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,
*
Departments of Surgery, Molecular Genetics and Biochemistry and
Pathology, University of Pittsburgh School of Medicine and University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213; and
University of Pittsburgh Mass Spectrometry Facility, University of Pittsburgh Center for Biotechnology and Bioengineering, Pittsburgh, PA 15219
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition to possessing the appropriate intracellular Ag-processing machinery, these highly efficient APCs also express cell surface markers that are shared with cells of the myeloid and lymphoid lineages (e.g., CD13), whose function has been associated with ectoenzymatic activity. Until recently, the biologic role of these ectoenzymes was poorly understood, but evidence is emerging that supports their role as markers for cellular activation and differentiation, signal-transducing molecules, molecules involved in cellular adhesion and migration, molecules that inactivate soluble mediators, and molecules involved in Ag processing (18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41). To date, there is no information concerning the existence or nature of cellular ectoenzymes on DCs other than the absence or the presence of the specific Ag as determined by phenotypic analysis. As DCs are increasingly appreciated as tools used in peptide vaccines, the analysis of DC ectoenzyme function becomes a critical limiting parameter for proper loading and presentation. In this study we report on the rapid and efficient degradation of synthetic class I peptides by endo- and exoproteases on the surface of human myeloid dendritic cells and offer alternatives to inhibit unwanted peptide degradation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PBMCs were obtained from a leukocyte research product (Leukopak, a by-product of platelet donation, Central Blood Bank, Pittsburgh, PA) by first washing the cells extensively in PBS to remove residual platelets, followed by centrifugation over Ficoll (Sigma, St. Louis, MO). The PBMC layer was washed resuspended in RPMI 1640 (Life Technologies, Grand Island, NY) and plated at a density of 5 x 106 cells/ml in tissue culture flasks. Monocytes from this population were allowed to adhere for 2 h at 37 C in a humidified incubator with 5% CO2. After 2 h, the nonadherent cells were washed in PBS several times, and the adherent cells were recultured in serum-free medium (AIM V, Life Technologies) containing 1000 U/ml of human recombinant GM-CSF and IL-4 (Schering-Plough, Kenilworth, NJ). Loose clumps of DCs became apparent after 2 or 3 days and peaked after days 5 to 7 in culture. The cells exhibited a typical DC morphology by light microscopy. Cell populations were consistently >80% DCs by FACS analysis. Cells were typically CD3-, CD14-, CD20-, HLA-DR+, CD40+, CD80+, and CD86+. These cells also displayed high levels of LFA-1 and ICAM-1 on their surface.
Synthetic peptides
The following peptides were synthesized at the University of Pittsburgh Peptide Synthesis Facility: Melan-A/MART-1(27, 28, 29, 30, 31, 32, 33, 34, 35), AAGIGILTV; tyrosinase(368–376), YMDGTMSQV; gp100(280–288), YLEPGPVTA; and angiotensin, DRVYIHPFHL (Sigma).
Assay for cell surface enzymatic activity
DCs were washed several times in PBS and resuspended to a concentration of 1 x 106/250 µl. They were preincubated in PBS at 37°C for 15 min before the addition of peptide. Peptide was added at a final concentration of 48 nmol and incubated for varying lengths of time. At the indicated time periods, supernatants were harvested by microcentrifugation and immediately frozen on dry ice and acetone or were loaded directly onto an analytical C18 column.
High performance liquid chromatography
Potential cleavage fragments were separated on an analytical C18 column (Waters Associates, Millipore, Bedford, MA) and separated with a linear gradient (3–60% B, 60 min) using a buffer system consisting 0.1% trifluoroacetic acid/H2O (buffer A) and 100% acetonitrile containing 0.1% trifluoroacetic acid using a Rainin HPLC system (Rainin, Emeryville, CA). 1.0 ml fractions were collected. Absorbance was at 214 nm.
Mass spectrometry/sequence analysis
Portions of the UV-absorbing HPLC fractions were concentrated and directly injected into a Fisons Quattro II triple quadrupole mass spectrometer (Fisons, Loughborough, U.K.) equipped with an electrospray ionization source. The electrospray needle was operated at a voltage differential of 3.5 keV using a sheath flow of 5 µl/min consisting of 50% acetonitrile/H2O containing 1% acetic acid. Mass spectra were obtained by scanning the range of mass to charge values of 300 to 1350 every 2.7 s and summing the individual spectra. Collision-induced dissociation spectra were obtained by selecting the appropriate mass ion and scanning at 500 amu/s using 3 mtorr Ar in the collision chamber.
