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Juvenile idiopathic arthritis (JIA) is a heterogeneous autoimmune disease characterized by chronic joint inflammation. Knowing which antigens drive the autoreactive T-cell response in JIA is crucial for the understanding of disease pathogenesis and additionally may provide targets for antigen-specific immune therapy. In this study, we tested 9 self-peptides derived from joint-related autoantigens for T-cell recognition (T-cell proliferative responses and cytokine production) in 36 JIA patients and 15 healthy controls. Positive T-cell proliferative responses (stimulation index > or =2) to one or more peptides were detected in peripheral blood mononuclear cells (PBMC) of 69% of JIA patients irrespective of major histocompatibility complex (MHC) genotype. The peptides derived from aggrecan, fibrillin, and matrix metalloproteinase (MMP)-3 yielded the highest frequency of T-cell proliferative responses in JIA patients. In both the oligoarticular and polyarticular subtypes of JIA, the aggrecan peptide induced T-cell proliferative responses that were inversely related with disease duration. The fibrillin peptide, to our knowledge, is the first identified autoantigen that is primarily recognized in polyarticular JIA patients. Finally, the epitope derived from MMP-3 elicited immune responses in both subtypes of JIA and in healthy controls. Cytokine production in short-term peptide-specific T-cell lines revealed production of interferon-gamma (aggrecan/MMP-3) and interleukin (IL)-17 (aggrecan) and inhibition of IL-10 production (aggrecan). Here, we have identified a triplet of self-epitopes, each with distinct patterns of T-cell recognition in JIA patients. Additional experiments need to be performed to explore their qualities and role in disease pathogenesis in further detail.

Therapeutic intervention in experimental autoimmune diseases by modulation of the T cell mediated autoimmune response has been accomplished in the past using altered peptide ligands (APLs). These peptides are usually created by applying alterations to the T cell epitope recognized by the autoaggressive T cells. In this study, we investigated whether it was possible to design APLs in a rational way, using knowledge of molecular interaction in the MHC-peptide-T cell receptor (TCR) complex, for the therapeutic intervention in experimental autoimmune encephalomyelitis (EAE). Additionally, the value of peptidomimetic modification and alterations based on posttranslational modifications for the design of APLs was examined. Based on a molecular model of the MHC-peptide complex, the T cell receptor contact residues were identified and selected alterations were applied. The designed APLs were tested for MHC binding capacity, T cell recognition, blocking of the autoreactive T cell response, immunogenicity, encephalitogenicity, and therapeutic activity. Based on the results of the in vitro assays, it was expected that some of our APLs would be able to modulate EAE. Nevertheless, none of these APLs displayed clear therapeutic activity in vivo. Thus, rational design of modified peptides for immunotherapy has to await further insights into the relationships between structure and peptide/peptidomimetic induced T cell activation, and until that, there is no possibility to take advantage of the tailor made origin of peptidomimetics.

T cells have an important role during the development of autoimmune diseases. In adjuvant arthritis, a model for rheumatoid arthritis, we found that the percentage of CD4+ T cells expressing the activation marker CD134 (OX40 antigen) was elevated before disease onset. Moreover, these CD134+ T cells showed a specific proliferative response to the disease-associated epitope of mycobacterial heat shock protein 60, indicating that this subset contains auto-aggressive T cells. We studied the usefulness of CD134 as a molecular target for immune intervention in arthritis by using liposomes coated with a CD134-directed monoclonal antibody as a drug targeting system. Injection of anti-CD134 liposomes subcutaneously in the hind paws of pre-arthritic rats resulted in targeting of the majority of CD4+CD134+ T cells in the popliteal lymph nodes. Furthermore, we showed that anti-CD134 liposomes bound to activated T cells were not internalized. However, drug delivery by these liposomes could be established by loading anti-CD134 liposomes with the dipalmitate-derivatized cytostatic agent 5'-fluorodeoxyuridine. These liposomes specifically inhibited the proliferation of activated CD134+ T cells in vitro, and treatment with anti-CD134 liposomes containing 5'-fluorodeoxyuridine resulted in the amelioration of adjuvant arthritis. Thus, CD134 can be used as a marker for auto-aggressive CD4+ T cells early in arthritis, and specific liposomal targeting of drugs to these cells via CD134 can be employed to downregulate disease development.

