Correspondence to: Navreet K. Nanda, Department of Microbiology and Immunology, 835 South Wolcott Ave., Chicago, IL 60612-7344. Tel:312-413-1446 Fax:312-996-6415 E-mail:navreetn{at}uic.edu.
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Abstract |
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The ability of the immune system to focus T cell responses against a select number of potential epitopes of a complex antigen is termed immunodominance. Epitopes that trigger potent T cell activation, after in vivo priming, are classified as immunodominant. By contrast, determinants that fail to elicit any response are called cryptic. DM, a major histocompatibility complex (MHC) heterodimer, plays a pivotal role in the presentation of MHC class IIrestricted epitopes by catalyzing the exchange of class IIassociated invariant chain peptide with the antigen-derived peptides within the MHC class II binding groove. Using L cells transfected with genes for MHC class II, invariant chain, and DM, we have studied the contribution of DM in the presentation of two cryptic (peptide 1125 and peptide 2035) and one dominant (peptide 106116) epitope of hen egg white lysozyme (HEL). Cells lacking DM heterodimers efficiently display the determinants HEL 1125 and HEL 2035 to T cells. Strikingly, however, cells expressing DM are severely compromised in their ability to present the cryptic HEL 1125/Ad and 2035/Ad epitopes. DM-mediated antagonism of HEL 1125/Ad and 2035/Ad presentation could thus be central to 1125/Ad and 2035/Ad being cryptic epitopes in the HEL system. Interestingly, the display of the immunodominant epitope of HEL, 106116/Ed, and of a dominant epitope of sperm whale myoglobin (SWM), 102118/Ad, is entirely dependent on the expression of DM. Thus, cells lacking DM molecules are unable to efficiently express HEL 106116/Ed and SWM 102118/Ad determinants. We conclude that the DM heterodimers direct the immunodominant and cryptic fate of antigenic epitopes in vivo.
Key Words: antigen determinant, antigen processing, H-2M, immunodominance, major histocompatibility complex class II
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Introduction |
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The T cell response in the H-2d haplotype mice immunized with hen egg white lysozyme (HEL)1 is almost exclusively directed towards a single immunodominant epitope of HEL, peptide 106116, bound to Ed MHC molecules (106116/Ed; references 14). Immunization with HEL does not elicit T cells specific for HEL 1125 or HEL 2035 bound to Ad MHC (2) (3) (4). However, HEL 1125/Ad and to some degree, HEL 2035/Adspecific T cells, can be activated by immunization with the peptides 1125 and 2035, respectively (2). Peptides 1125 and 2035, which require no further processing in order to be efficiently presented in the groove of the Ad molecules, thus encode cryptic determinants in the BALB/c (H-2d) mice. Immunodominance is a distinctive and classic characteristic of most T cell immune responses (1) (5) (6). Nevertheless, the molecular mechanisms dictating the immunodominant and cryptic fate of T cell epitopes remain unknown.
Processing and presentation of exogenous protein antigens taken up via the endocytic pathway involve the trafficking of MHC class II molecules from the constitutive secretory pathway to the endocytic compartment (containing the antigen-derived peptides), en route to the cell surface (7) (8). This task is accomplished by a glycoprotein called invariant chain (Ii) that enhances the transport of MHC class II chains to the endocytic compartment (7) (8). In addition, a fragment of Ii, called class IIassociated invariant chain peptide (CLIP), occupies the peptide-binding groove of MHC class II molecules (9) until DM, a nonclassical MHC class II heterodimer, induces its release and replacement with peptides derived from foreign or endogenous antigens (7) (10) (11) (12). Due to a lysosomal targeting signal encoded within the ß chain of DM heterodimers, DM is localized to endocytic MHC class II compartments (13) where it has been shown to transiently bind MHC class II molecules (12) (13). DM, in addition to catalyzing the release of CLIP, has been shown to influence the binding of several non-CLIP peptides to the MHC class II molecules (11) (12) (14) (15) (16) (17). DM mutant mice and APC lines have been used to show that DM is required for presentation of certain foreign antigens (12). We have shown previously that expression of DM can increase, diminish, or have a neutral effect on the presentation of different intracellular self proteins (14) (18) (19). But the biological consequence of the DM-mediated modification of the repertoire of peptides displayed by the MHC molecules on expression of the epitope specificity of T cell immune responsiveness to a foreign or self antigen remains unexplored. We present evidence here that DM directs the cryptic and immunodominant fate of antigen determinants.
