MHC class II-derived peptides can bind to class II molecules, including self molecules, and prevent antigen presentation.

Seven synthetic peptides corresponding to the polymorphic regions of the alpha and beta chains of the I-Ak molecule were examined for their ability to inhibit the presentation of foreign antigens to antigen-specific, I-A-restricted T cell hybridomas. Two of the peptides, representing the sequences found in the first and third polymorphic regions (PMR) of the A alpha k chain (alpha k-1 and alpha k-3) were capable of inhibiting the presentation of three different HEL-derived peptide antigens to their appropriate T cells. In addition, the alpha k-1 peptide inhibited the presentation of the OVA(323-339) immunodominant peptide to the I-Ad-restricted T cell hybridomas specific for it. Prepulsing experiments demonstrated that the PMR peptides were interacting with the APC and not with the T cell hybridomas. These observations were confirmed and extended by the demonstration that the alpha k-1 and alpha k-3 peptides blocked the direct binding of HEL(46-61) to purified I-Ak and that the alpha k-1 peptide blocked the binding of OVA(323-339) to I-Ad. The binding competition experiments suggest that the alpha k-1 peptide binds to the I-Ak molecule from which it was derived with a Kd approximately 10(-5) M, while the alpha k-3 peptide binds slightly less well. These combined data, suggesting that class II-derived peptides can bind to MHC class II molecules, including the autologous molecule from which they are derived, have important implications for the molecular basis of alloreactivity and autoreactivity. Further, they suggest a possible mechanism by which selecting elements, involving only MHC molecules, may be generated in the thymus.

An HLA-DR1 transgene confers susceptibility to collagen-induced arthritis elicited with human type II collagen.

Rheumatoid arthritis (RA) is an autoimmune disease that is strongly associated with the expression of several HLA-DR haplotypes, including DR1 (DRB1*0101). Although the antigen that initiates RA remains elusive, it has been shown that many patients have autoimmunity directed to type II collagen (CII). To test the hypothesis that HLA-DR1 is capable of mediating an immune response to CII, we have generated transgenic mice expressing chimeric (human/mouse) HLA-DR1. When the DR1 transgenic mice were immunized with human CII (hCII), they developed a severe autoimmune arthritis, evidenced by severe swelling and erythema of the limbs and marked inflammation and erosion of articular joints. The development of the autoimmune arthritis was accompanied by strong DR1-restricted T and B cell responses to hCII. The T cell response was focused on a dominant determinant contained within CII(259-273) from which an eight amino acid core was defined. The B cell response was characterized by high titers of antibody specific for hCII, and a high degree of cross-reactivity with murine type II collagen. These data demonstrate that HLA-DR1 is capable of presenting peptides derived from hCII, and suggest that this DR1 transgenic model will be useful in the development of DR1-specific therapies for RA.

Accretion of biopsy specimens of vaginal adenosis from patients exposed in utero to diethylstilbestrol, when transplanted to athymic nude mice.

Vaginal adenosis biopsy specimens from 10 patients exposed in utero to diethylstilbestrol were transplanted for 30 days into athymic (nude) mice. Almost all grafts were recovered, and they had morphologic features closely resembling those of the original biopsy specimens, i.e., cystic, complex, and simple occult glands covered mainly with an endocervical type of epithelium showing extensive squamous metaplasia. Autoradiographic analysis of these grafts after pulse administration of [3H]thymidine into the mice revealed extensive labeling of epithelial cells. These results imply that female athymic (nude) mice are compatible hosts for accretion of the human adenosis.

Comparative growth of SJL/J lymphomas in irradiated and normal SJL/J mice: cell cycle distribution and tumor cell quantification.

Flow cytometric and biological quantification of tumor cells revealed that 650 rad gamma-radiation 1 day prior to iv injection of H-2Ds negative lymphoma cells into SJL/J mice resulted in approximately a fourfold (day 3) to twelvefold (day 7) decrease in the tumor cell content of lymphoid organs as compared to that in unirradiated mice. Approximately 1.6-fold less tumor growth was noted (day 7) in 700-rad gamma-irradiated as compared to growth in unirradiated (SJL/J X CBA/J)F1 mice. Distributions of tumor cells in S-phase of the cell cycle were comparable at days 3, 5, and 7 in irradiated and unirradiated mice. Although approximately 26% of splenic tumor cells were in S-phase at days 5 and 7 in irradiated SJL/J mice, splenic tumor cell content did not increase during this time period. The data indicate that early (prior to day 3) and late (after day 5) events are responsible for decreased tumor growth in irradiated mice.

