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The Journal of Immunology, 2003, 171: 2183-2186.
Copyright © 2003 by The American Association of Immunologists

CUTTING EDGE

Cutting Edge: H-2DM Is Responsible for the Large Differences in Presentation Among Peptides Selected by I-Ak During Antigen Processing 1

Scott B. Lovitch, Shirley J. Petzold and Emil R. Unanue2

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
We quantitated the amounts of peptides from hen egg-white lysozyme presented by I-Ak molecules in APC lines. The large chemical gradient of presentation of the four hen egg-white lysozyme epitopes observed in cell lines expressing HLA-DM or H-2DM (referred to in this study as DM) was significantly diminished in the T2.Ak line lacking DM. Differences in levels of presentation between wild-type and DM-deficient APC were observed for all four epitopes, but differences were most evident for the highest affinity epitope. As a result of these quantitative differences in display, presentation of all four epitopes to T cells was impaired in the line lacking DM. The binding affinity of the pool of naturally processed peptides from DM-expressing lines was higher than that from the DM-deficient line. Thus, using a direct biochemical approach in APC, we demonstrate that DM influences the selection of peptides bound to MHC class II by favoring high affinity peptides.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
In a previous study from our laboratory, we quantitated the levels of presentation of the various hen egg-white lysozyme (HEL)3 peptides displayed by the MHC class II molecule I-Ak during processing of the protein by APC. We found a very large differential in the amounts of the various epitopes bound to I-Ak; one peptide containing the core segment 52–60 was highly abundant, whereas the others were present in much lower amounts, from 60- to 300-fold (1). One factor that determined the relative levels of presentation was the binding affinity of peptides for I-Ak, which was related to the presence in the peptide of favorable MHC anchor residues and the lack of amino acids with hindering side chains (2). However, the correlation between affinity and presentation was not precise, indicating that additional, as yet unidentified, factors play a critical role in epitope selection.

We have now examined chemically the display of the four HEL epitopes in APC lines that lack the auxiliary molecule H-2DM (referred to in this study as DM) to determine the extent that it influences the display of the various MHC-bound peptides. All previous studies of the role of DM in peptide selection from APC have depended on T cell assays, which are not strictly quantitative and which can be influenced by factors unrelated to peptide display. Of note, several studies have claimed to show differential DM dependence of various epitopes, with some dependent on DM for presentation and others for which DM has no effect or is even antagonistic (3, 4, 5, 6, 7, 8). Specifically, in studies of presentation of HEL epitopes by I-Ak, it has been claimed that the chemically dominant 48–61 epitope is uniquely dependent on DM and invariant chain, with other epitopes independent of these auxiliary molecules (9, 10, 11, 12). In contrast, recent studies in DM-deficient mice have come to a different conclusion, namely that DM dependence of presentation is a function of MHC haplotype and not of any particular epitope (13).

DM has been shown to favor the release of bound class II-associated invariant chain peptide (CLIP) from the binding groove of MHC class II molecules, allowing for binding of new peptides, and therefore editing the repertoire of selected peptides (14, 15, 16, 17, 18). In addition, DM acts as a molecular chaperone, because in its absence MHC class II molecules rapidly aggregate in the absence of bound peptide (19, 20, 21). In this study, we have quantitated the presentation of HEL epitopes in wild-type and DM-deficient APC. We find that DM affects levels of presentation of all epitopes, but most significantly impacts the chemically dominant epitope, resulting in the observed chemical gradient of presentation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Cell lines, culture conditions, and assays

T1.Ak and T2.Ak (obtained from P. Cresswell, Yale University, New Haven, CT) are human B cell lymphoma lines transfected with murine I-Ak; T2.Ak has a chromosomal deletion that spans the genes encoding HLA-DM{alpha} and DM{beta} (22). The T cell hybridomas 3A9 and A6A2 have been described; these recognize the 48–63 and 31–47 epitopes of HEL, respectively (23, 24). The 3F12 and 125.7 hybridomas, which recognize the 20–35 and 114–129 epitopes of HEL, respectively, were generated as in Refs. 25 and 26 . All cell lines were maintained in DMEM containing 5% FCS. T hybridoma assays were performed in 96-well flat-bottom tissue culture plates containing a final volume of 200 µl of DMEM plus 5% FCS. The cells (5 x 104/well) were incubated with graded doses of HEL or peptides and T1.Ak or T2.Ak APC (5 x 104/well) for 18 h at 37°C in 5% CO2. The 100-µl aliquots of culture supernatants were then tested for their ability to induce proliferation of the IL-2-dependent cell line CTLL-2. HEL was obtained from Sigma-Aldrich (St. Louis, MO) and purified to eliminate contaminants. Synthetic peptides representing the major member of each of the four HEL peptide families (Fig. 1) were produced in our laboratory (1).



