Presentation of the Goodpasture Autoantigen Requires Proteolytic Unlocking Steps That Destroy Prominent T Cell Epitopes
Juan Zou,
Lorna Henderson,
Vicky Thomas,
Patricia Swan,
A. Neil Turner and
Richard. G. Phelps
MRC Centre for Inflammation Research (Renal Autoimmunity), University of Edinburgh, Edinburgh, United Kingdom
Address correspondence to: Dr. Richard G. Phelps, MRC Centre for Inflammation Research (Renal Autoimmunity), The Queens Medical Research Institute, 47 Little France Crescent, EH16 4TJ, UK. Phone: +44-131-242-9164; Fax: +44-131-242-9168; E-mail: richard.phelps{at}ed.ac.uk
Received for publication September 27, 2006.
Accepted for publication December 29, 2006.
The most abundant autoreactive T cells in patients with Goodpasture’sdisease are specific for peptides in the autoantigen that havehigh affinity for the disease-associated HLA class II molecule,DR15. How can such T cells escape self-tolerance mechanisms?This study showed that these peptides are highly susceptibleto destruction in the earliest stages of antigen processing,and some must be cleaved for antigen digestion to be possible("unlocking"). Goodpasture autoantigen [collagen 3(IV)NC1; approximately31 kD] that was incubated with B cell lysosomes was cleavedwithin a few minutes to form approximately 9- and approximately22-kD fragments, then increasing quantities of smaller peptides.The processing was completely abrogated by pepstatin A, a specificinhibitor of cathepsin D/E, even though lysosomal extracts containa rich array of proteases. Purified cathepsin D generated thesame major 3(IV)NC1 fragments as entire lysosomes, suggestingthat cathepsin D cleavages are required to initiate 3(IV)NC1processing. The initial unlocking cleavages destroyed two majorself-epitopes, and subsequent preferred cleavages destroyedall of the other T cell epitopes that are recognized by mostpatients’ autoreactive T cells. The responses of T cellclones that are specific for a major disease-associated peptideto antigen-pulsed intact antigen-presenting cells were substantiallyenhanced by pepstatin A treatment. Therefore, cathepsin D activitysignificantly diminishes presentation of 3(IV)NC1 peptides thatare recognized by patients’ T cells by destroying thepeptides in early processing. These observations can explainwhy the mature T cell repertoire includes reactivity towardthese self-peptides and suggests that a key factor in diseaseinitiation is likely to be a shift in antigen processing.
In autoimmune diseases, the immune system targets moleculesto which tolerance is normally maintained by a variety of mechanisms.A number of hypotheses explain how this may occur, including,for example, previous "unawareness" of the antigen because ofits protected location, haptenization, a change in autoantigensequence or folding, molecular mimicry, altered thresholds forproinflammatory signaling, or impaired normal tolerance mechanisms.The last two mechanisms are likely to predispose to autoimmunityto multiple autoantigens and so are less likely to be relevantto organ-specific diseases.
Goodpasture’s (anti–glomerular basement membrane[anti-GBM]) disease is the best understood renal organ–specificautoimmune disease. It is characterized by antibody and T lymphocyte(T cell) reactivity to the NC1 domain of a tissue-specific isoformof type IV collagen, 3(IV)NC1 (1). Patients with recent-onsetGoodpasture’s disease have in their peripheral blood CD4+T lymphocytes that proliferate and make IFN- when incubatedwith the Goodpasture autoantigen (2,3). Recent work has demonstratedthat the fine specificity of patients’ 3(IV)NC1-reactiveT cells is highly consistent between patients and that the predominant3(IV)NC1-reactive T cells are specific for peptides that havehigh affinity for the disease-associated HLA DR15 class II molecule(3–5). These data raise the important question of howsuch T cells avoid deletion or immunoregulation by the normaltolerance mechanisms.
It is possible to rule out that the restricted tissue distributionand very slow turnover of 3(IV) collagen might lead to a relativelack of immunoregulatory mechanisms. We and others have demonstratedthat the Goodpasture antigen is expressed in human thymus (6,7).Therefore, any failure to develop tolerance to high-affinitypeptides from this antigen is likely to be a consequence ofthe failure of antigen-presenting cells (APC) to generate themor a failure to generate them in a context that leads to immunoregulation.