Ceramide signalling and the immune response.

Ceramide, produced through either the induction of SM hydrolysis or synthesized de novo transduces signals mediating differentiation, growth, growth arrest, apoptosis, cytokine biosynthesis and secretion, and a variety of other cellular functions. A generalized ceramide signal transduction scheme is shown in Fig. 2 in which ceramide is generated through the activation of distinct SMases residing in separate subcellular compartments in response to specific stimuli. Clearly, specificity of cellular responses to ceramide depends upon many factors which include the nature of the stimulus, co-stimulatory signals and the cell type involved. Ceramide derived from neutral SMase activation is thought to be involved in modulating CAPK and MAP kinases, PLA2 (arachidonic acid mobilization), and CAPP while ceramide generated through acid SMase activation appears to be primarily involved in NF-kappa B activation. While there is no apparent cross-talk between these two ceramide-mediated signalling pathways, there is likely to be significant cross-talk between ceramide signalling and other signal transduction pathways (e.g., the PKC and MAP kinase pathways). Other downstream targets for ceramide action include Cox, IL-6 and IL-2 gene expression, PKC zeta, Vav, Rb, c-Myc, c-Fos, c-Jun and other transcriptional regulators. Many, if not all, of these ceramide-mediated signalling events have been identified in the various cells comprising the immune system and are integral to the optimal functioning of the immune system. Although the role of the SM pathway and the generation of ceramide in T and B lymphocytes have only recently been recognized, it is clear from these studies that signal transduction through SM and ceramide can strongly affect the immune response, either directly through cell signalling events, or indirectly through cytokines produced by other cells as the result of signalling through the SM pathway. An overview of the signalling mechanisms coupling ceramide to the modulation of the immune response is depicted in Fig. 3 and shows how ceramide may play pivotal roles in regulating a number of complex processes. The SM pathway represents a potentially valuable focal point for therapeutic control of immune responses, perhaps for either enhancement of the activity of T cells in the elimination of tumors, or the down-regulation of lymphocyte function in instances of autoimmune disease. The recent explosion of knowledge regarding ceramide signalling notwithstanding, a number of critical questions need to be answered before a comprehensive, mechanistic understanding can be formulated relative to the incredibly varied effects of ceramide on cell function. For example, (i) how is a structurally simple molecule like ceramide able to mediate so many different, and sometimes paradoxical, physiological responses ranging from cell proliferation and differentiation to inhibition of cell growth and apoptosis, (ii) what are the molecular identities and modes of activation of the various SMase isoforms, (iii) what determines the distribution of the unique isoforms of SMase in cells of different lineages or at different stages of differentiation, (iv) what is the relative contribution of ceramide generated through SM hydrolysis versus de novo synthesis, and (v) by what means does ceramide interact with specific intracellular targets? Although a number of ceramide-activatable kinases, phosphatases, and their protein substrates have been identified, a more extensive search for additional cellular targets will be indispensable in determining the phosphorylation cascades linking the activation of the SM pathway to the regulation of nuclear events. Clearly, cross-talk between ceramide-induced signal transduction cascades and other signalling pathways adds to the inherent difficulty in distinguishing the specific effects of complex, intertwining signalling pathways.

The cartilage collagens: a review of their structure, organization, and role in the pathogenesis of experimental arthritis in animals and in human rheumatic disease.