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Materials and Methods |
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APCs.
L cells were initially transfected with genes encoding Ad alone, and a positive clone was isolated and then supertransfected with genes encoding genomic Ii (Ii or gIi) or Ii and murine DM. Derivation and characterization of these cells have been described previously (14) (18). Ad-positive cells expressing either no cofactor, Ii alone, or Ii and H-2M (or murine DM, hereafter referred to as DM) were then additionally transfected with genes encoding Ed, and positive cells were obtained by preparative sorting using the E-specific Ab 14-4-4S and magnetic beads. Sorted and other transfected cells were grown in selection media with appropriate drugs: Ad and Ad-Ii L cells were cultured in the presence of G418 (200 µg/ml; GIBCO BRL), Ad-Ii+DM, in G418 and blasticidin (100 µg/ml; Invitrogen), AdEd and AdEd-Ii, in G418 and HAT (GIBCO BRL), and finally AdEd-Ii+DM, in HAT, G418, and blasticidin. Cells were transferred into normal media (14) containing no drugs 2448 h before their use in T cell assays.
Flow Cytometry.
mAbs reacting with Ad (MKD6) or Ed (14-44S) were obtained from the American Type Culture Collection and used as culture supernatants. Cell surface expression of MHC class II molecules was evaluated by flow cytometry as described (19). In brief, cells were incubated successively with mAbs, followed by secondary step FITC-conjugated goat antimouse Igs (IgA, IgG, and IgM, and FITC-conjugated goat antimouse; Sigma-Aldrich), and were then analyzed on a FACScanTM cytofluorimeter (Becton Dickinson). Background fluorescence was determined by incubating cells with media alone or an irrelevant mAb, and then with FITC-conjugated goat antimouse.
Western Blots.
Cells were harvested from culture by mild trypsin treatment, washed extensively, and then counted. 8 x 106 cells of each type (cells expressing AdEd alone, AdEd Ii, or AdEd Ii+DM) were solubilized in 1.0 ml 0.5% Triton X-100 (Sigma-Aldrich) with protease inhibitors for 45 min on ice as described previously (19). Nuclei and other insoluble debris were pelleted by centrifugation and the supernatant was transferred to a fresh tube. An equivalent aliquot of each cell lysate, equal to 8 x 105 cell equivalents, was then adjusted to 2.0% SDS, 62 mM Tris, pH 6.8, and 10% glycerol, boiled for 3 min, and then fractionated by SDS-10% PAGE. After electrophoresis, proteins were transferred to nitrocellulose, then processed for Western blotting as described previously (19). Blots were first probed and developed using nonspecific rat mAb or normal rabbit serum Abs, and then probed specifically for Ii and DM protein expression, as indicated in the legend to Fig 1 b. Ii was detected by probing with the rat mAb In-1 (20), and DMß was detected by probing with a rabbit antisera raised against a synthetic peptide corresponding to the COOH-terminal tail of the mouse DMß chain, conjugated to KLH. Binding of primary Abs to the blot was detected with horseradish peroxidasecoupled secondary goat Abs specific for either rat Ig, purchased from KPL Laboratories, or rabbit Ig, purchased from Life Technologies. Western blots were processed for chemiluminescence and autoradiography as described previously (18).
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Metabolic Labeling, Immunoprecipitation, and SDS-Gel Analyses.
