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Parasite proteins containing repeats are essential invasion ligands, important for their ability to evade the host immune system and to induce immunosuppression. Here, the intrinsic suppressive potential of repetitive structures within parasite proteins was exploited to induce immunomodulation in order to establish self-tolerance in an animal model of autoimmune neurological disease. We tested the tolerogenic potential of fusion proteins containing repeat sequences of parasites linked to self-antigens. The fusion constructs consist of a recombinant protein containing repeat sequences derived from the S-antigen protein (SAg) of Plasmodium falciparum linked to a CD4 T cell epitope of myelin. They were tested for their efficacy to control the development of experimental autoimmune encephalomyelitis (EAE), In addition, we used the DO11.10 transgenic mouse model to study the immune mechanisms involved in tolerance induced by SAg fusion proteins. We found that repeated sequences of P. falciparum SAg protein linked to self-epitopes markedly protected mice from EAE. These fusion constructs were powerful tolerizing agents not only in a preventive setting but also in the treatment of ongoing disease. The tolerogenic effect was shown to be antigen-specific and strongly dependent on the physical linkage of the T cell epitope to the parasite structure and on the action of anti-inflammatory cytokines like IL-10 and TGF-β. Other mechanisms include down-regulation of TNF-α accompanied by increased numbers of FoxP3+ cells. This study describes the use of repetitive structures from parasites linked to defined T cell epitopes as an effective method to induce antigen-specific tolerance with potential applicability for the treatment and prevention of autoimmune diseases.

Despite tremendous improvements in short-term renal allograft survival, many patients still have chronic rejection or side effects of nonspecific immunosuppression. The discovery of Foxp3(+) regulatory T cells (Tregs) has revolutionized the concepts in immunoregulation and offers perspectives for overcoming rejection. Recently, a subset of Foxp3(+)CD39(+) effector/memory-like Tregs (T(REM)) was identified. The role of CD39(+) Tregs in immunoregulation is supported by the occurrence of alopecia areata and experimental autoimmune encephalomyelitis in CD39-deficient mice and by the failure of CD39(-) Tregs to suppress contact hypersensitivity. In humans, CD39 polymorphisms have been associated with diabetes and nephropathy, and multiple sclerosis patients have reduced numbers of blood CD39(+) Tregs. Preliminary experiments in a murine transplantation model showed that CD39(+) Tregs can determine allograft outcome. CD39 degrades the extracellular adenosine triphosphate (ATP) released during tissue injury, which otherwise would trigger inflammation. Currently, our groups are assessing the role of CD39(+) Tregs and extracellular ATP metabolism in clinical transplantation and whether tolerogenic Treg profiles possess immunopredictive value, envisioning the development of clinical trials using CD39(+) Treg-based vaccination for autoimmunity or transplantation. This is a comprehensive review on the fundamentals of Treg biology, the potential role of ATP metabolism in immunoregulation, and the potential use of Treg-based immunotherapy in transplantation.

Immunotherapy by using multimerized self-peptides has demonstrated a clear protective effect on experimental models of autoimmune diseases. However, the mechanisms involved remain ill-defined. Here we have evaluated the therapeutic efficacy of multimerized self-peptides at the effector phase of autoimmune diabetes and examined their mechanisms of action. Diabetes was induced in rat insulin promoter-hemagglutinin (HA) mice expressing HA in pancreatic β-cells by adoptive transfer of HA 110–119 -specific T helper 1 cells. Complete protection was provided by low doses of the HA 4-mer consisting of four covalently linked linear HA 107–119 peptides. In vivo , the 4-mer appeared to act directly on the pathogenic HA-specific T helper 1 cells and indirectly by activation/recruitment of lymphocytes with regulatory properties so that mice became resistant to a second transfer of diabetogenic T cells. This effect was associated with a recruitment of Foxp3 + CD4 T cells around islets. Moreover, we show that dominant protection from autoimmunity was transferable by spleen cells, and that development of this regulatory population was crucially dependent on the lymphocytes from treated rat insulin promoter-HA mice. Thus, immunotherapy using multimerized epitopes emerges as a promising strategy in view of the current identification of self-epitopes that are major targets of the pathogenic CD4 T cell response in autoimmune diseases.

