% of the mean.
Flow cytometry
For cell surface staining, 2 x 105 cells
± IFN- were washed and then incubated in 10% rabbit serum (Life
Technologies) in PBS with 0.1% azide. Primary Ab (see below) was
added, and the cells were incubated on ice for 40 min. The cells were
then washed repeatedly in PBS and resuspended in FITC-conjugated rabbit
anti-mouse IgG Fab-2 fragment, or FITC-conjugated donkey
anti-sheep IgG (DAKO, High Wycombe, U.K.) for 30 min on ice before
washing and fixing in 3% paraformaldehyde. For staining of
intracellular molecules, cells were first fixed in paraformaldehyde
(2%) for 10 min on ice and then incubated in PBS containing 0.2% BSA
and 0.2% Triton X-100 for 10 min. Cells were then processed as for
surface staining, except that an extra incubation of 16 h was
introduced into the washing procedure after the primary Ab, so as to
reduce background binding. Primary Abs used in this study were: TIB93,
anti-mouse I-Ak, IgG mAb from American Type Culture
Collection; L243, monomorphic anti-human HLA-DR, IgG mAb from P.
Beverley (UCL); LN2, anti-human invariant chain, IgG mAb from
Biotest AG (Reinher, Germany); W6/32, anti-human class I IgG mAb
from SeroTech (Oxford, U.K.); anti-human IgM, IgG mAb from P.
Lydyard (UCL); MAB442, anti-human cathepsin D, mouse mAb from
Chemicon (Harrow, U.K.); anti-HLA-DM, mouse IgG mAb from J.
Trowsdale, Department of Pathology, Cambridge University); CE1.1,
anti-human cathepsin E, mAb, produced in the Department of
Immunology, UCL (10); anti-human type II collagen (used as a
negative control), mouse IgG mAb from R. Holmdahl (Lund University,
Lund, Sweden); anti-human cathepsin B, sheep polyclonal from
Binding Site (Birmingham, U.K.). All mAbs were used at a 1/10 dilution
of culture supernatant. Flow cytometry was performed using the Becton
Dickinson FACScan, and FACScan software. A minimum of 5000 events was
collected for each sample.
Endocytosis/exocytosis assay
This assay was performed essentially as described previously (11). Briefly, 106 WMPT cells for each group were cultured in 50 µl of Lucifer Yellow (Sigma, St. Louis, MO) at 3 mg/ml at 37°C for the following time points: 180 min, 60 min, 30 min, 15 min, 10 min, and 5 min. Cells were then incubated on ice, washed twice with cold PBS, and then analyzed by flow cytometry. Lucifer Yellow uptake was expressed as mean fluorescence minus background fluorescence of unlabeled cells. To follow exocytosis, cells loaded with Lucifer Yellow for 5 or 180 min were washed at 4°C, then further incubated at 37°C for various time points, and analyzed by flow cytometry as before.
Immunoprecipitation of class II MHC
WMPT3.3 cells (4 x 107) were washed and incubated in methionine-free medium (Life Technologies) at 37°C in 5% CO2 for 1 h to remove all free intracellular methionine. [35S]Methionine, 18.5 MBq (ICN Biomedicals, Thame, U.K.), was added, and the cells were then incubated at 37°C for 1 h. Cells were then washed and incubated in full medium at 37°C for 15 min, 1 h, 3 h, or 6 h chase period in complete medium.
After repeated washes, cells were resuspended in 1 ml of cold lysis buffer (150 mM NaCl, 5 mM EDTA, 50 mM Tris/HCl, pH 8.0, with 0.26 mg/ml Pefabloc (Pentapharm, Basel, Switzerland) and 0.1 mg/ml leupeptin (Sigma)) and incubated at 4°C for 15 min. The lysate was centrifuged at 10,000 x g for 10 min, and the supernatant was collected. Preclearing was conducted by addition of 5 µl of rabbit serum with 50 µl of a 50% suspension of protein A-Sepharose to the cell lysate. The mixture was rotated at 4°C for 1 h. This procedure was repeated three times, and the supernatant was then stored at -20°C until required. For immunoprecipitation, protein A-Sepharose was preloaded with rabbit anti-mouse Ig (DAKO) followed by monoclonal anti-HLA-DR (Tal 14.1, Imperial Cancer Research Fund, London, U.K.) anti-I-Ak (TIB93), or anti-human Ii (LN2). Twenty microliters of preloaded Sepharose were added to 500 µl of lysate and rotated at 4°C for 1 h. The Sepharose was then washed sequentially in normal salt buffer (10 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.5% Nonidet P-40), low salt buffer (10 mM Tris-HCl (pH 8.0) with 0.5% Nonidet P-40), high salt buffer (10 mM Tris-HCl (pH 8.0), 500 mM NaCl, with 0.5% Nonidet P-40), and then twice more in normal salt buffer. An equal volume of sample buffer, containing 0.5% 2-ME with bromphenol blue was then added to each of the pellets. The samples were analyzed on a 12.5% SDS-polyacrylamide gel and exposed to x-ray-sensitive film (X-Omat, Kodak) for 7 days at -70°C.
