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The Journal of Immunology, 2000, 164: 129-135.
Copyright © 2000 by The American Association of Immunologists

Modification of the Amino Terminus of a Class II Epitope Confers Resistance to Degradation by CD13 on Dendritic Cells and Enhances Presentation to T Cells1

Xin Dong*, Bing An*, Lisa Salvucci Kierstead{dagger}, Walter J. Storkus{dagger},{ddagger}, Andrew A. Amoscato*,§ and Russell D. Salter2,*,{ddagger}

Departments of * Pathology and {dagger} Surgery and {ddagger} University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213; and § University of Pittsburgh Mass Spectrometry Facility, University of Pittsburgh Center for Biotechnology and Bioengineering, Pittsburgh, PA 15219


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Dendritic cells and human B cell lines were compared for ability to present synthetic peptides corresponding to residues 145–159 and 188–203 of human Ig {kappa}-chains to peptide-specific mouse T cell hybridomas restricted by HLA-DR4Dw4. B cell lines presented both peptides, but dendritic cells could only efficiently present the latter epitope. In this paper, we show that dendritic cells degrade the 145–159 peptide, removing four residues from the amino terminus. Binding of the peptide to the class II restriction element is not required for this process. The degradation product is resistant to further cleavage, accumulates in the culture supernatant, and does not bind to HLA-DR4Dw4 or stimulate T cell reactivity. Cleavage can be blocked with bestatin, but not with other protease inhibitors tested, or by a mAb directed against aminopeptidase N (CD13). Addition of an acetyl group to the amino terminus of peptide 145–159 also blocks degradation, and allows dendritic cells to present the peptide to specific T cells with greatly increased efficiency. These results demonstrate that CD13 on dendritic cells is able to selectively and efficiently degrade exogenously provided peptide Ags, in a process that can be blocked by addition of an acetyl group to the amino terminus of the peptide. Modification of the amino terminus of peptide epitopes susceptible to degradation may prove to be useful as a general strategy for enhancing their immunogenicity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Dendritic cells are highly potent stimulators of T cell immunity and are crucial in vivo for initiating primary responses to T cell-dependent Ags (reviewed in Ref. 1). To present Ags to T lymphocytes, dendritic cells in the periphery of the body first internalize Ags by macropinocytosis, receptor-mediated endocytosis, or phagocytosis and generate peptide fragments which bind to both class I and class II MHC molecules (2, 3, 4, 5, 6, 7, 8, 9). Following Ag uptake, dendritic cells increase cell surface levels of class I MHC, class II MHC, and several important costimulatory molecules, including CD80 and CD86, to become highly potent stimulators of Ag-specific T cells. Maturation is triggered by inflammatory stimuli, such as LPS, TNF-{alpha}, or in some cases by Ag uptake itself, and afterwards the dendritic cell migrates to a lymph node where Ag-specific T cells may be encountered (10, 11, 12, 13, 14, 15, 16, 17, 18, 19).

As with other cell types, dendritic cells are able to stimulate Ag-specific T cells following exposure to an appropriate peptide Ag (20, 21, 22, 23). This bypasses the requirement for intracellular processing of the Ag and has been used to direct the immune response toward a particular antigenic epitope. However, short peptides representing the minimal epitope for T cell stimulation may be critically affected by proteolysis because most residues of the peptide are essential for either MHC or TCR binding. As previously shown, some class I epitopes are degraded very rapidly by dendritic cells, suggesting that their utility as vaccines might be limited unless degradation can be inhibited (24).

In this study, we have investigated the ability of two peptide Ags which bind to class II MHC molecules to be presented by dendritic cells or B cell lines to Ag-specific T cell clones. Although B cell lines were able to present both peptides to these T cells, dendritic cells efficiently presented only one of the peptides and cleaved the other to an inactive fragment. We report that the observed degradation is mediated by CD13 expressed on dendritic cells and that modification of the amino terminus of the peptide rendered the epitope resistant to degradation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and peptides

Bestatin, DTT, and tosyl lysine chloromethyl ketone were purchased from Sigma (St. Louis, MO). Peptides were made by the Peptide Synthesis Facility of the University of Pittsburgh Cancer Institute (Pittsburgh, PA) using an Applied Biosystems (Foster City, CA) model 430A synthesizer. Acetylated peptides were prepared by reaction with acetic anhydride after peptide synthesis was completed using a standard protocol. Synthetic peptides used in this study are: {kappa}-chain 145–159 KVQWKVDNALQSGNS (mass = 1674), {kappa}-chain 149–159 KVDNALQSGNS (mass = 1132), and {kappa}-chain 188–203 KHKVYACEVTHQGLSS (mass = 1787). The {kappa}-chain 145–159 peptide was also prepared with an acetyl group at the amino terminus (mass = 1715).

Propagation of dendritic cells from human PBMC

PBMC were obtained from normal donors by venipuncture and then by isolation by density centrifugation on Ficoll-Hypaque gradients (Pharmacia, Uppsala, Sweden). Next, they were resuspended in serum-free AIM-V medium (Life Technologies, Gaithersburg, MD) in T75 flasks. After 1 h at 37°C, nonadherent cells were gently decanted and washed out with HBSS (Life Technologies). Plastic-adherent cells were then cultured in AIM-V medium containing 1000 U/ml rIL-4 and 1000 U/ml rGM-CSF (Schering-Plough, Kenilworth, NJ) for 5–12 days. After 5 days in culture, 75–90% of cells had a dendritic cell morphology and exhibited the following phenotype: CD3-, CD13+, CD14-, CD16-, CD32+, CD40+, CD80+, CD86+, and class I and class II MHC+. Class II HLA typing was performed on all donors either by Ab-based flow cytometry or by molecular typing (performed by Dewayne Falkner and Dr. Penny Morel, University of Pittsburgh School of Medicine).