Major histocompatibility complex (MHC) molecules are a key element of the cellular immune response. Encoded by the MHC they are a family of highly polymorphic peptide receptors presenting peptide antigens for the surveillance by T cells. We have shown that certain organic compounds can amplify immune responses by catalyzing the peptide loading of human class II MHC molecules HLA-DR. Here we show now that they achieve this by interacting with a defined binding site of the HLA-DR peptide receptor. Screening of a compound library revealed a set of adamantane derivatives that strongly accelerated the peptide loading rate. The effect was evident only for an allelic subset and strictly correlated with the presence of glycine at the dimorphic position beta86 of the HLA-DR molecule. The residue forms the floor of the conserved pocket P1, located in the peptide binding site of MHC molecule. Apparently, transient occupation of this pocket by the organic compound stabilizes the peptide-receptive conformation permitting rapid antigen loading. This interaction appeared restricted to the larger Gly(beta86) pocket and allowed striking enhancements of T cell responses for antigens presented by these "adamantyl-susceptible" MHC molecules. As catalysts of antigen loading, compounds targeting P1 may be useful molecular tools to amplify the immune response. The observation, however, that the ligand repertoire can be affected through polymorphic sites form the outside may also imply that environmental factors could induce allergic or autoimmune reactions in an allele-selective manner.

The asparagine-specific endoprotease (AEP) controls lysosomal processing of the potential autoantigen myelin basic protein (MBP) by human B lymphoblastoid cells, a feature implicated in the immunopathogenesis of multiple sclerosis. In this study, we demonstrate that freshly isolated human B lymphocytes lack significant AEP activity and that cleavage by AEP is dispensable for proteolytic processing of MBP in this type of cell. Instead, cathepsin (Cat) G, a serine protease that is not endogenously synthesized by B lymphocytes, is internalized from the plasma membrane and present in lysosomes from human B cells where it represents a major functional constituent of the proteolytic machinery. CatG initialized and dominated the destruction of intact MBP by B cell-derived lysosomal extracts, degrading the immunodominant MBP epitope and eliminating both its binding to MHC class II and a MBP-specific T cell response. Degradation of intact MBP by CatG was not restricted to a lysosomal environment, but was also performed by soluble CatG. Thus, the abundant protease CatG might participate in eliminating the immunodominant determinant of MBP. Internalization of exogenous CatG represents a novel mechanism of professional APC to acquire functionally dominant proteolytic activity that complements the panel of endogenous lysosomal enzymes.

Astrocytic production of nerve growth factor (NGF) is increased during inflammation of the central nervous system (CNS). Here we show that cell–cell interaction between primary murine astrocytes and myelin basic protein (MBP)-specific T cell receptor (TCR) transgenic Th1 and Th2 cells significantly increased production of NGF. This upregulation was found to be dependent on antigen recognition. Neutralization of cytokines produced in cocultures did not affect NGF production. This novel finding suggests a neuroprotective role of astrocytes during T cell-mediated inflammation in the CNS.

Small-molecular compounds with hydrogen bond (H-bond) donor function are able to trigger exchange reactions of MHC class II ligands. Here, we show that their effect is not limited to short peptides. Also encephalitogenic myelin basic protein (MBP) is transferred with great efficiency onto HLA-DR molecules when H-bond donor molecules such as parachlorphenol (pCP) are present. The effect was observed not only with soluble MHC class II but also with HLA-DR1 and HLA-DR2 molecules on the cell surface. The improved loading of APC translates directly into improved T cell activation. In the presence of pCP T cells reacted at significantly lower antigen concentrations, an effect observed with purified MBP protein as well as with crude spinal cord homogenate. The 'accidental' transfer of autoantigens such as MBP onto activated APC might trigger fatal autoimmune reactions and small molecules as catalysts of this process could represent risk factors, which had not been accounted for as yet.