Treatment with diazotized sulfanilic acid (DASA)
DASA was prepared as previously described (42). Briefly, NaNO2 was dissolved in cold H2O and sulfanilic acid added (1 x 10-4 mol) until dissolved. Concentrated HCl was added, and the mixture was chilled on ice. The product precipitated upon standing. The product was washed, and the concentration was determined by the addition of excess resorcinol and monitoring the OD at 385 nm of the colored product. Cells were treated with 3.0 mM DASA for 20 min, and the cells were washed and processed for enzyme assays as described above.
Inhibition of ectoenzyme function
Cells were incubated in the presence of a variety of enzyme
inhibitors, including soybean trypsin inhibitor, PMSF, pepstatin A,
EDTA, phophoramidon, aprotinin, ortho-phenanthroline, ubiquitin,
5,5'-dithiobis-2-nitrobenzoic acid, N-ethylmaleimide,
iodoacetamide, DTT, leupeptin, benzamidine iodoacetic acid,
N-
-p-tosyl-L-lysine chloromethyl
ketone, and L-p-tosylamino-2-phenylethyl
chloromethyl ketone (Sigma) at varying concentrations for 15 to 30 min
at 37°C before the addition of peptide. For the purposes of the
figures presented in this study, the final concentration of inhibitors
was 1 mM unless stated otherwise.
DC membrane isolation
DC membranes were isolated using an aqueous two-phase polymer system as previously described (43). Purity was assessed by monitoring the activity of the following marker enzymes: aminopeptidase, (plasma membranes), n-acetyl-ß-D-glucosaminidase (lysosomes), lactate dehydrogenase (cytosol), cytochrome oxidase (mitochondria), thiamine pryophosphatase (Golgi), and NADH diaphorase (endoplasmic reticulum). A purity of 85 to 90% or better was estimated by marker enzyme analysis.
Methylation of class I peptides
When necessary, peptides were methylated by dissolving the peptide in a solution of 3 N methanolic HCl prepared by the addition of acetyl chloride to dry methanol. Methylated peptides were purified by reverse phase HPLC. Methylation was verified by mass increases of 14 amu or multiples thereof.
Peptide loading/functional assays
A2+ dendritic cells (1 x 106) were preincubated with ubiquitin (1 mM) and DTT (0.1 µM) or without inhibitors for 30 min before the addition of peptide. Tyrosinase peptide (48 nmol) was added, and the cells were incubated for an additional 60 min at 37°C. Supernatants were then pulsed onto chromium-labeled Jurkat A2/Kb target cells in the presence of ß2m (Sigma) for 2 h. A tyrosinase-specific effector cell line (CAM) was added (E:T cell ratio, 5:1), and the percent specific lysis was determined by a standard chromium release assay after 4 h at 37°C.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We wondered whether the synthetic peptides derived from tyrosinase
would persist in culture with DCs. The interaction of
GM-CSF/IL-4-cultured DCs with the tyrosinase peptide yielded a rapid
loss in area of the parent peak and the appearance of five additional
peaks upon HPLC analysis (Fig. 1
A). These peaks were not
present when supernatants of DCs were analyzed in the absence of
peptide (Fig. 1B) or when the peptide was incubated with
DC-conditioned medium (Fig. 1C). The retention times for the
products are shown in Table I. The nature
and identity of these species were initially not clear. Possibilities
included degradation of the added peptide from ectoproteases present on
the cell surface or from internalization and secretion. Alternatively,
it was possible that peptides were derived from displacement of
endogenous class I or class II peptides. When a portion of the material
was analyzed at 280 nm, only peaks with retention times at 4.7 and 15.7
min displayed strong absorbance, indicating the presence of the
tyrosine group (data not shown).
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The HPLC peaks were concentrated and subjected to mass
spectrometry analysis. All the fragment peaks yielded masses smaller
than the measured parent mass (Table I
). Mass analysis indicated that
the peak at 4.7 min contained a number of species in addition to a mass
ion at 182, suggesting the presence of a free tyrosine residue. Since
the elution time of this product was so early, the other mass ion
species may represent species that were carried over from the injection
peak. In addition, we confirmed the elution time of free tyrosine by
injection of tyrosine standards (not shown). Sequence analysis of the
remaining mass ions was performed through collision-induced
dissociation tandem mass spectrometry (Fig. 2, A–D, and Table I).
Sequence analysis indicated that mass 641 corresponded to MDGTMS, mass
737 to DGTMSQV, mass 804 to YMDGTMS, and mass 868 to MDGTMSQV. These
results suggested that N-terminal (exoprotease) as well as C-terminal
(exoprotease) and possible endoprotease activities were acting on the
peptide.