Incubation of synthetic peptides with dendritic cells or B cell lines

Cells (0.8 x 106) were washed twice with AIM-V medium and resuspended in 50 µl AIM-V medium containing 0.15 mM peptide. After incubation at 37°C for varying times, cells were removed by centrifugation, and supernatants were stored at -70°C. For some experiments, as indicated in the figure legends, protease inhibitors or anti-CD13 were added to cells in 50 µl AIM-V medium for 30 min at 37°C before adding peptide and were present throughout the incubation period. mAb clone 3D8, specific for human CD13, was purchased from LabVision (Fremont, CA), and mAb L243 directed against HLA-DR was obtained from the American Type Culture Collection (Manassas, VA) for these experiments.

HPLC and tandem mass spectrometry

Peptides and cleavage fragments were separated on an analytical C18 column (µ-Bondapak, 3.9 mm x 30 cm, Waters Associates, Bedford, MA) with a linear gradient (3–60% B, 55 min) using a buffer system consisting of 0.1% trifluoroacetic acid/H2O (buffer A), and 100% acetonitrile containing 0.1% trifluoroacetic acid (buffer B) using a Rainin (Emeryville, CA) HPLC system. The flow rate was maintained at 1 ml/min, and 1-ml fractions were collected with absorbance monitored at 214 nm. For mass spectometry and sequence analysis, peak fractions from HPLC were evaporated to near dryness and resuspended in 50% acetonitrile/water containing 1% acetic acid, and then injected into a Fisons (Loughborough, U.K.) Quattro II triple-quadrupole mass spectrometer (at 5 µl/min) equipped with an electrospray ionization source, as previously described (24). Source temperature was maintianed at 70°C. Mass spectra were obtained by scanning mass-to-charge values of 500-1700 every 3 s and summing the individual spectra. The instrument was operated at unit resolution.

HLA-DR4Dw4 peptide binding assay

A total of 3 x 105 T2 cells transfected with HLA-DRB1*0401 were incubated overnight with 10 µM biotinylated class II-associated invariant chain peptide (CLIP)3 (invariant chain residues 81–104) in the presence or absence of unlabeled competitor peptides in AIM-V medium at 37°C. Cells were washed in PBS containing 1% BSA and 0.02% sodium azide, and were then incubated with 0.1 µg streptavidin-PE (Sigma) for 1 h at 4°C, washed again, and fixed with 1% paraformaldehyde. Samples were analyzed by flow cytometry, and median fluorescence values were used to calculate the percentage of inhibition of binding according to the following formula: [1 - (median fluorescence experimental/median fluorescence biotinylated CLIP)] x 100.

T cell hydridoma culture and assays

T cell hybridomas specific for Ig {kappa}-chains were generated previously by immunizing HLA-DR4Dw4 transgenic mice with human IgG as previously described (25, 26). These T cells have been shown to recognize epitopes presented by human B cell lines expressing HLA-DR4Dw4 and Ig {kappa}-chains (26). For Ag-driven proliferation assays, dendritic cells from HLA-DR4Dw4 donors were cultured for 5 days in AIM-V with cytokines before addition of peptide Ag for 16–18 h at 37°C. Peptide was then removed by washing, and cells were further cultured for 3–4 days before adding T cell clones. B cell lines were incubated with peptides overnight the day before their use in T cell assays. Ag-pulsed stimulator cells (104/well) were added to 96-well flat-bottom tissue culture plates (Costar, Corning, NY) containing 104 T cells/well in a total volume of 250 µl of RPMI 1640 medium (Life Technologies) containing 10% FBS (HyClone, Logan, UT). After incubation at 37°C for 24 h, culture supernatants were collected and assayed for IL-2 using CTLL-2 or HT-2 (obtained from the American Type Culture Collection) as indicator cell lines (104 cells/well). Coincubation of T cells and APC were performed in triplicate. Indicator cells were incubated with varying amounts of supernatant in 200 µl total volume at 37°C for 18 h, and then 50 µCi of [3H]thymidine in 10% FBS/RPMI 1640 was added. Incorporation of radioactivity was converted to production of IL-2 for each assay based on a standardized curve.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Recognition of human {kappa}-chain peptides by T cell hybridomas

Transgenic mice expressing the human class II molecule HLA-DR4Dw4 were previously immunized with human IgG (26). Two T cell hybridomas called 1.21 and 2.18, which recognize peptides corresponding to amino acid residues 145–159 and 188–203 of human Ig {kappa}-chains, respectively, were obtained as described and bound to HLA-DR4Dw4. Consistent with a previous report (26), human B-LCL expressing HLA-DR4Dw4 and endogenous Ig {kappa}-chains are recognized by both T cell hybridomas, which produce IL-2 in response to stimulation (our unpublished data). To test the ability of synthetic peptide epitopes to stimulate these T cells, the B cell line Frev, which expresses HLA-DR4Dw4 but lacks endogenous {kappa}-chains, was used (26). In addition, the HLA-DM-deficient cell line T2 transfected with HLA-DRB1*0401, which has a defect in intracellular loading of peptide Ags into class II molecules, was analyzed (27). The latter cells synthesize endogenous {kappa}-chains (our unpublished data), but do not efficiently load peptides from endogenous protein sources other than the invariant chain into class II MHC proteins.