This contribution reviews the structure and organization of collagen molecules found in cartilage and the roles that they may play in rheumatic diseases. Cartilage is unique in its physical properties and molecular composition, and contains sufficient amounts of types II, IX, X, and XI collagen to deem these molecules as "cartilage-specific." The vitreous body of the eye, a "cartilage-like" tissue is also rich in the same collagens but is type X deficient. Types VI and XII collagen are present in cartilage as well as noncartilaginous tissues. Types II, IX, and XI collagen are organized into matrix fibrils, where type II constitutes the bulk of the fibril, type XI regulates fibril size, and type IX facilitates fibril interaction with proteoglycan macromolecules. Genetic defects in these collagens can produce mild to severe developmental abnormalities, including spondyloepiphyseal dysplasia often accompanied by an accelerated form of osteoarthritis. Sensitization with collagen can produce experimental rheumatic diseases. Type II collagen induces an erosive polyarthritis in certain strains of rats, mice, and higher primates which can resemble rheumatoid arthritis and relapsing polychondritis. Type XI collagen is arthritogenic in rats but not mice; type IX induces autoimmunity in both species but not arthritis. Arthritis is initiated by complement fixing antibodies that bind to type II collagen in autologous cartilage, and the production of these antibodies is MHC restricted and T cell dependent. It is unclear whether T cells alone can induce arthritis, although they probably help sustain it. Mapping and characterizing the of T cell epitopes on type II collagen has resulted in the synthesis of small homolog and substituted peptides of type II collagen which suppress arthritis in an antigen-specific manner by a variety of routes, including mucosal. Moreover, collagen-induced arthritis has proven a valuable model to study the contribution of cytokines and other biological agents in the pathogenesis of joint injury and how they might be used to develop new therapies. Collagen autoimmunity has been implicated in the pathogenesis rheumatoid arthritis and polychondritis. Circulating antibodies to type II collagen are found in both diseases. Antibodies to types IX and XI collagen are also present in rheumatoid sera but are less prevalent. Rheumatoid cartilage and synovium contain antibodies to type II collagen at a prevalence far greater than serum, suggesting an intra-articular antigen-driven immune process. Although effective in animal models, attempts to treat rheumatoid arthritis with orally administered type II collagen have proven elusive. Different approaches using newer formulations and selected or modified oligopeptides remain to be tested and could prove effective in the treatment of the human rheumatic diseases.

Functional analysis of the antigen binding region of an MHC class II molecule.

The MHC class II molecules bind antigenic peptides and present them to T cells. Their ability to carry out these functions depends, in a critical way, on the detailed structure of the membrane-distal alpha 1 and beta 1 domains of these molecules. Using the I-Ak molecule and a series of hen egg lysozyme (HEL) peptide-specific, I-Ak-restricted T cell hybridomas as a model, we have examined the effect of altering essentially all of the polymorphic residues of the murine class II molecule on its ability to present Ag. Our results support the following conclusions: (1) both the location and the structural alteration introduced in a specific amino acid interchange are important in determining the effect the interchange will have on Ag presentation; and (2) changes in amino acids in the floor of the putative Ag binding cleft of the class II molecule can exert a major influence on the presentation of peptides to T cells. By carrying out direct binding experiments between the HEL(46-61) peptide and two mutant I-A molecules that fail to present HEL(46-61) to appropriate T cells, we were able to assess, in a quantitative fashion, the role played by peptide binding in the failure to present Ag. Our results suggest that, in the two cases studied, the failure to bind the HEL(46-61) peptide was not primarily responsible for the failure of the mutant class II molecule to present that peptide. Specifically, an A beta chain mutant that possesses d allelic residues at positions 65-67 in the second PMR of the Ak beta chain actually binds HEL(46-61) at wild type (I-Ak) levels. In contrast, an A alpha chain chimera in which b allelic residues are inserted in the third PMR of the Ak alpha chain, binds HEL(46-61) about three- to four-fold less well than wild type. While this decrease in binding affinity may be partially responsible for the inability of the latter chimeric molecule to present HEL(46-61), it can not be the total explanation because increasing the peptide concn even by an order of magnitude does not restore Ag presentation by APC expressing this chimeric molecule. These results are discussed in terms of the currently accepted model of the class II molecule.

Synthetic peptides that inhibit binding of the collagen type II 261-273 epitope to rheumatoid arthritis-associated HLA-DR1 and -DR4 molecules and collagen-specific T-cell responses.

