1 Section of Immunobiology, Department of Ophthalmology, Ludwig-Maximilians-University, Mathildenstrasse 8, 80336 Munich, Germany
Correspondence to: G. Wildner; E-mail: Gerhild.Wildner{at}ak-i.med.uni-muenchen.de
Transmitting editor: T. Hünig
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Abstract |
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Keywords: autoimmunity, epitope, MHC presentation, oral tolerance, uveitis
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Introduction |
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For autoimmune uveitis, an inflammatory disease of the eye, we have described mimicry of a peptide from the ocular autoantigen S-Ag (PDSAg, amino acids 341354) and a peptide derived from the sequence of disease-associated HLA-B molecules (B27PD, amino acids 125138) (8). The pathogenic cells in uveitis are CD4+/ßTCR+ and MHC class II-restricted T cells; however, statistical associations with HLA class I molecules [B27 (9,10), B51 (11)] are well known. In our model, the HLA class I molecule serves as an autoantigen itself, being presented as a peptide on HLA class II and mimicking the retinal peptide. This offers an explanation for the role of HLA class I in human uveitis.
Lewis rats develop uveitis after immunization with the HLA peptide, although the conventional autoantigens for experimental uveitis in rats are retinal proteins such as S-Ag (12), interphotoreceptor retinoid-binding protein (IRBP) (13) or peptides thereof. Molecular mimicry of S-Ag- and HLA-derived peptide was demonstrated for pathogenic T cells and for T cells, which act as suppressor cells in oral tolerance in rat EAU (14). Thus, oral administration of the HLA peptide could ameliorate and prevent uveitis in rats that were immunized with the whole S-Ag, peptide PDSAg or IRBP (8). Lymphocytes from uveitis patients can proliferate in response to both peptides (8) and therefore we performed a successful therapeutic trial for uveitis patients with oral administration of the HLA peptide B27PD (15).
The 14mer peptides PDSAg and B27PD share only five identical or similar amino acids in discontinuous order. Both peptides have the same tolerogenic potency; however, the uveitogenicity of PDSAg drastically exceeds that of B27PD. The latter induces only mild disease (average score 0.5) in 85% of rats, whereas peptide PDSAg induces severe uveitis (average score
3) in almost 100% of rats after immunization. To elucidate the different properties of pathogenic and tolerogenic epitopes we analyzed C- and N-terminally truncated peptides as well as chimeric variants consisting of amino acids from both peptides. In vitro we investigated the ability of these peptides to stimulate PDSAg-specific T cells, the binding to MHC class II molecules was tested by inhibiting the presentation of PDSAg to a PDSAg-specific class II-restricted T cell line. In vivo we determined the uveitogenic and tolerogenic potential of variant peptides in Lewis rats.
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Methods |
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Immunization
Groups of three or four rats were immunized s.c. into both hind legs with a total volume of 200 µl emulsion containing 50 µg peptide (PDSAg: 20 µg) and complete Freunds adjuvant, fortified to a final concentration of 2.5 mg/ml with Mycobacterium tuberculosis strain H37RA (Difco/Becton Dickinson, Heidelberg, Germany).
Induction of oral tolerance
Groups of three or four rats were fed with peptides (200 µg of peptide in PBS, 34 times at 2-day intervals) and uveitis was induced by immunization with 20 µg PDSAg 2 days after the last oral tolerization. All experiments were repeated at least 2 times to confirm the results.
Grading of uveitis
Clinical examination was performed daily from day 10 post-immunization. For histological grading, eyes were snap frozen in Tissue Tec OCT compound (Paesel and Lorey, Frankfurt/Main, Germany) in methylbutane, chilled to 70°C. Cryo sections of rat eyes were stained for CD4 with antibody W3/25 (Biozol, Eching, Germany) and the APAAP (alkaline phosphataseanti-alkaline phosphatase) technique as published previously (8). Uveitis was graded histologically as described elsewhere (17). Briefly, a score of 0.5 describes the infiltration of a few cells in the retina and vitreous; the maximum score of 4 describes the total destruction of the retina. Values are given as mean ± SE.
