Steven P. Fling

Peptide and protein molecular weight determination by electrophoresis using a high-molarity tris buffer system without urea☆

Various buffer systems were examined for their ability to resolve and provide molecular weight determinations of proteins and peptides over a wide size range using electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. Sharp bands and high resolution were achieved in the 1300 to 100,000 molecular weight range using a modified Laemmli discontinuous buffer system with high concentrations of Tris in the resolving gel (0.75 M) and in the running buffer (0.05 M). Linear gradient gels (8 to 25% acrylamide) were tested with and without varying concentrations of urea and/or glycerol and/or sucrose. At this high molarity of Tris, the inclusion of urea, glycerol, or sucrose proved unnecessary for successful peptide electrophoresis. Gels run without these reagents showed superior resolution throughout the entire molecular weight range when run with Tris at 0.75 and 0.05 M, respectively, obviating the need for urea or other additives as used in other systems. A single gel is thus able to resolve an entire range from large proteins to small peptides.

Identification of T cell recognition sites in S-antigen: Dissociation of proliferative and pathogenic sites

Experimental autoimmune uveoretinitis (EAU) is a predominantly CD4+ T cell-mediated autoimmune inflammatory disease of the retina and uveal tract of the eye and the pineal gland. S-antigen, a protein found in retinal photoreceptor cells and pinealocytes, is a potent agent for the induction of EAU in susceptible species and strains. In order to identify the T cell recognition sites of S-antigen responsible for its uveitogenicity and proliferative responses, cyanogen bromide (CB) fragments as well as synthetic peptides were used to test the proliferative responses of two uveitogenic T cell lines, R9 and R17, prepared against native bovine and human S-antigen, respectively. Two nonoverlapping synthetic peptides which are known to actively induce EAU, amino acid residues 286-297 and 303-314 of the bovine sequence, were unable to induce proliferative responses in either S-antigen-specific T cell line. However, both of these sites were adjacent to synthetic peptides, residues 273-292 and 317-328, respectively, which were unable to actively induce EAU, but elicited strong proliferative responses from T cell lines raised to bovine and human S-antigen. Repeated in vitro selection of the R9 T cell line with a synthetic peptide containing one of these proliferative sites, residues 317-328, gave rise to a transiently uveitogenic T cell line. Several species-specific T cell epitopes were identified, but none of these were found to be involved in a uveitogenic response. Our results indicate that spatially separated and distinct T cell epitopes are present in S-antigen which are responsible for the active induction of EAU, lymphocyte proliferation, and the ability to adoptively transfer EAU.

Conserved T cell receptor V gene usage by uveitogenic T cells

Retinal S-antigen is widely used to study the LEW rat model of experimental autoimmune uveoretinitis (EAU). In this report, we have examined the T cell receptor V gene usage of several T cell lines recognizing either pathogenic or nonpathogenic sites on S-antigen to determine whether the V alpha 510 and V beta 510 rat homologues of the murine V alpha 2 and V beta 8 families, respectively, are used by uveitogenic T cells. Using cDNA probes for a LEW rat T cell receptor specific for the encephalitogenic determinant of myelin basic protein, we have found that in the retinal S-antigen/EAU model for autoimmune disease, pathogenicity correlates with usage of those rat V genes. Thus, all of the pathogenic lines were found to express T cell receptors of the V beta 510 and V alpha 510 families; conversely, V beta 510 usage was not detected in any of the nonpathogenic lines. Usage of these V regions has been associated with pathogenicity in the murine and rat models of experimental autoimmune encephalomyelitis, and now with S-antigen-induced EAU.

Characterization of immunologically active cyanogen bromide peptide fragments of bovine and human retinal S-antigen *

