Characterization of a new, potent, immunopathogenic epitope in S-antigen that elicits T cells expressing V beta 8 and V alpha 2-like genes.

Experimental autoimmune uveoretinitis (EAU) is a predominantly T cell-mediated autoimmune disease induced in susceptible animals by active immunization with human or bovine retinal S-Ag or by passive transfer of activated S-Ag or peptide-specific CD4+ T cells. During the course of studies aimed at the identification of T cell and B cell recognition sites in bovine and human S-Ag, a new potent uveitogenic region, located near the carboxy terminus of the molecule, was identified and characterized. Analysis of several synthetic peptides from this region showed that a 14 amino acid residue peptide, BSAg339-352, was highly uveitogenic when injected with adjuvants into Lewis rats. A uveitogenic T cell line, R737, was raised by in vitro selection of lymphocytes from animals immunized with peptide BSAg333-352. Northern blot analysis of mRNA from the R737 T cell line was positive for the rat homologs of murine V beta 8 and V alpha 2 T cell receptor gene probes. Whereas peptide BSAg339-352 defined the pathogenic site, nonpathogenic, proliferative sites were found in close physical association. This region is immediately adjacent to previously characterized pathogenic and proliferative sites contained in residues BSAg352-364. These results, as well as our previous observations, show S-Ag to be a complex molecule with several highly conserved amino acid sequences that can elicit pathogenic T cells with restricted T cell receptor V gene usage capable of active and passive elicitation of experimental autoimmune uveoretinitis.     

Characterization of a T suppressor cell line that downgrades experimental allergic encephalomyelitis in mice.

A T-suppressor (Ts) cell line of CD8 phenotype was isolated from spleens of SJL/J mice that had recovered from experimental allergic encephalomyelitis (EAE) induced by injection of MBP-activated T cells. The Ts cell line inhibited the proliferation of MBP-sensitized T cells in vitro. Addition of recombinant IL-2 enhanced the Ts-mediated suppression. Adoptively transferred Ts line was able to downgrade EAE in mice subsequently challenged with MBP-activated T cells. The mechanism of suppression appeared to involve neither direct cytolysis of the effector T cells nor the production of a soluble suppressor factor. The findings suggest an in vivo role for suppressor T cells in the regulation of EAE.     

Differential effects of cyclosporins A and G on functional activation of a T-helper-lymphocyte line mediating experimental autoimmune uveoretinitis

The effect and relative efficiency of cyclosporin A (CsA) and cyclosporin G (CsG) on suppressing the activation of primed autoimmune rat T-helper lymphocytes were assayed. The autoimmune T-helper cells (ThS) are a long-term line specific to the retinal soluble antigen (SAg) and can adoptively transfer experimental autoimmune uveoretinitis (EAU), after in vitro reactivation with antigen or mitogen, to naive syngeneic hosts. Antigen-driven production of interleukin-2 (IL-2) and antigen-driven proliferation were inhibited in a dose-dependent manner and to a similar extent at each of the respective cyclosporin concentrations. CsA was 8-10 times more potent than CsG, with ID50-CsA occurring at 0.5 to 2 ng/ml, and ID50-CsG at 5 to 20 ng/ml, depending on the experiment and the cyclosporin batch. Addition of exogenous lymphokines in the form of rat spleen concanavalin A (Con A)-conditioned medium (SCM) or recombinant IL-2 (but not recombinant IL-1) was able to reverse only about half of the inhibition, as measured along the linear part of the dose-response curve. Inhibition of IL-2 production was lost if a maximally inhibitory dose of cyclosporin was added to the cultures later than 8 hr after antigen stimulation, while proliferation was still suppressed to 50% by cyclosporin added as late as 12 hr and could not be restored by addition of SCM. Both cyclosporins at concentrations that blocked proliferation and IL-2 production significantly suppressed the generation of high-affinity and low-affinity IL-2 receptors by ThS in response to antigen (as assayed by direct binding of 125I-IL-2). These results suggest that CsA and CsG inhibit antigen-induced expansion of ThS by interfering with more than one activation step. In contrast, the in vitro activation of the uveitogenic potential of ThS cells, incubated with antigen in the presence of CsA or CsG and adoptively transferred into untreated recipients, was not affected by the cyclosporins. Thus, triggering of the pathogenic potential of primed autoimmune T-helper lymphocytes can take place in the presence of cyclosporin and in the absence of cellular proliferation.     