MHC class II-peptide multimers are a valuable tool for antigen-specific detection of CD4(+) T cells. However, it has been proposed that T cells in a hypo-responsive state can have diminished binding of such multimers. In the present study, we investigated this phenomenon at the clonal level. We found that anergic CD4(+) T cells had a reduced capacity to bind MHC class II-peptide multimers compared to their non-anergic counterparts. Increasing the incubation temperature, time, or MHC-peptide valency could not equalize multimer binding by anergic and non-anergic T cells. Neither anergic T cells nor non-anergic T cells internalized the MHC class II-peptide dimers efficiently, and in both cases the dimers bound to the plasma membrane at locations containing a low amount of raft-associated lipids. Disruption of lipid rafts, however, led to decreased dimer binding by non-anergic T cells and to a lesser extent by anergic T cells. Finally, we show that the depth of the anergic state of the T cell, which determines its ability to regulate other T cell responses, correlates with the reduced dimer binding. We here demonstrate for the first time differential MHC class II-peptide multimer binding by regulatory (anergic) and effector T cells with identical TCR.

T cells presenting antigens in the context of MHC class II can induce anergy. In rat CD4+ T cell clones we have shown that depending on the depth of anergy other T cell responses can be inhibited in the presence of professional antigen presenting cells (APCs). This inhibition is cell-contact dependent, and APCs recovered from co-cultures with suppressive anergic T cells are modulated in their capacity to activate T cells. No changes in cell surface expression of MHC molecules, B7-1/B7-2, and OX40L were detected. Remarkably cell clusters formed by anergic T cells appeared to be more tight than clusters of activated T cells, and after fluorescent cell surface labelling of T cells, transfer of label was more profound in co-cultures of anergic T cells and APCs compared to activated T cells and APCs. Previously, it has been shown that activated T cells can absorb molecules from APCs in a unidirectional process. We now have evidence that also APCs can absorb cell surface molecules from T cells during APC-T cell co-cultures. We speculate that the quantity and quality of molecule reshuffling during cross-talk between T cells and APCs play a role in the regulation of the T cell response.

In the present study, the relation between the efficacy of immunotherapy, and the strength and site of T cell activation during immunotherapy was evaluated. We used a model of allergic asthma in which OVA-sensitized and OVA-challenged mice display increased airway hyperresponsiveness, airway inflammation, and Th2 cytokine production by OVA-specific T cells. In this model, different immunotherapy strategies, including different routes of administration, or treatment with entire OVA or the immunodominant T cell epitope OVA(323-339), or treatment with a peptide analogue of OVA(323-339) with altered T cell activation capacity were studied. To gain more insight in how immunotherapy affects allergen-specific T cells, the site of Ag-specific T cell activation and the magnitude of the T cell response induced during different immunotherapy strategies were determined using an adoptive transfer model. Our data suggest that amelioration of airway hyperresponsiveness and inflammation is associated with the induction of a strong, synchronized, and systemic T cell response, resulting in a decreased OVA-specific Th2 response. In contrast, deterioration of the disease after immunotherapy is associated with the induction of a weak nonsynchronized T cell response, resulting in the enhancement of the OVA-specific Th2 response after challenge.

