T cell recognition of penicillin G: Structural features determining antigenic specificity

Penicillin G (Pen G) and other beta-lactam antibiotics frequently induce allergic reactions constituting typical examples of human immune responses to haptens. In fact, penicillins represent a unique set of haptens with outstanding structural variability on the basis of an identical protein-reactive beta-lactam containing backbone. Although both cellular and humoral responses are involved in drug-induced allergies, little is known about the T cell reactivity to penicillins. To understand which structural features determine antigenic specificity, we isolated a panel of MHC-restricted, Pen G-reactive T cell clones from different penicillin-allergic patients and tested them for their capacity to proliferate in the presence of other penicillin derivatives. We found that the antigenic epitope consists of both the amide-linked side chain, which is different in every member of the penicillin family, as well as the thiazolidine ring common to all penicillin derivatives. We also demonstrated the presence of two different types of penicillin-specific T cells, one dependent, and the other independent of antigen processing by autologous antigen-presenting cells. Our data strongly suggest that penicillins form part of the epitopes contacting the antigen receptors of T cells.     

Evidence that HLA class II-restricted human CD4+ T cells specific to p53 self peptides respond to p53 proteins of both wild and mutant forms

By stimulating peripheral blood mononuclear cells of four healthy donors with a mixture of overlapping peptides representing the core domain of p53, we established two CD4+ α β T cell clones and four lines that recognized wild-type and mutant p53 proteins as well as p53 self peptides in an HLA class II-restricted fashion. Two T cell lines established from two unrelated donors reacted to the p53 peptide (p)153 – 166 and p108 – 122, respectively, in the context of DP5 molecules. Two T cell clones established from two other unrelated donors were specific for p193 – 204 in the context of DRB1*1401 and for p153 – 165 in the context of DP5, respectively. These two T cell clones responded almost equally to both wild-type and four mutant recombinant p53 proteins. The proliferative responses of these T cell clones to p53 recombinant proteins were augmented by heat denaturing, thereby suggesting that altered conformation of the protein facilitates proteolytic processing to produce antigenic peptides. The DRB1*1401-restricted T cell clone specific for p193 – 204 killed a B lymphoblastoid cell line homozygous for HLA-DRB1*1401 when the cell line was pre-pulsed with p53 protein as well as peptide. These results indicate that CD4+ T cells reactive to p53 do exist in healthy individuals and the epitopes are probably ignored by the immune system under physiological conditions. It is suggested that such epitopes stimulate T cells to induce anti-p53 antibody production in cancer patients as previously reported by others. The possible involvement of p53-reactive T cells in anti-tumor immunity is discussed.     

Hepatitis B surface antigen presentation and HLA-DRB1*- lessons from twins and peptide binding studies

The aim of this study was to investigate the underlying mechanisms of the genetic association between certain HLA-DRB1* alleles and the immune response to HBsAg vaccination. Therefore, HBsAg peptide binding to HLA-DR molecules was measured in vitro by peptide binding ELISAs. Additionally, HBsAg-specific T cell reaction and cytokine profile of immune response were analysed ex vivo in ELISPOT assays and DR-restriction of T-cell proliferative responses was investigated with HBsAg specific T cell clones. In addition, we compared HBsAg specific T cell responses of 24 monozygotic and 3 dizygotic twin pairs after HBsAg vaccination. Our results showed that the peptide binding assays did not reflect antigen presentation in vivo. DR alleles associated with vaccination failure like DRB1*0301 and 0701 efficiently presented HBsAg peptides. In 11 of 24 investigated monozygotic twin pairs we observed pronounced differences in the recognition of HBsAg peptides. This study indicates that HLA-DR associations with HBsAg vaccination response are not caused by differences in peptide binding or by a shift in the Th1/Th2 profile. Our findings strongly argue for differences in the T cell recognition of peptide/MHC complexes as the critical event in T cell responsiveness to HBsAg.     