S-antigen-specific rat T cell lines recognize peptide fragments of S-antigen and mediate experimental autoimmune uveoretinitis and pinealitis

Two S-antigen-specific rat T cell lines expressing the T helper cell surface phenotype (W 3/25+, OX 8-) have been isolated from the spleen and lymph node cells of retinal S-antigen-immunized Lewis rats, one of which displayed neither clinical nor histopathologic signs of experimental autoimmune uveoretinitis. The other rat had recovered from severe experimental autoimmune uveoretinitis for 2 mo before isolation of the cell line. Both lines are specific for S-antigen presented by histocompatible antigen-presenting cells, and also respond in vitro to several of the peptides produced by cyanogen bromide cleavage of bovine retinal S-antigen. The lesions induced by the i.v. transfer of from 1 to 10 X 10(6) viable line cells involve the retina and pineal gland, as is found when Lewis rats are immunized with immunopathogenic doses of S-antigen. Histologic examination of the eyes and pineal glands revealed pathologic lesions typical of experimental autoimmune uveoretinitis, and consisted of marked infiltration of the retina and surrounding tissues and the pineal gland by lymphocytes and inflammatory cells. T cells capable of mediating autoimmune disease are clearly present and readily isolated from both asymptomatic and convalescent animals. No significant differences in specificity for the cyanogen bromide peptides of S-antigen or cell surface phenotype were found in the T cell lines isolated from these two rats, nor was any difference found in the specificity or titer of serum antibodies taken from the original rats for the cyanogen bromide peptides of S-antigen.     

Pertussis toxin inhibits induction of tissue-specific autoimmune disease by disrupting G protein-coupled signals.

Pertussis toxin (PTX) has been used for many years as an adjuvant that promotes development of tissue-specific experimental autoimmune diseases such as experimental autoimmune encephalomyelitis, experimental autoimmune uveitis (EAU), and others. Enhancement of vascular permeability and of Th1 responses have been implicated in this effect. Here we report a surprising observation that, in a primed system, PTX can completely block the development of EAU. Disease was induced in B10.RIII mice by adoptive transfer of uveitogenic T cells, or by immunization with a uveitogenic peptide. A single injection of PTX concurrently with infusion of the uveitogenic T cells, or two injections 7 and 10 days after active immunization, completely blocked development of EAU. EAU also was prevented by a 1-h incubation in vitro of the uveitogenic T cells with PTX before infusing them into recipients. Uveitogenic T cells treated with PTX in vitro and lymphoid cells from mice treated with PTX in vivo failed to migrate to chemokines in a standard chemotaxis assay. Neither the isolated B-oligomer subunit of PTX that lacks ADP ribosyltransferase activity nor the related cholera toxin that ADP-ribosylates G(s) (but not G(i)) proteins blocked EAU induction or migration to chemokines. We conclude that PTX present at the time of cell migration to the target organ prevents EAU, and propose that it does so at least in part by disrupting signaling through G(i) protein-coupled receptors. Thus, the net effect of PTX on autoimmune disease would represent an integration of enhancing and inhibitory effects.     

Regulation of myelin basic protein-specific helper T cells in multiple sclerosis: generation of suppressor T cell lines.

Suppressor T cell (Ts) lines specific for myelin basic protein (MBP)-reactive helper T cell (Th) clones were generated from two patients with multiple sclerosis (MS) following a primary culture of peripheral blood mononuclear cells (PBMC) with MBP and cyclosporine A (CsA). These suppressor T cell lines were maintained in culture by alternate stimulation with MBP and antigen-presenting cells (APC). The Ts lines expressed preferentially the CD4 phenotype (5/6 Ts lines tested) and exhibited potent antigen-specific suppressor activity on the proliferation of MBP-specific Th clones and not on the T cell lines with other antigen specificity. For some Ts lines, a Ts-to-Th ratio of 1 was sufficient to inhibit the proliferation of MBP-specific T cells by 90%. The suppressor T cells obtained were weakly responsive to MBP and required the presence of the autologous PBMC for proliferation. Furthermore, proliferation of these suppressor T cell lines was restricted by HLA-DR molecules (for CD4+ Ts lines) and HLA class I (for a CD8+ Ts line). The suppressor T cell lines generated and the techniques described in this study may be helpful in our understanding of the events involved in the immune regulation in MS and other autoimmune diseases.     

Exogenous nitric oxide inhibits experimental autoimmune uveoretinitis development in Lewis rats by modulation of the Th1-dependent immune response.