Cell cultures
Draining inguinal, popliteal and para-aortal lymph nodes from immunized rats were collected and assayed as described (8). T cell lines were generated by alternating cycles of antigen stimulation with irradiated thymocytes (10 Gy) as antigen-presenting cells followed by expansion in culture medium supplemented with rat spleen conditioned medium (18). For proliferation assays, 2 x 104 PDSAg- or KerS-specific T cells were cultivated with 2 x 105 irradiated (10 Gy) thymocytes per well as antigen-presenting cells and 10 µg/ml peptide. Cultures were set up in triplicates and proliferation was determined by [3H]thymidine incorporation for the last 1216 h of a 3-day cell culture. Values are given as mean ± SE.
Determination of PDSAg-presenting MHC molecules by antibodies
PDSAg- or KerS-specific CD4+ T cell lines were cultured with irradiated thymocytes, the respective antigen peptides, and serial dilutions of MHC class II-specific antibodies Ox17 (RT1.D; Becton Dickinson Heidelberg, Germany) and Ox6 (RT1.B; Becton Dickinson) for 3 days. Proliferation was determined as described above.
Competitive inhibition of PDSAg binding to MHC class II by variant peptides
Irradiated (10 Gy) thymocytes (2 x 105/microwell) from 4- to 6-week-old naive rats were pre-cultured in serum-free medium with increasing concentrations of competitor peptides (dilutions from 2.5 to 75 µM) for 2 h at 37°C before addition of 1.5 µM PDSAg and the PDSAg-specific T cell line or KerS and KerS-specific T cell line respectively, and were further incubated as described above. MHC-binding was defined as reduced proliferation of the T cell line due to inhibition of PDSAg or KerS presentation. In parallel, proliferation of both T cell lines in response to the competitor peptides only was determined.
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Results |
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Proliferative response of PDSAg-specific as well as KerS-specific T cell lines and competition assays defining the binding of variant peptides to MHC class II were repeated several times. We tested different T cell lines and increasing concentrations of inhibitor peptides, as described in Methods. Since it is impossible to obtain T cell clones in the rat system, we had to use highly specific T cell lines. Despite this limitation, we obtained comparable, but slightly variable, results with PDSAg-specific (Fig. 2b and c) and oligoclonal KerS-specific T cell lines (Fig. 2j and l), and show representative experiments for proliferation assays and MHC binding competition, both from the same PDSAg- or KerS-specific cell line. We also tested various KerS-specific lines in independent experiments and found similar inhibition patterns, as with the PDSAg-specific lines.
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Truncated peptide PDSAgV11 did not efficiently stimulate PDSAg-specific T cell lines; however, in our assay this peptide was able to compete with PDSAg for MHC class II-binding. No binding of PDSAgV11 to RT1.B was observed in an in vitro binding assay with isolated RT1.B molecules (K. De Graaf, pers. commun.), suggesting that the competition assay is more sensitive.
Peptide B27PD and its N-terminally truncated variants (B27V4B27V6) bound to MHC class II, but did not stimulate proliferation of PDSAg-specific T cell lines (Fig. 2a and b). The chimeric peptide B-S consisting of the N-terminal part of B27PD and the C-terminal part of PDSAg was strongly stimulating the proliferation of PDSAg-specific T cell lines in vitro (Fig. 2e and f). This indicates that the major epitope for a PDSAg-specific TCR is located at the C-terminus of PDSAg. With the exception of B-S and Chi1(10), chimeric peptides did not bind to RT1.B in vitro (Fig. 2f). The octamer peptides PDSAgS, B27PDS, ConsS and ConsT bound strongly to RT1.B, but were not recognized by T cells in vitro and in vivo. Decreased proliferation caused by competitor peptides was not due to the toxicity of the peptides itself as demonstrated by co-incubation of high concentrations (75 µM) of variant peptides with PDSAg-specific T cells and PDSAg (data not shown). Despite the important role of the C-terminal amino acids of PDSAg, some truncated peptides were even more effective than their elongated form with respect to MHC-binding [PDSAgV11 versus PDSAgV12 (Fig. 2b), Chi1(10) versus Chi1(11) (Fig. 2f)].