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Degradation of the tyrosinase peptide was rapid, with the majority
(70%) of the degradation occurring within 10 min (Fig. 3). Degradation rates did vary from donor
to donor, but usually not more than 15 to 20% (not shown). Continuous
increases were not seen with any of the degradation products, since
they also serve as additional substrates. Upon increased incubation
times (15 min), masses of 717 (YMDGTM) and 554 (MDGTM) were also
identified (not shown).
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The finding of enhanced peptide degradation strongly suggested
that the degradative activity was at least cell associated. To evaluate
this possibility, DCs were incubated in the presence of the tyrosinase
peptide for 4 min, and the cells were rapidly removed by
microcentrifugation. The conditioned supernatant was incubated for an
additional 56 min, and the amount of tyrosinase remaining was
quantitated by HPLC. The results (shown in Fig. 4A) indicated that degradation
ceased upon cell removal. In control cultures containing DCs, the
parent peptide was reduced from 48 to 1.3 nmol within 15 min. These
results further support the idea of cell-associated enzymatic activity
and also indicates that the interaction of the peptide with the DC does
not stimulate the secretion of degradative enzymes.
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, B and C), and the amounts of the parent peptides
were quantitated by HPLC. Only the peptide that was in contact with the
DCs showed substantial amounts of degradation. These results indicate
that the major degradative events are not stimulated by
peptide-specific interactions that result in the release of enzymes
into the extracellular medium. To further investigate whether these
degradative events were truly due to ectoenzymes, the DC cell surface
was modified with DASA, a nonpermeable membrane-modifying reagent that
forms azo, diazoamino, S-azo, or thio ether derivatives with
proteins and lipids without passing through the membrane (42, 44). The
results shown in Figure 4D indicate that 70 to 80% of the
degradative activity could be abrogated by this treatment. Total
inhibition could not be achieved due to a loss in cell viability upon
prolonged incubation times (>20 min). The reaction time for DASA
modification was kept at 20 min (at concentrations ranging from 0.3–3
mM) to assure viability of 98% or greater and to avoid the potential
leaking of intracellular proteases into the extracellular medium.
Additional evidence for the membrane association of DC-degradative
enzymes comes from isolated DC membranes that were tested for
peptide-degradative activity. Table II
compares the tyrosinase degradation products identified by tandem mass
spectrometry for intact cells and isolated membranes. All the species
identified using intact cells were identified with purified DC
membranes. These results strongly support a plasma membrane association
and an ecto orientation for the degradative events.
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E). No loss was detected, suggesting that uptake during the
assay (<15 min) was not responsible for the observed loss. If uptake
was required, a lag period would have been noted before the detection
of products. The literature estimates that DCs can accumulate solute
molecules at a rate of 2400 fl/h (45, 46). This would translate
into a loss of <1.2% from the DC/peptide mixture, which would fall
within the error associated with analytical HPLC and could not account
for the loss of parent peptide observed in these studies. Inhibition of DC ectoenzyme activity using protease inhibitors
To determine the class of enzyme(s) responsible for the
degradation of tyrosinase, cells were incubated with a variety of
protease inhibitors (Table III) before
the addition of peptide. Only four reagents were capable of affecting
DC ectoenzyme cleavage; ubiquitin, EDTA, o-phenanthroline,
and DTT. All other inhibitors of serine, aspartic, metallo-, and thiol
proteases were without effect. Inhibition of the N-terminal degradative
activity could be completely inhibited by ubiquitin, a specific
competitive aminopeptidase inhibitor, EDTA, and
o-phenanthroline (all >95% inhibition). CD13,
aminopeptidase N, is known to exist on a variety of cell types,
including DCs and may play a role in migration and Ag presentation
processes (41, 47, 48). These data suggest that CD13 or CD13-like
enzymes are responsible for the N-terminal degradative activity. The
remaining major cleavage product of the tyrosinase peptide is the 7-mer
YMDGTMS. The only reagent capable of inhibiting this activity was DTT
(83% inhibition). This suggests that this type of enzymatic activity
requires intact disulfide bonds for proper function. Studies are
ongoing to determine the precise nature of this enzyme. We also
investigated the possibility that tyrosinase degradation could be
altered by the addition of protein in the form of either serum (5%) or
ß2m (10 µg/ml), conditions often used during peptide
pulsing. Neither manipulation significantly altered tyrosinase peptide
degradation by DCs (not shown).
