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APCs Present A^k-Derived Peptides That Are Autoantigenic to Type B T Cells¹

Scott B. Lovitch^*, James J. Walters, Michael L. Gross and Emil R. Unanue^2,*

^* Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110; and Department of Chemistry, Washington University, St. Louis, MO 63130

	Abstract

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Type B T cells recognize peptide provided exogenously but are ignorant of the same epitope derived from intracellular processing. In this study, we demonstrate the existence of type B T cells to an abundant autologous peptide derived from processing of the I-A^k -chain. T cell hybridomas raised against this peptide fail to recognize syngeneic APC despite abundant presentation of the naturally processed epitope but react in a dose-dependent manner to exogenous peptide. Moreover, these hybridomas respond to A^k peptide extracted from the surface of I-A^k-expressing APC. This peptide was isolated from B cell lines where it was found in high abundance; it was also present in lines lacking HLA-DM, but in considerably lower amounts. Therefore, type B T cells exist in the naive repertoire to abundant autologous peptides. We discuss the implications of these findings to the potential biological role of type B T cells in immune responses and autoimmune pathology.

	Introduction

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We previously reported the identification of a novel subset of CD4⁺ T cells that recognize their peptide epitope when provided exogenously to MHC class II-bearing APCs but fail to respond to the same peptide generated as a result of intracellular processing of the native protein from which it is derived. We refer to these cells as type B T cells, in contrast to the conventional type A T cells which recognize peptide whether given exogenously, as free peptide, or generated through intracellular processing (reviewed in Ref. 1). Type B T cells were first identified following immunization with peptides derived from processing of hen egg white lysozyme (HEL)³: the HEL_84–96 epitope presented by I-E^k (2) and the HEL_46–61 epitope presented by I-A^k (3). Type B T cells can also be detected to minor epitopes of HEL presented by I-A^k and to epitopes presented by I-A^g7, the diabetogenic class II allele of the nonobese diabetic mouse (A. Suri and E. R. Unanue, unpublished observation).

To explain this phenomenon, we have proposed that exogenously provided peptides bind class II in a conformation distinct from that resulting from peptide binding during intracellular processing (4). Peptides derived from processing of native protein bind to class II in a late endosomal or lysosomal compartment characterized by acidic pH and the presence of HLA-DM, whereas exogenously provided peptides bind by peptide exchange at the cell surface or in early endosomal compartments at neutral pH and in the absence of DM; these two pathways result in antigenically distinct conformations, the latter of which is specifically recognized by type B T cells. This model is supported by our recent finding that the TCR contact residues required for type B T cell reactivity are distinct from those required by type A T cells, with either the N- or C-terminal contact residues dispensable for peptide recognition by type B T cells (4). In contrast, alternative explanations for the type B phenomenon, including postsynthetic and posttranslational modification of the bound peptide, were investigated and found not to correlate with type B reactivity (5, 6, 7).

Type B T cells may play an important biological role. We previously showed that type B T cells comprise a significant proportion (30–50%) of the CD4⁺ T cell response induced by peptide immunization (7); this fact by itself calls into question strategies of vaccination that rely on peptide immunization, because many of the T cells primed by such protocols will not react with the native immunogen. More importantly, type B T cells escape negative selection and are detected in the periphery following peptide immunization of mice in which the native protein is transgenically expressed and in which type A T cells are completely eliminated by negative selection (7). These findings suggest that in situations in which free peptides are generated in vivo and bind to class II on the surface of APCs, naive type B T cells may become primed and contribute to an autoimmune response. This could occur through mechanisms of extracellular proteolysis at sites of inflammation, as have been described for neutrophils (8) and dendritic cells (9, 10). Such a model, however, assumes that type B T cells that are reactive to autoantigens are present in the naive CD4⁺ T cell repertoire and available to be primed at sites of inflammation.