The pattern of inhibition observed with truncated peptides and the KerS-specific line (Fig. 2j and l) was similar to that observed with the PDSAg-specific cells. The inhibition of proliferation of the KerS-specific line by N- and C-terminally truncated PDSAg-derived peptides was weaker than the inhibition of the PDSAg line, probably due to the fact that the KerS-specific T cells were more vigorously proliferating to KerS (SI = 82) than the PDSAg-specific cells to PDSAg (SI = 43). The assays were done at different time points with T cell lines obtained from rats of a different source and, in part, with different batches of peptides, which might explain some variations.
The 14mer peptide B27PD was less efficient in competing for MHC-binding with KerS than with PDSAg; however, N-terminal truncated peptides B27V4V6 (Fig. 2j) and the chimera with the C-terminus of B27PD, S-B (Fig. 2l), were highly competitive and efficiently inhibited (75%) the proliferation of KerS-specific T cells. This points to an important role of the C-terminal sequence of B27PD in binding to the MHC groove and competing for KerS. Peptide B-S, which was able to stimulate proliferation of PDSAg-specific T cells and thus efficient binding to RT1.B, did not compete for KerS-binding. The highly efficient blockade of the MHC groove by octamer peptides PDSAgS, B27PDS, ConsS and ConsT was also not observed with KerS-specific lines.
In vivo testing of variant peptides
In several independent experiments the peptides were tested for their uveitogenicity as well as their capacity to induce oral tolerance to PDSAg-mediated disease. Because of interexperimental variants in disease activity, the effectivity of oral tolerance induction was calculated for each experiment as relative intensity (percent severity) of uveitis in relation to the respective mock control (defined as 100%). Only variants of PDSAg with N-terminal truncations of up to 3 amino acids (PDSAgV2V4) or truncation of one C-terminal amino acid (PDSAgV13) retained the ability to induce uveitis and oral tolerance (Fig. 2c and d). Truncated peptide PDSAgV11 induced severe uveitis, but with a very low incidence (only 10% of immunized rats developed disease). Peptide B27PD and its N-terminally truncated variants induced oral tolerance; however, they were not or only weakly uveitogenic (B27PD and B27V5). Two chimeric peptides, S-1-B (N-terminus from PDSAg and C-terminus from B27PD) and Chi 2(10) (with the C-terminus similar to B27PD) did not induce uveitis, but were weakly tolerogenic (Fig. 2g and h). Peptides S-B and B-S were not effective in vivo, although B-S induced proliferation of T cells in vitro. The differences of effector T cell recognition versus tolerogenicity underline our previous data of T cells as effector cells in oral tolerance: they are CD4 and thus believed to recognize peptides in a different way than the effector cells, probably not presented by MHC class II (14). The four octamer peptides, PDSAgS, B27PDS, ConsS and ConsT, were not recognized by T cells in vivo, which is in concordance with their inability to stimulate T cell lines in vitro.
Potential MHC-binding motifs
We wanted to define the binding motifs of the peptides to RT1.Bl (Fig. 3) according to the anchor positions described by Reizis et al. (19). Amino acids at positions 4 (no negative charge) and 9 (negative charge preferred) are suggested to be the most important anchors, therefore the sequences from amino acids 5 to 13 of the 14mer peptides PDSAg and B27PD (LTSSEVATE and DLSSWTAAD respectively) should represent the optimal binding motif (Fig. 3a). This region is expressed in all truncated forms of PDSAg that still showed strong immunological activity or bound to MHC (PDSAgV2V5, PDSAgV13 and B27V4V5). Peptide PDSAg V5V13, which only consists of this core motif, bound to RT1.Bl, as shown by competition for PDSAg-binding in Fig. 2(b), but was not properly recognized by T cells (Fig. 2a). Peptide PDSAgV11 bound to RT1.B, although it did not have the proposed anchors at positions 4 and 9 (Fig. 2b). A potential motif in this peptide spans the first 9 amino acids of PDSAg (FLGELTSSE) and includes a negatively charged amino acid at position 9, but has an unfavored acidic side-chain at position 4 (Fig. 3b). Further suggested binding motifs of PDSAg covering amino acids 210 (LGELTSSEV) or 311 (GELTSSEVA) have a more favorable residue at position 4 (leucine or threonine), but lack the negative charge at position 9 (Fig. 3b).