As shown in Fig. 1GoA, both {kappa}-chain peptides 145–159 and 188–203 could be presented by Frev B cells, although with varying efficiency. Dendritic cells derived from an HLA-DR4Dw4-positive donor (DF) presented the 188–203 epitope with similar efficiency, but had a reduced ability to present the 145–159 epitope at 0.5 or 2.0 µM concentrations. Additional experiments shown in Fig. 1GoB extended these findings. The B cell line T2-DR4Dw4 or dendritic cells from a second DR4Dw4-positive donor (WS) were analyzed, and again only the 188–203 epitope was presented efficiently by dendritic cells. Presentation of both epitopes could be blocked by incubation with a DR4-specific Ab, confirming that both T cell clones were restricted by DR4Dw4. A B cell line derived from the same donor which synthesizes endogenous {kappa}-chains could be recognized by both T cell clones in the absence of exogenous peptides (our unpublished data).



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  		FIGURE 1. Presentation of synthetic peptide epitopes from human {kappa}-chains by either dendritic cells or B cell lines to Ag-specific murine T cell hybridomas. A, Dendritic cells were prepared from a healthy donor DF typed as HLA-DRB1*0401 as described in Materials and Methods. After 5 days in culture, dendritic cells were pulsed for 18 h with the indicated concentration of peptides 145–159 or 188–203. The HLA-DR4Dw4-positive B cell line Frev was incubated with peptides in parallel cultures. Peptides were removed by washing and cells were cocultured in triplicate wells at an E:T ratio of 1 with T cell hybridomas 1.21 or 2.18, which are mouse T cell hybridomas reactive with Ig {kappa}-chain peptides 145–159 and 188–203, respectively. After 24 h of culture, supernatants were collected and assayed for IL-2 using proliferation of HT-2 cells as measured by [3H]thymidine uptake. A standard curve was generated and used to relate cpm to picograms of IL-2 produced. Dendritic cells cultured for 1–3 days after peptide pulsing before addition of T cells gave comparable results to those shown (our unpublished data). B, Dendritic cells from a second healthy donor typed as HLA-DRB1*0401 (WS) or the BxT cell hybrid line T2 transfected with HLA-DRB1*0401 were incubated in the presence or absence of 1 µM peptide as described in A. After washing, cells were cocultured in triplicate with T cell hybridomas for 24 h at an E:T ratio of 1 in the presence or absence of 10 µg/ml of mAb 359.13F10, specific for HLA-DR4 (obtained from Dr. Janice Blum, University of Indiana School of Medicine, Indianapolis, IN). Supernatants were collected and assayed for IL-2 using CTLL-2 as an indicator cell line. Addition of peptide to DR4Dw4-transfected T2 cells enhanced stimulation of 1.21 T cells by ~10-fold relative to controls with no peptide added (our unpublished data).

 
Degradation of the 145–159 peptide by dendritic cells

The reduced ability to present the 145–159 epitope suggested that dendritic cells might selectively degrade the peptide. To test this possibility, peptide 145–159 was incubated in serum-free AIM-V medium for increasing time with either dendritic cells or the B cell line Frev, which is HLA-DR4Dw4 positive but does not synthesize Ig {kappa}-chains. Supernatants were collected by centrifugation and were added to fresh Frev cells, which were then incubated with 1.21 T cells. As shown in Fig. 2GoA, preincubation of the peptide with dendritic cells markedly reduced the antigenic activity in the supernatant. After 2 h incubation time, very little Ag could be detected in the supernatant compared with control samples with no peptide added. This reduction does not appear to depend upon binding of the peptide to MHC molecules because dendritic cells from both HLA-DR4Dw4 positive and negative donors gave similar results (our unpublished data). In contrast, preincubation with equivalent numbers of Frev cells did not alter the subsequent presentation of the peptide over the same time period (Fig. 2Go).



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  		FIGURE 2. Selective degradation of peptide 145–159 by dendritic cells. Peptides 145–159 or 188–203 were incubated at 150 µM at 37°C for the times indicated in AIM-V medium with either non-HLA-DR4Dw4 dendritic cells (day 5 culture) or the B cell line Frev. Supernatants were collected after removing the cells, and they were frozen immediately at -70°C. A, Aliquots of supernatants containing peptide 145–159 were added to Frev cells at a final peptide concentration of ~2 µM (calculated from the initial input). After overnight incubation, Frev cells were washed and incubated with 1.21 T cells at an E:T ratio of 1 for 24 h. Supernatants were then collected and assayed for IL-2 as described in Fig. 1Go. As controls, Frev cells were incubated overnight without peptide or with 2 µM peptide before addition of T cells (open symbols). Parallel experiments performed with supernatants containing peptide 188–203 showed no decrease in recognition after incubation with dendritic cells (our unpublished data). B, Supernatants used in A were analyzed by C18 reversed phase HPLC. Only data obtained with dendritic cells are shown. Peaks corresponding to the intact 145–159 and 188–203 peptides were identified and confirmed by direct sequencing using tandem mass spectrometry. An unidentified component of the AIM-V medium which comigrates with peptide 145–159 was quantitated and uniformly subtracted from the experimental values in these calculations. This component was present both before and after culture with cells and therefore was not a product of or removed by the dendritic cells during culture (our unpublished data). The amount of peptide remaining in individual samples is shown as a percentage of the input peptide detected at the start of incubation. Neither peptide was degraded by Frev cells, and dendritic cells from individuals typed as HLA-DR4Dw4 positive or negative gave comparable results (our unpublished data).