The use of mass spectrometry (MS) to isolate and sequence peptides eluted from class II MHC molecules has allowed identification of much of the repertoire of autologous peptides naturally processed and presented by class II MHC. In particular, the identity, tissue distribution, and relative abundance on the cell surface of peptides naturally processed and presented by I-A^k is now known in considerable detail as a result of studies in several laboratories, including our own (Refs. 11, 12, 13 ; see also Table I). This knowledge has allowed us to evaluate the generality of the type B phenomenon by investigating the presence of type B T cells to these autologous peptides.

View this table: [in this window] [in a new window]   Table I. Naturally processed Ak peptides isolated from APC lines expressing I-Aka

	Materials and Methods

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Peptide synthesis

The A^k_37–53 peptide (YVRFDSDVGEYRAVTEL) and its variants were synthesized using fluorenylmethoxycarbonyl chemistry on a Symphony Multiplex peptide synthesizer (Protein Technologies, Tucson, AZ). Peptides were purified to homogeneity by C₁₈ reverse phase HPLC, and the molecular mass and purity of each peptide were verified by matrix-assisted laser desorption ionization time-of-flight MS.

Mice

B10.BR mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and maintained under specific pathogen-free conditions in the Washington University School of Medicine (St. Louis, MO) animal facility. These mice are of the H-2^k haplotype and express the I-A^k and I-E^k class II molecules on APCs.

T cell hybridomas, APCs, and assays

T cell hybridomas were raised by immunization of B10.BR mice in the hind footpad with 10 nmol of peptide emulsified in 100 µl of CFA. Single-cell suspensions were prepared from draining lymph nodes 7 days postimmunization and restimulated ex vivo for 72 h with the peptide used for immunization, then fused with the BW5147^-- thymoma line. Hybridomas were screened for reactivity with peptide-pulsed syngeneic APC and maintained in DMEM supplemented with 5% FCS. The C3.F6 B cell lymphoma line used in many of these experiments was previously described (14) and was maintained in DMEM supplemented with 5% FCS, as were the DM-deficient cell line T2.A^k and its parent line T1.A^k (15), obtained from P. Cresswell (Yale Medical School, New Haven, CT).

T cell hybridoma assays were performed in 96-well flat-bottom tissue culture plates containing a final volume of 200 µl of DMEM and 5% FCS. T hybridoma cells (5 x 10⁴/well) were cultured in the presence of C3.F6 (5 x 10⁴/well) or irradiated (3000 rad) syngeneic B10.BR splenocytes (5 x 10⁵/well) in the presence of the indicated doses of Ag. After incubation for 18 h at 37°C in 5% CO₂, 100-µl aliquots of culture supernatant from each well were assayed for IL-2 production using the IL-2-dependent cell line CTLL-2.

Purification of naturally processed peptides

Cells were lysed in PBS containing 40 mM octanoyl-N-methylglucamide (MEGA-8) and nonanoyl-N-methlyglucamide (MEGA-9) detergents in the presence of protease inhibitors (1 mM PMSF, 10 mM iodoacetamide, and 40 µM leupeptin). After 1 h at 4°C, the lysate was spun at 8000 rpm for 30 min. I-A^k-peptide complexes were purified using 40F (anti-I-A^k) mAb coupled to cyanogen bromide-activated Sepharose beads (Sigma-Aldrich, St. Louis, MO). Sepharose beads were loaded onto disposable chromatography columns (Bio-Rad, Hercules, CA) and washed sequentially with 10 mM MEGA-8/MEGA-9 (40 ml), 2.5 mM MEGA-8/MEGA-9 (40 ml), PBS (100 ml), and distilled water (100 ml). Complexes were then eluted in 15 ml of 0.1% trifluoroacetic acid (TFA; pH 1.9), and the peptides were separated from MHC molecules by membrane separation on Centriprep YM-10 concentrators (Millipore, San Jose, CA; molecular mass cutoff, 10 kDa). Peptide extracts were lyophilized and stored at -80°C.