There is clear evidence that processing factors exert a powerfulinfluence on the presentation of 3(IV)NC1 peptides. Biochemicalanalysis of the 3(IV)NC1 peptides that bound to HLA-DR15 onthe surface of antigen-pulsed APC found that the most abundantlypresented 3(IV)NC1 peptides were not those with highest affinityfor HLA-DR15 (8,9). Therefore, processing factors must preventpeptides with higher affinity for DR15 from gaining access toMHC class II molecules within APC. Similar conclusions havebeen reached in experiments with other antigens (10).
We examined the hypothesis that the disease-associated peptidesin Goodpasture’s disease are particularly susceptibleto proteolytic destruction in APC. We focused on the four 3(IV)NC1peptides that contain epitopes that are recognized by T cellsfrom all six patients (the two disease-associated peptides dap131–150and dap71–90) and from four of six patients (dap1–20and dap31–50) (3). The results confirmed that the disease-associatedpeptides are destroyed early and furthermore showed that cleavagesof two of them were obligate early steps in the sequential proteolysisof the antigen by lysosomes.
Antigens, Enzymes, and Cells
Cathepsin D that was purified from human liver was obtainedfrom Athen Research & Technology (Athens, GA). Asparagineendopeptidase (AEP) was donated by Prof. C. Watts (Universityof Dundee, Dundee, UK). Production of recombinant 3(IV) NC1[r-3(IV) NC1] was as described before (8). The synthetic peptideswere as described before (9). Lysosomal extracts were preparedfrom Epstein-Barr virus (EBV) transformed B cells as describedpreviously (11). The DR15-expressing EBV-transformed B celllines LCL013, 041, and 061 were donated by Prof. D. Crawford(University of Edinburgh, UK).
Digestion of r-3(IV) NC1
Lysosomal extracts were incubated at 37°C with r-3(IV)NC1in 10 mM dithiothreitol (DTT), 50 mM citrate (pH 4.6), and theindicated protease inhibitors at the following concentrations:Pepstatin A 10 µM, iodoacetic acid 14 mM, and PMSF 14mM. Digests were analyzed by SDS-PAGE. For digests with purifiedenzymes, r-3(IV)NC1 (100 µg) was dissolved in 8 M ureaand coated onto S-Sepharose 6B beads (Sigma, St. Louis, MO)in 8 M urea and 50 mM Tris (pH 7.8) carried in 1.5 ml of Mobicol(VH Bio, Gateshead, UK) spin columns. Adsorbed protein was reducedby incubation in 10 mM DTT at 56°C for 45 min; washed withUHQ water (Millipore, Billerica, MA); and then suspended eitherwith 1 µg of human liver cathepsin D (Athen Research &Technology) in 50 µl of tetramethylammonium formate (25%wt, pH 4.5; Sigma-Aldrich, St. Louis, MO) or with 2 mU of AEPin 50 µl of 80 mM citrate, 240 mM Na2HPO4, 1 mM EDTA,and 2 mM DTT (pH 5.8). Peptides that were released by digestionafter 5 min to 2 h were recovered from the supernatant by centrifugationand from the beads by washing with 20% acetonitrile 1% tri-fluoroaceticacid (Romil, Cambridge, UK) and 1 M NH4OH. Recovered peptideswere combined, then divided into aliquots, dried under vacuum,and stored at –80°C.
Sequence Determinations
For N-terminal sequencing of fragments that were visualizedby SDS-PAGE, gels were blotted onto polyvinylidene difluoridemembranes and selected fragments were cut out and submittedfor Edman degradation. Peptides were sequenced by a batteryof mass spectrometric techniques, including matrix-assistedlaser desorption ionization time of flight with Mass and Compositionanalysis (12) and daughter ion generation in a Finnigan LCQmass spectrometer (ThermoFisher Scientific, Waltham, MA) followedby database and manual interpretation of daughter ion spectra.
Generation of Hybridomas
DR15-transgenic Balb/c mice were immunized with 50 µgof dap131–150 in complete Freund adjuvant with appropriateUK Home Office approvals. Splenocytes and lymph nodes were recoveredafter 7 to 10 d and expanded in vitro with three cycles of re-stimulationwith dap131–150, expansion with rIL-2 at 20U/ml, and restingwith rIL-2 at 2 U/ml. Surviving T cells were fused BW5147 [Tcell recptor ] (13) cells (a donation from Dr. John Robinson,Newcastle University, Newcastle, UK) using the polyethyleneglycol method and cloned by limiting dilution. Clones that expressedT cell receptor and CD4 were screened for IL-2 production inresponse to dap131–150 presented by mitomycin C–treatedDR15-expressing splenocytes. IL-2 was measured by ELISA (R&DDuoSet, Minneapolis, MN).