 
To directly determine whether peptide 145–159 is cleaved by dendritic cells, supernatants were analyzed by reversed phase HPLC as shown in Fig. 2GoB. Peaks were detected by absorbance at 214 nm corresponding to peptides 145–159 and 188–203, which eluted from the gradient after 26 min and 21 min, respectively, when run separately. No reduction in peak height was observed when either peptide was incubated with the Frev B cell line for 0–2 h (our unpublished data). In contrast, less than 10% of peptide 145–159 is detectable after incubation for 2 h with dendritic cells (Fig. 2GoB). In contrast, peptide 188–203 is not degraded significantly over the 2-h period. An example of the profiles obtained for peptide 145–159 is shown in Fig. 3Go. In addition to the reduction of the 145–159 peak eluting at 26 min, a novel peak eluted at about 16 min with a mass of 566 (M+2H)2+, which presumably could be a cleavage product. Sequence analysis using tandem mass spectrometry was performed, which showed that the major peptide species present was KVDNALQSGNS, corresponding to positions 149–159 of the {kappa}-chain. These data confirm that peptide 145–159 is cleaved by dendritic cells to yield the shorter fragment 149–159. There was no evidence of further degradation given that the latter peak accumulated in the medium and no candidate smaller fragments were visible on the HPLC profiles at any time point evaluated (Fig. 3Go).



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  		FIGURE 3. Detection of a fragment of peptide 145–159 following incubation with dendritic cells. Peptide 145–159 was incubated with dendritic cells and analyzed by HPLC as described in Fig. 2Go. Intact peptide 145–159 is the major peak (Parent) eluting from the gradient at 26.0 min, which is seen to decrease after 120-min incubation with dendritic cells (bottom panel). A new peak appears at the latter time points eluting at 16.2 (middle panel) or 15.7 min (bottom panel). Fractions containing this peak were shown to contain as their major constituent a peptide with the sequence KVDNALQSGNS, corresponding to residues 149–159 of human {kappa}-chains. No change in profiles were seen when peptide 145–159 was incubated with Frev cells (our unpublished data).

 
Peptide 149–159 does not bind to HLA-DR4DW4 and is not presented to 1.21 T cells

To determine whether the observed cleavage of peptide 145–159 was likely to result in loss of MHC binding, we aligned the 145–159 sequence with a peptide binding motif for HLA-DR4Dw4 determined by Rammensee et al. (28) several years ago. The tryptophan at position 148 of the peptide 145–159 sequence is predicted to be the first position within the HLA-DR4Dw4 binding motif and to represent an important anchor residue. This suggested that peptide 149–159, which lacks this tryptophan, likely would not bind to HLA-DR4Dw4. To test this experimentally, an assay was developed which measures binding of a reference biotinylated CLIP of invariant chain to DR4Dw4-expressing T2 cells in the presence of unlabeled test peptides, including synthetic 145–159 and 149–159 peptides. As shown in Fig. 4GoA, unlabeled CLIP and peptide 145–159 effectively inhibit binding of the biotinylated CLIP, whereas peptide 149–159 does not compete for binding. In addition, incubation of peptide 149–159 with Frev B cells did not result in their recognition by 1.21 T cells (Fig. 4GoB). These results demonstrate that removal of the four amino-terminal residues of the 145–159 peptide abrogates its ability to bind and to be presented by HLA-DR4Dw4 molecules.



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  		FIGURE 4. The degradation product 149–159 does not bind to HLA-DR4Dw4 and does not stimulate 1.21 T cells. A peptide was synthesized corresponding to the cleavage fragment 149–159. A, Peptide binding to HLA-DR4Dw4 was determined using a competitive binding assay. T2 cells transfected to express HLA-DR4Dw4 were incubated with 10 µM biotinylated CLIP 81–104 in the presence or absence of unlabeled competitor peptides. After washing, cells were incubated with 0.1 µg streptavidin-PE, washed again, and analyzed by flow cytometry. Competitor peptides are indicated. Results are shown as the percentage of inhibition of biotinylated CLIP binding, determined using mean fluorescence channel values. B, Indicated peptides were incubated with Frev cells overnight at a concentration of 2 µM. Cells were washed and incubated with 1.21 T cells at an E:T ratio of 1. Supernatants collected after 24 h were assayed for IL-2 as described in Fig. 1Go.

 
Characterization of the mechanism of cleavage of peptide 145–159

The proteolytic activity of dendritic cells responsible for cleaving peptide 145–159 appeared to reside either inside the cells or at the cell surface, but not in a secreted protease because supernatants from dendritic cell cultures were unable to degrade the peptide (our unpublished data). However, it seemed unlikely that a protease contained inside the cells was responsible because cleavage of 145–159 appeared to be nearly complete. Such efficient degradation presumably would require virtually quantitative internalization of the extracellular fluid or active uptake of the peptide by dendritic cells. Neither of these possibilities appears likely because we observed no differences in ability to degrade peptide between dendritic cell cultures that were highly endocytic (i.e., less mature) and those that were nonendocytic (i.e., fully mature) as measured by uptake of ligands through mannose receptor (our unpublished data). These observations suggest that a cell-surface protease would represent the most likely candidate for causing the observed cleavage.

To test which proteases in dendritic cells might cleave the 145–159 peptide, we preincubated the cells with various protease inhibitors before peptide pulsing. Of the compounds tested, only bestatin was able to significantly reduce cleavage as measured by HPLC analysis (Fig. 5Go, A and B) or T cell assays (our unpublished data). This is consistent with degradation mediated by a cell surface aminopeptidase such as CD13 which is present on dendritic cells, as was suggested earlier for degradation of certain class I epitopes (24). To test this directly, Abs reactive with CD13 or HLA-DR were preincubated with dendritic cells before addition of peptide. The latter was used as a control because dendritic cells express similar levels of the two proteins. Supernatants were then analyzed by HPLC or by T cell assay as shown in Fig. 6Go. After 2 h, significant inhibition of cleavage was observed in the presence of anti-CD13, but not with anti-HLA-DR, and was correlated with enhanced presentation to 1.21 T cells. Both Abs bound to dendritic cells at comparable levels (our unpublished data).



