By
From the * Institute of Biochemistry, University of Lausanne, BIL Research Centre, CH-1066
Epalinges, Switzerland; and Clinical Immunology, Department of Internal Medicine, University
Hospital, CH-8044 Zürich, Switzerland
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Abstract |
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Human Fas ligand (L) (CD95L) and tumor necrosis factor (TNF)- undergo metalloproteinase-mediated proteolytic processing in their extracellular domains resulting in the release of soluble trimeric ligands (soluble [s]FasL, sTNF-
) which, in the case of sFasL, is thought to be implicated in diseases such as hepatitis and AIDS. Here we show that the processing of sFasL
occurs between Ser126 and Leu127. The apoptotic-inducing capacity of naturally processed
sFasL was reduced by >1,000-fold compared with membrane-bound FasL, and injection of
high doses of recombinant sFasL in mice did not induce liver failure. However, soluble FasL
retained its capacity to interact with Fas, and restoration of its cytotoxic activity was achieved
both in vitro and in vivo with the addition of cross-linking antibodies. Similarly, the marginal
apoptotic activity of recombinant soluble TNF-related apoptosis-inducing ligand (sTRAIL),
another member of the TNF ligand family, was greatly increased upon cross-linking. These results indicate that the mere trimerization of the Fas and TRAIL receptors may not be sufficient
to trigger death signals. Thus, the observation that sFasL is less cytotoxic than membrane-bound FasL may explain why in certain types of cancer, systemic tissue damage is not detected,
even though the levels of circulating sFasL are high.
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Introduction |
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Tumor necrosis factor (TNF)- is a potent proinflammatory and immunomodulatory agent. However, elevated concentrations of circulating TNF-
seen during
many host responses may be harmful or even fatal to the
organism. Membrane-bound TNF-
is synthesized as a
type II membrane protein that acts locally through cell-to-cell contact. Soluble TNF-
(s)TNF-
1 is released from
the cell surface as the result of metalloproteinase cleavage
(1, 2). Although both cell surface and secreted TNF-
appear to be biologically active, it is the sTNF-
released into
circulation that appears to be primarily responsible for the
deleterious physiological responses such as cachexia and endotoxic shock.
Fas ligand (L), a member of the TNF family, is also processed and shed from the surface of human cells. FasL has
been implicated in one of the major cytolytic pathways of
cytotoxic T cells (3), and is a key element in the elimination of activated T cells during the downregulation of the
immune response in the periphery (6, 7). Moreover, expression of FasL in nonlymphoid and tumor cells contributes to the maintenance of immune privilege of tissues by
preventing the infiltration of Fas-sensitive lymphocytes (8- 12). Presently, it is not clear whether the activities described for the ligand are primarily due to the cell surface
form of FasL or whether sFasL is likewise implicated. As
seen for TNF-, naturally processed and recombinant
sFasLs were found to be active, inducing apoptosis in
mouse cells at a concentration of <10 ng/ml (13). Elevated levels (up to 10 ng/ml) of sFasL were found in sera
from patients with large granular lymphocytic leukemias
and natural killer cell lymphomas (17). Since the administration of recombinant FasL or agonistic Fas antibodies into
mice leads to liver failure and to rapid death of the animals
(18, 19), it has been proposed that sFasL is implicated in the
pathogenesis of various diseases such as hepatitis or AIDS
(20). The possible implication of sFasL in pathological
processes prompted us to investigate the structure and activities of naturally processed sFasL in more detail. Here we
report that, in contrast to membrane-bound FasL, the soluble ligand is only poorly active. A model is suggested whereby the apoptotic activity of the membrane-bound
ligand is downregulated upon processing and release. This
assures that circulating sFasL does not cause systemic tissue
damage.
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Materials and Methods |
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Materials.