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Peptide KerS has only three favored amino acids at anchor positions 1, 5 and 9 (Fig. 3a).
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Discussion |
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The binding competition assay revealed a minimal nonamer motif for RT1.Bl according to the definition of Reizis et al. (18) represented by peptide V5V13 of PDSAg (LTSSEVATE). This peptide was sufficient to inhibit PDSAg binding, but was still not able to stimulate the proliferation of a PDSAg-specific T cell line. This argues for the importance of amino acids flanking the core motif, recently described to be necessary for the interaction with the TCR (20). Truncating peptide PDSAg by 1 amino acid N-terminally (PDSAgV13) or C-terminally (PDSAgV2) resulted in peptides with reduced capability to stimulate PDSAg-specific T cell lines. Whereas truncation of up to 4 N-terminal amino acids (PDSAgV3V5) did not completely abrogate T cell proliferation, the peptide lacking 2 C-terminal amino acids (PDSAgV12) had lost its immunological activity, suggesting that the epitope for TCR binding must be located at the C-terminus of peptide PDSAg. This was confirmed by the chimeric peptide B-S, which is composed of the N-terminal part of peptide B27PD and the C-terminal part of PDSAg. B-S stimulated PDSAg-specific T cells, but less efficiently than PDSAg itself. However, it did not induce uveitis in vivo suggesting that it is either not binding well enough to MHC class II to compete with other peptides in vivo, or it is unable to activate naive, potentially pathogenic T cells due to insufficient avidity of the TCR. For different allelic MHC class II variants it has been found that encephalitogenicity in rat EAE correlates with the affinity of the myelin basic protein peptides for the restricting MHC class II molecules (21).
On the other hand, it has been reported for the mouse model of EAE that peptides binding to MHC with high affinity were not pathogenic (22), as well as pathogenic peptides that had only a very low affinity to MHC class II molecules (23). For most, but not all, of our tested peptides, we observed a correlation between binding to MHC class II and stimulation of T cell lines in vitro, and uveitogenicity and oral tolerogenicity in vivo. Except for peptide B-S, we found some other peptides with distinct functions in vitro and in vivo: the chimeric peptide Chi1(10), composed of single amino acids of both founder peptides and including a stretch of 4 amino acids that corresponds to the sequence of B27PD, showed good binding to MHC class II (RT1.Bl) without being recognized by PDSAg-specific T cells. In contrast, peptide Chi2(10), inducing uveitis in one of 10 immunized rats, was unable to stimulate T cell lines in vitro. This peptide lacks postulated favored amino acids at the most important anchor positions 4 and 9. However, positions 2, 5 and 8, which are thought to be recognized by the TCR, are identical to those of peptide PDSAg [Fig. 3c (Chi2(10)-a)]. This motif has tryptophan (W) at position 4, where, according to Reizis et al. (18), phenylalanine (F) is preferred, indicating that tryptophan might function as an anchor as well. The lack of an aliphatic or negatively charged anchor at position 9 might explain the weak MHC-binding, resulting in very low pathogenicity, i.e. in only one of 10 immunized rats. The latter might be due to a rare TCR with strong avidity that could compensate for the weak MHC-binding of the antigen peptide. Nevertheless, we could not exclude the presentation of peptide Chi2(10) by RT1.D, for which the binding motifs are still unknown. Chi2(10) might stimulate a non-dominant T cell response restricted for RT1.D.