For analysis of Ii, and DM expression within the APC, 0.5 x 106 cells were plated in two 100-mm tissue culture dishes for 24 h. The adherent cells were then prelabeled for overnight incubation in MEM deficient in leucine with added [3H]leucine obtained from NEN Life Science Products at 25 µCi/ml. Ii, DM, and MHC class I molecules were isolated from 1% Triton detergent lysates by immunoprecipitation with either rabbit antisera specific for the cytosolic tail of DMß, the rat mAb In-1 (20) reactive with the cytosolic tail of Ii, or the class Ispecific mAb 16.1.11N (21). Immunoprecipitated proteins were fractionated by SDS-10% PAGE and gels were stained with Coomassie brilliant blue, then destained with 30% methanol/10% acetic acid. Stained and destained gels were then washed with water, treated with Flouro-Hance (Research Products International), dried, and exposed to film at -80°C.
Antigens and Peptides.
HEL was either purified as described previously (22) or was obtained from Sigma-Aldrich as a purified preparation. Both preparations of HEL gave analogous results. Sperm whale myoglobin (SWM) was obtained form Accurate Chemicals and Scientific Corp. Peptides HEL 1125, HEL 2035, HEL 106116, and SWM 102118 were synthesized and purified by Macromolecular Resources.
T Cell Hybrids.
The cloned T cell hybridomas ISG-9-49, ISG-9-69, and 17.B.8.1 were obtained from continuously growing HEL-specific T cell lines derived from LN cells of HEL-primed B10.GD mice as described previously (22) (23). The cloned T hybrids D2-4-18 and D2-4-8 were obtained from HEL-specific T cell lines derived from LN cells of B10.D2 mice immunized with HEL (22) (23). In brief, T cell blasts from T cell lines were fused with the TCR-ß- variant of BW5147 as a fusion partner and cloned by limiting dilution in the presence of HEL. HEL-reactive hybrids were further analyzed for recognition of peptides. The hybrid C7-R14, specific for SWM 102118 (24) (25), was a gift from Dr. Jay Berzofsky (National Institutes of Health, Bethesda, MD).
Assay of T Cell Responses.
T hybridoma cells (105) were cultured with 15 x 104 of either Ad or AdEd APCs, Ad-Ii or AdEd-Ii APCs, or Ad-Ii+DM or AdEd-Ii+DM APCs with different concentrations of HEL, SWM, or peptide (22) (23), as indicated in the figure legends (Fig 2 Fig 3 Fig 4 Fig 5). Fixed APCs, where indicated, were treated with 1% formaldehyde in PBS for 12 min, then washed extensively before use. All cultures were done in triplicate. The culture supernatants collected 24 h later were assayed for IL-2 by ELISA as described previously (26). The substrate used was O-phelylenediamine dihydrochloride (Sigma-Aldrich). Absorption at 490 nm was measured using a microplate reader (Molecular Devices).
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Results |
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Cloned populations of L cells deficient in MHC class II, Ii, and DM heterodimers (DM) were transfected with genes encoding Ad MHC molecules followed by either Ii or Ii and DM (Ii+DM), as described previously (14) (19). The Ad, Ad-Ii, and Ad-Ii+DM L cell lines were further transfected with genes for Ed MHC molecules. Fig 1 shows the characterization of L cells transfected with Ad and/or AdEd MHC molecules, together with genes for either Ii only or with Ii and the DM heterodimers (Ii+DM). It is clear that the expression of MHC class II is optimal in cells expressing both the Ii and DM (Fig 1 a), as has been observed previously (14) (27). Protein expression of Ii and DM are shown in Fig 1b and Fig c, confirming that the original cell lines used for transfection lack the constitutive expression of Ii and DM glycoproteins, and that protein expression of Ii and DM was readily detected in the appropriate cells.
DM Extinguishes Presentation of the Cryptic Epitopes of HEL
Presentation of Cryptic Epitope HEL 1125.