The anti-Flag M2 monoclonal antibody and the anti-Flag M2 antibody coupled to agarose were purchased from Eastman Kodak Co. (Rochester, NY). Protein A-Sepharose and cyanogen bromide-activated Sepharose were purchased from Pharmacia (Uppsala, Sweden). Affinity-purified anti-FasL rabbit polyclonal antibody (anti-PE62) was obtained as described (23). Biotinylated anti-Fas antibody Jo-2 was purchased from PharMingen (San Diego, CA), and dichlorotriazinglaminofluorescein (DTAF)- labeled streptavidin and anti-mouse antibodies were purchased from Jackson ImmunoResearch (Milan, La Roche, Switzerland). Cell culture reagents were obtained from Life Sciences (Basel, Switzerland). Human (h) TNF-related ligand that weakly induces apoptosis (TWEAK) cDNA (24) and recombinant hIFN-Cells.
Murine B lymphoma A20 and A20R cells (11) were maintained in DMEM containing 5% heat-inactivated FCS. The human T lymphoblastoma Jurkat cells, colon adenocarcinoma HT-29 cells, BJAB Burkitt lymphoma, and murine fibrosarcoma WEHI-164 cells were grown in RPMI supplemented with 10% FCS. Human embryonic kidney cells 293 were cultured in DMEM-nutrient mix F12 (1:1) supplemented with 2% FCS. 293T cells were grown in DMEM, 10% FCS. All media contained antibiotics (penicillin and streptomycin at 5 µg/ml each and neomycin at 10 µg/ml). The IL-2-dependent murine cytotoxic T cell line CT6 (27), a gift of P. Vandenabeele (University of Ghent, Ghent, Belgium), was grown in RPMI supplemented with 10% FCS, 1 mM sodium pyruvate, 50 µM 2-mercaptoethanol, 10 mM Hepes, and 10% conditioned EL-4 cell supernatant.Vector Construction and Establishment of Cell Lines Expressing Ligands of the TNF Family.
hFasL was amplified by PCR from activated peripheral blood lymphocyte cDNA using the following oligonucleotides: JT289 5'-CCTCTACAGGACTGAGAAGAAG-3' and JT290 5'-CAACATTCTCGGTGCCTGTAAC-3'. muFasL was amplified by PCR from mouse testis cDNA with primers JT277 5'-AAGGCTGTGAGAAGGAAACC-3' and JT271 5'-AAGGATCCTAGCTGACCTGTTGGACCTTGC-3'. The PCR products were subcloned into PCR-II TA-cloning vector and then resubcloned as EcoRI fragments into the PCR-III mammalian expression vector (Invitrogen, NV Leek, the Netherlands). Point mutations in FasL were obtained by two rounds of PCR using the following oligonucleotide pairs: for L126E, T7/ JT915 5'-GCTTCTCGAGCTCTGATGCTGTGTGCATC-3' and JT914 5'-CAGAGCTCGAGAAGCAAATAGGC-3'/Sp6; for S127E, T7/JT917 5'-CAGAGCTCGCTGTGTGCATCTGG-3' and JT916 5'-GCACACAGCGAGCTCTGAGGAGAAGCAAATAGGC-3'/Sp6; for SL126-127EE, T7/JT963 5'-GCTTCTCCTCCTCGCTAGCTGTGTGCATCTGG-3' and JT962 5'-AGCGAGGAGGAGAAGCAAATAGG-3'/Sp6. The extracellular domains of hTWEAK (amino acids [aa] 141-284), muTNF-Purification of Recombinant Proteins.
Flag tagged recombinant proteins were purified on M2-agarose and eluted in 50 mM citrate-NaOH, pH 2.5, followed by neutralization with 0.2 volumes of 1 M Tris-HCl, pH 8. The buffer was exchanged to PBS in concentrators (Centrikon-30; Amicon Corp., Easton, TX). Soluble hFasL released from cells transfected with full-length FasL was subjected to phase separation in Triton X-114 (28) and the aqueous phase was then purified using human Fas with human immunoglobulin Fc (hFas/Fc)-Sepharose. hFas/Fc-Sepharose was prepared by coupling 4 mg of Fas/Fc to 1 ml cyanogen bromide-activated Sepharose according to the manufacturer's instructions. Bound FasL was eluted as described above for M2-agarose. The concentration of purified proteins was determined by the bicinchoninic acid method (Pierce Chemical Co., Rockford, IL) using bovine serum albumin as standard, and the purity of the samples was assessed by SDS-PAGE and Coomassie blue staining. The concentration of FasL in Neuro-2a cells supernatants was estimated by Western blotting with anti-FasL antibodies using known amounts of recombinant sFasL as standard.Protein Sequencing.