To identify clones that recognized 3(IV)NC1 epitopes that arenaturally processed from intact 3(IV)NC1, DR15 B cells thatwere intact or fixed by incubation in 0.5% paraformaldehydefor 30 min at room temperature were pulsed with 7 µM AS346or 3(IV)NC1 for 4 to 6 h, then washed and added to 0.5 x 105of selected T hybridomas. IL-2 production at 24 h was measuredby ELISA as above. The clone ha3p132.2 was selected as representativeof a number of clones that produced larger amounts of IL-2 whenincubated with dap131–150 or intact 3(IV)NC1 and usedfor all of the assays reported herein.
Fine Specificity of Patients’ T Cells
The harvesting and initial preparation of T cells from patientswith recent-onset Goodpasture’s disease was as we havedescribed before (3). For assessment of fine specificity of3(IV)NC1-specific T cells, 2 x 105 purified peripheral bloodmononuclear cells were incubated with 10 µg/ml selected3(IV)NC1 peptides for 72 h at 37°C, then IFN- and IL-2 weremeasured in the culture supernatant by BD cytometric bead array(BD Bioscience, San Jose, CA) and proliferation measured fromincorporation of 3H thymidine during an 18-h period.
Antigen Presentation Assays
DR15+ve EBV transformed B cells (1 x 105) or phorbyl myristateacetate–differentiated IFN-–activated THP1 cellswere pretreated with pepstatin A (Sigma) for 30 min at 4°C,then 3.5 µM dap131–150 or 3(IV)NC1 was added andthe cells were incubated at 37°C for 2 to 12 h. At the endof the incubation periods, processing was terminated by fixationwith 0.5% paraformaldehyde for 30 min, the APC were extensivelywashed, 0.25 x 105 ha3p132.2 T hybridoma cells were added, andthe cultures were incubated for an additional 24 h, at the endof which IL-2 production was measured by ELISA as described.
3(IV)NC1 Is Rapidly Cleaved by Lysosomal Proteases
To examine endolysosomal processing of 3(IV)NC1 under controlledconditions, we used the system described by Watts et al. (11,14).Antigen was incubated at 37°C with freshly disrupted lysosomesthat were purified from human EBV-transformed B cells and degradationfollowed by SDS-PAGE and mass spectrometric peptide analysis.
SDS-PAGE (Figure 1) demonstrated that intact 3(IV)NC1 was progressivelydepleted during the 120-min incubation with early appearance(by 5 min) of two strongly stained fragments (at approximately22 and 9 kD) and subsequent appearance of two additional distinctbands (at 4.5 and 6 kD) and numerous weaker bands. It was strikingthat several large fragments were momentarily very distinctin the digests because it suggested that processing might proceedto some extent along a consistent pathway such that fragmentsthat were relatively resistant to further processing could accumulate.
Figure 1. Time course of processing of 3(IV)NC1 by human lysosomes. SDS-PAGE analysis of purified recombinant 3(IV) NC1 [r-3(IV)NC1] and lysosomes from human B cells run separately (rightmost two lanes) or after combination for the indicated intervals (lanes headed Lys). Several darker staining higher mobility bands that were seen only in combined samples were presumed to be fragments of 3(IV)NC1. The mobility and time scale of appearance of the 3(IV)NC1 fragments was similar when 3(IV)NC1 was digested with lysosomes or purified human cathepsin D (interspersed lanes labeled CD).
Cathepsin D/E Activity Is Required to Unlock 3(IV)NC1 to Further Lysosomal Processing
To identify the responsible proteases, we determined the influenceof class-specific protease inhibitors on the appearance of earlyfragments during lysosomal processing. Pepstatin A, an inhibitorof aspartate proteases, but not inhibitors of cysteine or serineproteases prevented formation of early fragments (Figure 2A).Indeed, in the presence of pepstatin A, no discernible digestionof 3(IV)NC1 had occurred by 120 min, even though lysosomes containa rich array of proteases that are not sensitive to this proteaseinhibitor. Therefore, processing of 3(IV)NC1 by the host ofproteases in human B cell lysosomes requires a critical aspartateprotease activity that unlocks the otherwise protease-resistantmolecule for further processing by lysosomal proteases.