Eosinophils in tissues are often present in intimate contact with T cells in allergic and parasitic diseases. Resting eosinophils do not express MHC class II proteins or costimulatory B7 molecules and fail to induce proliferation of T cells to Ags. IL-5 and GM-CSF induce MHC class II and B7 expression on eosinophils and have been reported in some studies to induce eosinophils to present Ag to T cells. The cytokine IL-3, like IL-5 and GM-CSF, is a survival and activating factor for eosinophils and the IL-3 receptor shares with the IL-5 and GM-CSF receptors a common signal transducing β-chain. IL-3-treated eosinophils expressed HLA-DR and B7.2, but not B7.1 on their surface and supported T cell proliferation in response to the superantigen toxic shock syndrome toxin 1, as well as the proliferation of HLA-DR-restricted tetanus toxoid (TT) and influenza hemagglutinin-specific T cell clones to antigenic peptides. This was inhibited by anti-B7.2 mAb. In contrast, IL-3-treated eosinophils were unable to present native TT Ag to either resting or TT-specific cloned T cells. In parallel experiments, eosinophils treated with IL-5 or GM-CSF were also found to present superantigen and antigenic peptides, but not native Ag, to T cells. These results suggest that eosinophils are deficient in Ag processing and that this deficiency is not overcome by cytokines that signal via the β-chain. Nevertheless, our findings suggest that eosinophils activated by IL-3 may contribute to T cell activation in allergic and parasitic diseases by presenting superantigens and peptides to T cells.

In a previous study we reported that oligomerized T cell epitopes "superactivated" CD4+ T cells. These oligomers, consisting of 12-16 copies of a peptide epitope derived from the hemagglutinin protein of influenza virus (HA306-318), induced a specific T cell response in amounts as little as 5 pg/ml. We now show that the improved antigenicity of these multimerized epitopes can also be utilized to induce "high zone tolerance". Tolerization, similar to activation, occurred at about 3 logs lower concentration of oligomer than of peptide. HA306-318-specific T cell cultures became nonresponsive to stimulation with peptide after incubation with 0.5-5 microg/ml HA306-318 12-mer. The nonresponsiveness was accompanied by a drastic down-regulation of the TCR and by T cell elimination by apoptotic cell death. In contrast, stimulation with peptide even at 50 microg/ml led to temporary induction of anergy. Consequently, induction of tolerance with the oligomer was permanent and no recovery of the cultures was seen in recall experiments 12-14 days after high zone exposure to the 12-mer.

T cell epitope peptides derived from proteolipid protein (PLP139–151) or myelin basic protein (MBP86–100) induce experimental autoimmune encephalomyelitis (EAE) in “susceptible” strains of mice (e.g., SJL/J). In this study, we show that the encephalitogenic effect of these epitopes when injected subcutaneously in complete Freund's adjuvant was significantly enhanced if administered to the animal in a multimerized form as a T cell epitope oligomer (i.e., as multiple repeats of the peptide epitope, such as 16-mers). Oligomer-treated SJL/J mice developed EAE faster and showed a more severe progression of the disease than animals treated with peptide alone. In addition, haplotype-matched B10.S mice, “resistant” to EAE induction by peptide, on injection of 16-mers developed a severe form of EAE. Even more striking, however, was the dramatic suppression of incidence and severity of the disease, seen after single intravenous injections of only 50 μg of the PLP139–151 16-mer, administered to SJL/J mice 7 d after the induction of the disease. Although relapse occurred at about day 45, an additional injection several days before that maintained the suppression. Importantly, the specific suppressive effect of oligomer treatment was also evident if EAE was induced with spinal cord homogenate instead of the single peptide antigen. By contrast, the PLP139–151 peptide accelerated rather than retarded the progression of disease.