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  		FIGURE 5. Degradation of peptide 145–159 is prevented by bestatin, a selective inhibitor of cell-surface aminopeptidases. Dendritic cells were preincubated with the indicated compounds for 45 min before addition of 150 µM peptide 145–159 as described in Fig. 2Go. Supernatants were collected after the indicated times and stored at -70°C before analysis by HPLC as described in Fig. 3Go. Each inhibitor was used at two concentrations (H or L) corresponding to 200 µM and 100 µM (for tosyl lysine chloromethyl ketone and bestatin) or 100 µM and 30 µM (N-ethylmaleimide). Additional tested protease inhibitors which had no effect on degradation of peptide as determined by T cell assay included DTT, iodoacetamide, leupeptin, antipain, E-64, and o-phenanthroline (our unpublished data). A, The amount of the parent peptide 145–159 remaining is shown as a percentage of the input peptide at 0 h incubation. An unidentified molecule present in AIM-V medium which comigrates with peptide 145–159 was quantitated by analyzing supernatants from dendritic cells cultured in medium without added peptide and was subtracted from each experimentally determined value. This component was present both before and after culture with cells, and therefore was not a product of or removed by the dendritic cells during culture (our unpublished data). B, Generation of the fragment 149–159 was quantitated and is expressed in arbitrary units with the amount of the fragment produced in 2 h in the absence of added inhibitors corresponding to 100. Experiments shown in both A and B are representative of three independent experiments and were replicated in part several additional times.

 


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  		FIGURE 6. Cleavage of peptide 145–159 is mediated by CD13. Dendritic cells were preincubated with the Abs reactive with CD13 or HLA-DR for 45 min before addition of 150 µM peptide 145–159. Abs were also present during incubation with peptides. Supernatants were collected after the indicated times and stored at -70°C before analysis. A, Supernatants were analyzed by HPLC as described in Fig. 3Go. The amount of the parent peptide 145–159 remaining is shown as a percentage of the input peptide at 0 h incubation. B, Aliquots of supernatants containing peptide 145–159 were added to Frev cells at a final peptide concentration of ~2 µM (calculated from the initial input). After overnight incubation, Frev cells were washed and incubated with 1.21 T cells at an E:T ratio of 1 for 24 h. Supernatants were then collected and assayed for IL-2 as described in Fig. 1Go. The decreased T cell response seen in samples containing anti-HLA-DR Ab is due to direct inhibition of T cell reactivity with Frev cells. In control experiments, 1.21 T cells incubated with unpulsed Frev cells for 24 h produced 5.3 ± 3.1 pg/ml IL-2.

 
The 145–159 epitope is protected from degradation by acetylation of the amino terminus

If an exopeptidase such as CD13 were responsible for the observed cleavage, it might be possible to block degradation by modification of the amino terminus of the peptide. When an acetylated analogue of 145–159 was incubated with dendritic cells, no significant cleavage was seen in supernatants as analyzed by HPLC (Fig. 7Go). The acetylated peptide and unmodified peptides bind to HLA-DR4Dw4 with similar affinity, as evidenced by the fact that they were able to inhibit binding of the biotinylated CLIP to T2-DR4Dw4 cells to a comparable degree, showing less than 5% difference in inhibition at all concentrations tested (our unpublished data). However, the acetylated peptide was able to stimulate 1.21 T cells at a much lower concentration than the unmodified peptide when dendritic cells, but not B cell lines, were used as APC (Fig. 8Go). This suggests that artificial modification of the amino terminus can greatly facilitate the presentation of epitopes by cells which would otherwise degrade them. These results support the hypothesis that CD13 cleaves the peptide at the amino terminus and suggest a strategy for enhancing the antigenicity of peptides which are susceptible to cleavage by dendritic cells.



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  		FIGURE 7. N-acetylation of peptide 145–159 blocks its degradation by dendritic cells. Acetylated and unmodified peptides were incubated with dendritic cells as described in Fig. 2Go. After the indicated times, supernatants were collected and stored at -70°C before HPLC analysis. The amount of peptide remaining was calculated at each time point. Similar results were obtained in two additional experiments.

 


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  		FIGURE 8. T cell recognition of peptide 145–159 presented by dendritic cells is enhanced by N-acetylation. Peptides were incubated overnight with either dendritic cells obtained from donor DF (5 day culture) (A) or with Frev B cells (B). After washing the cells to remove peptide, 1.21 T cells were added immediately at an E:T ratio of 1, and incubation continued for 24 h. Supernatants were collected and assayed for IL-2 using HT-2 indicator cells. Dendritic cells pulsed with peptides and then cultured for an additional 1–3 days before T cell assay gave comparable results to those shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The data presented strongly support the hypothesis that dendritic cells degrade some peptides quite efficiently via CD13. However, the observed pattern of cleavage is somewhat unexpected because the {kappa}-chain 145–159 peptide is degraded nearly quantitatively to a fragment lacking the first four amino acid residues, corresponding to positions 149–159. We could not detect any partial cleavage products with fewer residues missing. This initially suggested to us that an endopeptidase might be responsible, but other evidence did not support this, particularly the resistance to cleavage noted for the acetylated peptide. One possible explanation for these results is that the first four residues are sequentially cleaved very rapidly following association with CD13 and that the remaining fragment either dissociates from the protease or is intrinsically resistant to further cleavage. In support of the first part of this hypothesis, an analogue of peptide 145–159 with substitution of Tyr for Trp at position 148 was cleaved to generate a nested set of degradation products lacking residues at positions 1, 2, and 3 from the amino terminus, which are detectable by HPLC and mass spectrometry sequencing (our unpublished data). This suggests that Trp at position 148 facilitates the rapid removal of residues from the amino terminus of the peptide by CD13.