Analysis of HEL peptides generated by WMPT3.3
WMPT3.3 were cultured overnight with 300 µg/ml of HEL in complete RPMI (as detailed above). 2 x 107 cells were washed extensively and then lysed in 0.5 ml of 0.7% trifluoroacetic acid (Sigma) on ice for 30 min. Insoluble material was removed by centrifugation, and the resulting supernatant was collected and fractionated by reverse phase liquid chromatography on a Pharmacia Biotech (Piscataway, NJ) Smart System, using a 0–50% water/acetonitrile gradient with 0.05% trifluoroacetic acid. Five microliters of each fraction were then added to 5 x 105 freshly isolated murine CBA mouse-derived splenocytes and 5 x 104 T cell hybridomas. T cell hybridoma response was assayed as previously described.
Measurement of apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP-nick end-labeling (TUNEL) method (12)
WMPT3.3 cells were treated with cytokine-containing or control medium and centrifuged onto a glass slide using a cytospin centrifuge. The cells were fixed in a freshly prepared solution of 4% paraformaldehyde (w/v) for 20 min at RT followed by three washes in PBS each for 5 min. Fixed sections were then incubated in a permeabilization solution consisting of 0.1% Triton X-100 in a 0.1% solution of sodium citrate for 2 min on ice. Slides were rinsed in PBS and then incubated with 50 µl of TUNEL reagent (Boehringer Mannheim, Lewes, U.K.) for 60 min at 37°C. After a further rinse in PBS, the sections were incubated in POD reagent (anti-fluorescein Ab Fab fragment from sheep, conjugated with horseradish peroxidase) (Boehringer Mannheim) for 30 min at 37°C. The slides were washed twice in PBS, and then 100 µl of 3,3-diaminobenzidine-0.3% hydrogen peroxide (Sigma) added for 10 min. Slides were rinsed three times in PBS, counterstained with hematoxylin, dehydrated, cleared, and mounted in Eukitt (BDH, Poole, U.K.). Apoptotic cells could be readily distinguished by the strong signal in the nucleus.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The EBV B cell line WMPT was transfected with the murine
I-Ak and ß genes under the control of a CMV promoter.
As shown in Fig. 1
a, a
transfected clone, WMPT3.3, but not the parental cell line, expresses
the murine MHC molecule as detected with a haplotype-specific Ab,
TIB93. Immunoprecipitation, using TIB93 Ab, detected a trimer in the
transfectants, composed of and ß MHC chains (identified
previously by sequential immunoprecipitation and Western blotting
(13)), and the human invariant chain (Ii), confirming previous reports
(14) that the murine MHC class II chains associate correctly with the
human Ii (Fig. 1b). In contrast, the untransfected cells
expressed equal amounts of invariant chain (and HLA-DR; see Fig. 3),
but no I-AK molecules. The transfected cells, but not the
parental line, were able to present two distinct epitopes of the
lysozyme molecule, 46–61 and 74–86, to appropriate murine hybridomas
(Fig. 1c). The concentrations of Ag required were comparable
with other similar studies examining B cell Ag processing by
nonspecific fluid phase endocytosis (15).
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down-regulates surface expression of class II MHC
Surface levels of HLA-DR, as assessed by flow cytometry were
down-regulated after IFN- treatment in two independent WMPT
transfectants (Fig. 2, a and
b). The surface expression of the I-Ak transgene
was also decreased in parallel in both transfectants. The
down-regulation of class II MHC was selective, since surface levels of
IgM were unchanged (Fig. 2a, column A), while
surface class I was up-regulated (Fig. 2b, column A). To
confirm that the effect on class II MHC levels was not specific simply
to the WMPT cell line, the results were repeated with two other EBV B
cell lines, which showed similar down-regulation in HLA-DR expression
in the presence of IFN- (Fig. 2, c and d). The
inhibition of HLA-DR was reversed by the addition of neutralizing
anti-IFN- rabbit antiserum but not by control rabbit antiserum
(Fig. 2e).
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). In three experiments,
no consistent differences were apparent at any point of the time chase
analysis, although small differences between control and
cytokine-treated cells could sometimes be observed (Fig. 3b).
