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Antigen1
*
Laboratory of Molecular and Cellular Immunology, Fondazione Andrea Cesalpino, Istituto I Clinica Medica, Università degli Studi di Roma "La Sapienza", Rome, Italy;
Laboratory of Medical Bacteriology and Mycology, Istituto Superiore di Sanità, Rome, Italy;
Division of Gastroenterology, Ospedale Molinette, Turin, Italy;
§
Chiron Corporation, Emeryville, CA 94608; and
¶
Institute Pasteur-Cenci Bolognetti, Rome, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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virus is a human pathogen that is responsible for a
severe form of hepatitis affecting hepatitis B envelope Ag carriers. We
have previously identified a series of hepatitis Ag (HDAg) epitopes
that are recognized by CD4+ T cell clones isolated
from infected subjects. Herein, we show that the presentation of
soluble HDAg to CD4+ T cell clones that are specific for
the HDAg(106–121) epitope was exceptionally
unaffected by the inhibition of the APC-processing machinery when APCs
were fixed with glutaraldehyde before Ag pulsing or treated with
chloroquine or brefeldin A. Interestingly, 5 h of pulsing was
strictly required for the efficient presentation of the
HDAg(106–121) epitope by fixed APCs, suggesting that some
form of extracellular processing had occurred. Indeed, fixed APCs were
able to present HDAg after only 1 h of pulsing when HDAg was
preincubated with serum for 5 h. More important, presentation was
completely abrogated when Ag was previously incubated in medium
containing serum in the presence of a potent inhibitor of trypsin
activity such as the secretory leukoprotease inhibitor. These results
show that HDAg may undergo extracellular processing and suggest that
the generation of immunogenic epitopes directly by serum proteases
could play a role in the immune response against hepatitis virus
during infection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Hepatitis virus (HDV)3 is
a defective RNA virus that is responsible for severe forms of hepatitis
in humans. HDV may either cause coinfection in combination with
hepatitis B virus or may superinfect hepatitis B envelope Ag (HBenvAg)
carriers (5, 6, 7). Hepatitis Ag (HDAg) is the only known protein
product of HDV, existing as two isoforms of 24 and 27 kDa (p24 and p27,
respectively) that are both crucial for virus assembly and infectivity
(8). We have recently analyzed the HDAg-specific CD4+ T
cell response in subjects infected with HDV and identified a number of
epitopes that are relevant for this response using a panel of specific
T cell clones (9). Here, we report the identification of an epitope,
HDAg(106–121), which was generated through an
extracellular cleavage mediated by serum proteases, and discuss the
possible relevance of such a unusual pathway of Ag processing.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The complete medium (CM) used in this study consisted of RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 2 µM L-glutamine, 1% nonessential amino acids, 1 µM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin (Flow Laboratories, Irvine, U.K.), and 10% human AB serum (HS) (Sigma-Aldrich, Milan, Italy).
Recombinant forms of both large (p27) and small (p24) HDAg were expressed in yeast (Saccharomyces cerevisiae), and the reconstituted proteins were at least 80% pure as judged by SDS-PAGE and Coomassie blue staining.
Synthetic peptides corresponding to amino acids 26–41 and 106–121 of the HDAg sequence (HDV genotype I) were kindly provided by Chiron Mimotopes (Clayton, Australia). Each peptide contained free amine N termini and free acid C termini and was purified by HPLC to >80% purity. Lyophilized peptides were reconstituted at 20 mg/ml in DMSO and diluted to 1 mg/ml with PBS.
Recombinant vaccinia viruses (VVs) expressing HDAg (VV-HDAg) and wild-type VV (VV-wt) as a control were used to infect APCs as previously described (9).
T cell clones and APCs
Two well-characterized T cell clones (TB238 and TB3) that were specific for HDAg(106–121) (KALENKKKQLSAGGKN) and HDAg(26–41) (KLEELERDLRKTKKKL), respectively, were used. As previously described, these clones were obtained from an HBenvAg carrier with HDV superinfection, autologous Epstein Barr-immortalized B lymphoblastoid cell lines (B-LCLs) while served as APCs (9). In particular, the TB238 clone recognized Ag in the context of multiple HLA-DR molecules, including DRB1*1101, DRB1*1102, DRB1*1201, DRB1*0101, DRB1*0701, DRB1*1401, and DRB5*0202, as previously described (9), so that the 106–121 peptide was defined as promiscuous (10). On the contrary, the TB3 clone recognized Ag in the context of DPB1*1701 (9). For Ag-presentation experiments, both T cell clones were tested against either HDAg-pulsed autologous or partially HLA-matched B-LCLs; Sweig cells (DRB1*1101) in particular were used as APCs for the TB238 clone, while the cell line LBS (DPB1*1701) was used for the TB3 clone (9).