Reverse phase HPLC and MS

Peptide extracts were fractionated by reverse phase HPLC on a 2-mm C₁₈ column with an injection loop volume of 1 ml (Waters, Milford, MA). Before injection of peptide extract, the column was washed with methanol followed by a mixture of 98% solvent A (0.06% TFA) and 2% solvent B (0.052% TFA plus 80% acetonitrile). Fractions were collected at 30-s intervals beginning 22 min after sample injection; 60 fractions were collected per run. When testing fractions for T cell reactivity, 25 µl of each fraction were transferred to 96-well flat-bottom plates, and the solvent was allowed to evaporate overnight; fractions were then reconstituted in DMEM plus 5% FCS for analysis by T cell hybridoma assay.

MS and MS/MS were performed on a Finnigan LCQ-Deca ion trap mass spectrometer with XCalibur 1.1 software (Thermo Finnigan, San Jose, CA). HPLC fractions were desalted using three ZipTip pipet tips containing C₁₈ reverse phase medium (Millipore, Bedford, MA) and then lyophilized and reconstituted in 45 µl of 3% acetonitrile, 0.1% formic acid solution (solvent A); 6 µl were loaded onto a Delta-Pak C₁₈ 0.075- x 100-mm, 5-µm, 300-Å capillary column (Waters) connected online to the mass spectrometer. The gradient was from 0% solvent B (97% acetonitrile, 0.1% formic acid) to 5% solvent B over 3 min and then to 50% solvent B during 70 min. Eluent flow was 6 µl/min and split before the column at a 1:30 ratio to maintain a flow rate of 280 nl/min. For MS, scan range was m/z 600-1400 in profile mode, in which every three microscans were averaged to one scan; acquisition was started 15 min after the start of the liquid chromatography run. For MS/MS, a scan range of m/z 600-1400 in centroid mode was used for the MS, and the MS/MS range was from 30% of the parent ion to m/z 2000. The isotopic parent ions were dynamically selected and were isolated with a 2.2-m/z window. Collision energy was set to 28% of the maximum. For analysis of T1.A^k and T2.A^k peptide extracts, multiple MS/MS analysis were performed on each sample; one run isolating the first and second most abundant ions and another isolating the third and fourth most abundant ions.

MS/MS spectra were analyzed, and peptide sequences were determined automatically using SEQUEST software provided by the instrument manufacturer (Waters). All automatically determined sequences were manually verified against the experimental product-ion spectra.

	Results

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The A^k_37–53 peptide family is abundantly presented on I-Ak-expressing cell lines

The autologous peptide Ag selected for use in these studies was the peptide spanning residues 37–53 of the I-A^k -chain (A^k). In the study of Marrack et al. (11), one member of this peptide family occupied 9.3% of I-A^k molecules on splenic APC, making it the single most abundant autologous peptide identified on splenocytes, and 1.1% of I-A^k molecules on thymic APC. Our own studies of naturally processed peptides presented by I-A^k (12, 13) showed that several members of this peptide family were presented in abundance by the C3.F6 B cell lymphoma line, used as the APC in our T cell hybridoma assays (see below).

To investigate further the endogenous processing of the I-A^k -chain, and in particular to determine whether presentation of theA^k_37–53 epitope is dependent on the catalytic function of H2-DM, we analyzed the spectrum of naturally processed A^k peptides presented by I-A^k on two additional cell lines by MS: T2.A^k, which contains a disruption in the gene for DM and is therefore deficient in DM-dependent Ag processing; and T1.A^k, the parent cell line of T2.A^k, which expresses functional DM (15). A^k peptides constituted the most abundant peptide family detected from T1.A^k (Table I). We identified 18 A^k peptides from T1.A^k, with a hierarchy of abundance similar to that seen for those from C3.F6 (13); in both cell lines, the most abundant A^k peptide spanned residues 37–52. In contrast, only six A^k peptides were detected from T2.A^k, the hierarchy of abundance was altered, and the overall relative abundance of the family was reduced. This result indicates that A^k peptides are naturally processed and presented by multiple I-A^k-expressing APC lines and that processing of this epitope is dependent on function of DM.