Figure 2. Influence of protease inhibitors on digestion of 3(IV)NC1 by lysosomal extracts and cathepsin D. SDS-PAGE of 30 min digests with the indicated components. The principal 3(IV)NC1 fragments that were generated within minutes of exposure to purified lysosomes have the same mobility as those that were generated by cathepsin D. Inhibition of cathepsin D/E activity with pepstatin A (PepA) prevents formation of the fragments, whereas other broad-specificity protease inhibitors of serine (PMSF) or cysteine proteases (iodoacetic acid [IAA]) have no effect.
Cathepsin D and cathepsin E are the major pepstatin A–sensitiveaspartate proteases that are known to occur within lysosomes.We chose first to investigate cathepsin D because it is moreprevalent than cathepsin E, and it had a similar but slightlywider specificity for substrates within 3(IV)NC1 when the twoproteases were compared with 3(IV)NC1 peptides (manuscript inpreparation). Incubation of 3(IV)NC1 with purified human cathepsinD yielded fragments with very similar sizes and time courseof appearance as with lysosomal extracts (Figure 1). Moreover,Edman degradation of lysosome and cathepsin D–generatedfragments that appeared similar by SDS-PAGE showed identicalor probably identical N-terminal amino acid sequences (Table 1,Figure 3B). For example, the N-terminal sequence that was determinedfor the 22-kD band 1 that was recovered from the lysosomal andcathepsin D digests was in both cases FVQGN. Similarly, thesequences of the heavier components of the 9-kD band 2 (band2a) were identical and those of the 6-kD (band 3) and 4.5-kD(band 4) bands probably identical from both types of digest.The N-terminal sequences of the lighter approximately 9-kD bands2 (band 2b) were TSAGS from the lysosome digest and IMFTSAGSfrom the cathepsin D digest. Because the TSAGS fragment didnot occur in lysosomal digests that were treated with pepstatinA, and neither cathepsin D nor E cuts 3(IV)NC1 peptides at IMFTSAG(manuscript in preparation), it is likely that the TSAGS fragmentwas generated by an initial cathepsin D/E cleavage generatingIMFTSAGS, followed by removal of the IMF residues by one ofthe exopeptidases that are active in lysosomal extracts.
Figure 3. Locations of processing sites and T cell epitopes within 3(IV)NC1. Bars are used in B and C to depict the 3(IV)NC1 fragments that were detected in lysosomal () or purified cathepsin D digests ( and ; all fragments that were found in lysosome digests were also found in cathepsin D digest) relative to the sequence of the intact 3(IV)NC1 molecule shown in A. Vertical dotted lines highlight preferred cleavages that cut within disease-associated peptides. Numbering is of amino acid residues relative to the sequence SPAT at the beginning of the NC1 domain of 3(IV); shaded insets in the bar, representing intact 3(IV)NC1, in A indicate the locations of epitopes that are recognized by patients’ autoreactive T cells and the core sequences of major (biochemically identified) naturally processed and presented nested sets of peptides (see key); hatching at the C-termini of the larger fragments indicates the uncertainty as to the exact C-terminus of fragments that were identified by SDS-PAGE + Edman degradation. (D) Amino acid sequences in the vicinity of peptides bonds inferred to be cut in early processing. The boxed motifs are reasoned to be important in unlocking the molecule to further processing.
Therefore, lysosomal processing of 3(IV)NC1 proceeds only afterinitial unlocking proteolytic events that are mediated by apepstatin A–sensitive lysosomal protease. Because theunlocking protease is either cathepsin D itself or a proteasewith indistinguishable substrate specificity, we hereafter referto this activity simply as cathepsin D.
Major Epitopes Recognized by Patients’ Autoreactive T Cells Are Destroyed during Early Processing In Vitro
Next we inferred the sites of early processing of 3(IV)NC1 andexamined their location relative to epitopes that are recognizedby patients’ T cells (Figure 3, A and B). Cleavage atSFL38 FVQ was clearly identified as an early event that givesrise to the N-termini of two prominent early fragments and verylikely the C termini of two other prominent fragments. Notethat the C-termini of the larger fragments were not directlydetermined but located to within 5 to 10 amino acids from theirknown N-termini, and from their molecular weights estimatedby SDS-PAGE. Similarly, cleavages at FSF147 IMF, LFC69 NVN,and SFW196 LAS were inferred to be early processing events.Strikingly, two of the early cleavages mapped within two ofthe four peptides that stimulated patients’ T cells. Specifically,the peptide bond SFL38 FVQ is within dap31–50 and FSF147IMF within dap131–150, potentially destroying T cell epitopes.