Copolymer 1 [Cop 1, poly (Y, E, A, K)] is a random synthetic amino acid copolymer effective in the treatment of relapsing forms of multiple sclerosis (MS), a disease that is linked to HLA-DR2 (DRB1*1501). Another copolymer [poly (Y, A, K)] was also identified that binds to rheumatoid arthritis (RA)-associated HLA-DR1 (DRB1*0101) or HLA-DR4 (DRB1*0401) molecules and inhibits the response of HLA-DR1- and -DR4-restricted T cell clones to an immunodominant epitope of collagen type II (CII) 261-273 (a candidate autoantigen in RA). In the present study various peptides have been synthesized based on binding "motifs" of Cop 1 for HLA-DR1 and -DR4 molecules. Those peptides with K at P-1 or K at P8 were particularly effective as inhibitors of binding of CII 261-273, of Cop 1 and of the influenza virus hemagglutinin peptide 306-318 to these class II proteins. Moreover, several of them were also potent inhibitors of the CII 261-273-reactive T cell clones. These findings suggest that small peptides or their more stable derivatives may be able to substitute for copolymers in the treatment of RA, and by implication of MS.

I-Aq and I-Ap bind and present similar antigenic peptides despite differing in their ability to mediate susceptibility to autoimmune arthritis.

Susceptibility to collagen induced arthritis (CIA) in the murine model is linked to expression of the MHC class II alleles, I-Aq and I-Ar. We have examined the molecular basis for this MHC-linked susceptibility by studying the antigen presentation function of two class II molecules, I-Aq and I-Ap, that are closely related yet differ in mediating susceptibility to CIA. These class II molecules differ by only 4 amino acids, yet only mice expressing I-Aq develop CIA. Although the I-Ap molecule can bind the same immunodominant determinant from type II collagen as I-Aq, H-2p APC have difficulty generating I-Ap:CII peptide complexes when processing of CII is required. Immunization of H-2p mice with type II collagen (CII) generated only a weak T cell response when compared to H-2q mice, whereas immunization with the a CII peptide containing the dominant determinant induced a strong T cell response in both strains. In antigen presentation assays, H-2p APC were very inefficient in stimulating T cells when native CII was used as antigen, however they presented CII synthetic peptides with similar efficiency as H-2q APC. Processing and presentation of other antigens by H-2p APC was not affected. Using soluble class II binding assays, the affinity of I-Ap for the CII dominant peptide was 10 to 50 fold lower than I-Aq, however, this reduced affinity was not a general defect in I-Ap function. I-Aq and I-Ap had virtually identical affinities for binding other antigenic peptides. These data indicate that MHC-based susceptibility to autoimmunity may involve more than simple determinant selection and that the successful generation of an antigenic peptide by processing may be related to the overall affinity of the peptide for the MHC molecule.

Collagen-induced arthritis, an animal model of autoimmunity.

Collagen induced arthritis (CIA) is an autoimmune model that in many ways resembles rheumatoid arthritis (RA). Immunization of genetically susceptible strains of rodents and primates with type II collagen (CII) leads to the development of a severe polyarticular arthritis that is mediated by an autoimmune response. Like RA, synovitis and erosions of cartilage and bone are hallmarks of CIA, and susceptibility to both RA and CIA is linked to the expression of specific MHC class II molecules. Although not identical to RA, CIA clearly establishes the biological plausibility that an autoimmune reaction to a cartilage component can lead to a chronic, destructive, polyarthritis. Although it is induced in susceptible animals by immunization with heterologous CII, it is the autoreactive component of the immune response that leads to disease. A wealth of evidence indicates that synovitis is initiated by the production of pathogenic autoreactive antibodies capable of fixing and activating complement. The elucidation of the specific amino acid sequences of collagen that are recognized by the MHC molecules has enabled at least two approaches to specific immunotherapy to be considered. Firstly, small synthetic peptides representing dominant epitopes have been used as effectively as the original antigen as a tolerogen. The rather fastidious physicochemical properties of collagen that make it difficult for its routine use in therapy are thereby circumvented by the use of oligopeptides. Secondly, analysis of the specific amino acid side chains that are involved in MHC contact and TCR recognition enables analog peptides to be devised which can specifically and exquisitely inhibit the response to CII, preventing the onset of arthritis. Further investigations involving this model may contribute to the development of specific immunotherapies in the human disorder.

Epitopic analysis by anti-I-Ak monoclonal antibodies of I-Ak-restricted presentation of lysozyme peptides.

