Cell-based gene therapy experiments in murine experimental autoimmune encephalomyelitis.

With the ultimate goal of developing a novel treatment for multiple sclerosis (MS), we have developed a cell-based gene therapy protocol for the treatment of murine experimental autoimmune encephalomyelitis (EAE), a powerful animal model for MS. We have determined that transduced fibroblasts secreting encephalogenic epitopes, when injected into mice with EAE, cause a striking abrogation of disease. Both myelin basic protein (MBP) and proteolipid protein mini-gene constructs expressed in syngeneic fibroblast cells were capable of ameliorating ongoing EAE induced by MBP protein. These experiments are crucial since they suggest that not all encephalogenic epitopes need be secreted for the control of disease. We also demonstrate the success of this protocol when transduced syngeneic, and most importantly, allogeneic cells are sequestered within an implantable chamber. Furthermore, we find that through modifying antigen expression by changing the signal sequence of the mini-gene construct, we were able to significantly reduce the dose of cells required for treatment. These improvements to the mini-gene delivery system are critical for the eventual translation of our protocol to the clinic.

Vaccination with the T cell antigen Mtb 8.4 protects against challenge with Mycobacterium tuberculosis.

The development of an effective vaccine against Mycobacterium tuberculosis is a research area of intense interest. Mounting evidence suggests that protective immunity to M. tuberculosis relies on both MHC class II-restricted CD4(+) T cells and MHC class I-restricted CD8(+) T cells. By purifying polypeptides present in the culture filtrate of M. tuberculosis and evaluating these molecules for their ability to stimulate PBMC from purified protein derivative-positive healthy individuals, we previously identified a low-m.w. immunoreactive T cell Ag, Mtb 8.4, which elicited strong Th1 T cell responses in healthy purified protein derivative-positive human PBMC and in mice immunized with recombinant Mtb 8.4. Herein we report that Mtb 8.4-specific T cells can be detected in mice immunized with the current live attenuated vaccine, Mycobacterium bovis-bacillus Calmette-Guérin as well as in mice infected i.v. with M. tuberculosis. More importantly, immunization of mice with either plasmid DNA encoding Mtb 8.4 or Mtb 8.4 recombinant protein formulated with IFA elicited strong CD4(+) T cell and CD8(+) CTL responses and induced protection on challenge with virulent M. tuberculosis. Thus, these results suggest that Mtb 8.4 is a potential candidate for inclusion in a subunit vaccine against TB.

CD8+ T cells recognize an inclusion membrane-associated protein from the vacuolar pathogen Chlamydia trachomatis.

During infection with Chlamydia trachomatis, CD8(+) T cells are primed, even though the bacteria remain confined to a host cell vacuole throughout their developmental cycle. Because CD8(+) T cells recognize antigens processed from cytosolic proteins, the Chlamydia antigens recognized by these CD8(+) T cells very likely have access to the host cell cytoplasm during infection. The identity of these C. trachomatis proteins has remained elusive, even though their localization suggests they may play important roles in the biology of the organism. Here we use a retroviral expression system to identify Cap1, a 31-kDa protein from C. trachomatis recognized by protective CD8(+) T cells. Cap1 contains no strong homology to any known protein. Immunofluorescence microscopy by using Cap1-specific antibody demonstrates that this protein is localized to the vacuolar membrane. Cap1 is virtually identical among the human C. trachomatis serovars, suggesting that a vaccine incorporating Cap1 might enable the vaccine to protect against all C. trachomatis serovars. The identification of proteins such as Cap1 that associate with the inclusion membrane will be required to fully understand the interaction of C. trachomatis with its host cell.

Classically restricted human CD8+ T lymphocytes derived from Mycobacterium tuberculosis-infected cells: definition of antigenic specificity.

Previous studies in murine and human models have suggested an important role for HLA Ia-restricted CD8(+) T cells in host defense to Mycobacterium tuberculosis (Mtb). Therefore, understanding the Ags presented via HLA-Ia will be important in understanding the host response to Mtb and in rational vaccine design. We have used monocyte-derived dendritic cells in a limiting dilution analysis to generate Mtb-specific CD8(+) T cells. Two HLA-Ia-restricted CD8(+) T cell clones derived by this method were selected for detailed analysis. One was HLA-B44 restricted, and the other was HLA-B14 restricted. Both were found to react with Mtb-infected, but not bacillus Calmette-Guérin-infected, targets. For both these clones, the Ag was identified as culture filtrate protein 10 (CFP10)/Mtb11, a 10.8-kDa protein not expressed by bacillus Calmette-Guérin. Both clones were inhibited by the anti-class I Ab and anti-HLA-B,C Abs. Using a panel of CFP10/Mtb11-derived 15-aa peptides overlapping by 11 aa, the region containing the epitopes for both clones has been defined. Minimal 10-aa epitopes were defined for both clones. CD8(+) effector cells specific for these two epitopes are present at high frequency in the circulating pool. Moreover, the CD8(+) T cell response to CFP10/Mtb11 can be largely accounted for by the two epitopes defined herein, suggesting that this is the immunodominant response for this purified protein derivative-positive donor. This study represents the first time CD8(+) T cells generated against Mtb-infected APC have been used to elucidate an Mtb-specific CD8(+) T cell Ag.