Peptides which account for most, if not all, of the cyanogen bromide (CNBr)-generated peptide fragments of bovine retinal S-antigen have been identified and examined for their immunoreactivity with antisera raised to bovine and human S-antigen and with immune lymphocytes further selected twice in vitro with either bovine or human S-antigen. Amino-acid sequencing of a large fragment of S-antigen missing a small N-terminal peptide revealed the location of three overlapping CNBr peptides near the N-terminus. Amino-acid sequencing of several other CNBr peptides has allowed their position in a partial DNA-predicted sequence of the carboxy terminal half of the antigen to be determined. The total CNBr digest of human S-antigen was also prepared and compared with the fragments of the bovine antigen. Sera from rats immunized with bovine or human S-antigen were similar in their specificity in the enzyme-linked immunosorbent assay (ELISA) for purified bovine peptides except for the CB21 peptide which was not significantly bound by anti-human S-antigen sera. All of the other bovine peptides recognized by anti-bovine S-antigen sera were also bound by antibodies in the sera raised to the human antigen. The CNBr peptides of human and bovine S-antigen were extracted from gel slices and also assayed in the ELISA. Peptides of bovine S-antigen purified by HPLC were tested for their ability to stimulate an in vitro proliferative response in lymphocytes from Lewis rats immunized with either bovine or human S-antigen. Only quantitative differences in the proliferative response to human vs. bovine S-antigen and CNBr peptides were found. Methodology for the purification and analysis of the peptides is presented as well as the properties of the peptides.

A new perspective of S-antigen from immunochemical analysis

Elucidation of the amino acid sequences of retinal S-antigens from several species has allowed the fine dissection of T cell and antibody epitopes using synthetic peptides. S-antigen, isolated from retinal rod photoreceptor cells, elicits experimental autoimmune uveoretinitis (EAU), a predominantly CD4+ T-cell mediated autoimmune disease of the retina and uveal tract of the eye and pineal gland. Three uveitogenic T cell lines, R9, R17 and R208, prepared against native bovine S-antigen, human S-antigen and cyanogen bromide peptide CB123, respectively, were used to identify the T cell recognition sites responsible for uveitogenic and proliferative responses. T cell epitopes were found to be clustered into 6 regions, some of which were species-specific. The two synthetic peptides known to actively induce EAU, residues 286-297 and 303-314 of bovine S-antigen, were unable to induce significant proliferative responses in any of the three T cell lines. However, both of these sites were adjacent to synthetic peptides, residues 273-292 and 317-328, respectively, which were unable to actively induce EAU, but elicited proliferative responses from the T cell lines. We also report the presence of a new pathogenic site, also associated with an adjacent proliferative site, together in residues 343-362 of bovine S-Ag. Our results indicate that spatially separate and distinct T cell epitopes are present in S-antigen which are responsible for the active induction of EAU, lymphocyte proliferation, and adoptive transfer of EAU.

Multiple, autoreactive TCR Vβ genes utilized in response to a small pathogenic peptide of an autoantigen in EAU☆

The restricted usage of particular T cell receptor beta chain genes in autoimmune disease was studied in LEW rats using T cell hybridomas specific for an immunodominant sequence of bovine retinal S-Ag, which induces experimental autoimmune uveoretinitis. T cell hybridomas from a pathogenic T cell line, R858, specific for residues 273-289 of bovine retinal S-Ag were analyzed in order to determine the contribution of their TCR V beta to self specificity as determined by recognition of the pathogenic epitope represented in the autologous rat S-Ag sequence. Six different, functional TCR rearrangements were expressed by the panel of hybridomas, including two distinct V beta 8.2 rearrangements and functional V beta 10, V beta 14, V beta 19 rearrangements, and an unidentified V beta gene. All hybridomas were Ag specific and reacted both to nonself-peptide derivatives as well as to self-peptide homologues. No unique pattern of peptide reactivity distinguished V beta 8.2+ hybridomas from V beta 8.2- hybridomas; all of the hybridomas were most reactive to the nonself sequences and reacted to self peptide with one to three orders of magnitude less sensitivity. However, all V beta 8.2+ hybridomas were much better responders overall and were activated by lower concentrations of all peptides than were V beta 8.2- hybridomas. Although V beta 8.2 gene usage is strongly associated with autoimmune pathology, these data show that in LEW rats several different TCR V beta genes are utilized in response to a short pathogenic sequence of this autoantigen and show that V beta 8.2 receptors are not uniquely self-reactive. However, the enhanced reactivity to Ag of V beta 8.2+ hybridomas relative to V beta 8.2- hybridomas specific for the same peptide may help explain the close association of V beta 8.2 TCR gene usage with pathogenicity found in autoimmune disease models.

The use of synthetic peptides in the study of experimental autoimmune uveitis

S-antigen is a highly pathogenic retinal autoantigen for the induction of experimental autoimmune uveitis (EAU). EAU is predominantly a T cell mediated autoimmune disease of the uveal tract and retina of the eye and the pinealocytes of the pineal gland. Using synthetic peptides it has been possible to identify several B cell and T cell epitopes in the molecule. In addition, synthetic peptides derived from proteins of diverse origin with amino acid sequence homology to pathogenic regions of S-antigen induce an EAU which is indistinguishable from the disease induced by native S-antigen. These studies aid in the understanding of immune mechanisms in EAU and provide a basis for the pathogenesis of uveitis in humans.