Experimental autoimmune uveoretinitis (EAU) is a T cell-mediated autoimmune disease of posterior uvea that closely resembles a human disease. The uveitogenic effector T cell has a Th1-like phenotype [high interferon-gamma (IFN-gamma), low interleukin-4 (IL-4)], and genetic susceptibility to EAU that is associated with an elevated Th1 response. Suppression of CD4+ Th1 cells for the treatment of autoimmune disease is an attractive potential therapeutic approach. Nitric oxide (NO) has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. In this study, we investigated the potential role of NO as an immunoregulator to alter Th1/Th2 cytokine production, as well as to inhibit the interphotoreceptor retinoid binding protein (IRBP)-induced EAU, a CD4+ Th1 cell-mediated autoimmune disease. Injection of IRBP (100 microg) into two footpads resulted in severe EAU. The beginning peak of the disease was days 12 to 15 after immunization. Oral treatment with molsidomine (MSDM), a NO donor, began 24 h before IRBP immunization to the end of the experiments, which resulted in a significant inhibition of the disease by clinical and histopathological criteria. When MSDM was administered until day 21, a complete reduction of incidence and severity of EAU was observed. To investigate the cytokine alterations from Th1 to Th2 cytokines by MSDM, the cytokines were assayed in a culture medium of IRBP-stimulated inguinal lymphocytes. IRBP-immunized rats secreted a high concentration of IFN-gamma and a low concentration of IL-10. In contrast, MSDM treatment enhanced IL-10 secretion and tended to decrease IFN-gamma secretion. In conclusion, we show that the administration of NO suppresses EAU by altering the Th1/Th2 balance of inflammatory immune responses. We suggest that NO may be useful in the therapeutic control of autoimmune uveitis.     

Antigen-induced T suppressor cells regulate the autoreactive T helper-B cell interaction

The T suppressor (Ts) cell population that functions to regulate antigen-specific MHC-restricted T helper (Th)-B cell interactions also regulates the activation of B cells by cloned autoreactive Th cells. Activated Ts cells were generated by in vivo priming and restimulation in vitro with high concentrations of the specific priming antigen. Once generated, this Ts population inhibits the Th-dependent activation of primed B cells by both antigen-specific and autoreactive T cells in an antigen-nonspecific manner. This suppression requires the participation of both Lyt-1+2- and Lyt-1-2+ T cells. It was also demonstrated that accessory cells were required for the induction of Ts cells. Moreover, the generation of suppression was MHC-restricted and required the recognition by T cells of Ia antigens on accessory cells. These studies demonstrate that the same or a very similar Ts cell population can function to inhibit the activation of B cells by antigen-specific as well as autoreactive T cells.     

The role of suppressor factors in the regulation of immune responses by ultraviolet radiation-induced suppressor T lymphocytes. I. Activity of suppressor cell culture supernatants

The purpose of this study was to determine whether soluble suppressor factors are involved in the regulation of immune responses by ultraviolet radiation-induced suppressor T lymphocytes (UV Ts). The UV Ts were induced by applying contact allergens to the ventral, unirradiated skin of mice that had been exposed 5 days earlier to UVB radiation. Supernatants from cultures that contained a mixture of UV Ts, normal responder lymphocytes, and hapten-modified stimulator cells were injected iv into normal recipients at the time of sensitization; they inhibited the induction of contact hypersensitivity (CHS) in vivo in an hapten-specific manner. The supernatants similarly suppressed the generation of specific cytotoxic T lymphocytes (CTL) in vitro. Moreover, supernatants from cultures that contained either UV Ts alone or UV Ts in combination with either the responder or the stimulator cells failed to suppress the CHS and CTL responses. These results suggest that hapten-specific inhibitory factors may participate in the regulation of immune responses by suppressor cells generated by epicutaneous sensitization of UV-irradiated mice.     

Absence of suppression in natural and induced tolerance to F antigen

The role of suppression in natural and induced tolerance to F antigen was investigated in two sets of experiments. In the first, CBA mice were submitted to pretreatments which decrease suppression and the antibody response to self- or allo-F type was investigated. The second set of experiments involved the transfer of spleen cells from tolerized or from naturally tolerant mice into normal mice which were then primed with allo-F, as well as the co-transfer of tolerant and primed lymphocytes into normal mice, to test whether tolerant lymphocytes present suppressor cells. The results indicate that the immune response against allo-F antigen is normally kept in a low level by a suppressive mechanism, and that F-specific suppressor T cells are absent from tolerant mice.Abbreviations used in this paper ATx adult thymectomy - BSS buffered salt solution - CFA Freund's complete adjuvant - CY cyclophosphamide - F.1 type-1 F antigen - F.2 type-2 F antigen - PBS phosphate-buffered saline - RIA radioimmunoassay - Th T helper cell - Ts T suppressor cell     