T cell hybridomas raised against A^k_37–53 display type B reactivity

To assess whether type B T cells reactive to A^k peptide exist in the naive CD4⁺ T cell repertoire, we immunized B10.BR mice with 10 nmol of A^k_37–53 peptide emulsified in CFA and generated a panel of T cell hybridomas from the draining lymph nodes isolated 7 days postimmunization. Hybridomas were screened for recognition of APC pulsed with A^k_37–53 peptide and for recognition of the same APC in the absence of exogenous peptide. Of a total of 240 hybridomas examined, 9 were reactive to peptide-pulsed APC but ignorant of unpulsed APC. These hybridomas were expanded and further tested in T cell hybridoma assays for their response to graded doses of the A^k_37–53 peptide.

The responses of four representative hybridomas are shown in Fig. 1. Each hybridoma responded to the presence of A^k peptide in a dose-dependent manner, producing IL-2 sufficient to stimulate maximally the CTLL-2 cells used in the assay at the highest doses of peptide tested. The hybridomas failed to respond to normal APC; none responded in the absence of exogenous peptide or when pulsed with 0.1 µM peptide (Fig. 1, left). Yet the normal APC had abundant levels of naturally processed A^k peptides presented by I-A^k (Table I). Therefore, each hybridoma displays a type B pattern of reactivity. The same concentration-dependent response to peptide was observed when B10.BR splenocytes were used as the APC (Fig. 1A, left). In addition, the same pattern of reactivity was observed when using T2.A^k cells as APC, indicating that presentation of the peptide is DM independent (Fig. 1, right). Although the nine hybridomas selected from the initial screen varied in their sensitivity to the A^k peptide, as assessed by half-maximal stimulation, all displayed this same pattern of reactivity (data shown for four of the nine).

View larger version (22K): [in this window] [in a new window]   FIGURE 1. T cell hybridomas raised against Ak37–53 display type B reactivity. Data are for the hybridomas Ak.11 (A), Ak.18 (B), Ak.36 (C), and Ak.73 (D); these four hybridomas constitute a representative sample of a total of nine that were isolated. Left, C3.F6 B lymphoma cells (5 x 104/well, ) or irradiated (3000 rad) B10.BR splenocytes (5 x 105/well, ) were cocultured with the T cell hybridoma (5 x 104/well) in the presence of serial dilutions of the Ak37–53 peptide in 96-well flat-bottom plates for 18 h, and supernatants were assayed for their ability to induce proliferation of the IL-2-dependent CTLL-2 cell line (1 x 104/well). The CTLL-2 response to 500 U/ml IL-2 () is shown as an indicator of their maximum proliferation. Right, DM-deficient T2.Ak cells (5 x 104/well, ) were cocultured with the T cell hybridoma in the presence of serial dilutions of Ak peptide, and supernatants assayed for their ability to induce CTLL-2 proliferation as described.

A^k-reactive type B hybridomas respond to naturally processed peptide extracted from the cell surface of I-A^k-bearing APC

Although T cell hybridomas raised against A^k_37–53 peptide clearly displayed type B reactivity, it remained possible that these hybridomas were specific for postsynthetic modifications present in the preparation of synthetic peptide used for immunization, a phenomenon detected in studies of the CD8⁺ T cell response to synthetic peptide (16, 17) and which we have observed for CD4⁺ T cells (Ref. 5 and D. A. Peterson, unpublished data). To address this possibility, we determined whether the hybridomas raised against synthetic A^k peptide could respond to peptide extracted from the surface of the I-A^k-expressing C3.F6 B cell lymphoma line, which presents multiple members of the A^k peptide family (12). I-A^k-peptide complexes were isolated from a lysate of 7.5 x 10⁹ C3.F6 cells by immunoaffinity chromatography, and the peptides were extracted and fractionated by reverse phase HPLC. Individual HPLC fractions of peptide extract were then tested against one of the hybridomas (A^k.11; see Fig. 1A). Three fractions stimulated the hybridoma, indicating that they contained A^k peptides (Fig. 2). Assuming no peptide loss in the fractionation procedure, we estimate that the peptide concentration in each positive fraction in Fig. 2 was 0.2 µM, a concentration compatible with that obtained with exogenous peptide in Fig. 1.