Anti-I-A mAb were used as probes of functional epitopes for both the presentation of hen egg lysozyme (HEL) peptides to I-Ak-restricted T cell hybridomas and the direct binding of the HEL (46-61) peptide. When mAb directed to polymorphic regions of I-Ak were used as inhibitors of Ag presentation, several different patterns of inhibition were observed among T cells specific for the same HEL peptide as well as among T cells specific for different fragments of HEL. Although there appears to be a conserved usage of some TCR V beta gene segments among the T cell hybrids specific for the same HEL peptide, no correlation is evident between a single V gene usage and susceptibility to blocking of Ag presentation by a particular anti-I-Ak mAb. Several of the mAb demonstrated T cell "clonotypic blocking" of Ag presentation, whereas others blocked presentation to every T cell hybrid tested, regardless of the peptide specificity. When mAb directed to nonpolymorphic regions of the I-A molecule were tested for their ability to block Ag presentation, little or no inhibition was observed. In addition, Fab' fragments of inhibitory mAb functioned identically to their intact homologous counterparts in their ability to block Ag presentation indicating that "nonspecific" steric hindrance was not playing a major role in the inhibitions observed. When the polymorphic region-directed anti-I-A mAb were tested for their ability to block the direct binding of the lysozyme peptide HEL(46-61) to I-Ak, those mAb that block HEL presentation to all T cell hybrids were found to block the binding of this peptide. However, anti-I-A mAb that demonstrate selective inhibition of T cell hybrid stimulation during Ag presentation, i.e., those directed to polymorphic serologic specificities Ia.15 and Ia.19, do not block the binding of HEL(46-61) to I-Ak. These data indicate that functionally independent epitopes exist on the I-Ak molecule for the binding of antigenic peptides and for interaction with the TCR.

Induction of T-cell proliferation and enhancement of NK activity by supernatants from Con A-stimulated human peripheral blood mononuclear cells: a new lymphokine.

Supernatants from human peripheral blood mononuclear cells activated by Con A contain a factor(s) that stimulates blastogenic activity of normal human peripheral blood mononuclear cells. This Con A supernatant (CAS) contains stimulatory activity for E-rosette positive lymphocytes (T cells) and requires adherent cells for stimulation of T-cell proliferation. CAS does not contain detectable amounts of IL-2 as determined by its inability to support CTLL cell growth. Nor does it contain IL-1 or interferon. Examination of functional activity of lymphocytes stimulated for 3 days by CAS revealed that NK activity is augmented. This supernate does not appear to have any direct effect on B-cell function, although it induces suppression of polyclonal PWM stimulation of immunoglobulins. Thus, CAS appears to contain a new cytokine with immunomodulating potential.

A visual display board used for efficient breeding and utilization of an athymic (nude) mouse colony.

A visual display board was designed to aid in the management of a barrier sustained athymic (nude) mouse colony. The board displayed pertinent information for breeding and weaning, including phenotype and age for each animal in the colony. In addition, the board showed the availability and current status of experimental groups. This system provided an efficient means of organizing production and planning utilization of animals in the colony.

Synthetic amino acid copolymers that bind to HLA-DR proteins and inhibit type II collagen-reactive T cell clones.

Copolymer 1 [poly(Y,E,A,K)] is a random synthetic amino acid copolymer of L-tyrosine, L-glutamic acid, L-alanine, and L-lysine that is effective both in suppression of experimental allergic encephalomyelitis and in the treatment of relapsing forms of multiple sclerosis. Copolymer 1 binds promiscuously and very efficiently to purified HLA-DR molecules within the peptide-binding groove. In the present study, YEAK and YEAK-related copolymers and type II collagen (CII) peptide 261-273, a candidate autoantigen in rheumatoid arthritis (RA), competed for binding to RA-associated HLA-DR molecules encoded by DRB1*0101 and DRB1*0401. Moreover, these copolymers (particularly YEAK, YAK, and YEK) inhibited the response of DR1- and DR4-restricted T cell clones to the CII epitope 261-273 by >50%. This direct evidence both for competitive interactions of these copolymers and CII peptide with RA-associated HLA-DR molecules and for inhibition of CII-specific T cell responses suggests that these compounds should be evaluated in animal models for rheumatoid arthritis.

Restricted TCR V alpha repertoire in the T cell response to a tolerogenic determinant of type II collagen.