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  		FIGURE 1. The chemical gradient of presentation of HEL epitopes is abrogated in the absence of DM. Peptides were isolated from lysates of T1.Ak ({square}) or T2.Ak ({blacksquare}) as described in Materials and Methods. Numbers over brackets indicate the fold excess of 48–63 relative to each of the two minor epitopes. The ratio for each in T1.Ak over T2.Ak is also indicated. Information in the box includes the sequences, registers, and affinities of the four epitopes (see Ref. 2 ); the primary anchor residue of each epitope (D at P1 or N at P4) is underlined. Data represent mean values from three experiments, each conducted with a separate preparation of peptide extract.

 
Biochemical assays

Peptides were isolated from T1.Ak and T2.Ak as described (1, 26, 27, 28). Briefly, cells were grown in the presence or absence of HEL, then lysed in PBS containing 40 mM octanoyl-N-methylglucamide and 40 mM nonanoyl-N-methylglucamide (both from Sigma-Aldrich) in the presence of PMSF, iodoacetamide, and leupeptin. I-Ak-peptide complexes were isolated using 40F (anti-I-Ak) mAb (29) coupled to Sepharose beads, washed, and eluted in 0.1% trifluoroacetic acid. Peptides were separated from MHC molecules by membrane separation (Centricon YM-10; Millipore, San Jose, CA). Total peptide content was measured by bicinchoninic acid-based colorimetric assay (Ref. 30 ; Pierce, Rockford, IL) using the 48–61 peptide from HEL (DGSTDYGILQINSR) as the standard. Comparison of various standard peptides indicated variability of ~20%, reflecting length and content of tyrosines, cysteines, and tryptophans.

HEL peptides extracted from APC were quantitated by competitive ELISA as described (1) using a panel of mAbs that recognize peptides in free solution; 96-well plates were coated with streptavidin and incubated with biotinylated peptide. Serial dilutions of standard peptide (synthetic 48–61, 20–35, or 31–47; Ref. 2) or extract were applied to the plate along with a fixed amount of anti-peptide Ab. Bound Ab was detected using HRP-conjugated goat anti-mouse IgG and developed with ABTS plus 0.03% H2O2. Quantitation was performed using a standard curve of synthetic peptide.

Binding assays were performed using recombinant I-Ak-CLIP molecules (1) and a 125I-labeled standard peptide (YEDYGILQINSR). Serial dilutions of peptide extract or the standard unlabeled peptide were combined with 0.125 pmol of radiolabeled peptide and 0.75 µg of I-Ak-CLIP in binding buffer (20 mM MES, 150 mM NaCl, pH 5.5) in total volumes of 30 µl and incubated overnight at room temperature. MHC-peptide complexes were then isolated on Bio-Spin P6 gel filtration columns (Bio-Rad, Hercules, CA), and the bound radioactivity was quantitated on a gamma counter (Wallac, Turku, Finland). All assays used concentrations of peptide that bound ~20–30% of input cpm.

SDS-stability of I-Ak-peptide complexes was assessed by metabolic labeling and immunoprecipitation. A total of 2 x 107 cells were incubated for 1 h at 37°C in 1 ml of methionine- and cysteine-free DMEM plus 5% FCS, then pulsed with 200 µCi of 35S-TransLabel (ICN Pharmaceuticals, Costa Mesa, CA) for 30 min at 37°C, after which excess unlabeled methionine and cysteine were added. Following the appropriate chase interval (4 or 24 h), cells were lysed as described above. Lysates were precleared with protein A-Sepharose (Sigma-Aldrich), then immunoprecipitated with the mAb 40F followed by protein A-Sepharose. Adsorbed complexes were washed, resuspended in sample buffer containing SDS, fractionated by SDS-PAGE, and analyzed by autoradiography. Quantitation was performed using a PhosphorImager and ImageQuant software (Molecular Dynamics, Sunnyvale, CA).


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
For our analysis, we used the DM-deficient T2.Ak line and its DM-expressing parent T1.Ak (22). Whereas T1.Ak efficiently processes and presents HEL, T2.Ak fails to present the dominant 48–63 epitope (3, 25, 31, 32). Total MHC-bound peptides were isolated from large cultures of T1.Ak and T2.Ak (~5 x 109 cells); the total concentration of peptides in the extract was determined by bicinchoninic acid assay (see below), whereas the concentration of each of the HEL epitopes was measured by the competitive ELISA method used previously in Ref. 1 (Fig. 1). The concentration of peptides isolated from each cell line was similar, 4.2 nmol/109 cells for T1.Ak and 3.6 nmol/109 cells for T2.Ak. (The data represent the mean of three experiments with essentially identical results.) These numbers correspond to recovery of 2.5 x 1015 and 2.2 x 1015 I-Ak molecules from 109 T1.Ak and T2.Ak, respectively. We estimate that ~2.2 x 1015 MHC molecules are present per 109 cells; thus, these figures are within the limits of the various assays and indicate that most MHC molecules should be occupied by peptides.