T cell proliferation assay and Ag-processing experiments
Routine proliferation assays of T cell clones were performed by incubating 3 x 104 T cells for 48 h in 200 µl in 96-well flat-bottom microtiter plates (Falcon Labware, Oxnard, CA) in the presence or absence of autologous or partially HLA-matched B-LCLs, used as APCs (5 x 104), that were previously pulsed either with the entire HDAg for 6 h, or with HDAg-derived peptides for 1 h, or left unpulsed (1). At 18 h before the harvesting of cultures, 1 µCi [3H]thymidine was added, and the radioactivity incorporated by the cells measured by liquid scintillation counting.
In some experiments, APCs (autologous or partially HLA-matched B-LCLs) were treated with chloroquine or brefeldin A (Sigma-Aldrich). For chloroquine treatment, APCs were preincubated for 10 min in CM containing 80 µM chloroquine before Ag-pulsing (10 µg/ml p27 HDAg), and the same concentration of chloroquine was maintained throughout pulsing including washes. After treatment, APCs were fixed with 0.05% glutaraldehyde (Sigma-Aldrich) in PBS for 1 min. The reaction was stopped with 0.2 M glycine (Sigma-Aldrich) in PBS v/v for 10 min at room temperature. Cells were washed three times and incubated with T cells. For brefeldin A treatment, B-LCLs were incubated with 2 µM brefeldin A at the time of Ag-pulsing, fixed as described above, and used as APCs.
Extracellular processing experiments
In a series of experiments to determine whether extracellular processing was required for HDAg(106–121) peptide generation, glutaraldehyde-fixed APCs (autologous or partially HLA-matched B-LCLs) were pulsed with 10 µg/ml HDAg and washed after different pulsing times (10, 30, 60, 120, 240, and 300 min). After washing, APCs were incubated with responder T cells.
In some experiments, HDAg was incubated either in CM with FCS or HS at 10, 1, 0.1, and 0.01% or in serum-free medium for 5 h. Afterward, these HDAg preparations were used independently to pulse-fixed APCs in 10% CM-HS or CM-FCS for 1 h. APCs were then washed extensively and incubated with responder T cells.
Furthermore, to determine whether the HDAg(106–121) peptide could also be generated through the conventional processing pathways, nonfixed (live) autologous B-LCLs were either infected with VV-HDAg or VV-wt at a multiplicity of infection of 10 in CM or pulsed with soluble HDAg (10 µg/ml) in serum-free medium; this process was intended to rule out the possibility that soluble HDAg was extracellularly processed by serum proteases. Infected cells were incubated for 2 h at 37°C, washed twice, and then incubated overnight at 37°C in 5% CO2, while soluble HDAg-pulsed APCs were directly incubated overnight. On the following day, the cells were washed three times, irradiated at 12,000 rad, and used as APCs in a 3-day proliferation assay (9).
Treatment with protease inhibitors
In experiments that were set up in an attempt to identify the serum proteases possibly involved in extracellular HDAg processing, Ag (10 µg/ml p27 HDAg) was incubated with various protease inhibitors in 10% CM-FCS or CM-HS for 5 h at 37°C. Glutaraldehyde-fixed APCs were subsequently pulsed with those Ag preparations for 1 h at 37°C, extensively washed, and incubated with 105 TB238 T cells for the proliferation assay. As a control for nonspecific toxicity, B-LCLs were treated as described above, and their ability to present the HDAg(106–121) peptide (10 µg/ml) to the TB238 T cell clone was determined. The following inhibitors were used: EDTA (Sigma-Aldrich), leupeptin (Sigma-Aldrich), captopril (Sigma-Aldrich), phenanthroline (Sigma-Aldrich), Pefablock (Sigma-Aldrich), pepstatin A (Sigma-Aldrich), and recombinant human secretory leukocyte protease inhibitor (SLPI) (R&D Systems, Minneapolis, MN) (11). Each inhibitor was tested at four concentrations.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Experiments aimed at investigating the pathways required for the
processing of the entire HDAg revealed that HDAg presentation to the
HDAg(106–121)-specific TB238 T cell clone was not
inhibited when autologous B-LCLs, used as APCs, were
glutaraldehyde-fixed and then pulsed with p27 for 6 h. Figure 1
A clearly shows that the
TB238 clone proliferated in response to either fixed or nonfixed cells,
whereas the control HDAg(26–41)-specific TB3 clone
proliferated only in response to nonfixed pulsed APCs (Fig. 1B), as was previously demonstrated for a large panel
of HDAg-specific CD4+ T cell clones that were tested in
glutaraldehyde-fixation experiments (9). Comparable results were shown
when allogeneic HLA-matched B-LCL Sweig cells (DRB1*1101), were used as
APCs for the TB238 clone and allogeneic HLA-matched B-LCL LBS cells
(DPB1*1701) were used for the TB3 clone (data not shown). Moreover,
similar results were also obtained when the small (p24) HDAg form was
used and by using five different HDAg(106–121)-specific T
cell clones derived from different patients (data not shown).