Regulation of hapten-specific T-cell response. II. Functional analysis of helper T cells and cytotoxic T cells in animals suppressed by azobenzenearsonate (ABA)-specific suppressor T cells

The administration of azobenzenearsonate-modified syngeneic spleen cells (ABA-SC) intravenously induces a population of first order hapten-specific inducer suppressor T cells (Ts1), which downregulate various aspects of T-cell-mediated immune responses via a well defined suppressor-T-cell pathway. In this study, we investigated the effects of these suppressor cells on the generation of ABA-specific cytolytic T lymphocytes (CTL) and helper T cells (Th) in vivo. We found evidence for functional impairment of ABA-activated Th and ABA-specific CTL precursors (CTLp) in the suppressed animals by a number of different in vitro criteria. Functional analysis of ABA-specific CTLp and ABA-activated Th in suppressed animals revealed that ABA-specific Ts inhibit the generation of CTL by impairing the antigen-specific activation of Th, which may in turn, prevent the clonal expansion of antigen-specific CTLp. The significance of these findings in relationship to our understanding of the cellular interactions necessary for the generation of CTL and the mode of action and mechanisms of suppressor T cells is discussed.     

Immunoregulation of lysozyme-specific suppression. III. Epitope-specific amplification of immunosuppression induced by monoclonal suppressor-T-cell products

The hen egg-white lysozyme (HEL)-specific suppression induced by soluble molecules produced by a monoclonal T-cell lymphoma line (LH8-105) obtained from HEL-specific suppressor T lymphocytes has been examined. Injection of I-J+ molecules from LH8-105 cell culture supernatant (TsFa) in HEL-primed mice during the afferent phase of the response induced Lyt-2+ second order suppressor T (Ts) cells which, upon transfer into HEL-CFA-primed syngeneic recipients, inhibit the delayed-type hypersensitivity (DTH) response to HEL. Transfer of spleen cells from TsFa-injected mice primed with HEL or human lysozyme suppresses the DTH response to HEL in recipient mice whereas this response is not affected by cell transfer from ring-necked pheasant egg-white lysozyme (REL)-primed and TsFa-injected mice, indicating that induction of second order Ts by TsFa is specific for a lysozyme epitope including phenylalanine at position 3. Fine antigenic specificity of second order Ts-cell induction is confirmed by similar results obtained upon injection of TsFa in mice primed with HEL N-terminal synthetic peptide or with an analog in which, as in REL, phenylalanine has been substituted by tyrosine at position 3. The same fine antigenic specificity observed in the induction of second order Ts cells is also present in the expression of TsFe suppressive activity. The similar antigenic specificity of Tsa and Tse suggests that Tse cells could result from amplification of the Tsa cell population or these two cell subsets could reflect different maturation stages of the same cell type rather than distinct T-cell populations activated in cascade.     

Dissociation between lymphocyte activation for proliferation and for the capacity to adoptively transfer uveoretinitis

We have shown previously that immunization with bovine interphotoreceptor retinoid-binding protein (IRBP) induces in rats severe eye disease, experimental autoimmune uveoretinitis (EAU). This study examined the uveitogenic capacity of IRBP of another species, the monkey, and tested the cross-antigenicity between these two proteins by a battery of immunological assays. Monkey IRBP was found to be approximately 20 times less uveitogenic in Lewis rats than bovine IRBP. High levels of cross-reactivity between bovine and monkey IRBP were demonstrated by antibodies as measured by the enzyme-linked immunosorbent assay, and by the radiometric ear test of delayed-type hypersensitivity, by using rats immunized with either one of the IRBP. On the other hand, lymphocytes from these rats failed to detect the cross-reactivity between the two IRBP by the proliferation response in culture. Yet, such lymphocytes did recognize the nonimmunizing IRBP when activated in culture for acquiring the capacity to adoptively transfer EAU into naive recipients. The data are discussed with regard to the limited usefulness of the lymphocyte proliferation assay for detection of immunopathogenic processes and the role of cross-reacting antigens in initiation of autoimmune responses.     

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.     

Suppression of experimental autoimmune uveitis in mice by induction of anterior chamber-associated immune deviation with interphotoreceptor retinoid-binding protein.