Fig 2 shows the display of the cryptic HEL determinant 1125 by AdEd L cells used as APCs at different concentrations of HEL. The determinant display was examined by analyzing activation of two different cloned T cell hybridomas, ISG9-49 and ISG-9-69, that recognize HEL 1125/Ad and were derived as described previously ((22); see Materials and Methods). It is clear that the AdEd-Ii APCs, lacking expression of DM heterodimers, can efficiently process HEL and present the cryptic epitope 1125. The display of 1125 epitope by the AdEd-Ii APCs can be observed at even low (0.22 µM) concentrations of HEL. Most strikingly, the presentation of 1125 was nearly extinguished, even at >30-µM concentrations of HEL, when presented by APCs that express DM in addition to Ii (Fig 2). The peptide 1125, which requires no further processing, is equivalently presented by APCs with Ii alone and with Ii+DM (insets, Fig 2, a and b), despite the fact that APCs that express Ii alone display lower levels of MHC class II molecules than Ii+DM APCs (Fig 1 a). To further confirm DM-mediated antagonism of presentation of the epitope 1125, we used an additional set of APCs, the Ad APCs, expressing either Ii alone or Ii+DM. Once again, the presentation of the cryptic epitope 1125 was dramatically diminished when DM molecules were expressed by the APCs. Ad-Ii and AdEd-Ii APCs lacking DM molecules were clearly very active at presenting this cryptic epitope in the absence of DM (Fig 2 and Fig 3). In addition, we used fixed APCs for presentation of the cryptic epitope 1125 to address the question whether the Ad-Ii APCs were able to effectively present the cryptic HEL 1125 simply by virtue of being more efficient in taking up either denatured HEL or any other contaminants of HEL. As is evident in Fig 3b and Fig d, the fixed Ad-Ii or Ad-Ii+DM APCs are unable to present HEL even at the highest concentration, 100 µM, of HEL. The competence of the Ad-Ii APCs to present the cryptic epitope is thus solely due to proficient intracellular processing of HEL. Results obtained using a third 1125/Adspecific cloned T hybrid, GD-5-42, additionally validated the DM-mediated antagonism of presentation of 1125/Ad, even at high concentrations (25100 µM) of HEL (data not shown).
Presentation of Cryptic Epitope HEL 2035. Fig 4 shows the display of an additional cryptic epitope of HEL, 2035/Ad, by Ad APCs. Interestingly, presentation of this second cryptic epitope of HEL was also antagonized by the expression of DM within APCs. Once again, Ad-Ii APCs were able to present this epitope very efficiently to 2035specific T cells, 17B.8.1. However, Ad-Ii+DM APCs were severely compromised in their ability to display this epitope. The fixed Ad-Ii or Ad-Ii+DM APCs, as shown for the 1125/Ad cryptic epitope, were unable to present this epitope from HEL at even the highest concentration of HEL tested (Fig 4 b), thus ruling out the possibility that the expression of the epitope 2035/Ad was a result of efficient uptake of denatured HEL.
DM Stimulates Presentation of the Immunodominant Determinants of HEL and SWM. We then examined the display of the immunodominant determinant of HEL, 106116/Ed, by AdEd APCs using the cloned T cell hybrids D2-4-18 and D2-4-8. Fig 5 shows that in sharp contrast to our observations for the HEL 1125 and 2035 determinants (Fig 2), AdEd-Ii APCs, lacking DM molecules, were very poor at displaying this immunodominant epitope (Fig 5), whereas cells additionally expressing DM showed efficient presentation. The half-maximal response of the T cell hybrid D2-4-18 was obtained at 2 µM HEL using AdEd-Ii+DM APCs. However, the activation of this T hybrid essentially remained at the baseline levels at concentrations of up to 33.3 µM HEL, using AdEd-Ii APCs lacking DM. Optimal processing and display of this immunodominant determinant is thus dependent on the expression of DM molecules. Furthermore, these results attest that the AdEd-Ii+DM APCs are functionally competent and the abrogation of presentation of the cryptic 1125 determinant in the presence of DM was not due to some non-DMrelated factor expressed by the Ii+DM APCs. The data presented in the insets (Fig 5, a and b) show that the AdEd-Ii and AdEd-Ii+DM APCs were able to stimulate the 106116/Ed T cells equivalently when the peptide HEL 106116, which requires no further processing (data not shown), was used. These results indicate the lack of any intrinsic deficiency in AdEd-Ii APCs to interact with 106116/Edspecific T cells.