Purified soluble FasL was blotted onto polyvinylidene difluoride membrane (BioRad Labs., Hercules, CA) as previously described (29), and then sequenced using a gas phase sequencer (ABI 120A; Perkin Elmer, Foster City, CA) coupled to an analyzer (ABI 120A; Perkin Elmer) equipped with a phenylthiohydantoin C18 2.1 × 250 mm column. Data was analyzed using software (ABI 610; Perkin Elmer).Triton X-114 Extraction of Cells and Phase Separation.
293T cells transiently transfected with full-length hFasL (wild type and mutants), muFasL, or empty plasmid were transferred in Opti-MEM medium for 5 d. Supernatants were harvested, and the cells were lysed in 2% precondensed Triton X-114 in PBS (28) containing a protease inhibitor cocktail (CømpleteTM; Boehringer Mannheim GmbH, Mannheim, Germany) and subjected to phase separation. Proteins were recovered free of detergent by precipitation with 1.25 volume CHCl3/MeOH (1:4, vol/vol) before Western blotting analysis.Western Blotting.
FasL blotted onto nitrocellulose membranes was detected with anti-PE62 (2 µg/ml, in blocking buffer: PBS, 0.5% Tween 20, and 4% skimmed milk), followed by goat anti- rabbit immunoglobulins coupled to horseradish peroxidase. Peroxidase activity was detected by enhanced chemiluminescence.Gel Permeation Chromatography.
The supernatant of Neuro-2a cells expressing FasL was concentrated 5 times, and then 200 µl was loaded onto a column (Superdex-200 HR10/30; Pharmacia). Proteins were eluted in PBS at 0.5 ml/min and fractions were analyzed by Western blotting and cytotoxic assays. The column was calibrated with standard proteins ferritin (440 kD), bovine serum albumin (67 kD), and ribonuclease A (13.7 kD).Cytotoxic Assay.
The cytotoxic assay was performed essentially as described previously (26). In brief, 50,000 cells were incubated for 16 h in 100 µl medium containing the indicated concentrations of ligands in the presence or absence of 1 µg/ml M2 antibody (2 µg/ml for TRAIL). For HT-29 cells, incubation was carried out for 40 h in the presence of 50 ng/ml of hIFN-Cell Proliferation Assay.
CT6 cells were grown for 4 d in the presence of a reduced concentration of EL-4 supernatant (2.5%) before the proliferation experiment. Cells were incubated in 96-well plates (40,000 cells/well) for 16 h with the indicated concentrations of muTNF-In Vitro Fas-FasL Binding Assay.
ELISAs were performed as previously described (26) except that FasL incubation was done in the presence or in the absence of 1 µg/ml of M2 antibody.FACS® Staining.
Cells expressing Fas (A20, Jurkat) or not expressing Fas (A20R) were stained in 25 µl FACS® buffer (PBS, 10% FCS, 0.02% NaN3) with 2.5 µg of Flag tagged soluble hFasL for 20 min at 4°C, followed by anti-Flag M2 (1.5 µg) and DTAF-labeled anti-mouse antibody. A20 cells were also stained with 1 µg biotinylated Jo-2 antibody followed by streptavidin-DTAF.Treatment of Mice with sFasL, sTRAIL, sTNF-, and
-Flag M2
Antibody.
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Results and Discussion |
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An uncharacterized metalloproteinase cleaves the 40-kD
membrane-bound FasL to generate the 26-29 kD-soluble
fragment (13, 14, 17). In agreement with these published
results, we found that processed sFasL was detectable in supernatants of 293 cells transiently or stably transfected with
the full-length human FasL (Fig. 1 A) and that this release
was inhibited upon addition of metalloproteinase inhibitors
(data not shown). Murine FasL underwent a similar conversion to soluble FasL (Fig. 1 B). By gel filtration chromatography, native sFasL of both species had an apparent molecular size of ~70 kD, indicating that the released ligand
had a homotrimeric structure (Fig. 2 B, and data not
shown). Subsequent isolation of sFasL by Fas/Fc affinity
chromatography and NH2-terminal sequence analysis revealed that sFasL begins with Leu127, suggesting that the
cleavage occurs between Ser126 and Leu127 (Fig. 1 C).