Because early processing seemed to correlate with the specificitiesof patients’ autoreactive T cells, we sought to identifymore fully the earliest cleaved peptide bonds in 3(IV)NC1 byexamining the digests for small 3(IV)NC1 fragments that areinvisible to SDS-PAGE. For these experiments, 3(IV)NC1 was incubatedwith purified lysosomal proteases rather than entire lysosomalextracts because of the difficulty in discerning peptide fragmentsof 3(IV)NC1 in complex lysosomal digests. Cathepsin D was chosenbecause of its important role in early 3(IV)NC1 processing andAEP because it is crucial in early processing of other globularantigens, including tetanus toxoid c-fragment and myelin basicprotein (MBP) (15).
To infer the earliest cut peptide bonds, we identified 3(IV)NC1peptides that were released after just 5 min of digestion withcathepsin D. Sequence was determined for 23 3(IV)NC1 peptides,shown in full in Supplementary Information and diagrammaticallyin Figure 3C. Twelve of the early-released peptides had NH2-or COOH-termini generated by the already-identified early cleavages(10 by SFL38 FVQ, one by FSF147 IMF, and one by SFW196 LAS).Ten had sequences that began FVQ (within dap31–50), re-confirmingthe SFL38 FVQ peptide bond as highly preferred by cathepsinD, but all had different C-termini, suggesting that cathepsinD has little preference among several potentially scissile peptidebonds in 3(IV)NC1 immediately C-terminal to SFL38 FVQ. In contrast,only one peptide fragment was found indicative of the earlycleavages FSF147 IMF and SFW196 LAS, suggesting that cathepsinD has strong preference to make subsequent cuts at particularnearby peptide bonds, specifically, ISL141 WKG N-terminal toFSF147 IMF releasing the fragment WKGFSF and ERM205 FRK C-terminalto SFW196 LAS releasing the peptide LASLNPERM. The peptide bondsISL141 WKGFSF147 IMF were never found intact in any fragmentsmaller than 22 kD, so it is likely that both peptide bondsare similarly highly susceptible to early proteolysis. Also,one of the prominent early-released peptides indicated thatcleavage at RGF10 VFT, destroying dap1–20, is an earlyevent and possibly indispensable because no peptide was foundwith RGF10 VFT intact.
Taken together, the data identified 11 peptide bonds that werecleaved in early processing (Figure 3D). Among them, they destroyall conceivable DR15-binding subsequences of all 4 3(IV)NC1peptides that stimulate most patients’ T cells. Importantly,no fragment of 3(IV)NC1 smaller than 6 kD was found to containany of the T cell epitopes intact.
The techniques similarly were applied to investigate 3(IV)NC1processing by AEP. Four peptides were released by AEP treatmentof intact 3(IV)NC1, indicative of cuts after five of the nineasparagines in 3(IV)NC1 (Figure 4). Remarkably, all of the scissileasparagines were in the vicinity of (two of five) or within(three of five) the sequence of the T cell self-epitope thatcontained dap71–90. The other four asparagines are presumablyless accessible to AEP within intact 3(IV)NC1 because all nineasparagines were substrates for AEP when presented in the formof short synthetic peptides (data not shown). Note that althoughAEP released fragments from intact 3(IV)NC1, its action wasinsufficient to unlock 3(IV)NC1 in the presence of pepstatinA (Figure 2). This contrasts with lysosomal processing of tetanustoxoid c-fragment, in which AEP action alone is sufficient tounlock the antigens to further processing (11).
Figure 4. Asparagine endopeptidase (AEP) cuts 3(IV)NC1 at multiple sites within dap71–90. The bars depict the four peptides that were identified in AEP digests of 3(IV)NC1 with layout and numbering as in Figure 3. The sequences of the peptides are shown in the bottom panel. Vertical dotted lines identify the nine asparagine residues in 3(IV)NC1. Cuts after asparagine residues 2, 3, 4, 5, and 6 accounted for all of the peptides observed.