Immunization with bovine interphotoreceptor retinoid-binding protein induces autoimmune uveitis in B10.A mice. We have examined whether this soluble retina-specific Ag can induce anterior chamber-associated immune deviation when injected into the anterior chamber (AC) of the eye, and whether this deviant immune response has any effect on uveitis is susceptible mice. The results of these experiments indicate that interphotoreceptor retinoid-binding protein (IRBP) injected intracamerally altered the subsequent immune response of B10.A mice such that a) they were not able to develop IRBP-specific delayed hypersensitivity, nor (b) were they able to express significant autoimmune uveitis following a uveitogenic regimen. Moreover, spleen cells from mice that received IRBP in the AC suppressed uveitis when adoptively transferred into naive recipients. The splenic suppressor cells were able to prevent autoimmune uveitis in recipient mice when administered after the uveitogenic regimen. Most important, IRBP-specific splenic cells from mice treated with IRBP in the AC when injected into mice with established uveitis caused an abrupt cessation of the intraocular inflammation. The ability of intracamerally-injected soluble Ag to induce suppressor T cells that act on the efferent limb of the immune response suggests that the anterior-chamber-associated immune deviation phenomenon may have physiologic relevance in terms of preservation of the integrity of ocular tissue and renders this approach particularly suitable for treating already established experimental autoimmune diseases of this type. These results are discussed in terms of other methods that have been devised experimentally to suppress and prevent autoimmune uveitis and encephalomyelitis.     

Loss of STAT3 in CD4+ T cells prevents development of experimental autoimmune diseases

Th17 cells are implicated in CNS autoimmune diseases. We show that mice with targeted-deletion of Stat3 in CD4(+) T cells (CD4(Stat3)(-/-)) do not develop experimental autoimmune uveoretinitis (EAU) or experimental autoimmune encephalomyelitis. Defective Th17 differentiation noted in CD4(Stat3)(-/-) mice is compensated by exaggerated increases in Foxp3-, IL-10-, IL-4-, and IFN-gamma-expressing T cells, suggesting critical roles of STAT3 in shaping Ag-specific CD4(+) T cell repertoire. In mice with EAU, a high percentage of IL-17-expressing T cells in their peripheral lymphoid organs also secrete IFN-gamma while these double-expressors are absent in CD4(Stat3)(-/-) and wild-type mice without EAU, raising the intriguing possibility that uveitis maybe mediated by Th17 and IL-17-expressing Th1 cells. Resistance of Stat3-deficient mice to EAU derives in part from an inability of uveitogenic Th17 and Th1 cells to enter eyes or brain of the CD4(Stat3)(-/-) mouse because of the reduction in the expression of activated alpha4/beta1 integrins on CD4(Stat3)(-/-) T cells. Adoptive transfer of activated interphotoreceptor retinoid-binding protein-specific uveitogenic T cells induced in CD4(Stat3)(-/-) mice a severe EAU characterized by development of retinal folds, infiltration of inflammatory cells into the retina, and destruction of retinal architecture, underscoring our contention that the loss of STAT3 in CD4(+) T cells results in an intrinsic developmental defect that renders CD4(Stat3)(-/-) resistant to CNS inflammatory diseases. STAT3 requirement for IL-17 production by Th17, generation of double positive T cells expressing IL-17 and IFN-gamma, and for T cell trafficking into CNS tissues suggests that STAT3 may be a therapeutic target for modulating uveitis, sceritis, or multiple sclerosis.     

Interaction of idiotype-specific T suppressor factor with the hapten-specific third-order T suppressor subset results in antigen-nonspecific suppression

The interaction between the third-order T suppressor (Ts3) cell and the idiotype (Id)-specific second-order Ts factor (TsF2) was studied in the phenyltrimethylamino (TMA) hapten system. The experimental system which we used allowed the independent analysis of induction and activation requirements of Ts3. The procedure consisted of inducing the Ts3 in vivo and activating the enriched T-cell populations containing Ts3 in vitro with TsF2. The suppressive potential was then tested in mice previously primed for delayed-type hypersensitivity responses which were also treated with cyclophosphamide to deplete Ts3 and other drug-sensitive Ts cell types. Using this experimental system, it was found that the Id-specific TsF2 was required for the in vitro activation of Ts3. Furthermore, the TsF2 activated only the homologous and not heterologous antigen-primed Ts3-containing T cells and moreover, the target of TsF2 was found to be the Ts cells bearing hapten-specific receptors. Once the TMA hapten-specific Ts3 was activated with TsF2, the ensuing suppression was antigen nonspecific. The data demonstrate that the Ts3 represents a final effector Ts cell type in the TMA system.     