Assignment of several epitopes to cyanogen bromide peptides of bovine retinal S-antigen by immunoblotting with peptide-specific antibodies

Peptide fragments of bovine S-antigen, an immunopathogenic retinal autoantigen which mediates experimental autoimmune uveoretinitis, were produced by cyanogen bromide cleavage and used to study antibody-defined epitopes, primarily those defined by antibodies from Lewis rats immunized with the intact antigen or various peptide fragments purified from the digests by HPLC. Antibodies from the sera have been affinity-purified on several of the peptides and examined by western blot analysis and enzyme-linked immunosorbent assay on S-antigen, digests and purified fragments. By immunoblotting it could be shown that five of the purified peptides, CB46, CB47, CB67, CB74 and CB123 were immunogenic, eliciting antibodies which recognized the peptides to which they were prepared; all, except for CB67, elicited antibodies which also bound intact S-antigen. Two more peptides, CB14 and CB27 were not immunogenic and did not contain epitopes. An epitope was also found in CB35, a previously uncharacterized peptide. Using these procedures together with amino acid sequence and composition data, we have been able to determine the origins of the peptides which contain antibody epitopes, including those which we have previously determined to possess epitopes recognized by class II MHC-restricted T cell lines raised to S-antigen and several of the peptides. A T cell line specific for the non-uveitogenic CB47 peptide was unable to transfer the disease.

Unresponsiveness to self-peptides of S-antigen in EAU: an overview of recent results

Several observations in the characterization of EAU are examined. First, sequences of heterologous S-Ag (bovine S-Ag in LEW rats) which induce strong in vitro T cell proliferative responses are dissociated from sequences which induce EAU. Strong in vitro responses were detected only to nonself peptide homologues. Second, T cells specific for self-sequences of S-Ag are unresponsive in vitro. Third, TCR V beta 8 gene usage is associated with pathogenic T cells. V beta 8.2 bearing hybridomas from a pathogenic line exhibited enhanced reactivity to pathogenic self-peptides, but were unresponsive unless presented Ag on nonirradiated, splenic APC. We propose that these findings reflect self, nonself discrimination of the epitopes on heterologous autoantigen, and examine the hypothesis that TCR containing V beta 8 have enhanced avidity for MHC complexed with autologous sequences, but that these V beta 8 autoreactive T cells appear unresponsive in vitro due to mechanisms of self-tolerance involving superantigen/coligand participation.

Preparation of overlapping peptides of bovine retinal S-antigen and their localization by immunoblotting with peptide-specific antibodies

Bovine retinal S-antigen was cleaved by three chemical cleavage procedures including o-iodosobenzoic acid (IBA), mile acid and cyanogen bromide. The resultant peptides were used to study antibody-defined epitopes. Treatment with IBA, which cleaves primarily at tryptophanyl peptide bonds, produced at least 4 major fragments and several minor fragments. The peptides have been identified by their migration on SDS-PAGE and tested for their immunoreactivity to several affinity-purified anti-CNBr-peptide antibodies and to affinity-purified anti-IBA peptide antibodies. The presence of a single tryptophan residue 194 residues from the amino-terminus should result in 2 fragments of approximately 23,000 and 26,000 molecular weight based on the known size of intact S-antigen. The additional fragmentation is due to the presence of acid labile bonds and cleavage at IBA-sensitive tyrosyl residues associated with a side reaction. Western immunoblots using affinity-purified antibodies against the various IBA and CNBr peptides have allowed location of these peptides within the intact molecule. Specifically, IBA23K and IBA21K are overlapping fragments on the carboxy end, mutally exclusive of all other peptides. IBA15K and IBA5.6K overlap, and IBA18K and IBA10K overlap within IBA26K which comprises the N-terminal half of S-Ag. Additionally, IBA10K contains an antibody epitope destroyed by CNBr cleavage of the methionyl residue between CB53 and CB56. Further characterization of these IBA peptides will expedite the location of possible additional uveitogenic epitopes in the amino-terminal half of S-Ag as well as epitopes lost by other peptide generating techniques.