Spontaneous onset of pancreatic beta cell destruction in the nonobese diabetic (NOD) mouse is preceded by the induction of autoreactive T cells, which recognize a variety of autoantigens. The 60-kDa endogenous (murine) heat shock protein 60 (hsp60) has been proposed to be one of the key autoantigens. Here we demonstrate that subcutaneous immunization of normoglycemic NOD mice with highly homologous mycobacterial or murine hsp60 activates T cells in the spleen that produce high levels of IL-10 upon restimulation in vitro with either hsp60 protein. In time, increasing levels of hsp60-induced IL-10 could be detected in NOD mice, but not in age- and MHC class II-matched BiozziABH mice, which lack any sign of pancreatic inflammation. These results suggest that the IL-10 responses in NOD mice are primarily driven by endogenous inflammation. Genetically protected NOD-asp mice, showing a less progressive development of insulitis, demonstrated a similar increase in hsp60-induced IL-10 in time compared with wild-type NOD mice. Taken together, our results suggest that endogenous hsp60 is not a primary autoantigen in diabetes but is possibly associated with regulation of insulitis. Moreover, the capacity to respond to (self) hsp60 is independent of the MHC class II-associated genetic predisposition to diabetes.

T cell anergy is one of the mechanisms leading to the establishment and maintenance of peripheral tolerance. Recent data from our and other laboratories indicate that anergic T cells are not functionally inert but in fact are capable of regulating the immune response in an active manner. In this review, we describe our viewpoint on how anergic self-reactive T cells could contribute to regulation of the immune response.

Purpose: The aim of this study was to use small unilamellar liposomes with incorporated MHC II/peptide complexes as a carrier system for multivalent antigen presentation to CD4 + T cells., Methods: Purified peptide pre-loaded MHC II molecules were incorporated into small unilamellar liposomes and tested for their ability to activate A2b T cells. The outcome of T cell activation by such liposomes in the absence of accessory cells was tested via flow cytometry and a T cell anergy assay., Results: Provided the presence of external co-stimulation, MHC II/ peptide liposomes were able to induce proliferation of the A2b T cell clone. More importantly incubation of these T cells with MHC II/ peptide liposomes in the absence of co-stimulation did not induce proliferation, however, a MHC/peptide ligand-density dependent down-regulation of the TCR was observed. Interestingly, when T cells after incubation with the MHC II/peptide liposomes were restimulated with their specific antigen in the presence of professional APC, these cells were anergic., Conclusions: We propose MHC II/peptide liposomes as a novel means to induce T cell anergy. The possibility to prepare 'tailor-made' liposomal formulations may provide liposomes with an important advantage for applications in immunotherapy.

Current therapies for autoimmune diseases and allergy involve general immune suppression. However, the ideal therapy should specifically eliminate or modulate the (auto)pathogenic immune response or, alternatively, it should reinforce the regulatory response, without affecting the overall function of the immune system. This could be achieved by antigen-specific immunotherapy. Antigen-specific immunotherapy has received ample attention in the last years, and several clinical trials attempting to treat autoimmune diseases or allergy through the induction of antigen-specific tolerance or immune deviation have been conducted, albeit with varying success. Recent advances in our understanding of peripheral tolerance, regulatory T cells, and routes of antigen administration have resulted in better insight into the different working mechanisms and potential target molecules of antigen-specific immunotherapy. The experimental animal models and new technological developments force the pace in the development of these immunotherapies. The current review addresses several aspects of antigen-specific immunotherapies and focuses on the mechanisms of the different approaches in experimental autoimmune and allergy models.

We compared the effects of antigen (Ag) presentation by T cells and professional antigen-presenting cells (APC) on T cell proliferation, cytokine production and surface molecule expression. Ag presentation by T cells (T-T presentation) induced an initial T cell activation phase as measured by proliferation and IL-2 production. These activated T cells became anergic upon antigenic restimulation by professional APC, as shown by a failure to proliferate or produce IL-2 or IFN-gamma. Interestingly, such T cells were not intrinsically defective in their signal transduction pathways since they did proliferate and produce cytokines upon restimulation with mitogenic stimuli. Flow cytometric analysis revealed a more profound TCR and CD3 down-regulation during T-T presentation than during APC-T presentation. However, no up-regulation of CD80, CD86, CD45RC and OX40 (CD134) was observed on T cells during T-T presentation or subsequent antigenic restimulation of anergic T cells in the presence of professional APC, whereas increased expression of these molecules was observed during professional APC-T presentation of non-anergic T cells. The impaired expression of co-stimulatory and activation molecules on T cells after T-T presentation of Ag might lead to altered interactions between T cells and professional APC upon antigenic restimulation. We propose that T cell anergy is a functional consequence of these altered T cell-APC interactions.