The peptides bound to class II major histocompatibility complex (MHC) molecules extend out both ends of the peptide binding groove. This structural feature provided the opportunity to design multivalent polypeptide chains that cross-link class II MHC molecules through multiple, repetitive MHC binding sites. By using recombinant techniques, polypeptide oligomers were constructed that consist of up to 32 copies of an HLA-DR1-restricted T cell epitope. The epitope HA306–318, derived from influenza virus hemagglutinin, was connected by 12- to 36-aa long spacer sequences. These oligomers were found to cross-link soluble HLA-DR1 molecules efficiently and, upon binding to the MHC molecules of a monocyte line, to trigger signal transduction indicated by the enhanced expression of some cell surface molecules. A particularly strong effect was evident in the T cell response. A hemagglutinin-specific T cell clone recognized these antigens at concentrations up to three to four orders of magnitude lower than that of the peptide or the hemagglutinin protein. Both signal transduction in the monocyte and the proliferative response of the T cell were affected greatly by the length of the oligomer (i.e., the number of repetitive units) and the distance of the epitopes within the oligomer (spacing). Thus, the formation of defined clusters of T cell receptor/MHC/peptide antigen complexes appears to be crucial for triggering the immune response and can be used to enhance the antigenicity of a peptide antigen by oligomerizing the epitope.

BACKGROUND: Autoimmune diseases result from a breakdown in self-tolerance to autoantigens. Self-tolerance is induced and sustained by central and peripheral mechanisms intended to deviate harmful immune responses and to maintain homeostasis, where regulatory T cells play a crucial role. The use of self-antigens in the study and treatment of a range of autoimmune diseases has been widely described; however, the mechanisms underlying the induced protection by these means are unclear. This study shows that protection of experimental autoimmune disease induced by T cell self-epitopes in a multimerized form (oligomers) is mediated by the induction of active suppression. PRINCIPAL FINDINGS: The experimental autoimmune encephalomyelitis (EAE) animal model for multiple sclerosis was used to study the mechanisms of protection induced by the treatment of oligomerized T cell epitope of myelin proteolipid protein (PLP139-151). Disease protection attained by the administration of oligomers was shown to be antigen specific and effective in both prevention and treatment of ongoing EAE. Oligomer mediated tolerance was actively transferred by cells from treated mice into adoptive hosts. The induction of active suppression was correlated with the recruitment of cells in the periphery associated with increased production of IL-10 and reduction of the pro-inflammatory cytokine TNF-{alpha}. The role of suppressive cytokines was demonstrated by the reversion of oligomer-induced protection after in vivo blocking of either IL-10 or TGF-{beta} cytokines. CONCLUSIONS: This study strongly supports an immunosuppressive role of repeat auto-antigens to control the development of EAE with potential applications in vaccination and antigen specific treatment of autoimmune diseases.

Here we report peptide motifs of five HLA-B molecules, B*5101, B*5102, B*5103 B*5201 and B*7801. Motifs were obtained by pool sequencing of natural ligands eluted from the respective molecules expressed in C1R cells upon transfection. A number of individual ligands that could be sequenced confirmed the motifs. All five HLA-B molecules belong to the HLA-B5, B35-cross-reactive group and are closely related as indicated by similar sequences. B51 and B52 are associated with Bw4, whereas B78 is associated with Bw6. One of the HLA-B molecules investigated here, HLA-B*5101, is associated with Behçet's disease, a multisystemic inflammatory disease affecting various organs. This disease is especially frequent among the Japanese population. The subtle differences between the peptide motifs of B*5101 as compared with the motifs of the four other closely related but not disease-associated molecules could shed light on the nature of the HLA-association of Behçet's disease.