Glutaraldehyde fixation did not affect the presentation of the relevant
peptide to Ag-specific T cells (see Fig. 6). In addition, neither
chloroquine nor brefeldin A treatment of APCs inhibited HDAg
presentation to the TB238 clone, but both drugs inhibited HDAg
presentation to the control TB3 clone (Fig. 2). It is known that the endocytic
presentation of Ags can be inhibited either by APC fixation, which
blocks all forms of Ag internalization, or by lysosomotropic agents
such as chloroquine, which inactivates endocytic proteases, or
brefeldin A, which blocks the transport of newly synthesized proteins
from the endoplasmic reticulum to the trans-Golgi, including the class
II molecule/invariant chain complexes (12, 13, 14). Our data strongly
suggest that the generation of the HDAg(106–121) peptide
does not require processing within the cells. We also tested the
capacity of fixed APCs pulsed at different times with HDAg to stimulate
the TB238 clone. Figure 3 shows that
5 h of pulsing was required for efficient Ag presentation by fixed
APCs, suggesting that some form of extracellular processing had
occurred and ruling out the possibility that HDAg preparation could
contain preformed cleaved fragments. The latter possibility was further
excluded by HPLC analysis (data not shown).
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A). Conversely, APCs
infected with VV-HDAg were able to generate and present the 26–41
peptide but not the 106–121 peptide (Fig. 4B).
However, control experiments revealed that APCs infected with VV-wt
were able to present the relevant peptides to both of the clones
used.
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To verify whether serum proteases were involved in extracellular
HDAg processing, we have first individually pulse fixed autologous
B-LCLs, used as APCs, with different preparations of HDAg that had
previously been treated with medium containing increasing
concentrations of HS. HDAg was incubated with different serum
concentrations for 5 h, which was a length of time previously
demonstrated to be required for extracellular Ag processing, and the
different Ag preparations were subsequently incubated with fixed APCs
for 1 h. Fixed APCs were able to present HDAg after 1 h of
pulsing only when HDAg was preincubated with serum, and a 0.01% HS
concentration was still sufficient to obtain presentation (Fig. 5). The same results were obtained using
FCS. This observation suggests that the HDAg had been processed by
serum proteases. Interestingly, attempts to block serum proteases with
different inhibitors of serine proteases, metalloproteases, and trypsin
(EDTA, leupeptin, captopril, phenanthroline, Pefablock, pepstatin A,
and SLPI) demonstrated that only the latter, which is known to be a
very strong trypsin inhibitor (11), affected extracellular Ag
processing. Indeed, fixed APCs were drastically inhibited in their
ability to present HDAg after 1 h of pulsing only when HDAg was
preincubated in 0.01% CM-HS (a concentration previously demonstrated
to be sufficient for the extracellular Ag processing) containing SLPI
(50 µg/ml) for 5 h (Fig. 6
); in no
case did the other inhibitors, used at different concentrations,
affected Ag presentation (data not shown). Experiments performed as a
control for nonspecific toxicity showed that B-LCLs that had been
treated with inhibitors as described above retained their ability to
present the HDAg(106–121) peptide to the TB238 T cell
clone (Fig. 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The finding that fixed APCs were able to present HDAg after 1 h of pulsing only when HDAg was preincubated with serum for 5 h supports the idea that HDAg had been processed by serum proteases. Consistent with this possibility is the demonstration that a potent inhibitor of the trypsin activity, such as SLPI (10), was the only one of a panel of protease inhibitors that was able to block HDAg presentation to the TB238 clone, strongly suggesting that serum trypsin is involved in the generation of the HDAg(106–121) peptide during extracellular HDAg processing. The finding that the N-terminal cleavage site of the 106–121 epitope appears to be located after dibasic residues (RRRKALENKKKQLSAGGKN), that are typical of many enzymes including trypsin-like enzymes, and that the C terminus is not flanked by such residues supports the role of trypsin in extracellular HDAg processing and possibly suggests the combined action of two different protease enzymes. Moreover, a sequence comparison study of four different HDV isolates showed that the putative protease cleavage sites around the 106–121 peptide are almost totally conserved among the isolates (data not shown). Therefore, these data indicate that 106–121 peptide processing by serum may apply to HDV isolates in general and further suggest that at least two proteases may be involved in this extracellular-processing mechanism.