Aspartate Protease Activity within Intact Human APC Affects Presentation of Goodpasture Antigen
Next we investigated whether the propensity of lysosomal aspartateprotease activity to destroy 3(IV)NC1 T cell epitopes in vitrohad discernible consequences in vivo. To detect presentationof 3(IV)NC1 peptides by human APC, we made 3(IV)NC1 peptide-specificDR15-restricted murine T cell hybridomas in mice that were engineeredto express HLA DR15 (donated by Dr. Altmann and described inreference [16]). Nine hybridomas, including ha3p132.2, 132.3,and 133.6, had the properties of responding to dap131–150with fine specificity that was indistinguishable (with 15merpeptides overlapping by 10) from patients’ T cells (Figure 5).DR15 restriction was confirmed by demonstrating responses todap131–150 presented by only DR15-expressing B cell lines(data not shown). Analysis of all possible interactions betweenthe common sequence of the stimulatory peptides and DR15 moleculesfound only one orientation with high predicted affinity (5),so the hybridomas and patients’ T cells almost certainlyrecognize the peptide 3(IV)NC1139–148 with sequence ISLWKGFSFIengaging pockets in the peptide binding groove as shown in Table 2.
Figure 5. Characteristics of T cell hybridomas compared with patients’ T cells. (A through C) Specificity: Comparison of the responses of a representative T cell hybridoma (ha3p132.2; A) and a patient’s (EB#01; B and C) peripheral blood T cells with four overlapping 3(IV)NC1 peptides presented by DR15-homozygous Epstein-Barr virus (EBV)-transformed B cell lines. Patient EB#01, like all patients whom we have studied with recent-onset Goodpasture’s disease (3), had peripheral blood T cells that proliferated to dap131–150 and dap71–90. Here responses are shown to short peptides that overlap dap131–150 (see Supplementary Table 1) to indicate the location of the principal epitope(s) more precisely. The hybridoma ha3p132.2 (selected from 12 with similar specificity) and the responding T cells from patient EB#1 most likely are responding to the core sequence ISLWKGFSF lying in the DR15 peptide-binding groove with side chains oriented into pockets as shown in Table 2. (D) The epitope that is recognized by ha3p132.2 is generated by human B cell processing of intact 3(IV)NC1. Note that processing is required because no response was observed when the antigen-presenting cells (APC) were lightly fixed before addition of intact 3(IV)NC1 or in other experiments (data not shown) in which APC were treated with chloroquine. The indicated concentrations of 3(IV)NC1 and AS346 equate to approximately equivalent molar concentrations of 7 µM.
The hybrids were first used to investigate whether B cells thatwere incubated with intact 3(IV)NC1 were able to present theISLWKGFSFI epitope at all, in view of its containing two peptidebonds that are known to be cut in early processing by B celllysosomes in vitro. As shown in Figure 5D, B cells that wereincubated with abundant (200 µg/ml, approximately 7 µM)3(IV)NC1 were able to present ISLWKGFSFI, as assessed by IL-2production by ha3p132.2. Presentation was processing dependentbecause it was inhibited by fixation (Figure 5D) or chloroquine-treatmentof the B cells (data not shown). Therefore, processing of 3(IV)NC1within intact cells does generate some ISLWKGFSFI, which ispresented on DR15 in the conformation that is recognized byha3p132.2.
Next the hybrids were used to interrogate the level of ISLWKGFSFIpresentation on the surface of living APC that were treatedwith pepstatin A. ISLWKGFSFI presentation from intact 3(IV)NC1was enhanced two- to four-fold by 10 mM pepstatin A treatmentof the APC. This was not an APC-specific or T hybrid–specificphenomenon because it was observed for all of the DR15-expressinghuman B cell lines that were studied and for a human macrophagecell line, using any of three of our ISLWKGFSFI-specific T hybridomas(Figure 6A). The effect of pepstatin A on presentation of peptidevaried from no effect, as expected for processing-independentpresentation of peptide, to up to two-fold enhancement. Enhancementwas most striking for ThP1 cells and almost certainly relatedto the high levels of free cathepsin D that were detectablein medium that was conditioned by these cells (data not shown).The effects of pepstatin A were in striking contrast to previousreports of the effect of pepstatin A (17–19) that haveshown epitope-specific effects that vary between strong inhibitionof presentation and no discernible effect, thought to reflectthe varying importance of aspartate proteases in generatingparticular epitopes (Figure 6A, bottom 8 pairs of bars). Theeffect of pepstatin A was further examined by determinationof the time course and dosage dependence of ISLWKGFSFI presentationby the LCL061 B cell line interrogated with the ha3p132.2 Thybrid. Presentation of ISLWKGFSFI from intact 3(IV)NC1 wastwo- to three-fold less than from molar equivalent quantitiesof ISLWKGFSFI-containing peptide and exhibited dosage-dependentenhancement by pepstatin A (Figure 6, B and C). Taken together,the results indicate that the aspartate protease activity intwo different human APC types is sufficient to reduce presentationof the ISLWKGFSFI epitope, at least under cell culture conditions.