Cleavage in TNF- occurs in the equivalent region between Ala76 and Val77, and is mediated by at least two
zinc metalloproteases (TNF-
converting enzyme [TACE],
and a disintegrin and metalloprotease 10 [AD10 or
ADAM10]; references 30). Interestingly, the processing
activity of TNF-
is only marginally affected by mutations
Ala76Ser and Val77Leu (32), suggesting that the Ser-Leu
site of FasL may also be a substrate of the TNF-
-converting proteases, in agreement with the observation that inhibitors known to block TNF-
conversion also inhibit FasL
release (14, 15, 17). In contrast, mutations of the flanking
amino acids Ala76 and Val77 to acidic residues influences
the processing of TNF-
. We performed similar mutation experiments with FasL (Fig. 1 D). Mutations at the P1
(Ser126Glu) and P1' (Leu127Glu) sites partially prevented
the release of sFasL, whereas the double mutation
(Ser126Glu, Leu127Glu) resulted in the loss of detectable
sFasL, indicating that the metalloproteinase present in 293 cells uses this cleavage site. While this manuscript was under revision, Tanaka et al. reported on the identification of
the cleavage of FasL transfected into WR19L cells (33).
Their analysis revealed that membrane-bound human FasL
expressed in the WR19L murine T cell line was cleaved
between Lys129 and Gln130, three amino acid residues
downstream of the cleavage site determined in this paper.
Surprisingly, the mutation of their P1 residue Lys130 into
Ala had no effect on the processing of FasL, and FasL resistant to cleavage was only generated after deletions of several amino acid residues, which probably affect or even delete the Ser126-Leu127 processing site. This suggests either
that the murine metalloproteinase present in WR19L cells
has a distinct specificity from its human homologue present
in 293 cells, or that the sFasL found in WR19L cell supernatants has undergone further proteolytic processing by a
trypsin-like protease.
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Unexpectedly, the specific activity of sFasL after its purification and removal of residual membrane-bound FasL by Triton X-114 extraction was marginal. Human Jurkat T cells, which are highly sensitive to apoptosis induced by anti-Fas antibodies (34), remained viable at sFasL concentrations exceeding 1 µg/ml (Fig. 1 E). Recombinant sFasL corresponding in length to the naturally processed form was equally inactive (Fig. 1, C and E). This low activity was surprising in view of several previous reports showing that recombinant sFasL or supernatants of cells overexpressing FasL were very active. One well-described example is the highly apoptotic supernatant of Neuro-2a cells transfected with murine FasL (19), which induces apoptosis in human Jurkat or mouse A20 cells at concentrations as low as 1 ng/ml (Fig. 2 A). Western blot analysis (Fig. 2 B) revealed that the recombinant FasL detected in these supernatants was not the processed sFasL, but corresponded to the membrane-bound, unprocessed form (Fig. 2 B). Accordingly, the active fractions were eluted in the void volume (>500 kD) of a gel filtration column (Superdex 200; Pharmacia) and not at the expected molecular weight of trimeric sFasL (70 kD), indicating that the high apoptotic activity was associated with aggregated forms of FasL. It is likely that Neuro-2a cells express little or no processing proteases, and that membrane fragments or vesicles containing the membrane-bound FasLs are released into the supernatant. This conclusion was corroborated by the observation that after ultracentrifugation (100,000 g for 2 h), the activity was recovered in the pellet (data not shown). Moreover, most of the activity present in some batches of recombinant sFasL was attributable to small quantities of contaminating aggregates of sFasL (data not shown).