View larger version (15K): [in this window] [in a new window]   FIGURE 2. Recognition of peptide extracted from the surface of C3.F6 by a type B hybridoma raised against Ak peptide. Class II I-Ak molecules from C3.F6 were purified by immunoaffinity chromatography using mAb 40F, and bound peptides were released with 0.1% TFA and fractionated by reverse phase HPLC. After solvent evaporation, reconstituted fractions were cocultured with Ak.11 hybridoma (5 x 104/well) in 96-well flat-bottom plates for 18 h. Supernatants were assayed for their capacity to induce CTLL-2 proliferation as described above. The three hybridoma-reactive fractions (fractions 26–28) represent retention times of 34.5–35, 35–35.5, and 35.5–36 min, respectively.

View larger version (19K): [in this window] [in a new window]   FIGURE 3. Detection of Ak peptides in hybridoma-reactive fractions by MS. Data are for one fraction representative of all three analyzed. A and B, Peptides from HPLC fraction 26 were reconstituted in 100 µl of 2% acetonitrile, 0.6% acetic acid, and 5 µl was injected into the reverse phase HPLC online with the mass spectrometer. XCalibur software was used to scan the total ion chromatogram for ions with m/z ± 0.5 of the predicted values for Ak37–52 (A, predicted m/z = 954.0) and Ak37–51 (B, predicted m/z = 889.5). The horizontal axis represents retention time in min; the vertical axis represents relative abundance normalized to the indicated value (NL; note the difference between A and B). C, Mass spectrum of all ions within the range of retention time corresponding to the peak shown in A and B. Distinct peaks indicating doubly charged ions can be observed corresponding to Ak37–52 (outline arrow) and Ak37–51 (filled arrow). The horizontal axis here represents m/z; the vertical axis represents relative abundance as in A and B.

	Discussion

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Type B T cells were initially described for cryptic epitopes derived from foreign Ags, which induce strong T cell responses upon peptide immunization but weak or undetectable responses upon immunization with the native protein (18). When we studied one such epitope derived from HEL and presented by I-E^k, we observed that many T cells reactive to the peptide failed to respond to HEL protein despite chemical evidence of abundant presentation of the peptide on MHC molecules (2); indeed, we proposed then that many so-called cryptic epitopes reported are in fact type B epitopes. Further studies also detected type B T cells against the dominant 48–61 epitope of HEL presented by I-A^k, with 30–50% of CD4⁺ T cells raised against this peptide displaying type B reactivity (3, 7); the family of 48–63 peptides can occupy up to 10% of I-A^k molecules (19). These findings, as well as the observation that type B T cells to HEL_48–61 escape negative selection in HEL-transgenic mice and can be detected in the periphery after peptide immunization (7), imply that the type B phenomenon is unrelated to epitope crypticity, instead reflecting a fundamental difference in the handling of peptides and native proteins by APC. We hypothesize that type B T cells exist in the periphery that are reactive to most, if not all, autologous peptides. The results reported here support this hypothesis by showing that A^k_37–53, an autologous peptide, can function as a type B epitope. Previous studies have identified this peptide as the single most abundant autologous peptide presented by I-A^k; it occupies nearly 10% of splenic I-A^k molecules and is also abundant on thymic APC (11, 12). Therefore, the failure of our type B hybridomas to recognize the naturally processed peptide cannot be explained by inadequate density of peptide-MHC complexes; the complexes recognized by type B T cells must be distinct from those formed by the naturally processed peptide.

From our studies of type B T cells to HEL peptides (2, 3, 4, 7), we proposed a model in which exogenous and endogenously generated peptides load onto class II MHC via different pathways: type A complexes are generated by processing of the native protein in acidified endosomal vesicles, where the peptide loads in its most stable conformer as a result of the catalytic function of H2-DM; whereas free peptide binds MHC by peptide exchange at the cell surface or in recycling vesicles where H2-DM is absent, resulting in the more flexible, less stable type B conformer. In support of this model, we demonstrated that type B presentation was unaffected by fixation or chloroquine treatment of the APC, and that it could occur on both wild-type and H2-DM-deficient APC (4). Type B presentation of A^k_37–53 likely occurs through the same mechanism. In particular, the fact that presentation of the A^k epitope is affected by the function of DM, as shown by our chemical analysis of presentation by T1.A^k and T2.A^k (Table I), supports this view.