To pursue the argument that one function of the DM molecules is to play a role in the manifestation of immune dominance upon immunization with protein antigens, we examined the effect of DM on the presentation of a dominant determinant of SWM (23). Fig 6 shows that just like the immunodominant epitope 106116/Ed of HEL, the optimal display of the immunodominant 102118/Ad determinant of SWM (23) (24) by Ad APCs was dependent on expression of DM in these APCs. The half-maximal response of SWM 102118specific C7-R14 T cell hybrids stimulated by the Ad-Ii+DM APCs was induced at only 1 µM SWM, while a similar response by the Ad-Ii APCs was induced at 60 µM SWM. Again, the results in Fig 6 not only support the conclusion that the immunodominant fate of T cell determinants is dependent on the expression of DM, but they also affirm that the failure of our set of Ad-Ii+DM APCs (Fig 3 and Fig 4) to present the cryptic epitope 1125 is unrelated to any non-DMrelated attribute of these cells.
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Discussion |
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The focusing of T cell responses directed to protein antigens to only a limited number of potential T cell epitopes is a classical feature of antigen-specific immune responses (1) (5) (6). The evolutionary development of this basic principle appears to be to accomplish (a) avoidance of activation of an army of T cells unnecessary for a successful attack on a pathogen; and (b) construction of an effective regulatory T cell system directed towards a small subset of T cells (28) specific for the immunodominant epitopes. T cell responses directed towards complex pathogens such as Leishmania have been shown to be, astonishingly, focused on a single immunodominant epitope (29) (30). A variety of concepts including determinant capture, nature of APCs, proteolytic enzymes in distinct intracellular microenvironments, lymphokines, and general antigen processing have been proposed to explain this classic feature of the immune system (1) (3) (31) (32) (33). The molecular mechanisms to explain these hypotheses still remain undetermined. We now show that DM, an MHC-encoded heterodimer, previously shown by us and others (14) (16) (17) to influence the repertoire of self-peptides loaded onto MHC class II molecules, may well be the guide that directs the antigen-derived peptides to have either an immunodominant fate or a cryptic fate. The two immunodominant determinants (HEL 106116/Ed and SWM 102118/Ad) examined by us are dependent on DM for presentation (Fig 5 and Fig 6). A third, the Leishmania-derived, immunodominant determinant Leishmania homologue of receptors for activated C-kinase (LACK [29, 30]) 161173/Ad, also requires the expression of DM molecules in order to be displayed on the surface of APCs (data not shown). In the case of HEL (and perhaps also of other protein antigens), DM determines the immunodominant fate of epitopes by strongly augmenting the presentation of the dominant determinant, 106116/Ed, on APCs, while simultaneously extinguishing the expression of the two cryptic epitopes studied in this report, 1125/Ad and 2035/Ad. DM-mediated antagonism was also observed for the display of 7496, a minor cryptic epitope (4) (23) of HEL (our preliminary observations, not shown). The results reported here also indicate that the cryptic fate of the 1125/Ad epitope is clearly not due to "determinant capture" (1). The concept of determinant capture predicts that during the creation of the dominant 106116/Ed determinant (1), the denatured HEL molecule would bind to Ed MHC, and the portion of HEL outside the binding groove (including the 1125 region) would get digested by proteolytic enzymes (1) (31). This outcome of the determinant capture is expected to be independent of expression of Ii and DM within APCs. Since the AdEd-Ii APCs are able to process and present this epitope remarkably efficiently (Fig 2), it is clear that the Ed MHC molecules, while "capturing" the dominant epitope HEL 106116/Ed, are not responsible for destroying the 1125 epitope. Instead, it is the expression, or lack of DM molecules in APCs, that determines the presentation of the cryptic epitope.