IFN- down-regulates Ag processing and presentation of lysozyme
by the transfected B cells
The effect of IFN- on WMPT3.3 Ag processing and
presentation was studied. After culture in cytokine, WMPT3.3 cells were
washed extensively to remove any residual cytokine and then cocultured
with whole HEL and IC5.1 or AOIT hybridomas for a further 24 h.
IFN--treated cells were found to be less efficient at processing and
presenting both 46–61 and 74–86 determinants from whole HEL, giving a
lower response at each Ag concentration (Fig. 4, a and b).
IFN--treated WMPT3.3 cells were also poorer at presenting the
peptide form of the epitope to both IC5.1 (Fig. 4c) and AOIT
(Fig. 4d) compared with untreated cells, although the
inhibition seen using the AOIT peptide 74–86 was always less than that
seen using the intact Ag.
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-treated B cells contain less processed Ag
To determine whether IFN- directly inhibited the ability of the
WMPT3.3 to generate the immunogenic fragments recognized by the T cell
hybridomas, B cells incubated with Ag were lysed, and the peptide
fragments within the cells were fractionated by reverse phase HPLC. The
presence of peptides containing the epitope recognized by each
hybridoma within each fraction was determined by measuring the ability
of the fraction to stimulate the hybridoma response in the presence of
spleen cells. As shown in Fig. 5, IFN-
treatment of WMPT3.3 cells decreased the levels of both epitopes tested
and appeared to qualitatively change the nature of the remaining
peptides recognized by AOIT cell line. To confirm that HEL fragments
within the HPLC fraction were not being further processed by the
splenocytes, the two active fractions recognized by IC5.1 (Fig. 5a) were also tested using glutaraldehyde-fixed splenocytes.
The IL-2 release induced by fraction 28 was 82% of that induced on
live splenocytes, and the IL-2 release induced by fraction 29 was
115%.
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does not alter endocytosis or apoptosis in WMPT 3.3 cells
IFN- is known to increase endocytosis/phagocytosis by
macrophages. Rates of fluid phase endocytosis (Fig. 6a) and exocytosis (Fig. 6) by
WMPT3.3 cells following IFN- treatment, and with no IFN-
treatment were measured using the soluble tracer Lucifer Yellow.
Although some small differences between control and IFN--treated
cells on the rates of exocytosis were sometimes observed, no consistent
effect was observed at any of the time points tested.
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was inducing apoptosis in the WMPT cells, the
proportion of cells undergoing apoptosis was measured using the
TUNEL method (12). The proportion of cells stained using this
method in untreated cultures was 4% (±2%), and was unchanged in the
presence of IFN-.
IFN- alters the levels of proteinases in WMPT 3.3 cells
The proteinases cathepsins B, D, and E have previously been
implicated in Ag processing (16). Levels of these enzymes in
IFN--treated and untreated WMPT3.3 cells were measured (Fig. 7a). Levels of cathepsins B
and D in IFN--treated WMPT3.3 cells were up-regulated consistently
in three experiments. Cathepsin E was slightly down-regulated in two of
three experiments. The levels of HLA-DM, another intracellular
component of the Ag processing machinery, showed only a very small
decrease following cytokine treatment (Fig. 7b).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, which in the context of the B cell is generally regarded as an
inhibitory cytokine, also down-regulates the processing of Ag for the
class II MHC pathway.
This study introduces a new model system that utilizes a chimeric
EBV-transformed B cell line, transfected with the murine
I-Ak genes to process and present lysozyme to two murine T
cell hybridomas of different specificity. Although the model does
introduce the added complexity of chimerism, as well as the question of
the relationship of the normal to the virally transformed B cell, it
does possess significant advantages for the type of study outlined in
this paper. First, since the transfected MHC molecule is under the
control of a minimal heterologous CMV promoter (8), it reduces the
likelihood that the effects of exogenous IFN- are mediated via
effects on MHC transcription (although this possibility was
specifically addressed in this study; see Fig. 3). Secondly, since the
processing line is of human origin, it allowed us to use available
reagents for detection of human processing enzymes, but still to use as
readout very well-characterized HEL-specific murine T cell hybridomas.