Surprisingly, we could observe an improvement of function of some peptides by further truncation of single amino acids [Chi1(11) versus Chi1(10) and PDSAgV12 versus PDSAgV11], although we expected that removal of amino acids would rather destroy than rescue epitopes. We suspect that flanking amino acids might stabilize the elongated structure of the respective motifs and enable the peptides to fit better into the groove of RT1.B. Chi1(10), in contrast to Chi1(11), bound to MHC, but was not recognized by T cells in vitro nor in vivo, indicating that it has respective MHC-binding residues without the suitable amino acids recognized by the TCR. Peptides PDSAgV12 and PDSAgV11 showed even more striking differences. The 12mer PDSAgV12 was poorly MHC-binding, and consequently did not stimulate T cells in vivo and in vitro. The 11mer PDSAgV11 was moderately binding to RT1.B, therefore weakly stimulating PDSAg-specific T cells, and poorly uveitogenic in vivo. Only 10% of immunized rats developed uveitis, however, it was a severe disease, indicating an all-or-nothing-phenomenon: when a pathogenic immune response was initiated, it was highly effective. T cells recognizing PDSAgV11 might as well utilize a different TCR than those activated by PDSAg. Primary in vitro responses of lymph node cells from rats immunized with PDSAgV11 frequently exhibit a heteroclitic proliferative response to PDSAg, whereas further re-stimulation in vitro with PDSAgV11 selected for exclusive PDSAgV11 specificity (data not shown).
Binding of variant peptides to Lewis rat MHC class II RT1.Bl was determined by in vivo competition for the binding groove for either peptide PDSAg or a control peptide, KerS, and the inhibition of proliferation of respective T cell lines. This system is very sensitive, but also affected by variables like the fact that T cell lines are inhomogeneous and composed of variable subpopulations with different TCR avidities which might recognize different contact residues on a presented peptide epitope (24). Furthermore, proliferative responses of some T cells recognizing peptide presented by RT1.Dl might quench the effect of blocking RT1.B. Several factors can influence the binding of peptides to MHC class II in vitro: the availability of free MHC grooves, competition with other peptides present in the culture medium, the pH of the culture, which can affect the hydrogenation and charge of the peptides amino acid side-chains, and finally further proteolytic degradation of the peptides. With respect to TCR-binding, little is known about the factors that could modify T cell recognition. Nevertheless, our experiments with two different T cell lines of completely different peptide specificities did not reveal contradictory results.
Our findings question the general opinion that peptides of any length exceeding the minimal binding motif can be used to detect naturally occurring T cell responses. Long peptides including more than one MHC-binding motif, several T cell epitopes or even epitopes with immunoregulatory properties are unpredictable in their function, which was previously shown for a region from the sequence of HLA-B27 (amino acids 6084). A peptide corresponding to the N-terminal part of this region (amino acids 6072) can induce proliferation of peripheral blood lymphocytes from patients with ankylosing spondylitis (25) and is arthritogenic in Lewis rats (27). The C-terminal part includes an immunoregulatory sequence (26) which can positively influence CD4+-mediated experimental autoimmune diseases such as EAU and has a negative effect on oral tolerance induction (28).
In conclusion, we could not clearly define MHC-binding motifs or amino acids responsible for recognition by uveitogenic ß TCR+ cells. Amino acids flanking the core epitope or certain combinations of amino acids can obviously influence the structure of a peptide in an unpredictable way (19,29), and therefore inhibit or favor MHC presentation and/or recognition by T cells. With respect to pathogenicity or tolerogenicity, none of the variant peptides had a higher immunological activity than the original 14mer peptides PDSAg or B27PD. For oral tolerance induction, which is based on
T cell activation in our rat model, no prediction for suitable peptide sequences could be made, since neither the presenting molecule nor the way of presentation of peptide antigens to these
TCR is known so far. Furthermore, our data point out the differences between in vitro and in vivo responses of T cells. In vitro, specific T cell lines can still be able to react to peptides that highly differ from their original antigen, whereas in vivo such putative cross-reactive peptides are unable to induce disease or tolerance. In the human situation, where only in vitro assays are available to test potentially pathogenic or tolerogenic epitopes, the results from cell cultures with T cell clones might be misleading and not representative of the in vivo situation.
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Acknowledgements |
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Abbreviations |
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IRBPinterphotoreceptor retinoid binding protein
S-Agretinal S-antigen
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References |
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