Pathogenesis of drug allergy – current concepts and recent insights

Drug hypersensitivity reactions (DHRs) may be caused by immunologic and non‐immunologic mechanisms. According to the World Allergy Organization, drug allergy (DA) encompasses the subgroup of immunologic DHRs which are mediated either by specific antibodies or specific T lymphocytes. Due to the immunologic memory, DA reactions bear an increased risk for dramatically enhanced reactions on re‐exposure. Some current concepts of DA were described decades ago. Drug allergies to soluble macromolecular protein drugs such as biopharmaceuticals are predominantly T cell‐dependent drug‐specific antibody responses leading to IgE‐or IgG‐mediated allergy. However, most drugs are too small to be directly recognized by specific B and T cells. Immune reactions to low‐molecular drugs have been explained by the hapten model: a hapten drug can bind covalently to soluble autologous proteins (e.g. serum albumin). Resulting compounds may then be recognized by matching B cell receptors (BCRs) and induce a specific T cell‐dependent IgE‐or IgG‐antibody production. Drug haptens may bind to extra‐ or intracellular proteins, which are processed and presented by various professional antigen‐presenting cells (APCs). Depending on the APC, they may induce not only specific antibody production, but also non‐immediate T cell‐mediated DA. More recently, a supplementary effector mechanism for non‐immediate DA to low‐molecular drugs has been described, namely the pharmacological interaction of native low‐molecular drugs with immune receptors (p‐i‐concept). Low‐molecular drugs may directly and reversibly attach to immune receptors. These non‐covalent interactions may modify the affinity between autologous major histocompatibility complex (MHC), presented peptides and specifically primed T cell receptors (TCRs) and thereby stimulate T cells. A special type of p‐i‐reaction has been recently described between the antiviral drug abacavir and the F pocket of HLA‐B*57:01. This interaction causes an alteration of the MHC‐presented self‐peptide repertoire and may consecutively lead to a kind of auto‐reactivity. Such types of reactions can explain the strong MHC‐HLA associations which have been found for some T cell‐mediated DHRs.     

T lymphocyte-mediated immune responses to chemical haptens and metal ions: implications for allergic and autoimmune disease

Chemical haptens and metal ions interact with proteins and thereby become recognizable by T and B lymphocytes. They induce the production of proinflammatory cytokines and chemokines by various cell types due to triggering of innate immune responses. This is an important prerequisite for the activation of the adaptive immune system and the development of diseases like allergic contact dermatitis and adverse drug and autoimmune reactions. Our increasing knowledge about the molecular basis of hapten and metal ion recognition by T cells and about the pathomechanisms of contact hypersensitivity and chemical-induced autoimmune reactions allows concomitant progress in the development of modern strategies for immunotherapy and will hopefully enable more specific intervention in hapten- and metal ion-induced human diseases in the future.     

Emerging principles for the design of promiscuous HLA-DR-restricted peptides: an example from the major bee venom allergen

Mechanisms underlying successful immunotherapy of allergic patients operate at the level of CD4+ helper T cells. T cell epitopes from allergens may thus constitute interesting molecules for immunotherapy, provided they are efficient for all patients and are not recognized by IgE. In an attempt to define such peptides for allergy to bee venom, we have investigated the capacity of peptides encompassing the sequence of the major bee venom allergen to stimulate PBMC from allergic patients and to react specifically with their IgE. The region 77-110 emerged as the most frequently T cell stimulating. We then analyzed the binding modes of the sequence 81-97 for ten different HLA-DR molecules and introduced punctual mutations to enhance the peptide affinity for these molecules. Six different modes have been identified on the sequence 81-97, one mode being common to eight HLA-DR molecules. Four HLA-DR molecules can bind the P85-97 peptide by two different modes with an equivalent affinity. The peptide N89L has a higher affinity for DRB1*0301 and DRB3*0101 and remains as active as the native peptide towards the other HLA-DR molecules.     

Attempts to modulate the immune response to a hapten-carrier complex with various hapten-containing compounds.

The immune response to a hapten-carrier conjugate appears to be a complex phenomenon where reactions of the T-cell population are not restricted to the carrier and where the reactions of the B-cell population are not limited to the hapten determinant of the antigen molecule. To get a better understanding of the different cell interactions during the immune response to a hapten-carrier complex, the effects of immunogenic or tolerogenic injections of various hapten-containing compounds on the responses induced by immunization with the same hapten coupled to protein carriers were studied. The results indicate that T cells involved in delayed hypersensitivity and T cells involved in contact dermatitis could belong to distinct subclasses and confirm that hapten and carrier moieties of the antigen molecule could compete, probably at the macrophage level, for both delayed hypersensitivity to the carrier and antibody synthesis to the hapten.     

Structural basis of metal hypersensitivity

Metal hypersensitivity is a common immune disorder. Human immune systems mount the allergic attacks on metal ions through skin contacts, lung inhalation and metal-containing artificial body implants. The consequences can be simple annoyances to life-threatening systemic illness. Allergic hyper-reactivities to nickel (Ni) and beryllium (Be) are the best-studied human metal hypersensitivities. Ni-contact dermatitis affects 10 % of the human population, whereas Be compounds are the culprits of chronic Be disease (CBD). αβ T cells (T cells) play a crucial role in these hypersensitivity reactions. Metal ions work as haptens and bind to the surface of major histocompatibility complex (MHC) and peptide complex. This modifies the binding surface of MHC and triggers the immune response of T cells. Metal-specific αβ T cell receptors (TCRs) are usually MHC restricted, especially MHC class II (MHCII) restricted. Numerous models have been proposed, yet the mechanisms and molecular basis of metal hypersensitivity remain elusive. Recently, we determined the crystal structures of the Ni and Be presenting human MHCII molecules, HLA-DR52c (DRA*0101, DRB3*0301) and HLA-DP2 (DPA1*0103, DPB1*0201). These structures revealed unusual features of MHCII molecules and shed light on how metal ions are recognized by T cells.     