Tolerization of B10.RIII mice (H-2r) with i.v.-injected type II collagen (CII) renders the animals resistant to induction of collagen-induced arthritis (CIA). The B10.RIII mouse is of particular interest, in that the T cell determinants that induce tolerance are different from those that induce arthritis. To characterize T cells that react with the tolerogenic determinant and play a role in regulation of arthritis, we have developed a panel of T cell hybridomas reactive with the tolerogenic T cell epitope, CII 607-621. None of the hybrids cross-reacted with either the arthritogenic CII 445-453 or murine CII. As determined by PCR and immunofluorescence, the T cell response to the tolerogenic determinant was oligoclonal, with evident preferential usage of V alpha. Through the analysis of a large panel of T cell hybridomas, preferential usage of V alpha2, J alpha44, J beta2.7, and D beta2.1 was observed. Characterization of T cells reactive with the immunodominant determinant, CII 607-621, responsible for the induction of tolerance should prove important in developing novel therapeutic approaches for the treatment of autoimmune diseases.

Characterization of signal transduction through the TCR-zeta chain following T cell stimulation with analogue peptides of type II collagen 260-267.

The immunodominant T cell determinant of type II collagen (CII) recognized by DBA/1 mice (I-Aq) is CII 260-267. The aims of this study were to determine the role of the amino acid residues within CII 245-270 in T cell signal transduction. To that end, we utilized I-Aq-restricted, CII-specific T cell hybridomas and examined tyrosine phosphorylation of TCR-zeta following stimulation with either wild-type CII 245-270 or a panel of analogue peptides. A variety of patterns occurred, ranging from increased phosphorylation of TCR-zeta to either partial or a complete abrogation of phosphorylation. Critical substitutions also completely abrogated the phosphorylation of ZAP70, a downstream molecule in TCR-zeta signaling. Evaluation of the supernatants of the T cell hybridomas for cytokine production in response to the peptides revealed a close correlation between the induction of phosphorylation of TCR-zeta and the amount of cytokine induced. Selected analogue peptides were tested as tolerogens in neonatal mice. Analogues that did not induce the phosphorylation of zeta chain, such as B3 (CII 251-270s263F-->N), were completely unable to induce tolerance, while analogues that caused a partial phosphorylation, such as B6 (CII 251-270s267Q-->T) and A3 (CII 245-270s269P-->A), induced partial tolerance judged by intermediate degrees of suppression of arthritis. We conclude that discrete alterations in specific amino acid residues of antigenic peptides had profound effects on T cell signaling and that the signaling correlated with T cell cytokine secretion and T cell function in the induction of tolerance and suppression of arthritis.

Efficacy of modified recombinant type II collagen in modulating autoimmune arthritis.

OBJECTIVE: Previous studies have shown that an analog peptide of the immunodominant T cell determinant of type II collagen (CII), i.e., CII(256-276)(N(263), D(266)), was able to suppress the immune response to CII and the development of arthritis in DR1-transgenic mice. The present study tested the hypothesis that introduction of the same amino acid substitutions into full-length CII might improve the efficacy of the mutant collagen in achieving suppression of autoimmune arthritis. METHODS: Using recombinant technology, full-length CII was modified, while the native conformation was retained. Two point mutations were introduced within the immunodominant T cell determinant to convert the F(263) to N and E(266) to D, using a baculovirus expression system that has previously been utilized in the production of recombinant CII (rCII). RESULTS: The mutant rCII(N(263), D(266)) was capable of reducing the incidence and severity of arthritis as well as the antibody response to CII when administered to DR1-transgenic mice that display susceptibility to collagen-induced arthritis. More importantly, it was significantly more effective than the synthetic analog peptide, CII(256-276)(N(263), D(266)). Its mechanism of suppression may be explained by the secretion of predominantly Th2 cytokines by the T cells immunized with rCII(N(263), D(266)). Administration of rCII(N(263), D(266)) was ineffective in suppressing arthritis in IL4(-/-) mice, suggesting that the profound suppressive effects of rCII(N(263), D(266)) were mediated through the production of interleukin-4. CONCLUSION: These findings describe a promising specific immunotherapy for patients with DR1-mediated autoimmunity to CII.

Truncation of the A alpha chain of MHC class II molecules results in inefficient antigen presentation to antigen-specific T cells.