Synergism between mouse thyroglobulin- and vaccination-induced suppressor mechanisms in murine experimental autoimmune thyroiditis.

Previous studies have shown that genetically susceptible mice can be rendered resistant to the induction of experimental autoimmune thyroiditis (EAT) by pretreatment with deaggregated mouse thyroglobulin (dMTg). This resistance is mediated by CD4+ suppressor T cells (Ts) which suppress the afferent/inductive phase of EAT. Recent work has also shown that resistance to EAT can be achieved by vaccination with irradiated spleen cells previously primed in vivo with MTg and cultured in vitro with MTg (gamma SC). The gamma SC-induced resistance also inhibits the afferent phase of EAT but is mediated by both CD4+ and CD8+ Ts. To determine if dMTg- and gamma SC-induced suppression can cooperate to prevent EAT, we pretreated mice with suboptimal doses of dMTg and gamma SC before challenge with MTg and adjuvant. Mice receiving dMTg or gamma SC only showed suppressed in vitro response to MTg, but the development of thyroid lesions was unaltered. However, mice given one or two subtolerogenic doses of dMTg followed by gamma SC not only showed suppressed in vitro response to MTg, but also little or no thyroiditis, indicating cooperation between these two mechanisms. The cooperation was not reciprocal since reversing the order, giving gamma SC first followed by dMTg, was not effective in suppressing EAT. Thus, suppressor mechanisms activated by pretreatment with dMTg and gamma SC can act synergistically to suppress EAT induction; the two mechanisms may cooperate in vivo to maintain self-tolerance provided that MTg-specific CD4+ Ts are initially activated.     

T cell lines mediating experimental autoimmune uveoretinitis (EAU) in the rat

Long-term S-antigen (S-Ag)-specific T lymphocyte lines were derived from the lymph nodes of immunized Lewis rats that had been pretreated with low-dose cyclophosphamide. The protocol consisted of functional selection by alternating cycles of stimulation with S-Ag presented on syngeneic accessory cells and proliferation in IL 2-containing spleen-conditioned medium, coupled with early phenotypic selection for cells bearing the helper/inducer membrane marker (W3/25), by panning on antibody-coated plastic dishes. This protocol consistently resulted in the rapid generation of in vivo functional cell lines capable of mediating experimental autoimmune uveoretinitis (EAU) when transferred into naive rats at 5 to 10 X 10(6) cells/rat systemically or 1 to 2 X 10(6) cells/rat intravitreally. The disease appeared within 6 to 8 days, usually with minimal anterior chamber involvement, and was often unilateral. Pathologic changes resembled those seen in EAU induced by active immunization. The disease could be transferred without concomitant formation of serum antibodies. The uveitogenic line cells were negative for Ia antigen and positive for the W3/25 membrane marker, which appeared to be stable in long-term culture. They proliferated vigorously in vitro and produced IL 2 in response to S-Ag or Con A, but not to unrelated antigens. The establishment of uveitogenic T helper lymphocyte lines will permit the analysis of the cellular mechanisms involved in EAU in a more defined system than has been available.     

Characterization of rat CD8+ uveitogenic T cells specific for interphotoreceptor retinal-binding protein 1177-1191

The uveitogenic T cells that mediate experimental autoimmune uveitis are commonly assumed to be exclusively CD4(+). In the present study, we showed that, although a panel of long-term cultured rat uveitogenic T cell lines specific for the interphotoreceptor retinal-binding protein peptide, R16, all expressed CD4, approximately 40% of the R16-specific uveitogenic T cells freshly prepared from Ag-immunized rats were CD8(+)alphabetaTCR(+), as demonstrated by CFSE staining. We showed that the expansion of these CD8(+)alphabetaTCR(+) T cells was Ag-specific and that highly purified CD8(+) R16-specific T cells were able to induce uveitis on transfusion into naive rats. Moreover, CD8(+) uveitogenic T cells more readily switched phenotype from, and to, TCR(-)CD8(-)CD4(-) during in vivo or in vitro activation compared with their CD4(+) counterparts. In a previous study, we showed that highly purified CD8(+) myelin oligodendrocyte glycoprotein-specific T cells induced more severe autoimmune encephalomyelitis than the corresponding CD4(+) T cells. In this study, we show that an interphotoreceptor retinal-binding protein peptide consistently activated a high proportion of CD8(+)alphabetaTCR(+) T cells, which were uveitogenic in Lewis rats.