T cell anergy has been proposed as one of the mechanisms underlying peripheral T cell tolerance. In recent years, the functional relevance of T cell anergy has been studied extensively in vitro and in vivo, using different species, cell systems, and ways to induce anergy. Although these studies concurred about the induction of unresponsiveness, conflicting findings were obtained with respect to the function of anergic T cells and to the persistence of T cell anergy. In the present study, T cell anergy was induced through T-T presentation of the specific Ag by rat MHC class II+ T cells in the absence of professional APC. We show that, depending on the Ag dose with which T cells were incubated, distinct anergic phenotypes were induced. Incubation of T cell clones with a low (suboptimal) Ag dose induced hyporesponsiveness. Incubation with a higher (optimal) Ag dose induced an anergic state capable of exerting immunoregulatory effects. Incubation with a high (supraoptimal) Ag dose led to an anergic suppressive phenotype that was persistent and was not reversed by APC, Ag, and rIL-2. These findings demonstrate that T cell anergy is not confined to a single state of functional inactivation. Instead, multiple levels of T cell anergy exist. Thus, anergic T cells can contribute to the regulation of the immune response either in a persistent and active manner or in a passive manner, depending on their level of T cell anergy.

Adjuvant arthritis (AA) in Lewis rats is T-cell mediated and seems to depend on T cells recognising the 180-188 epitope of mycobacterial heat-shock protein (hsp) 60. Analysis of arthritogenic T-cell clone A2b has revealed a mimicry of this particular epitope with an articular cartilage-associated target T-cell epitope. Nasal administration of synthetic peptides covering this 180-188 sequence led to epitope-specific tolerance and resistance to AA. Since this tolerisation protocol also inhibited avridine arthritis, one may conclude that this form of epitope-specific tolerance had effectuated a spreading tolerisation at the level of target antigens that included a diverse set of possible arthritis-associated antigens. In vitro anergised T cells exhibited suppressive activity in a co-culture system. As in this case--depending on the presence of the antigen of the anergic T cell--such T cells suppressed responder T cells of a different antigenic specificity, we postulated that anergic T cells may be responsible for a spreading of tolerance. It seemed that such spreading of tolerance was channelled through the antigen-presenting cells (APC) and was dependent on direct cell-cell contact. This and additional forms of spreading of tolerance could be responsible for specific nasal tolerance, causing inhibition of the development of an arthritogenic inflammatory response. This can be similarly the case for the arthritis protection that resulted from immunisation with hsps. Analysis of T-cell responses following hsp immunisations revealed that the arthritis inhibitory activity resided in T cells with specificity for a conserved part of microbial hsp 60. The same T cells cross-responded to rat self-hsp60. Low level expression of the latter molecule on non-professional APC could possibly have induced a suppressive anergic state in these autoreactive cells. Thus, immunisation with microbial hsp would have led to an expansion of such T cells, leading to raised disease-suppressive potential when selectively trapped and activated in the inflamed self-hsp-overexpressing joint. Alternatively, the cross-recognised self-hsp epitope could have the regulatory qualities of an altered peptide ligand or a partial agonist for T cells that see the microbial homologue as the full agonist.