To extend the model further, experiments are in progress to
supertransfect Ag-specific Igs into WMPT 3.3 thus converting the model
into an Ag-specific one. In addition, experiments on "normal" human
B cells will be required to generalize the results obtained in this
study further. Our initial observation was that IFN- inhibited the
surface levels of both the endogenous HLA-DR and the transgene
I-Ak in WMPT3.3 cells. This effect is the opposite to the
enhancement of class II MHC usually seen on macrophages, and on many
nonhemopoietic cell types, but has been reported previously on
untransformed B cells (6). The result is also consistent with the fact
that they have previously been shown to express at least one chain of
the IFN- receptor (18). Although an effect on the I-Ak
transgene is unlikely to be mediated via transcriptional regulation (as
discussed above), this was demonstrated directly, since rates of
biosynthesis of both the MHC - and ß-chains, and Ii were unchanged
in the IFN--treated cells. The reduced surface expression of MHC
must therefore result either from reduced transport to the cell surface
or from increased rate of removal from the cell surface. Either or both
these changes could be secondary to alterations in peptide/MHC loading
(see below). Studies of MHC stability, by pulse chase analysis, did not
reveal any major alterations in the presence of cytokine. Further
detailed studies of the patterns of intracellular MHC
transport/degradation will be required to resolve these questions.
Reduced levels of surface MHC were paralleled by decreased ability to
process and present two different epitopes of HEL, presented as either
intact Ag or synthetic peptides. The reduced presentation of peptide Ag
is likely to be the direct result of lower surface MHC expression.
However, IFN- could, in principal, inhibit several other stages of
the assay. IFN- could, for example, act directly on the T cell
hybridomas to inhibit release of IL-2, but this is not likely to be the
explanation in these experiments, since the B cells were thoroughly
washed to remove all exogenous cytokine before addition of T cells.
Alternatively, IFN- could down-regulate levels of costimulatory
molecules on the B cell surface. However, we confirmed that IFN- was
directly inhibiting the formation of the lysozyme epitopes within the B
cells, by demonstrating a decrease in the level of the two lysozyme
epitopes within B cell lysates. This result rules out an indirect
effect of IFN- on costimulator expression. The result suggests that
the reduced presentation of determinants from intact HEL by
IFN--treated WMPT3.3 cells may result from inhibiting the processing
of HEL by B cells directly, although it is very difficult to determine
whether an overall lower level of Ag processing is contributing to
decreased export of mature MHC or whether lower maturation of MHC
results in lower steady state levels of the lysozyme epitopes within
the cell. Intracellular HLA-DM was not much changed by IFN-
treatment, but we cannot rule out that there were parallel changes in
other components of the Ag-processing machinery such as HLA-DO (19), or
peptide chaperones.
IFN- alters many other parameters of function in
monocytes/macrophages, including the rates of endocytosis/phagocytosis
and the levels of lysosomal proteinases. Both endocytosis and
exocytosis were unchanged in the WMPT3.3 cell treated with IFN-.
Levels of two lysosomal proteinases tested, cathepsins B and D were
increased, as had previously been described for macrophages (20). Some
of the normally coordinately regulated effects of IFN- signaling
were therefore dissociated in the EBV-transformed B cells, in such a
way as to decrease the overall Ag-processing activity, but still permit
other responses which might perhaps be required to induce efficient
destruction of intracellular pathogens within the B cell. Any
causal relationship between decreased processing and increased
lysosomal function must remain speculative, but excess enzyme levels
could paradoxically decrease the amount of peptide available by
destroying the epitopes required for T cell recognition. Such an
antiprocessing activity has been documented previously, especially for
cathepsin B (21, 22). Down-regulation of MHC expression and Ag
processing on an activated B cell inhibits the ability of the cell to
receive Ag-specific help from a cognate T cell. This study therefore
identifies another level at which IFN- can inhibit the generation of
humoral immunity response, and further bias the immune response to a
Th1 cellular phenotype. More generally, these results provide more
evidence of the dynamic and plastic nature of the Ag-processing
machinery, and suggest that the outcome of the processing machinery may
depend critically on the immune microenvironment.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. B. M. Chain, Department of Immunology, Windeyer Institute of Medical Sciences, University College London, Cleveland St., London W1P 6DB. E-mail address:
3 Abbreviations used in this paper: HEL, hen egg lysozyme; UCL, University College of London.
Received for publication January 8, 1998. Accepted for publication October 7, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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downregulates the proliferative response of hapten-specific B cells stimulated by antigen and cytokines. Immunol. Cell Biol. 73:52.[Medline]
R expression and renders B cells sensitive to IFN--mediated inhibition. J. Immunol. 153:3369.[Abstract]
and B cell stimulatory factor-1 reciprocally regulate Ig isotype production. Science 236:944., or interleukin-4. Cell. Immunol. 171:1.[Medline]
suppresses B cell stimulation factor (BSF-1) induction of class II MHC determinants on B cells. J. Immunol. 137:3534.[Abstract]
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, results in the presence of IFN-
, results
in control medium.