Background Class II MHC molecules (MHC II) are cell surface receptors displaying short protein fragments for the surveillance by CD4+ T cells. Antigens therefore have to be loaded onto this receptor in order to induce productive immune responses. On the cell surface, most MHC II molecules are either occupied by ligands or their binding cleft has been blocked by the acquisition of a non-receptive state. Direct loading with antigens, as required during peptide vaccinations, is therefore hindered. Principal Findings Here we show, that the in vivo response of CD4+ T cells can be improved, when the antigens are administered together with ‘MHC-loading enhancer’ (MLE). MLE are small catalytic compounds able to open up the MHC binding site by triggering ligand-release and stabilizing the receptive state. Their enhancing effect on the immune response was demonstrated here with an antigen from the influenza virus and tumour associated antigens (TAA) derived from the NY-ESO-1 protein. The application of these antigens in combination with adamantane ethanol (AdEtOH), an MLE compound active on human HLA-DR molecules, significantly increased the frequency of antigen-specific CD4+ T cells in mice transgenic for the human MHC II molecule. Notably, the effect was evident only with the MLE-susceptible HLA-DR molecule and not with murine MHC II molecules non-susceptible for the catalytic effect of the MLE. Conclusion MLE can specifically increase the potency of a vaccine by facilitating the efficient transfer of the antigen onto the MHC molecule. They may therefore open a new way to improve vaccination efficacy and tumour-immunotherapy.

T lymphocytes recognize their antigen as peptides associated with major histocompatibility complex (MHC) molecules. Peptides naturally presented by MHC class I molecules are uniform in length and have a specific motif, both defined by the respective MHC allele (Falk, K. et al. Nature 1991. 351:290). These allele-specific motifs should allow exact prediction of natural T cell epitopes. H-2Kb-restricted epitopes, for example, have a length of eight amino acid residues and conserved anchor residues at positions 5 and 8. According to this information, we predicted the natural Kb-restricted epitope of ovalbumin, thought to be contained in the 19-mer IINFEKLTEWTSSNVMEER, to be SIINFEKL. Here we show that this prediction is correct. Thus, exact prediction of natural T cell epitopes is possible.

The strong reaction of T cells against foreign major histocompatibility complex (MHC) antigens, commonly termed "alloreactivity", is not only a nuisance for clinical organ transplantation; it also remains a puzzling question for immunologists. By making use of recent technical developments, alloreactive T cells nominally directed against a mutation in a single MHC class I molecule were found to fall into several major categories. One is recognizing peptides whose occurrence is dependent on one particular MHC allele, another is recognizing peptides supported by several MHC alleles, and a third is recognizing peptides occurring independently of MHC alleles. In a fourth category, the binding to MHC of any of a broad range of peptides appears sufficient. In addition, there are T cells for which no peptide involvement could be detected at all. Even within these categories, the heterogeneity of T cells is considerable: among 16 Kb-reactive T cells analyzed, 15 different modes of reactions were found.

Class II MHC molecules display peptides on the cell surface for the surveillance by CD4+ T cells. To ensure that these ligands accurately reflect the content of the intracellular MHC loading compartment, a complex processing pathway has evolved that delivers only stable peptide/MHC complexes to the surface. As additional safeguard, MHC molecules quickly acquire a ‘non-receptive’ state once they have lost their ligand. Here we show now that amino acid side chains of short peptides can bypass these safety mechanisms by triggering the reversible ligand-exchange. The catalytic activity of dipeptides such as Tyr-Arg was stereo-specific and could be enhanced by modifications addressing the conserved H-bond network near the P1 pocket of the MHC molecule. It affected both antigen-loading and ligand-release and strictly correlated with reported anchor preferences of P1, the specific target site for the catalytic side chain of the dipeptide. The effect was evident also in CD4+ T cell assays, where the allele-selective influence of the dipeptides translated into increased sensitivities of the antigen-specific immune response. Molecular dynamic calculations support the hypothesis that occupation of P1 prevents the ‘closure’ of the empty peptide binding site into the non-receptive state. During antigen-processing and -presentation P1 may therefore function as important “sensor” for peptide-load. While it regulates maturation and trafficking of the complex, on the cell surface, short protein fragments present in blood or lymph could utilize this mechanism to alter the ligand composition on antigen presenting cells in a catalytic way.