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DCs were incubated with two other class I-associated synthetic
peptides, AAGIGILTV and YLEPGPVTA, as well as with an unrelated 10-mer
peptide, angiotensin. Incubation was allowed to proceed for 7
min, the supernatants were harvested, the products were separated by
HPLC, and the cleavage sites were mapped and sequenced using
collision-induced dissociation tandem mass spectrometry. The cleavage
sites are shown in Figure 5. Both exo and
endo cleavage sites are present with the majority of the cleavage
events taking place with tyrosinase and angiotensin.
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We compared the rates of tyrosinase degradation by DC ectoenzymes
with a variety of cell populations, as shown in Figure 6. The nonadherent cell population
isolated after the adherence step in the myeloid DC preparation yielded
no degradation upon incubation with the tyrosinase peptide during the
15-min assay period. Tyrosinase degradation was detected within the
adherent peripheral blood monocyte population, which is used as the
starting cell type for myeloid DC preparation. The rate of tyrosinase
degradation was 1.0 nmol/min/106 cells, with only limited
specificities on the three class I peptides and no activity against
angiotensin (Figs. 5 and 6). Upon culture in AIM V medium for 7 days,
an increased rate of tyrosinase degradation was observed (2.9
nmol/min/106 cells), but with broader specificity toward
tyrosinase and angiotensin substrates. Day 7 DCs cultured in GM-CSF and
IL-4 displayed the highest tyrosinase degradation rate (4.3
nmol/min/106) and the broadest peptide specificity. These
results indicate that upon maturation in GM-CSF and IL-4, the myeloid
DCs not only express an increased amount of their baseline enzyme
activities, but additional enzyme class specificities begin to emerge.
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Human DCs (5 x 106) were incubated in the
absence and the presence of ubiquitin and DTT (1.0 and 0.1 mM,
respectively) for 30 min at 37°C before the addition of tyrosinase
peptide. After addition of peptide, incubation was allowed to proceed
for an additional 30 min. DCs were then removed by centrifugation, and
supernatants containing peptide (0.1 µM) were pulsed onto
51Cr-labeled A2/Kb target cells in the presence of
ß2m (10 µg/ml). A tyrosinase-specific effector cell
line (CAM) was used as the effector cell, and the percent specific
lysis was determined after 4 h at 37°C. Figure 7 demonstrates that the
tyrosinase-specific effector cells recognized only the targets that had
been pulsed with inhibitor-treated DC-peptide supernatants. In
conjunction with the above data, we conclude that the lack of
recognition of targets pulsed with DC-peptide supernatants is due to
efficient degradation by DC ectoenzymes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Sequence analysis by tandem mass spectrometry indicated that the tyrosinase peptide suffers substantial loss at the N-terminus, suggesting possible aminopeptidase activity. Inhibition studies indicated that the N-terminal degradative events could be abrogated by treatment with EDTA, ortho-phenanthroline, and ubiquitin (with >95% inhibition for N-terminal degradation for these three inhibitors). The two former reagents support the presence of a metalloprotease, while the latter inhibitor (a specific competitive inhibitor of aminopeptidases) supports the presence of the CD13 metalloprotease (aminopeptidase N) or CD13-like activity. CD13 is expressed by a number of cell types, including monocytes and dendritic cells and has been suggested to have important physiologic significance (20, 21, 30, 41, 42, 43). We have shown in this study that the overall cell surface enzymatic activity of the DC is increased four- to fivefold upon incubation in culture in the presence of GM-CSF/IL-4 compared with the starting peripheral blood monocyte population. While the enzymatic activities of the day 7 monocytes and the day 7 DCs are similar in substrate specificities, the day 7 DCs exhibit the highest enzymatic rate as well as the appearance of a C-terminal exoproteolytic event that is capable of removing the C-terminal residue from substrate angiotensin. Both the DC population and the day 7 monocyte population exhibit not only increased degradative activities toward neutral and hydrophobic N-terminals, but also the emergence of degradative activities toward acidic N-terminals compared with the starting monocyte population. This suggests the emergence of aminopeptidases of altered specificities (aminopeptidase A). In addition to the alterations in N-terminal degradative activities and specificities, new endoproteolytic activities emerge upon culture. This is clearly seen when angiotensin is used as the substrate.