The set of naturally processed peptides displayed by DR molecules is tuned by polymorphism of residue 86

The response to tetanus toxoid (TT) was studied in immune donors that carry two alleles of DR5 that differ only at DRβ residue 86: DRB1*1101 (G86, abbreviated 1101) and DRB1*1104 (V86, abbreviated 1104). A large number of TT-specific T cell clones was isolated and the epitopes recognized in association with 1101 and 1104 were mapped. We found that two epitopes (p2 and p32) can be recognized in association with both 1101 and 1104 while three epitopes (p23, p27 and p30) are recognized in association with 1101, but never in association with 1104. The sets of naturally processed self peptides displayed by 1101 and 1104 were characterized using alloreactive T cell clones. We found that all 1104 alloreactive clones recognize both 1104 and 1101, while ?30% of the alloreactive 1101 clones fail to recognize 1104. Thus it is apparent that both naturally processed TTand self peptides displayed on 1104 molecules represent a fraction of those displayed on 1101 molecules. The mechanism responsible for this differential presentation was investigated by comparing the capacity of 1101 and 1104 antigen-presenting cells to present TTor synthetic peptides to specific T cells and by measuring the binding of these peptides to DR molecules. Three sets of results suggest that V86 acts as a constraint to the binding of naturally processed peptides: (i) all 1104-restricted or alloreactive T cell clones recognize TT- or allo-epitopes presented by 1101 as well, thus ruling out a major effect of V86 as a residue determining T cell restriction specificity; (ii) presentation of naturally processed peptides correlates in general with the capacity of long synthetic peptides to bind to 1101 or 1104 and (iii) while the naturally processed p30 epitope discriminates between 1101 and 1104, a short synthetic peptide binds equally well to and is comparably recognized in association with both 1101 and 1104. Taken together these results suggest that the natural polymorphism at residue 86 might be a molecular switch that finely tunes the complexity of the peptide collection presented on DR molecules.     

Detection of low avidity desmoglein 3-reactive T cells in pemphigus vulgaris using HLA-DR beta 1*0402 tetramers

In the present study, we developed a HLA class II tetramer-based detection system utilizing DRB1*0402 tetramers loaded with recently identified immunodominant peptides of desmoglein 3 (Dsg3), the major autoantigen of pemphigus vulgaris (PV). Initial experiments demonstrated staining of a Dsg3-reactive T cell hybridoma which was derived from HLA-DR0402-transgenic mice with loaded PE-labeled DRbeta1*0402 tetramers. However, staining of autoreactive T cell clones (TCC) derived from PV patients resulted only in positive staining by addition of exogenous peptides to the staining reactions. There was a dose-dependent specific binding of TCC to the tetramers with the agonistic Dsg3 peptide which was not altered by exogenous unrelated Dsg3 peptide. Noteworthy, the TCC did not stain with HLA-DR4 tetramers complexed with unrelated Dsg3 peptides. The findings of this study suggest that HLA class II tetramers may provide a highly specific approach to monitor ex vivo the T cellular autoimmune response against Dsg3 in patients with PV.     

Suppression by mAbs against DQB1 peptides of in vitro proliferation of AChR-specific T cells from myasthenia gravis patients

It has been indicated that multiple genes, including HLA genes, are collectively involved in the susceptibility to myasthenia gravis (MG). DQB1 alleles represent one of those associated with MG. We have prepared B-cell hybridomas that produce mAbs against peptides corresponding to the tip of the MHC antigen-binding cavity (region 70–90) of alleles DQB1*02, *03, *05 and *06. The mAbs bound to DQ molecules isolated from cells. In the assays using peripheral blood lymphocytes (PBL) from patients with MG, the mAbs against peptides of the correlate HLA DQ sequences inhibited the in vitro proliferation of acetylcholine receptor (AChR)-specific T cells. The results indicate that the function of disease-related MHC alleles may be blocked by directly and selectively targeting the antigen-presenting region on these MHC molecules. The results also suggest that DQ molecules are one of those involved in the restriction of autoimmune anti-AChR responses in MG. The strategy could provide an effective means for immunointervention in MG. It may also potentially be adapted for down-regulation of undesirable immune responses such as in other autoimmune diseases, allergic reactions, or clinical conditions where immune responses to a therapeutic protein develop.     