Novel mutants define genes required for the expression of human histocompatibility leukocyte antigen DM: evidence for loci on human chromosome 6p.

We and others have shown that the products of the HLA-DM locus are required for the intracellular assembly of major histocompatibility complex class II molecules with cognate peptides for antigen presentation. HLA-DM heterodimers mediate the dissociation of invariant chain (Ii)-derived class II-associated Ii peptides (CLIP) from class II molecules and facilitate the loading of class II molecules with antigenic peptides. Here we describe novel APC mutants with defects in the formation of class II-peptide complexes. These mutants express class II molecules which are conformationally altered, and an aberrantly high percentage of these class II molecules are associated with Ii-derived CLIP. This phenotype resembles that of DM null mutants. However, we show that the defects in two of these new mutants do not map to the DM locus. Nevertheless, our evidence suggests that the antigen processing defective phenotype in these mutants results from deficient DM expression. These mutants thus appear to define genes in which mutations have differential effects on the expression of conventional class II molecules and DM molecules. Our data are most consistent with these factors mapping to human chromosome 6p. Previous data have suggested that the expression of DM and class II genes are coordinately regulated. The results reported here suggest that DM and class II can also be differentially regulated, and that this differential regulation has significant effects on class II-restricted antigen processing.

Differential APC requirements of self- and nonself-reactive T cells and T cell hybridomas specific for retinal S-antigen.

Autoreactive T cell hybridomas specific for pathogenic peptides of retinal S-Ag require a novel radiosensitive APC activity for IL-2 secretion that is distinct from Ag presentation by MHC class II. Antigen-dependent IL-2 secretion by self-reactive hybridomas was much more efficient with splenic APC than with thymic APC, although both provided similar levels of hybridoma TCR occupancy as measured by activation-induced cell death. Furthermore, thymic APC did stimulate IL-2 secretion by a non-self reactive hybridoma. To test the hypothesis that this activity was provided by a distinct cell population, fractionated splenocytes were tested for their ability to present Ag to these hybridomas. The most potent Ag presentation for IL-2 secretion was found to segregate with low-density, B cell-enriched fractions while adherent cells, or purified T cells were unable to support IL-2 production. Together with previous results, the data show that antigen presentation leading to IL-2 secretion by these autoreactive T cell hybridomas requires activated B cells, whereas TCR occupancy can be provided by several APC subsets.

A radiosensitive APC activity dissociates IL-2 secretion and activation-induced cell death by autoreactive T cell hybridomas.

T cell hybridomas were generated from a LEW rat T cell line specific for the uveitogenic peptide bov-B1 of bovine retinal S-antigen. Using these autoreactive hybridomas, IL-2 production and activation-induced cell death (AICD) were dissociated as outcomes of activation. The self-reactive hybridomas secrete IL-2 and undergo AICD in response to antigen presented by non-irradiated syngeneic splenocytes, whereas antigen presentation by irradiated splenocytes induced only AICD. IL-2 production by a non-self reactive hybridoma was unaffected by irradiation of the APC. Pretreatment of the APC with phorbol ester or lipopolysaccharide and IL-4 protected their ability to induce IL-2 secretion after gamma-irradiation. Although the co-stimulation-blocking reagent CTLA-4-Ig mimicked the effect of gamma-irradiation by preventing IL-2 secretion but not AICD, B7 expression on the APC was not radiosensitive, nor did co-stimulation, provided 'in trans' with a B7-expressing third-party cell, reconstitute antigen-specific hybridoma IL-2 secretion in response to irradiated APC. In summary, the data show that IL-2 secretion and AICD of a self-reactive T cell hybridoma can be dissociated as consequences of TCR occupancy in the presence of a functional co-stimulatory signal. It is proposed that the signals producing these events are transduced through the TCR-CD3 complex alone and reflect the differential outcomes of high- and low-affinity interactions.

HLA-DMA and -DMB genes are both required for MHC class II/peptide complex formation in antigen-presenting cells.

Major histocompatibility complex (MHC) class II molecules are highly polymorphic cell-surface glycoproteins that present antigenic peptides to CD4+ T lymphocytes. The normal assembly of class II molecules with cognate peptides for antigen presentation requires an accessory function provided by a gene mapping to the class II region of the HLA complex. The isolation of somatic cell mutants of antigen-presenting cells (APC) has shown that at least one gene which maps between HLA-DP and HLA-DQ, provisionally designated c2p-1 (ref. 3), mediates this process. Here we describe a unique new mutant 2.2.93, which manifests defective formation of class II/peptide complexes like that described in c2p-1 mutants. We show that (1) mutant 2.2.93 contains a mutation in HLA-DMA, and a representative c2p-1 mutant, 9.5.3, contains a mutation in HLA-DMB; and (2) transfection and expression of DMA complementary DNA in 2.2.93, and DMB cDNA in 9.5.3, reverses their mutant phenotypes. These results show that HLA-DMA and -DMB, genes of previously unknown function mapping between HLA-DP and HLA-DQ, are required for the normal assembly of peptides with MHC class II molecules. They suggest that HLA-DMA and -DMB encode subunits of a functional heterodimer which is critical in the pathway of class II antigen presentation.