In addition to the present results, one of us had previously analyzed the ability of dendritic cells to degrade peptides that can bind to class I MHC proteins (24). In that study, an aminopeptidase was suggested to participate in the degradation of several peptide epitopes, but truncations of the carboxy termini were also observed, suggesting the involvement of additional proteases. Cleavage of these class I-binding peptides was more extensive than that seen with {kappa}-chain 145–159 in the present study, leading to the production of nested sets of larger fragments and tri- and dipeptide species. However, no general conclusions regarding the selectivity or specificity of the cell surface exopeptidases involved in the cleavage of these peptide epitopes could be made based on these experiments. Although how CD13 is able to selectively cleave peptide 145–159 and why the 149–159 product is largely resistant to further degradation remain unclear in the present study, it should be possible to address these questions by further characterizing the specificity of the protease.

CD13, or aminopeptidase N, is a cell-surface proteinase with a wide range of biological activities (29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41), including the ability to trim the amino terminus of a peptide Ag bound to class II MHC molecules on the surface of the mouse B cell line CH12 (42). It was suggested that such trimming occurs when the ends of the peptide extend beyond the confines of the binding cleft, allowing the protease access to these unprotected residues. In such a model, the class II molecule would serve as a template for directing cleavage. This is consistent with an earlier demonstration that binding to class II MHC molecules protects peptides from cleavage (43). We observe that in humans dendritic cells, but not B cell lines, express CD13, consistent with the involvement of this protease in selective degradation of peptide by dendritic cells (our unpublished data). However, the previously reported epitope trimming directed by class II MHC is likely to differ from that described in the current study in an important way because dendritic cells lacking the restriction element HLA-DR4Dw4 are also able to cleave efficiently peptide 145–159 into the same truncated product. In addition, conversion of the peptide to truncated product is nearly complete after 1-h incubation with dendritic cells (Fig. 6Go), which is relatively rapid compared with the kinetics of peptide-binding to class II MHC molecules. Both observations support the idea that cleavage of peptide 145–159 by dendritic cells is not "directed" by class II MHC molecules.

Protection from cleavage by ectoenzymes on dendritic cells is likely to be an important strategy for allowing presentation of susceptible peptide epitopes to T cells. It is possible to enhance presentation of an epitope from tyrosinase by class I MHC molecules on dendritic cells using bestatin and DTT to block aminopeptidase activity and carboxypeptidase activities, respectively (24). Such a strategy should also be applicable to class II MHC-binding epitopes. However, more simply, as shown in the current study, modification of the amino terminus by acetylation can interfere with enzymatic cleavage and selectively enhance class II MHC presentation of peptides by dendritic cells more than 10-fold. This is consistent with an earlier report by Allen et al. (44) showing that modifications of the amino and carboxy termini of peptides which bind to class II MHC greatly enhanced their antigenicity both in vitro and in vivo. In that study, no mechanism to explain the enhanced presentation was offered. Our results suggest that inhibiting peptide degradation by modification of the amino terminus can significantly enhance presentation of at least some CD4+ T cell epitopes by dendritic cells, which will likely result in enhanced priming of epitope-specific T cell responses in vivo. In contrast, modification of the termini of peptides which bind to class I molecules is unlikely to be effective for enhancing their antigenicity because such alterations would be expected to result in loss of MHC binding due to interference with critical hydrogen bond formation between residues of the class I molecule and the amino terminus of the peptide (45).


   Acknowledgments
 
We thank Dr. Linda Wicker (Merck, Rahway, NJ) for providing T cell clones, Dewayne Falkner and Dr. Penny Morel (University of Pittsburgh School of Medicine) for HLA molecular typing, and Raymond Yurko and Dr. Frances Finn (University of Pittsburgh Cancer Institute Peptide Synthesis Facility) for peptide synthesis. We are especially grateful to Dr. Janice Blum for providing a number of critical reagents and for her advice and comments throughout the study.


   Footnotes
 
1 This work was supported by grants from the National Institutes of Health (P60 AR44811 to R.D.S and R01 CA57840 to W.J.S.). Back

2 Address correspondence and reprint requests to Dr. Russell D. Salter, Department of Pathology, University of Pittsburgh School of Medicine, W957 Biomedical Science Tower, 200 Lothrop Street, Pittsburgh, PA 15213. E-mail address: Back