One of the major cleavage products of the tyrosinase peptide is the hexapeptide YMDGTMS. The possibility exists that cleavage was produced by a carboxypeptidase-like activity. However, typical carboxypeptidases (A, B, P, and Y) are responsive to inhibition with metal chelators, sulfhydryl modifying reagents, or serine protease inhibitors. All these inhibitors were without effect. The only reagent that provided near complete inhibition was DTT. This suggests that the formation of disulfide bonds is critical for this enzymatic activity. This resistance to typical inhibitors is suggestive of a cathepsin-like enzyme, since human, bovine, and rabbit cathepsin D enzymes demonstrate DTT sensitivity (49). However, typical carboxyl proteases such as cathepsin D usually show sensitivity toward pepstatin, which was not seen in these studies. Additional work is in progress to determine the identity of this enzyme.
Since DCs are now regarded as the APC of choice and since they possess a repertoire of highly active enzymes on their cell surface, protein Ag loading becomes a critical issue, especially when low levels of previously processed peptides are being pulsed onto an APC. This problem can be circumvented in two ways. With respect to synthetic peptides, one can effectively overload the system such that sufficient peptide remains in an intact state for proper loading. However, the critical concentration may be different for each peptide, since degradation is enzyme specific and therefore sequence dependent. It is suggested that with synthetic peptides, degradative events should be investigated and controlled for. N- and C-terminal modifications of synthetic peptides can be performed to avoid unwanted degradation; however, once modified, the peptides will lose their ability to bind to class I molecules. One potential modification that will retain the peptide’s natural structure would be the substitution of D-amino acids at critical positions (i.e., at the N-terminus), since aminopeptidase activity is greatly reduced toward D-substituted substrates. However, such modifications may or may not be tolerated at the level of the class I molecule.
A more critical problem comes into play when low levels of eluted natural peptides and peptides isolated from tumor cell class I molecules are used for pulsing. These peptides are often in the femtomole to near picomole range, and efficient loading of these peptides will be dependent on several factors. First, differences in degradative rates of peptides by ectoenzymes may be attributable to species differences. Second, since some of these ectoenzymes have a somewhat ubiquitous nature, the cell type becomes a critical parameter (i.e., those that are highly active in migration and/or Ag processing displaying the broadest and most rapid degradative activities). Third, the state of cellular maturation and activation (including the effects of cytokines) may influence the degree and specificity of ectoenzymatic activity. Finally, degradation will be sequence dependent. The latter statement suggests that in instances where peptides are presented to the APC as extremely dilute mixtures in small volumes used for pulsing, only those peptides with the most resistant sequences will survive. This will allow for the APC system to become biased toward certain peptides and the T cells they elicit. It is suggested that for these conditions, loading in the presence of protease/peptidase inhibitors may be advantageous.
In this study near complete inhibition of tyrosinase degradation could be attained in the presence of DTT and ubiquitin. Since DTT is in the family of thiol compounds, similar results should be obtained with 2-ME. The latter compound has been used as a typical additive to cell culture media and therefore presents itself as a nontoxic additive during the loading process, although it remains to be seen whether the concentration used in cell culture is sufficient for inhibition of tyrosinase degradation. The remaining compound critical for the inhibition of tyrosinase degradation is ubiquitin. This competitive inhibitor is also nontoxic and has been used in vitro and in vivo for over a decade. The addition of these two inhibitors to DC cultures inhibited tryrosinase peptide degradation and allowed efficient pulsing onto target cells and recognition by tyrosinase-specific CTL. Predicting the outcome of peptide loading will ultimately depend on the affinity of peptide for class I molecules vs the rate of peptide degradation.
From a biologic point of view, the role of ectoenzymes on the surface of the DC may have important physiologic significance. First, upon cellular maturation in the presence of cytokines, an enhanced cell surface enzyme repertoire with increased activity may be necessary for the increased ability to inactivate soluble or inflammatory mediators (50). Second, this enhanced ectoenzymatic activity could enhance DC migration, since CD13 activity has been shown to play a role in adhesion and tumor cell infiltration (48). Third, the DC ectoenzymatic activity could function in extracellular Ag processing, since CD13 has already been implicated in the trimming of class II molecules (41). Finally, since CD13 has been described as the receptor for coronaviruses, and CD13-like surface structures have been shown to be involved in CMV infection (51, 52, 53), similar DC surface enzymes may serve as portals necessary for viral entry.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Andrew A. Amoscato, University of Pittsburgh Mass Spectrometry Facility, University of Pittsburgh Center for Biotechnology and Bioengineering, Suite 206, 300 Technology Dr., Pittsburgh, PA 15219. E-mail address:
3 Abbreviations used in this paper: DC, dendritic cell; GM-CSF, granulocyte-macrophage CSF; DASA, diazotized sulfanilic acid.
Received for publication November 25, 1997. Accepted for publication June 18, 1998.
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