We focused our chemical analysis on the dominant 48–63 epitope and two minor epitopes, 31–47 and 20–35 (Fig. 1). (We are unable to precisely quantitate the 114–129 epitope in peptide extracts.) Fig. 1 also shows the sequence of the four major peptides and their binding affinities estimated previously from IC50 assays. In previous studies using M12.C3-derived B lymphoma lines as APC, we observed a steep gradient in presentation of these epitopes, with presentation of 48–61 exceeding that of 31–47 by 60-fold, and that of 20–35 by >200-fold (1). Analysis of extracts from T1.Ak incubated with HEL protein yielded similar differences. However, this gradient was reduced in HEL peptides extracted from T2.Ak APC. The total variation in levels of presentation of the three major peptide families in the DM-deficient APC line was <7-fold, compared with a range of >600-fold in T1.Ak (Fig. 1 shows the results and also indicates the differences between the epitopes). Analyzing the same data in a different way, presentation of 48–63 was enhanced >1000-fold in T1.Ak relative to T2.Ak, whereas presentation of the minor 31–47 and 20–35 epitopes was increased much less dramatically (60-fold and 12-fold, respectively) in the presence of DM (see T1.Ak:T2.Ak ratio in Fig. 1). Thus, DM influences the gradient of presentation of both chemically dominant and subdominant epitopes from the same protein, but favors presentation of the peptides that bind MHC class II with the highest affinity.

The affinity of the peptide pool for binding to I-Ak was measured using a standard competitive binding assay in which the ability of peptides in the extract to compete with a radiolabeled standard peptide for binding to I-Ak was measured. The affinity of the pool of peptides extracted from T2.Ak was 4.2 µM, whereas that from T1.Ak was 1.73 µM (Fig. 2). Thus, the peptides isolated from T2.Ak had significantly lower affinity than those isolated from T1.Ak, indicating that without DM there is a higher representation of lower affinity peptides in the extract. The same results from T1.Ak were obtained from the C3.F6 line used in our previous quantitative analysis (1); we estimated 2.2 x 1015 peptide molecules per 109 cells containing ~2.0 x 1015 MHC molecules, the pool having an affinity of 1.83 µM.



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  		FIGURE 2. The overall affinity of peptides bound to I-Ak is reduced in DM-deficient APC. Dilutions of peptide extract from T1.Ak ({blacksquare}) or T2.Ak (•) were combined with fixed amounts of radiolabeled competitor and recombinant Ak-CLIP in 20 mM MES, 150 mM NaCl at pH 5.5. The IC50 is the peptide concentration that inhibits competitor binding by 50%.

 
These results with T2.Ak were also reflected in their content of SDS-stable molecules. In normal APC lines, the percentage of SDS-stable I-Ak molecules is a reflection of their content of high affinity peptides (33, 34). The percentage of SDS-stable complexes in T1.Ak after a 24-h chase following a 30-min pulse with [35S]methionine and [35S]cysteine was 40%, whereas in T2.Ak it was <10% (Fig. 3). However, SDS-unstable complexes in T2.Ak could be rescued by incubation with the high affinity 48–61 peptide (Fig. 3, far right lane), indicating that the lack of stability in the absence of exogenous 48–61 was caused by their occupation by low affinity peptides. These data are consistent with previous results from our laboratory (25) and from Karlsson and colleagues (35) and corroborate the result of Bikoff et al. (13) using splenocytes from DM-deficient mice. However, a previous study using T2.Ak concluded that DM was unnecessary for formation of SDS-stable complexes by I-Ak (36).



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  		FIGURE 3. SDS-stability of I-Ak-peptide complexes formed in normal and DM-deficient APC. T1.Ak or T2.Ak (2 x 107 cells/lane) were metabolically labeled with [35S]methionine and [35S]cysteine in the presence of HEL (1 mg/ml), 48–61 peptide (100 µM), or medium alone for 30 min and chased for 24 h. I-Ak and associated proteins were then immunoprecipitated and analyzed by SDS-PAGE without boiling. The positions of SDS-stable {alpha}{beta} dimers and of the dissociated A{alpha}k ({alpha}) and A{beta}k ({beta}) chains on the gel are indicated.