3 Abbreviation used in this paper: CLIP, class II-associated invariant chain peptide. Back

Received for publication June 24, 1999. Accepted for publication October 18, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Banchereau, J., R. M. Steinman. 1998. Dendritic cells and the control of immunity. Nature 392:245.[Medline]
  2. Sallusto, F., M. Cella, C. Danieli, A. Lanzavecchia. 1995. Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: down-regulation by cytokines and bacterial products. J. Exp. Med. 182:389.[Abstract/Free Full Text]
  3. Steinman, R. M., J. Swanson. 1995. The endocytic activity of dendritic cells. J. Exp. Med. 182:283.[Free Full Text]
  4. Cella, M., F. Sallusto, A. Lanzavecchia. 1997. Origin, maturation and antigen presenting function of dendritic cells. Curr. Opin. Immunol. 9:10.[Medline]
  5. Rubartelli, A., A. Poggi, M. R. Zocchi. 1997. The selective engulfment of apoptotic bodies by dendritic cells is mediated by the {alpha}vß3 integrin and requires intracellular and extracellular calcium. Eur. J. Immunol. 27:1893.[Medline]
  6. Prigozy, T. I., P. A. Sieling, D. Clemens, P. L. Stewart, S. M. Behar, S. A. Porcelli, M. B. Brenner, R. L. Modlin, M. Kronenberg. 1997. The mannose receptor delivers lipoglycan antigens to endosomes for presentation to T cells by CD1b molecules. Immunity 6:187.[Medline]
  7. Engering, A. J., M. Cella, D. M. Fluitsma, M. Brockhaus, E. C. Hoefsmit, A. Lanzavecchia, J. Pieters. 1997. The mannose receptor functions as a high capacity and broad specificity antigen receptor in human dendritic cells. Eur. J. Immunol. 27:2417.[Medline]
  8. Tan, M. C., A. M. Mommaas, J. W. Drijfhout, R. Jordens, J. J. W. Onderwater, D. Verwoerd, A. A. Mulder, A. N. van der Heiden, D. Scheidegger, L. C. Oomen, et al 1997. Mannose receptor-mediated uptake of antigens strongly enhances HLA class II-restricted antigen presentation by cultured dendritic cells. Eur. J. Immunol. 27:2426.[Medline]
  9. Albert, M. L., S. F. Pearce, L. M. Francisco, B. Sauter, P. Roy, R. L. Silverstein, N. Bhardwaj. 1998. Immature dendritic cells phagocytose apoptotic cells via {alpha}vß5 and CD36, and cross-present antigens to cytotoxic T lymphocytes. J. Exp. Med. 188:1359.[Abstract/Free Full Text]
  10. Regnault, A., D. Lankar, V. Lacabanne, A. Rodriguez, C. Thery, M. Rescigno, T. Saito, S. Verbeek, C. Bonnerot, M. Rescigno, et al 1998. Dendritic cell survival and maturation are regulated by different signaling pathways. J. Exp. Med. 188:2175.[Abstract/Free Full Text]
  11. Rovere, P., C. Vallinoto, A. Bondanza, M. C. Crosti, M. Rescigno, P. Ricciardi-Castagnoli, C. Rugarli, A. A. Manfredi. 1998. Bystander apoptosis triggers dendritic cell maturation and antigen-presenting function. J. Immunol. 161:4467.[Abstract/Free Full Text]
  12. Winzler, C., P. Rovere, V. S. Zimmermann, J. Davoust, M. Rescigno, S. Citterio, P. Ricciardi-Castagnoli. 1997. Checkpoints and functional stages in DC maturation. Adv. Exp. Med. Biol. 417:59.[Medline]
  13. Lutz, M. B., P. Rovere, M. J. Kleijmeer, M. Rescigno, C. U. Assmann, V. M. Oorschot, H. J. Geuze, J. Trucy, D. Demandolx, J. Davoust, P. Ricciardi-Castagnoli. 1997. Intracellular routes and selective retention of antigens in mildly acidic cathepsin D/lysosome-associated membrane protein-1/MHC class II-positive vesicles in immature dendritic cells. J. Immunol. 159:3707.[Abstract]
  14. Regnault, A., D. Lankar, V. Lacabanne, A. Rodriguez, C. Thery, M. Rescigno, T. Saito, S. Verbeek, C. Bonnerot, P. Ricciardi-Castagnoli, S. Amigorena. 1999. Fc{gamma} receptor-mediated induction of dendritic cell maturation and major histocompatibility complex class I-restricted antigen presentation after immune complex internalization. J. Exp. Med. 189:371.[Abstract/Free Full Text]
  15. Buelens, C., V. Verhasselt, D. De Groote, K. Thielemans, M. Goldman, F. Willems. 1997. Human dendritic cell responses to lipopolysaccharide and CD40 ligation are differentially regulated by interleukin. Eur. J. Immunol. 27:1848.[Medline]
  16. Verhasselt, V., C. Buelens, F. Willems, D. De Groote, N. Haeffner-Cavaillon, M. Goldman. 1997. Bacterial lipopolysaccharide stimulates the production of cytokines and the expression of costimulatory molecules by human peripheral blood dendritic cells: evidence for a soluble CD14-dependent pathway. J. Immunol. 158:2919.[Abstract]
  17. Morel, A. S., S. Quaratino, D. C. Douek, M. Londei. 1997. Split activity of interleukin-10 on antigen capture and antigen presentation by human dendritic cells: definition of a maturative step. Eur. J. Immunol. 27:26.[Medline]
  18. Poggi, A., E. Tomasello, E. Ferrero, M. R. Zocchi, L. Moretta. 1998. p40/LAIR-1 regulates the differentiation of peripheral blood precursors to dendritic cells induced by granulocyte-monocyte colony-stimulating factor. Eur. J. Immunol. 28:2086.[Medline]
  19. Randolph, G. J., S. Beaulieu, S. Lebecque, R. M. Steinman, W. A. Muller. 1998. Differentiation of monocytes into dendritic cells in a model of transendothelial trafficking. Science 282:480.[Abstract/Free Full Text]
  20. Wilson, C.C., W. C. Olson, T. Tuting, C. R. Rinaldo, M. T. Lotze, W. J. Storkus. 1999. HIV-1-specific CTL responses primed in vitro by blood-derived dendritic cells and Th1-biasing cytokines. J. Immunol. 162:3070.[Abstract/Free Full Text]