The structural basis for the distinction between type A and B complexes is now under investigation. Display of the complexes results in different interaction between the TCR contact residues and the TCR (4). Studies of SDS stability of type A and B complexes, however, failed to distinguish between the two (12). An analysis of the SDS stability of the A^k complexes was previously reported (12).

The studies reported here raise issues similar to those addressed by Janeway and colleagues (20, 21) in their studies of the E_52–68 epitope presented by I-A^b. Like the A^k peptide, the E epitope is an autologous Ag derived from the -chain of class II MHC. In mice expressing E, the epitope is expressed in abundance in both spleen and thymus (22). This system has been instrumental in defining the role of self peptide-MHC recognition in both positive and negative selection (23, 24, 25, 26, 27, 28). Two studies in particular by Janeway’s group merit discussion in light of the results reported here. Barlow et al. (29) isolated a T cell clone, E6, that recognizes APC pulsed with E peptide but is unreactive to APC that generate the epitope endogenously. The clone reacted preferentially with a C-terminal truncation of the naturally processed epitope, and immunization with this truncated peptide elicited T cells in mice tolerant to the longer peptide. We found no evidence in our studies for further truncation of exogenous peptides, although we observed preferential recognition of short peptides in a subset of type B T cells (4). Janeway and colleagues (29) interpret their results as indicating that T cells can distinguish between proteins synthesized within the cell and proteins that enter through endocytosis. In contrast, our view is that the same linear sequence can be presented in two conformational states dictated by the site of assembly in the APC: in a late processing vesicle, H2-DM imparts a fixed conformer; whereas without H2-DM loading of peptide in a recycling vesicle allows for a more flexible conformer (4). We also suggest that the report of Viret et al. (30) of positive selection mediated by a covalent complex of MHC and agonist peptide might be explained by similar conformation-dependent T cell reactivity.

The presence in the naive repertoire of type B T cells to self Ags has significant implications for their potential role in autoimmune responses. Several mechanisms have been identified that generate class II epitopes independently of intracellular processing. It is well known that neutrophils express ectoenzymes with proteolytic activity, and Potter and Harding (8) reported that neutrophils responding to acute inflammation can generate peptides that are regurgitated and presented on class II by professional APC; in addition, dendritic cells are reported to generate epitopes via extracellular proteolysis for presentation on both class I (9) and class II MHC (10). It was even suggested that circulating serum proteases can generate epitopes for presentation on class II, although this has only been observed in one study (31). We propose, therefore, that at sites of acute inflammation, catabolism of proteins from necrotic and/or apoptotic cells in the presence of these mechanisms could result in autologous peptides being presented by APC in a type B conformation; type B T cells reactive to these epitopes would then be primed and could induce an autoimmune response. The extent to which this occurs in vivo, and its role in autoimmune pathology, are important topics for further investigation.

	Acknowledgments

 
We gratefully acknowledge Shirley Petzold and Gina Filley for their technical assistance with multiple aspects of this study. We also thank Kevin Clark for synthesizing and purifying the A^k_37–53 peptide; Kathy Frederick for help in maintaining the mice used in these studies; Ilan Vidavsky for technical assistance with the mass spectrometric analysis of HPLC fractions; and Anish Suri, Osami Kanagawa, and other members of the laboratory for helpful discussion and critical reading of the manuscript.

	Footnotes

 
¹ This work was supported by grants from the National Institutes of Health. The mass spectrometry research was supported in part by the National Centers for Research Resources (Grant 2P41RR00954).

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

³ Abbreviations used in this paper: HEL, hen egg white lyzozyme; MEGA-8, octanoyl-N-methylglucamide; MEGA-9, nonanoyl-N-methylglucamide; MS, mass spectrometry; MS/MS, tandem MS; TFA, trifluoroacetic acid.

Received for publication November 27, 2002. Accepted for publication February 7, 2003.

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