Antigen presenting cells (APC) expressing MHC class II molecules composed of chains with part or all of the cytoplasmic domains deleted are inefficient at presenting hen egg lysozyme peptides to antigen specific T cell hybrids compared with APC that express wild-type MHC class II molecules. This effect is most apparent for mutants in which the alpha chain has been truncated. The inefficiency in antigen presentation can be amplified by pulsing the APC for 4 h with peptide rather than having peptide present throughout the presentation assay. Fixation of antigen-pulsed APC improves the capacity of APC with truncated class II molecules to stimulate T cell hybrids. Fixation of APC prior to exposure to antigen also leads to significant improvement in antigen presentation by the truncated class II molecules. Because the inefficiency of a given hybrid for antigen presentation does not correlate with its ability to transduce a signal as measured by protein kinase C translocation, we suggest that defects in this pathway are not the only cause of impaired antigen presentation. However, because previous studies have demonstrated the need for an intact cytoskeleton for successful antigen presentation, we propose that the carboxy truncated class II molecules are inefficient in antigen presentation because they are unable to generate the signal that ultimately leads to their interaction with the cytoskeleton. These observations underscore the complexity of the events that are required for achieving effective interactions between MHC class II molecules and TCR, and suggest, with regard to efficient antigen presentation, that the physical state of the class II molecules is at least as important as their signal transducing capacity.

Characterization of a peptide analog of a determinant of type II collagen that suppresses collagen-induced arthritis.

Immunization of susceptible strains of mice with type II collagen (CII) elicits an autoimmune arthritis known as collagen-induced arthritis (CIA). One analogue peptide of the immunodominant T cell determinant, A9 (CII245-270 (I260-->A, A261-->B, F263-->N)), was previously shown to induce a profound suppression of CIA when coadministered at the time of immunization with CII. In the present study, A9 peptide was administered i.p., orally, intranasally, or i.v. 2 to 4 wk following CII immunization. We found that arthritis was significantly suppressed even when A9 was administered after disease was induced. To determine the mechanism of action of A9, cytokine responses to A9 and wild-type peptide A2 by CII-sensitized spleen cells were compared. An increase in IL-4 and IL-10, but not in IFN-gamma, was found in A9 culture supernatants. Additionally, cells obtained from A9-immunized mice produced higher amounts of IL-4 and IL-10 when cultured with CII compared with cells obtained from mice immunized with A2, which produced predominantly IFN-gamma. Suppression of arthritis could be transferred to naive mice using A9-immune splenocytes. Lastly, phosphorylation of TCRzeta was not altered in the immunoprecipitates from the lysates of cells exposed to analogue peptides (A9 and A10) together with wild-type A2 in a T cell line and two I-Aq-restricted, CII-specific T hybridomas. We conclude that analogue peptide A9 is effective in suppressing established CIA by inducing T cells to produce a Th2 cytokine pattern in response to CII.

Intravenous tolerization with type II collagen induces interleukin-4-and interleukin-10-producing CD4+ T cells.

Intravenous (i.v.) administration of type II collagen (CII) is an effective way to induce tolerance and suppress disease in the collagen-induced arthritis (CIA) model. In this study, we demonstrated that a single i.v. dose of CII (as low as 0.1 mg/mouse) completely prevented the development of CIA. This suppression was accompanied by decreases in levels of antibody specific for the immunogen, bovine CII and autoantigen, mouse CII. Splenocytes obtained from CII-tolerized mice and stimulated with CII in vitro produced predominantly the T helper 2 (Th2)-type cytokines interleukin-4 (IL-4) and interleukin-10 (IL-10). In contrast, cells obtained from mice immunized with CII produced predominantly interferon-gamma (IFN-gamma). Two-colour flow cytometric analysis of cytokine expression and T-cell phenotype demonstrated that CD4+ cells and not CD8+ or gammadelta+ cells were the predominant regulatory cells producing IL-4 and IL-10. Transgenic mice bearing a T-cell receptor (TCR) specific for CII had a greater increase in the number of IL-4-secreting CD4+ cells, as well as a marked increase of IL-4 in culture supernatants. This cytokine was produced by transgene-bearing T cells. Elucidation of mechanisms for the induction of tolerance in mature T cells is an important line of study in autoimmune models because of the potential application for treating organ-specific autoimmune disease.