  21. Tjandrawan, T., D. Martin, M. J. Maeurer, C. Castelli, M. T. Lotze, W. J. Storkus. 1998. Autologous human dendriphages pulsed with synthetic or natural tumor peptides elicit tumor-specific CTLs in vitro. J. Immunother. 21:149.
  22. Zitvogel, L., J. I. Mayordomo, T. Tjandrawan, A. B. DeLeo, M. R. Clarke, M. T. Lotze, W. J. Storkus. 1996. Therapy of murine tumors with tumor peptide-pulsed dendritic cells: dependence on T cells, B7 costimulation, and T helper cell 1-associated cytokines. J. Exp. Med. 183:87.[Abstract/Free Full Text]
  23. Celluzzi, C. M., J. I. Mayordomo, W. J. Storkus, M. T. Lotze, Jr L. D. Falo. 1996. Peptide-pulsed dendritic cells induce antigen-specific CTL-mediated protective tumor immunity. J. Exp. Med. 183:283.[Abstract/Free Full Text]
  24. Amoscato, A. A., D. A. Prenovitz, M. T. Lotze. 1998. Rapid extracellular degradation of synthetic class I peptides by human dendritic cells. J. Immunol. 161:4023.[Abstract/Free Full Text]
  25. Woods, A., H. Y. Chen, M. E. Trumbaue, A. Sirotina, R. Cummings, D. M. Zaller. 1994. Human major histocompatibility complex class II-restricted T cell responses in transgenic mice. J. Exp. Med. 180:173.[Abstract/Free Full Text]
  26. Kovats, S., P. E. Whitely, P. Concannon, A. Y. Rudensky, J. S. Blum. 1997. Presentation of abundant endogenous class II DR-restricted antigens by DM-negative B cell lines. Eur. J. Immunol. 27:1014.[Medline]
  27. Denzin, L. K., N. F. Robbins, C. Carboy-Newcomb, P. Cresswell. 1994. Assembly and intracellular transport of HLA-DM and correction of the class II antigen-processing defect in T2 cells. Immunity 1:595.[Medline]
  28. Rammensee, H. G., T. Friede, S. Stevanoviic. 1995. MHC ligands and peptide motifs: first listing. Immunogenetics 41:178.[Medline]
  29. Balog, T., T. Marotti, M. Abramic, I. Svoboda-Beusen, I. Hrsak. 1999. The role of aminopeptidase N in Met-enkephalin modulated superoxide anion release. Immunopharmacology 41:11.[Medline]
  30. Stefanovic, V., P. Vlahovic, M. Mitic-Zlatkovic. 1998. Epidermal growth factor up-regulates aminopeptidase N and 5'-nucleotidase in human glomerular mesangial cells. Kidney Blood Press. Res. 21:310.[Medline]
  31. Ashmun, R. A., A. T. Look. 1990. Metalloprotease activity of CD13/aminopeptidase N on the surface of human myeloid cells. Blood 75:462.[Abstract/Free Full Text]
  32. Look, A. T., R. A. Ashmun, L. H. Shapiro, S. C. Peiper. 1989. Human myeloid plasma membrane glycoprotein CD13 (gp150) is identical to aminopeptidase N. J. Clin. Invest. 83:1299.
  33. Ramakrishnan, L., Q. Wu, A. Yue, M. D. Cooper, N. Rosenberg. 1990. BP-1/6C3 expression defines a differentiation stage of transformed pre-B cells and is not related to malignant potential. J. Immunol. 145:1603.[Abstract]
  34. Wu, Q., J. M. Lahti, G. M. Air, P. D. Burrows, M. D. Cooper. 1990. Molecular cloning of the murine BP-1/6C3 antigen: a member of the zinc-dependent metallopeptidase family. Proc. Natl. Acad. Sci. USA 87:993.[Abstract/Free Full Text]
  35. Amoscato, A. A., J. W. Alexander, G. F. Babcock. 1989. Surface aminopeptidase activity of human lymphocytes. I. Biochemical and biologic properties of intact cells. J. Immunol. 142:1245.[Abstract]
  36. Amoscato, A. A., R. M. Sramkoski, G. F. Babcock, J. W. Alexander. 1990. Neutral surface aminopeptidase activity of human tumor cell lines. Biochim. Biophys. Acta 104:317.
  37. Menrad, A., D. Speicher, J. Wacker, M. Herlyn. 1993. Biochemical and functional characterization of aminopeptidase N expressed by human melanoma cells. Cancer Res. 53:1450.[Abstract/Free Full Text]
  38. Saiki, I., H. Fujii, J. Yoneda, F. Abe, M. Nakajima, T. Tsuruo, I. Azuma. 1993. Role of aminopeptidase N (CD13) in tumor-cell invasion and extracellular matrix degradation. Int. J. Cancer 54:137.[Medline]
  39. Kanayama, N., Y. Kajiwara, J. Goto, E. el Maradny, K. Maehara, K. Andou, T. Terao. 1995. Inactivation of interleukin-8 by aminopeptidase N (CD13). J. Leukocyte Biol. 57:129.[Abstract]
  40. Yeager, C. L., R. A. Ashmun, R. K. Williams, C. B. Cardellichio, L. H. Shapiro, A. T. Look, K. V. Holmes. 1992. Human aminopeptidase N is a receptor for human coronavirus 229E. Nature 357:420.[Medline]
  41. Giugni, T. D., C. Soderberg, D. J. Ham, R. M. Bautista, K. O. Hedlund, E. Moller, J. A. Zaia. 1996. Neutralization of human cytomegalovirus by human CD13-specific antibodies. J. Infect. Dis. 173:1062.[Medline]
  42. Mouritsen, S., M. Meldal, O. Werdelin, A. Stryhn Hansen, S. Buus. 1992. MHC molecules protect T cell epitopes against proteolytic destruction. J. Immunol. 149:1987.[Abstract]
  43. Larsen, S. L., L. O. Pedersen, S. Buus, A. Stryhn. 1996. T cell responses affected by aminopeptidase N (CD13)-mediated trimming of major histocompatibility complex class II-bound peptides. J. Exp Med. 184:183.[Abstract/Free Full Text]
  44. Allen, P. M., G. R. Matsueda, S. Adams, J. Freeman, R. W. Roof, L. Lambert, E. R. Unanue. 1989. Enhanced immunogenicity of a T cell immunogenic peptide by modifications of its N and C termini. Int. Immunol. 1:141.[Abstract/Free Full Text]
  45. Bouvier, M., D. C. Wiley. 1994. Importance of peptide amino and carboxyl termini to the stability of MHC class I molecules. Science 265:398.[Abstract/Free Full Text]



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