Multiple, autoreactive TCR V beta 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.

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.

In vitro unresponsiveness to autologous sequences of the immunopathogenic autoantigen, S-antigen.

Previous analyses of T cell recognition sites on immunopathogenic neural autoantigens have demonstrated, using LEW rats, the functional dissociation of in vitro proliferative responses and the ability to actively induce autoimmune diseases. In experimental autoimmune uveoretinitis, immunization of LEW rats with bovine retinal S-Ag reveals the presence of three immunodominant T cell recognition sites located in regions containing sequence differences between bovine and rat S-Ag. Immune responses of LEW rats to self (rat) and nonself (bovine and human) peptide homologues representing these three sites were compared. The immunodominant sequences of heterologous S-Ag were found to predict new pathogenic T cell recognition sites in the corresponding autologous rat sequence. Furthermore, in vitro proliferative responses to the pathogenic autologous sequences are dramatically diminished relative to the responses of lymphocytes raised to the non-self homologues. A pathogenic T cell line, R858, efficiently transferred disease, but was unresponsive to the autologous S-Ag peptide in proliferation assays. However, responses to autologous peptides were readily detected using nonirradiated splenic APC. Detection of responses to non-self peptides was independent of this radiosensitive Ag-presenting activity. The lack of in vitro proliferative responses to pathogenic autologous sequences by T cells bearing self-specific receptors, contrasted with the strong proliferation induced by non-self peptide homologues, suggests a mechanism of unresponsiveness to self.

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.

Multiple, spatially distinct T cell epitopes within a pathogenic 123 residue cyanogen bromide peptide of bovine retinal s-antigen.

We have examined the T cell specificity of a Lewis rat T cell line (R208) specific for a pathogenic, 123 residue cyanogen bromide produced peptide of bovine S-antigen by using two independent sets of overlapping synthetic peptides representing the entire length of the 123 residue fragment. S-antigen, a 48 kDa immunopathogenic photoreceptor cell autoantigen induces T cell mediated experimental autoimmune uveoretinitis (EAU) in experimental animals. Extensive analyses revealed a heterogenous response by the R208 line to the panel of synthetic peptides, proliferating weakly to 4 distinct sites. Unexpectedly, peptides representing sequences (residues 286-297 and 303-320 of bovine S-antigen) known to actively induce the autoimmune pathology were unable to significantly stimulate the R208 line as assessed by proliferation assays. Similarly, attempts to isolate T cells specific for these sequences from the R208 line have proven unsuccessful. However, two sequences, residues 253-269 and 273-289, sufficiently stimulated R208 cells to allow isolation of sub-lines, R208:26 and R208:28, respectively. Neither of these peptides actively induce an autoimmune response. R208:26 does not transfer EAU and R208:28 transfers moderate EAU. As a control, we are able to isolate a pathogenic T cell line (R502) specific for the actively pathogenic sequence, residues 303-320, when this peptide is used as the immunogen. However, the R502 line proliferates to peptides (e.g. 305-322) which do not contain residues 303 and 304 which are critical for the active induction of disease. These results show a multiplicity of distinct T cell epitopes within a relatively small region of S-antigen.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

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.

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.

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.

Identification of a uveitogenic cyanogen bromide peptide of bovine retinal S-antigen and preparation of a uveitogenic, peptide-specific T cell line.

Peptide fragments of bovine retinal S-antigen produced by cyanogen bromide (CB) digestion have been purified and tested for their ability to induce experimental autoimmune uveoretinitis (EAU) and pinealitis (EAP) in Lewis rats. Following immunization with the various peptides in complete Freund's adjuvant, one of the peptides, CB123, was found to be potently uveitogenic. A CB123-specific, class II restricted T helper lymphocyte line (R208) prepared from one of the CB123 peptide-immunized animals by repeated in vitro selection with purified CB123 was able to transfer severe EAU and EAP to naive rats. Peptides CB36, CB46, CB51 and CB66 were immunogenic as assessed by the presence of antibodies detected in the enzyme-linked immunosorbent assay and sensitized lymphocytes found in [3H]thymidine incorporation assays using lymphocytes from the peptide-immunized rats, but they did not induce significant EAU or EAP. Antibodies induced by immunization with the peptides also bound intact S-antigen, unlike the lymphocytes which were only weakly responsive to S-antigen. Cross-reactivity of antibodies and lymphocytes from CB51 and CB66 immune animals suggested that these peptides overlap and contain a common epitope. No measurable immunity to any CB peptide or intact S-antigen was found in the animals immunized with peptide CB26. Although a T cell line (R17) raised to human S-antigen was also able to transfer EAU, it was weakly responsive to the CB123 peptide; also, the R208 line did not respond well to human S-antigen. Since both human and bovine S-antigen are uveitogenic, these results suggest that the uveitogenic epitope in CB123, which is of bovine origin, is not the same as the uveitogenic epitope of the human antigen.

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.