Since aggregated or membrane-bound FasL was considerably more active than trimeric sFasL, we reasoned that controlled cross-linking of sFasL, mimicking membrane-bound FasL (Fig. 3 D), should lead to an increase in activity. We took advantage of the presence of a Flag tag on the recombinant sFasL to induce sFasL aggregation. Fig. 3 A shows that the mere addition of cross-linking anti-FasL antibodies resulted in a >1,000-fold activity increase of recombinant sFasL on Jurkat cells. Other cell lines tested responded identically. Human cells that were insensitive to sFasL up to 1 µg/ml underwent apoptosis at concentrations as low as 1-10 ng/ml (BJAB B lymphoma cells, human colon carcinoma HT29) with aggregated sFasL. In agreement with the higher affinity of human sFasL to mouse Fas (26), we found that mouse cell lines were generally more sensitive to FasL-mediated apoptosis, as exemplified by the A20 B lymphoma cell. A20 cells underwent apoptosis in the absence of cross-linking antibodies at 100 ng/ml. This (and the presence of contaminating aggregates) may explain previous results showing that recombinant sFasL produced in yeast was active at 1-10 ng/ml concentrations on the murine W4 cell line (human cells were not tested; reference 17).
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We tested whether other members of the TNF ligand
family known to induce apoptosis also required cross-linking for their activity (Fig. 3 B). Similar to FasL, TRAIL
(35, 36) was active at low concentrations only in the presence of cross-linking antibodies (Fig. 3 B). No apoptosis
was observed with sTRAIL at concentrations as high as 5 µg/ml. In contrast, cross-linking of sTWEAK (24) did not
increase its activity (Fig. 3 B), suggesting that this ligand
can exert its apoptotic activity in the soluble form. Interestingly, the activity of sTNF- was differentially influenced
by the presence of cross-linking antibodies depending on
the receptor triggered. To distinguish between TNFR1-
and TNFR2-mediated effects, we took advantage of the
fact that human TNF-
only binds to murine TNFR1, but
not to murine TNFR2 (37). When TNF-
was added to
WEHI164 cells, which are known to undergo apoptosis
upon stimulation of TNFR1, the activity was independent
of the presence of cross-linking antibodies (Fig. 3 B). In
contrast, the proliferative effect of TNF-
on mouse CT6
cells, which is mediated through TNFR2-dependent signaling pathways and is therefore only observed in response to murine TNF-
, was highly increased in the presence of
cross-linking antibodies (Fig. 3 C). This is in line with results demonstrating that membrane-bound TNF-
is the
prime activating ligand for TNFR2-mediated responses
(38).
These results suggested that sFasL and sTRAIL should be either not or only poorly active in vivo. Indeed, mice injected with 12.5 µg of sFasL or sTRAIL survived and showed no signs of sickness (Fig. 4 A). In contrast, injection of sFasL followed by cross-linking anti-Flag antibodies was fatal and mice died within 3 h, similar to mice treated with agonistic anti-Fas antibodies (18) or with FasL contained in supernatants of transfected Neuro-2a cells (19). The cross-linked FasL induced lethal liver hemorrhages and hepatocyte apoptosis (Fig. 4 B), whereas in the absence of the anti-Flag antibody, hepatocytes had a normal morphology (Fig. 4 B). The administration of the anti-Flag antibody alone, or of sFasL with an isotype-matched irrelevant antibody, also had no effect. Interestingly, treatment of mice with cross-linked sTRAIL was not lethal, despite the broad expression of the two cytotoxic TRAIL receptors (39). Whether this is due to the expression of the inhibitory TRAILR3 (39, 43, 44), TRAILR4 (45), or other inhibitory molecules remains to be determined.
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There are two possibilities to explain the absence of apoptotic activity of sFasL. Either trimeric sFasL displays a low affinity for its receptor Fas, which is increased upon aggregation and multimeric binding as seen for many protein- protein interactions involving multivalent partners (46), or trimerization of Fas is not sufficient to transmit FasL-mediated death signal (in contrast to TWEAK receptor and TNFR1). We measured sFasL binding to Fas fused to the human constant region of human IgG (Fas/Fc) in the presence or in the absence of aggregating antibody (Fig. 5 A), and to cells (Fig. 5 B). Binding of trimeric sFasL was observed in both cases, and aggregation of sFasL did not significantly increase binding efficiency to Fas/Fc.