Major histocompatibility complex (MHC) class I molecules present peptides derived from cellular proteins to cytotoxic T lymphocytes (CTLs), which check these peptides for abnormal features. How such peptides arise in the cell is not known. Here we show that the MHC molecules themselves are substantially involved in determining which peptides occur intracellularly: normal mouse spleen cells identical at all genes but MHC class I express different patterns of peptides derived from cellular non-MHC proteins. We suggest several models to explain this influence of MHC class I molecules on cellular peptide composition.

Multiple Sclerosis (MS) is considered to be a T cell-mediated autoimmune disease. An attractive strategy to prevent activation of autoaggressive T cells in MS, is the use of altered peptide ligands (APL), which bind to major histocompatibility complex class II (MHC II) molecules. To be of clinical use, APL must be capable of resisting hostile environments including the proteolytic machinery of antigen presenting cells (APC). The current design of APL relies on cost- and labour-intensive strategies. To overcome these major drawbacks, we used a deductive approach which involved modifying proteolytic cleavage sites in APL. Cleavage site-directed amino acid substitution of the autoantigen myelin basic protein (MBP) resulted in lysosomal protease-resistant, high-affinity binding peptides. In addition, these peptides mitigated T cell activation in a similar fashion as conventional APL. The strategy outlined allows the development of protease-resistant APL and provides a universal design strategy to improve peptide-based immunotherapeutics.

In the immune system, extracellular ATP functions as a “natural adjuvant” that exhibits multiple proinflammatory effects. It is released by damaged cells as an indicator of trauma and cell death but can be inactivated by CD39 (nucleoside triphosphate diphosphohydrolase-1 [NTPDase 1]), an ectoenzyme that degrades ATP to AMP. Here, we show that CD39 is expressed primarily by immune-suppressive Foxp3+ regulatory T (Treg) cells. In mice, the enzyme is present on virtually all CD4+CD25+ cells. CD39 expression is driven by the Treg-specific transcription factor Foxp3 and its catalytic activity is strongly enhanced by T-cell receptor (TCR) ligation. Activated Treg cells are therefore able to abrogate ATP-related effects such as P2 receptor-mediated cell toxicity and ATP-driven maturation of dendritic cells. Also, human Treg cells express CD39. In contrast to mice, CD39 expression in man is restricted to a subset of Foxp3+ regulatory effector/memory-like T (TREM) cells. Notably, patients with the remitting/relapsing form of multiple sclerosis (MS) have strikingly reduced numbers of CD39+ Treg cells in the blood. Thus, in humans CD39 is a marker of a Treg subset likely involved in the control of the inflammatory autoimmune disease.

Using a polypeptide oligomer harboring 16 repeats of the neuritogenic epitope (aa 58–73) of myelin P2 protein separated by spacers, enhancement of the immune response to the P2 protein, an important neuritogenic autoantigen in experimental autoimmune neuritis (EAN), was attempted. In contrast to a previous study with PLP-16-mer antigen-specific response of T cells was attenuated at all doses examined to a variable degree. Treatment of Lewis rats with the P2-16-mer up to 2 months before immunization with P253–78(vaccination) or after immunization but before appearance of disease (prevention) had a strong tolerizing effect against the induction of EAN on immunization with P253–78. Moreover, rats injected with 200 μg of the P2-16-mer i.v. on day 11 after disease induction, at which time the initial signs of disease had appeared, were almost completely protected against progression of clinical disease, whereas animals treated with the same amount of monomeric control peptide developed severe disease (treatment). Similar results were obtained by i.v. treatment of adoptive-transfer EAN with the P2-16-mer. The lack of clinical signs of disease after 16-mer therapy could be correlated with a reduced proliferative response of P253–78-specific lymph node cells. The frequency of apoptotic T cells in sciatic nerve or in lymph node cells, however, was not increased by the 16-mer treatment, suggesting that induction of anergy or other forms of peripheral tolerance may be responsible for the effect. Thus, the oligomerized P2 peptide antigen was highly effective in all three treatment modalities examined in this specific autoreactive T cell-mediated immune response.