Figure 6. Pepstatin A enhances the presentation of 3(IV)NC1138–147 by B cells. (A) Production of IL-2 by T cell hybridomas was used as a measure of presentation of specific peptide by the indicated APC that were incubated with intact protein () or short peptide (). The bars show the percentage change in IL-2 production with addition on 10 µM pepstatin A. The top five pairs of bars show presentation of ISLWKGFSFI by DR15-expressing human EBV B cell lines (LCL013, LCL041, and LCL061) and ThP1 (a human monocyte/macrophage cell line). The bottom eight pairs of bars summarize results of published experiments in which pepstatin A was added to presentation assays using DR1-bearing mouse macrophages to present collagen II to the HCII-9.1 and 9.2 hybridomas (19) or A20 murine B cells to present ovalbumin to the DO11.10 hybridoma (17), or the anthrax protective antigen (PA) to five hybrids with different PA peptide specificity (18). (B) Time course of ISLWKGFSFI presentation by LCL061 that were incubated for 4 to 12 h with approximately 3.5 µM 3(IV)NC1 peptide (dotted lines) or the same molar concentration of intact 3(IV)NC1 (solid lines) as assessed by IL-2 production from ha3p132.2. Pepstatin A substantially increased ISLWKGFSF presentation from intact 3(IV)NC1 but had little effect on presentation of peptide. (C) Summary of results of four experiments using LCL061 as APC and ha3p132.2 to detect ISLWKGFSF presentation showing the change in IL-2 production when 1 or 10 µM pepstatin A was added to 6-h cultures of ha3p132.2. The figure depicts the percentage change in IL-2, compared with that produced in the absence of pepstatin A, as the median (solid bar), 25th and 75th centiles (box) and limits of data (whiskers), and the significance of the indicated comparisons as assessed by the unpaired t test.
The results demonstrate that the level of aspartate proteaseactivity within human EBV-transformed B cells is sufficientto diminish substantially the presentation of a key epitopein Goodpasture’s disease, despite the epitope’shaving high affinity for HLA DR15 molecules. This echoes theobservation of Manoury et al. (20) that AEP activity withinAPC destroys a key epitope in multiple sclerosis and adds credenceto the hypothesis that destructive processing may direct thespecificities of autoreactive T cells by preventing constitutivepresentation of self-epitopes at sufficient levels to drivesecure self-tolerance (10,21). Whereas AEP activity seems criticalin shaping autoimmunity to MBP, our data suggest that aspartateproteases, almost certainly cathepsin D, are critical in shapingautoimmunity to 3(IV)NC1. It is interesting that a prominentrole for cathepsin D in destructively limiting antigen presentationwas also found in a study of myoglobin processing (22). Thus,cathepsin D cleavage of 3(IV)NC1 and AEP cleavage of MBP areantigen-processing mechanisms that diminish presentation ofpotential epitopes within antigens, analogous to the processingmechanisms that are thought to account for certain epitopes’behaving as poorly presented "cryptic epitopes" in animal models(23).
We suggest that the influence of destructive processing couldrun much deeper. In the case of 3(IV)NC1, all of the major self-epitopescontain highly scissile peptide bonds, and fragments that areindicative of cuts within all but one were detected at the earlieststages of processing. Therefore, destructive processing coulddiminish presentation of all of the important self-epitopesin 3(IV)NC1. However, some anomalies from our previous workthat examined peptides that were eluted from DR15 moleculesindicate that other processing factors must also be influential.For example, we eluted from 3(IV)NC1-pulsed B cells peptidesthat contained intact the highly scissile sequence SFL38 FVQ(8). The peptides comprised a nested set that seemed to bindto DR15 via VPLYSGFSF38. A possible explanation is suggestedfrom consideration of how DR15 might interact with intact 3(IV)NC1.The DR15-binding VPLYSGFSF core sequence is almost entirelyexposed on the surface of 3(IV)NC1, where it might be able tobind DR15 before substantial proteolysis, gaining steric protectionfor YSG34 FSF and SFL38 FVQ, and presumably directing processingvia a different route (24).