Tolerance for self has appeared incomplete for many self antigens. We have obtained experimental evidence that both for self heat shock proteins and T cell receptor V-gene products, reactive T cells are part of the normal immune repertoire. Furthermore, it has become apparent that stimulation of T cell responsiveness to these antigens, by using peptide immunisation or by transfer of activated T cells, raises resistance to experimentally induced autoimmune arthritis. In addition, available evidence has suggested that these reactivities may be functional during natural processes of disease remission. The observations with regard to heat-shock proteins have indicated that mechanism leading to disease resistance are most efficiently triggered by exposing the immune system to non-self antigens such as bacterial hsp's, which are similar to, but not identical to, self. Experimental evidence has been obtained, that conserved bacterial hsp peptides, may trigger self hsp reactive T cells, with disease suppressive regulatory potential. It is possible that such self hsp reactive T cells, being expanded by recognising bacterial peptides as full agonists, do, in fact, perceive the self epitopes as partial agonists, and therefore have the possibility of displaying downregulatory activity at the site of inflammation. Experiments with peptide analogs of self epitopes, being variants of disease critical T cell epitopes, have indeed suggested that also their activity in modulating disease may comply with the principles of dominant immunological tolerance.

In the present report, we studied the presence and functional significance of major histocompatibility complex (MHC) class II antigen on rat T cells. Most rat T-cell lines cultured in vitro were found to be MHC class II+. Also, these T-cell lines were shown to synthesize MHC class II molecules. Immunohistochemical and flow cytometric double stainings for T-cell receptor (TCR) and MHC class II showed that in vivo as well a large proportion of T cells was MHC class II+. The immunohistochemical staining of spleen sections enabled us to characterize the MHC class II+ and MHC class II- T cells. It was shown that resting T cells in vivo were MHC class II-. In contrast, activated T cells, as determined by their localization in the marginal zone of the spleen, proved to be MHC class II+. Finally, T-cell clones were found to be able to present peptidic antigens, but could only poorly present more complex exogenous antigens, probably due to inefficient uptake of such antigens. These features would endow activated rat T cells with the capacity to present cell-specific self-proteins, such as TCR, to regulatory CD4+ MHC class II-restricted T cells, as was described by our group elsewhere.

New strategies applied in the treatment of experimental autoimmune disease models involve blocking or modulation of MHC-peptide-TCR interactions either at the level of peptide-MHC interaction or, alternatively, at the level of T cell recognition. In order to identify useful competitor peptides one must be able to assess peptide-MHC interactions. Several well described autoimmune disease models exist in the Lewis rat and thus this particular rat strain provides a good model system to study the effect of competitor peptides. So far no information has been available on the peptide binding characteristics of the Lewis rat MHC class II RT1.B1 molecule. We have now developed a biochemical binding assay which enables competition studies in which the relative MHC binding affinity of a set of non-labelled peptides can be assessed while employing detection of biotinylated marker peptides by chemiluminescence. The assay is sensitive and specific. We have used this assay to determine the binding characteristics of several disease associated T cell determinants and their sequence analogues in the Lewis rat. Notably, most of the autoimmune disease associated peptide sequences tested were found to be intermediate to poor binders. Single amino acid substitutions at defined positions were sufficient to turn certain peptides into good binders. These results are relevant to the design of competitor peptides in the treatment of experimental autoimmune diseases.

T cells specific for the mycobacterial 65 kDa heat-shock protein (hsp65) play a pivotal role in the development of adjuvant arthritis (AA) in Lewis rats. Upon adoptive transfer, CD4+ T cells recognizing a particular hsp65 epitope trigger the onset of disease. Activation of hsp65-reactive T cells can be achieved by immunization with heat-killed mycobacteria in mineral oil--complete Freund's adjuvant (CFA)--or with purified recombinant hsp65. Arthritis, however, will only develop after immunization with CFA. In fact, preimmunization with hsp65 protects against any subsequent attempt to induce AA. In this study, we examined polyclonal lymph node cell responses in Lewis rats, immunized with either CFA or purified recombinant hsp65 in incomplete Freund's adjuvant, to a set of hsp65 fragments generated by a mild digestion with cathepsin D. Proliferative responses to several hsp65 fragments varied with the type of antigen used for immunization. A cathepsin D-released fragment, identified as residues 376-408, preferentially triggered proliferation of rat T cells after hsp65 immunization. Preimmunization of Lewis rats with this peptide delayed the onset and reduced the severity of AA. Preimmunization with another fragment which was preferentially recognized after CFA immunization, representing residues 40-60, did not have such a protective effect. Our findings suggest the presence of mycobacterial hsp65 determinants that selectively trigger AA-regulating T cells and illustrate that cathepsin D may be used as an experimental tool to generate such determinants.