Our results are intriguing to consider in light of an earlier report that the dendritic cells, but not B cells, focus T cell responses against the immunodominant peptide of HEL and are unable to present the peptide HEL 731 (34). One possible explanation for this differential determinant display could be the cell typespecific expression within B cells, but not dendritic cells, of DO (35), an additional MHC class II heterodimer that has been shown to inhibit the function of DM molecules (35) (36). It has been shown that DM specifically induces the release of the MHC-bound peptides that display a lower binding stability, while favoring the binding of peptides that show a higher stability of binding MHC class II molecules (17). In view of the optimal DM function and expression in the acidic endosomal or lysosomal compartments (11) (16) (17), we examined MHC binding and display of these peptides using fixed APCs (37) at a variety of pH. We found that at a pH between 4 and 5, a 10-fold lower concentration of the immunodominant peptide (106116/Ed) was able to result in half-maximal stimulation compared with the stimulatory requirements for the cryptic peptide (1125/Ad). However, at pH 7.0, equivalent concentrations of both peptides were needed for half maximal stimulation (data not shown). These observations could be the result of distinct pH optima of peptide binding to Ed (pH 4.5) and Ad molecules (
pH 5.5), differential off-rates for the two peptides at the lower pH (38), or effects of DM expressed on the surface of cells, as has been shown in a recent report (39).
It has been proposed that the DM-induced editing of the repertoire of MHC class IIbound peptides could be a function of differential off-rates of the peptideMHC binding, leading to differences in the stability of the peptideMHC complexes (17) (40). In addition, it can be envisaged that DM would selectively enhance the peptide binding within the MHCpeptide complexes that show optimal binding at lower pH, leading to the immunodominance of such epitopes. In this context, it is interesting that the only immunodominant epitope in both the HEL/H-2d and cytochrome c/H-2k systems (41), and one of two dominant epitopes in the SWM/H-2d system (24), are restricted by I-E molecules (the latter with lower pH optima than the I-A molecules). The precise features that contribute to the DM-mediated effects on the differential display of peptides as seen in the current report, including the relative stability of the 1125/Ad and 106116/Ed complexes using purified MHC molecules, which is yet undetermined, are avenues of future investigations.
In summary, we propose that the cryptic and immunodominant fate of antigenic epitopes is a consequence of the ability of DM to skew the repertoire of peptides displayed by the MHC class II molecules. Our results that expression of DM within APCs is able to dictate the fate of two cryptic and three dominant epitopes testify that the DM-mediated decree of the nature of an antigenic epitope could be a general phenomenon. This function of DM may have evolved in parallel with the "immunodominance" attribute of the immune system as a mechanism to trigger only selective T cell responses essential to fight a pathogen. It is now becoming clear that one is tolerant to only the dominant determinants of self-proteins and that the T cell repertoire to the cryptic determinants remains intact in the host (5) (42) (43). It is the T cell repertoire to the cryptic self-epitopes that becomes activated in autoimmune disease (5) (42) (43). Any variation in the levels or activity of DM heterodimers thus may have consequences for autoimmunity as well as response to pathogens.
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Footnotes |
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1 Abbreviations used in this paper: CLIP, class IIassociated invariant chain peptide; HEL, hen egg white lysozyme; Ii, invariant chain; SWM, sperm whale myoglobin.
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Acknowledgements |
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We thank Georgia Angelakos, Frederick Krafcik, and Nasa Valentine for excellent technical assistance, and John Katz for assistance in the analyses of the L cell transfectants. We are grateful to Dr. J. Berzofsky for providing us with the SWM-specific T cell hybrids, and Dr. E. Sercarz for purified HEL. We also thank Drs. E. Sercarz and S. Scheider for providing us with recloned, recharacterized 17B.8.1 T cell hybrid.
This work was supported in part by National Science Foundation grant MCB 9808841 (to N.K. Nanda), and by National Institutes of Health grant PO DK49799 and Juvenile Diabetes Foundation International (to A.J. Sant).
Submitted: 22 November 1999
Revised: 9 June 2000
Accepted: 28 June 2000
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References |
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