If indeed destructive processing does suppress constitutivepresentation of some of the epitopes that are recognized bypatients’ T cells in autoimmune disease, how are thoseepitopes ever presented to drive pathogenesis? The results inthe context of previous studies of 3(IV)NC1 processing suggestan explanation, at least for the ISLWKGFSFI epitope for whichthe data are most complete. Peptides that contain this epitopehave the highest affinity of all of the 3(IV)NC1 peptides thatwe have studied but are not presented by DR15-bearing B cellsat sufficient level for biochemical detection, whereas otherlower DR15-affinity peptides are. The new data demonstrate thatrapid destruction by endosomal aspartate proteases can accountfor the deficit of ISLWKGFSFI but, importantly, that sufficientISLWKGFSFI to stimulate T cells can be presented by DR15-bearingB cells under particular conditions, such as culture in highconcentrations of 3(IV)NC1, and that partial inhibition of theactivity of endosomal aspartate proteases is sufficient to increaseits presentation substantially. Therefore, presentation of ISLWKGFSFIis likely to be low level rather than absent and so could bea sufficient stimulus to drive T cells that are already activatedby, for example, a cross-reactive epitope from an infectingorganism. Moreover, the level of ISLWKGFSFI presentation willbe modulated by the balance of positive factors, such as theabundance of 3(IV)NC1 and possibly the level of DR15 expression,and negative factors, including the activity of endosomal aspartateproteases. It is notable in this regard that many of the stimulithat are reported to trigger Goodpasture’s disease wouldbe expected to increase turnover of basement membrane 3(IV)NC1(25), and the levels of protease activity within APC are reportedto vary with cell type and activation status (26).
This is the second report to associate destructive processingwith the specificity of autoreactive T cells, so the questionarises as to the general importance of the mechanism. The twoautoantigens, MBP and 3(IV)NC1, share several features. Bothare tightly folded, extensively disulfide-bonded, protease-resistantcationic globular molecules that are processed only by lysosomesafter unlocking cleavages at particular peptide bonds. It isstriking that in both cases, the unlocking cleavages destroypeptides that have high affinity for HLA DR15 and are majortargets of autoimmune attack. This set of circumstances maynot be rare, because many autoantigens are globular with disulfidebonds, and a requirement for unlocking cleavages has now beenreported for several proteins. Clearly, evaluation of the generalimportance of destructive processing in defining the specificityof autoreactive T cells will require analysis of processingof more autoantigens, but of all of the ways processing couldinfluence presentation, unlocking deserves earnest focus becauseit more than any other processing event is likely to be a consistentoccurrence that could credibly influence the scope of self-tolerancethat is built up during a life time and credibly be subverted,in a stimulating milieu, only infrequently to precipitate autoimmunedisease at close to the low frequency with which disease occursin human.
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J. Zou, S. Hannier, L. S. Cairns, R. N. Barker, A. J. Rees, A. N. Turner, and R. G. Phelps Healthy Individuals Have Goodpasture Autoantigen-Reactive T Cells
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[Abstract][Full Text][PDF]
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Abstract
Introduction
3(IV)NC1; approximately 31 kD] that was incubated with B cell lysosomes was cleaved within a few minutes to form approximately 9- and approximately 22-kD fragments, then increasing quantities of smaller peptides. The processing was completely abrogated by pepstatin A, a specific inhibitor of cathepsin D/E, even though lysosomal extracts contain a rich array of proteases. Purified cathepsin D generated the same major 
when incubated with the Goodpasture autoantigen (2,3). Recent work has demonstrated that the fine specificity of patients’ 
] (13) cells (a donation from Dr. John Robinson, Newcastle University, Newcastle, UK) using the polyethylene glycol method and cloned by limiting dilution. Clones that expressed T cell receptor 
) or purified cathepsin D digests (
) or short peptide (