What could be the relevance of this phenomenon in vivo? We can envisage the following scenario. The initiation of HDV superinfection or coinfection (5) is absolutely dependent upon HBenvAg coating, which occurs in hepatitis B virus-infected cells such as hepatocytes or monocytes. These cells, which can act as APCs or scavengers, could cleave and regurgitate complex viral proteins (21) which, in turn, may be processed by serum enzymes to generate immunodominant peptides. The availability of large quantities of the 106–121 peptide after extracellular processing for binding to class II molecules could either enhance the presentation of this peptide to specific CD4+ T cells for mounting a protective response against HDV or play a crucial role in the pathogenesis of HDV-mediated liver disease (8). Indeed, the 106–121 peptide could bind to MHC class II molecules that are expressed on hepatocytes activated by inflammatory cytokines (22), causing extensive lysis of bystander class II+ hepatocytes by liver-infiltrating HDV-specific cytotoxic CD4+ T cells (1, 8). This mechanism could be relevant for HDV immunopathogenesis in a large cohort of patients, because 106–121 is a promiscuous, immunodominant peptide that is recognized by T cells in the context of multiple HLA-DR molecules (8, 9). In addition, HDAg(106–121)-specific CD4+ T cells, in contrast to control HDAg(26–41)-specific T cells, were stimulated only when the relevant peptide was generated by the extracellular or endocytic processing of exogenous HDAg and were not stimulated by the endocytic processing of endogenous HDAg. This discrepancy may occur either because the 106–121 peptide is not generated at all by the processing of endogenous HDAg, or because it is generated at subthreshold levels to act as a T cell epitope (23, 24, 25). It is tempting to hypothesize that the "exogenous" 106–121 epitope, which binds to several class II molecules (and possibly to some class I molecules) of all cells irrespective of virus infection, could act in different ways. Indeed, it may function both as a "blocking" peptide inhibiting MHC binding and the presentation of "endogenous" HDAg epitopes (26), and as a some kind of decoy inducing the exhaustion of HDAg(106–121)-specific T cells (27), and consequently facilitating the persistence of virus-infected cells. Alternatively, the extracellular processing of the 106–121 peptide by serum proteases may represent a mechanism of natural immunity against HDV, which would indirectly allow more efficient and prompt mounting of a protective, specific immune response through the rapid generation and presentation of this peptide. The viral load (low vs high), the type of APCs involved (professional vs nonprofessional), and the strength of the host-specific T cell response (high vs low memory T cell frequency) could all be crucial for the preferential establishment of either an immunity or an immunopathology state against HDV. In conclusion, ours is the first report demonstrating in humans the extracellular processing of a complex pathogen-derived Ag that is recognized by Ag-specific T cell clones, which have been isolated from virus-infected individuals. The extracellular-processing pathway for the presentation of HDV epitopes that is described herein provides clues for understanding HDV immunopathogenesis and suggests its possible relevance in vivo.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Vincenzo Barnaba, Istituto I Clinica Medica, Università di Roma "La Sapienza", Policlinico Umberto I, viale del Policlinico, 155, 00161 Roma, Italy. E-mail address:
3 Abbreviations used in this paper: HDV, hepatitis virus; HBenvAg, hepatitis B envelope Ag; HDAg, hepatitis Ag; CM, complete medium; HS, human AB serum; VV, vaccinia virus; wt, wild-type; B-LCL, B lymphoblastoid cell line; SLPI, secretory leukocyte protease inhibitor.
Received for publication October 22, 1997. Accepted for publication January 30, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) viral genome. Nature 323:508.[Medline]
-chain does not require processing. J. Immunol. 140:1063.[Abstract]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) or nonfixed (•) APCs
were then pulsed with increasing concentrations of Ag, washed three
times, and incubated with either the TB238 (A)
or TB3 (B) clone, the last of which was used
as a control. T cell proliferation was evaluated by measuring
[3H]thymidine uptake in a standard proliferative assay
and expressed as mean cpm of triplicate determinations.
),
0.1% (
), 1% (
), and 10% (
). These mixtures were used
individually to pulse 2 x 105 fixed APCs for
1 h in CM containing HS or FCS at a final concentration of 10%.
Next, APCs were extensively washed and incubated in increasing
concentrations with cloned TB238 cells. T cell proliferation was
evaluated by measuring [3H]thymidine uptake in a standard
proliferative assay and expressed as mean cpm of triplicate
determinations.