The immune system has the potential to utilize a diverse T cell repertoire for the recognition of Ag in the context of MHC molecules. Here we describe the analysis of two rat T cell clones, both of which recognize a synthetic peptide comprised of the arthritis-associated 180-188 amino acid sequence of the mycobacterial 65-kDa heatshock protein (hsp65 180-188), but which differ in the recognition of the naturally processed hsp65. The arthritogenic T cell clone A2b, generated by immunization with whole Mycobacterium tuberculosis, recognized the hsp65 180-188 synthetic peptide as well as the processed hsp65, whereas T cell clone ATL11, generated after immunization with a single amino acid substituted peptide analog of hsp65 180-188, recognized peptide hsp65 180-188 but not the processed hsp65. For both T cell clones the minimal stimulatory sequence was hsp65 180-186. However, within this minimal stimulatory sequence marked differences between the clones were found with regard to peptide residues interacting with the TCR. Furthermore, addition of extra residues at the N terminus of the hsp65 180-186 sequence abrogated the recognition by clone ATL11, but not by A2b. These findings demonstrate that, upon in vivo immunization with a synthetic peptide comprised of a single amino acid variant of a T cell epitope, T cells can be triggered that recognize a peptide comprised of the original epitope sequence, but that do not recognize this epitope in its naturally processed protein fragment. The possibility of triggering such T cells by immunization with synthetic peptides, may well have consequences for the design of peptide vaccines or peptide immunomodulatory agents.

Previously, fusion of established T cell lines or clones has been claimed to be difficult. We now report our experiences in the fusion of both long term cultures of rat T cell clones and mouse T cell lines to rat W/Fu (C58NT)D. Upon fusion of rat T cell clones the hybrids obtained expressed antigen specificities identical to those of the parent clones. In addition, C58 was used for interspecies hybridisation of murine T cell lines. The specificity of intra- and inter-species hybrids was maintained by subcloning. We conclude that the C58 cell line can be used to generate continuously growing monoclonal T-cell reagents of sufficient stability using both intra- and inter-species hybridisation.

Adjuvant arthritis (AA) can be induced in genetically susceptible rats by immunization with heat-killed mycobacteria suspended in mineral oil. From our analysis of arthritogenic T cell clone A2b, obtained from an arthritic Lewis rat and specific for the 180-188 epitope of mycobacterial 65-kDa heat-shock protein (hsp 65), the possible origin of AA was explained by the existence of a molecular mimicry of the 180-188 epitope with a cartilage-associated self antigen. We now have shown that Lewis rats respond to the 180-188 epitope after Mycobacterium tuberculosis immunization and that arthritis-resistant Fisher and (Lewis x Fisher)F1 rats, although major histocompatibility complex class II identical with Lewis, do not respond to this epitope. However, in rare cases of arthritis in Fisher rats, responses to the epitope were seen. We obtained no evidence for a defect at the level of antigen processing and presentation or for suppression in Fisher rats. Thus, non-responsiveness in Fisher rats was likely due to a difference at the level of the T cell repertoire. Previously, we have reported that pretreatment with hsp 65 in experimental arthritis, and not only in AA, caused resistance to arthritis induction. We now present evidence that immunization with hsp 65 or in vitro stimulation with hsp 65 may lead to inhibition of responses specific for epitope 180-188. Thus the hsp 65-induced resistance to arthritis is probably caused by the induction of regulatory control specifically targeted at the 180-188 epitope. Especially in rats that tend to focus their responses on the critical 180-188 sequence, such as Lewis, regulation seems to develop following immunization with hsp 65. Since recent evidence suggests that hsp 65 and also the 180-188 epitope have a role in human arthritic conditions, the present findings are expected to contribute to further experimentation directed at exploiting hsp 65 or its epitopes for the development of new therapeutical approaches in humans.