 
Taken together, our results suggest that peptides bound by I-Ak in T2.Ak are of lower affinity than those in T1.Ak. In experiments in progress we are reporting on the content and features of peptides bound to I-Ak in T1.Ak and T2.Ak examined by tandem electrospray mass spectrometry. In our previous analysis of one peptide family presented by I-Ak (derived from the I-Ak {beta}-chain), we found reduced abundance of this high affinity peptide family on T2.Ak relative to T1.Ak (26).

The biochemical results were confirmed by functional assays. We tested hybridomas reactive to each of the four epitopes for their ability to recognize peptide presented by either T1.Ak or T2.Ak APC following processing of HEL. The presentation of exogenous peptide was unaffected by DM; however, the presentation of all four epitopes from intact HEL was poor in T2.Ak compared with T1.Ak APC (Fig. 4). Whereas the difference in presentation by T1.Ak and T2.Ak varied somewhat among epitopes, all showed markedly reduced presentation in the absence of DM (recognition was completely abolished for one epitope), indicating that all epitopes are DM-dependent. These results contrast with the earlier studies mentioned above, but agree with the more recent findings in DM-deficient mice bearing the H-2k haplotype (13) while extending the findings to all four HEL epitopes presented by I-Ak.



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  		FIGURE 4. Processing of all four HEL epitopes is DM dependent. Hybridomas recognizing each of the four epitopes from HEL presented by I-Ak (5 x 104/well) were incubated for 18 h with T1.Ak or T2.Ak cells as APC, and graded doses of either HEL or the appropriate peptide ({blacksquare}, HEL presented by T1.Ak; {square}, HEL presented by T2.Ak; •, peptide presented by T1.Ak; {circ}, HEL presented by T2.Ak). Production of IL-2 was determined by CTLL assay. Each hybridoma was tested in three to five independent experiments, and each point represents the mean of triplicate wells; error bars represent SD.

 
In summary, in the presence of DM there is a gradient of HEL peptides bound to I-Ak with a pronounced skewing to the chemically dominant peptide family centered around core residues 52–60 that form a high affinity, long-lived (t1/2 > 96 h) interaction with the I-Ak binding cleft (1). The other three peptide families (31–47, 20–35, and 114–129) are presented at much lower levels. We find that this differential is markedly reduced in the absence of DM, with all epitopes presented at nearly equivalent levels. These results, together with the demonstration that the pool of peptides bound in the absence of DM has lower affinity than the pool from cells containing DM and is less able to form SDS-stable complexes with I-Ak, offer direct biochemical proof that in APC DM exerts a marked influence on the relative content of strongly or weakly binding peptides. These studies thus confirm the peptide-editing role that many have cogently argued for based on binding analyses and structural studies (4, 5, 6, 14, 15, 16, 17, 18, 37), i.e., that DM serves to edit the peptide repertoire by inducing, through its interaction with the MHC class II molecule, more rapid dissociation of weakly binding peptides, causing the MHC molecule to become receptive to interaction with higher affinity and more stably binding peptides. Notably, the CLIP peptide dissociates from I-Ak with very rapid kinetics compared with other haplotypes (Refs. 38 and 39 , and our unpublished studies). It would seem that with I-Ak the DM molecule influences less the dissociation of CLIP, which in itself is quite fast, and may serve best to stabilize the I-Ak molecule, allowing it to interact with HEL-derived and other peptides. Indeed, in the presence of DM, nascent I-Ak molecules can effectively process HEL and present HEL epitopes, whereas this process is restricted to the early endosome in the absence of DM (32). By thus prolonging the window of opportunity for I-Ak to sample the repertoire of peptides present in the endosomal compartment, DM favors binding of high affinity peptides to I-Ak.


   Acknowledgments
 
We thank Kevin Clark for synthesizing the peptides used in these studies, Ravi Veeraswamy for generating the 125.7 hybridoma, Gina Filley for technical assistance, and Zheng Pu and Craig Byersdorfer for helpful discussions.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant 5R01AI022033. S.B.L. is a trainee of the Medical Scientist Training Program. Back

2 Address correspondence and reprint requests to Dr. Emil R. Unanue, Department of Pathology and Immunology, Washington University School of Medicine, Box 8118, 660 South Euclid Avenue, St. Louis, MO 63110. E-mail address: unanue{at}pathbox.wustl.edu Back

3 Abbreviations used in this paper: HEL, hen egg-white lysozyme; CLIP, class II-associated invariant chain peptide. Back

Received for publication May 14, 2003. Accepted for publication June 27, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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