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Most of the members of the TNF ligand family are initially membrane bound, but can undergo processing and
shedding by metalloendoproteinases. The efficacy of the
ligand release varies. Membrane-bound TNF- is immediately processed by proteolytic cleavage and the soluble form released into the circulation can exert its various activities locally and systemically. On the other end of the
spectrum stands CD40L, whose physiological activities,
such as B cell help, completely depend on the membrane
form, and for which processing has not been reported (47).
Local cell to cell contact appears to be critical for T cell-
specific immunosurveillance and, indeed, FasL-mediated
killing of virus-infected or tumor target cells is a highly
specific process (4) that assures that neighboring cells or tissues are not affected. This specificity can only be guaranteed if the cytotoxic ligand remains associated with the lymphocytes. However, FasL is rapidly released from cells
and detectable levels of sFasL have been found in sera from
patients with large granular lymphocytic leukemias, NK
lymphomas (17), or melanoma (11). Considering the hepatic toxicity of FasL, circulating sFasL, if active, would be
pathological. Neither patients with tumors nor individuals
with high numbers of activated T cells (for example, after a
viral infection) do suffer from hepatitis, implying that sFasL
in vivo is at best poorly active, in agreement with our results. We cannot exclude, however, that high local concentrations of sFasL, in conjunction with other cytotoxic
agents, may lead to cell death.
Our experiments also suggest that the mere trimerization
of Fas by FasL may not suffice to transmit death signals. It is
thought that the trimeric FasL induces Fas trimer formation, followed by the binding of Fas-associated death domain-containing protein (FADD) to the death domain of
Fas (48). FADD then recruits the caspase FADD homologous IL-1 converting enzyme (ICE)-like protease (FLICE)/MACH/caspase 8 (51, 52), providing the connection of death receptors to caspases (53). The exact stoichiometry of adapters mediating caspase activation has,
however, never been determined, and it is possible that
multiple Fas trimers are required to allow the assembly of
multiple FADD adapters bringing several FLICE proteins
together and allowing reciprocal cleavage and subsequent activation of the caspase. The requirement of the formation
of large aggregates of Fas for signal transduction is also suggested by the efficacy of agonistic antibodies of the IgM
and IgG3 type, which are known to be multimeric or to
form aggregates (54).
It could be predicted from the results presented here that
circulating cells expressing membrane-bound FasL are
more potent inducers of systemic tissue damage, and that
the conversion of the ligand into the soluble form serves to
downregulate the potentially harmful apoptotic activity. If
true, the proposed use of metalloproteinase inhibitors to
treat systemic tissue damage caused by circulating sFasL
(17), which has proved efficient in the case of TNF- (1,
2), could therefore result in an exacerbation rather than an
amelioration of the disease in the case of FasL.
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Footnotes |
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Address correspondence to Jürg Tschopp, Institute of Biochemistry, University of Lausanne, Ch. des Boveresses 155, CH-1066 Epalinges, Switzerland. Phone: 41-21-692-5738; Fax: 41-21-692-5705; E-mail: jurg.tschopp{at}ib.unil.ch
Received for publication 11 December 1997 and in revised form 11 February 1998.
1Abbreviations used in this paper: aa, amino acids; DTAF, dichlorotriazinglaminofluorescein; FADD, Fas-associated death domain-containing protein; Fas/Fc, Fas with human immunoglobulin Fc; h, human; L, ligand; mu, murine; s, soluble; TRAIL, TNF-related apoptosis-inducing ligand; TWEAK, TNF-related ligand that weakly induces apoptosis.We thank S. Hertig (University of Lausanne, Epalinges, Switzerland), T. Bornand (University of Lausanne, Epalinges, Switzerland), S. Muljana (University Hospital, Zürich, Switzerland), and S. Aslan (University of Lausanne, Epalinges, Switzerland) for technical and secretarial assistance, S. Foley (Biogen, Cambridge, MA) and J. Browning (Biogen, Cambridge, MA) for protein sequence analysis, and S. Belli (University of Lausanne, Epalinges, Switzerland) for reading the manuscript.
This work was supported by grants from the Swiss National Science Foundation (to J. Tschopp and A. Fontana; NFP38), the Swiss Federal Office of Public Health (to P. Schneider and J. Tschopp), and from the Human Frontier Science (to M. Hahne).
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