To the editor: We read with interest the article by Zhao et al[1][1] on the specific abilities of murine CD103+ T regulatory (Treg) cells to ameliorate ongoing chronic graft-versus-host disease (cGVHD). The authors report here that only in vivo–activated effector/memory-like Tregs that express

Cytolytic T lymphocyte (CTL) clones have previously been derived from peripheral blood of melanoma patient SK29(AV). They lyse autologous melanoma cells but not autologous Epstein-Barr virus (EBV)-transformed B lymphocytes. Immunoselection experiments indicate that these CTL clones recognize 4 different antigens (Aa, Ab, B, C) in association with a single HLA restriction element, HLA-A2.1. While the expression of antigens B and C appears to be confined to SK29-melanoma cells, antigens Aa and Ab are shared by a high proportion of allogeneic HLA-A2-positive melanoma lines. HLA-A2.1 and total HLA class I molecules have now been purified from SK29-melanoma cells using affinity chromatography and associated peptides have been eluted. Peptide pools eluted from HLA-A2.1 and total class I were separated by reversed phase high performance liquid chromatography (HPLC). Individual HPLC fractions were tested for their ability to sensitize target cells for recognition by SK29-CTL clones. The presence of antigens Aa, Ab, B and C was detected in distinct HPLC fractions that were identical for both peptide pools. As target for detection of peptide antigens in HPLC fractions, the use of the HLA-A2.1-positive antigen processing mutant cell line CEM x 721.174.T2 (T2), pre-incubated with anti-HLA-A2 monoclonal antibody (MAb) MA2.1, was shown to be essential. Single-peak target-sensitizing activity was found for antigens Ab and B, whereas multi-peak sensitizing activity was reproducibly detected for antigens Aa and C. We reason that at least some of these melanoma peptide antigens might occur in biochemically distinct isoforms.

HeLa cells, derived from a human cervix carcinoma line, were transfected with a mouse MHC class I gene, H-2Kb, and chicken ovalbumin. H-2Kb-restricted cytotoxic mouse T cells specific for ovalbumin recognized the double-transfected human cells with similar efficiency as ovalbumin-transfected EL4 mouse thymoma cells (H-2b). The naturally processed ovalbumin T cell epitope was eluted from H-2Kb molecules from double-transfected HeLa cells and was biochemically compared to a synthetic peptide, SIINFEK, known to be the natural Kb ligand of ovalbumin-transfected H-2b mouse cells. The results indicate that the ovalbumin-derived Kb-ligand of double-transfected HeLa cells is also SIINFEKL. Thus, both human cervix carcinoma cells and mouse thymoma cells expressing Kb and ovalbumin process the same octapeptide. Together with previous data, derived by comparing Kb ligands of unknown sequences from both human and mouse cells expressing Kb, it can be concluded that both mouse and human cells are capable of processing the same ligands for mouse MHC class I molecules. Hence, the general specificity of the peptidegenerating mechanism for class I ligands is apparently conserved between evolutionary distant species.

Polypeptide constructs containing up to four copies of the T cell epitope 306-318 of influenza virus haemagglutinin have been synthesized on solid phase. Between the copies, a non-natural PEG-based spacer amino acid has been introduced. The oligomeric epitopes were analysed by RP-HPLC and ES-MS. The arrangement of the epitopes within the peptide constructs was either linear or comb-like. The proliferative response in a T helper cell assay induced by these oligomerized epitopes has been tested, showing that the linearly arranged epitopes are more effective than the comb-like oligomers. Copyright © 2001 European Peptide Society and John Wiley & Sons, Ltd.