Suppression by mAbs against DQB1 peptides of in vitro proliferation of AChR-specific T cells from myasthenia gravis patients

It has been indicated that multiple genes, including HLA genes, are collectively involved in the susceptibility to myasthenia gravis (MG). DQB1 alleles represent one of those associated with MG. We have prepared B-cell hybridomas that produce mAbs against peptides corresponding to the tip of the MHC antigen-binding cavity (region 70-90) of alleles DQB1*02, *03, *05 and *06. The mAbs bound to DQ molecules isolated from cells. In the assays using peripheral blood lymphocytes (PBL) from patients with MG, the mAbs against peptides of the correlate HLA DQ sequences inhibited the in vitro proliferation of acetylcholine receptor (AChR)-specific T cells. The results indicate that the function of disease-related MHC alleles may be blocked by directly and selectively targeting the antigen-presenting region on these MHC molecules. The results also suggest that DQ molecules are one of those involved in the restriction of autoimmune anti-AChR responses in MG. The strategy could provide an effective means for immunointervention in MG. It may also potentially be adapted for down-regulation of undesirable immune responses such as in other autoimmune diseases, allergic reactions, or clinical conditions where immune responses to a therapeutic protein develop.     

Peptide-based vaccination: where do we stand?

PURPOSE OF REVIEW: Allergen-specific immunotherapy represents the only causative approach towards allergy treatment. Specific immunotherapy can, however, include allergic reactions and occasionally life-threatening anaphylaxis. Peptides have been evaluated as a potential therapeutic approach in atopic allergic disease because they have the potential to inhibit T-cell function but not induce anaphylaxis. RECENT FINDINGS: Data from early clinical trials of peptide vaccination revealed that therapy was associated with a modest improvement in allergic disease, and was accompanied by a high frequency of adverse reactions. More recent studies have demonstrated improved clinical outcomes, improved safety, and have defined the mechanisms of adverse events observed in earlier studies. Mechanisms of peptide vaccination include the hyporesponsiveness of allergen-specific responses and the induction of regulatory T cells and cytokines. Novel peptide design has allowed the generation of fragments that contain T-cell stimulatory epitopes, lack B cell epitopes, and can induce protective IgG responses in both mice and humans. Other approaches have focused on hypoallergenic B-cell epitopes that induce inhibitory IgG antibodies. Peptides that specifically induce regulatory cytokine production would also enhance peptide vaccines. Several recent studies have described immunodominant epitopes from major allergens that may form candidate peptides for use in peptide vaccination. SUMMARY: The manipulation of peptide epitopes may provide a strategy for the rational design of peptide allergy vaccines further improving safety and efficacy.     

T cell epitopes of type II collagen in HLA-DRB1*0101 or DRB1*0405-positive Japanese patients with rheumatoid arthritis

Rheumatoid arthritis (RA) is a T cell-mediated autoimmune disease, but target antigens (autoantigens) responsible for T cell activation remain unclear. Type II collagen (CII) is a candidate autoantigen that is largely confined to the articular cartilage. To investigate whether CII is an important antigen in patients with RA, we examined peripheral blood T cell reactivity to CII in HLA-DRB1*0101 and DRB1*0405-positive RA patients. Reactivities to candidate T cell epitopes of CII were also examined. Peripheral blood T cell reactivity to CII and CII peptides (256-271, 429-442, 593-610, 1064-1081) were detected by measurement of IL-2, IFN-gamma, and IL-4 in culture supernatant of PBMC after in vitro antigen stimulation. Cytokine concentration was measured by ELISA. In DRB1*0101-positive patients, T cell reactivity to CII as detected by measurement of IL-2 production in culture supernatant, was present in 4 out of 9 patients. IL-2 production upon stimulation with CII 256-271 peptide was found in all of these 4 patients. In DRB1*0405-positive patients, high frequency of positive T cell response to CII was detected in 9 out of 11 patients. IFN-gamma production was also detected in 4 out of 6 patients producing IL-2 by stimulation with CII. T cell response to CII 256-271 and/or CII 1064-1081 was detected in these patients. In DRB1*0101-positive RA patients, CII 256-271 peptide might function as a T cell epitope, whereas either CII 256-271 or CII 1064-1081 peptide may be a major T cell epitope in DRB1*0405-positive RA patients. In DRB1*0405-positive RA patients, CII reactive T cells might play a crucial role in the development of RA through IFN-gamma production.     

O-glycosylated versus non-glycosylated MUC1-derived peptides as potential targets for cytotoxic immunotherapy of carcinoma

Due to the fact that many cellular proteins are extensively glycosylated, processing and presentation mechanisms are expected to produce a pool of major histocompatibility complex (MHC) class I-bound protein-derived peptides, part of which retain sugar moieties. The immunogenic properties of the presented glycosylated peptides in comparison to their non-glycosylated counterparts have not been determined clearly. We assessed the cellular immunogenicity of MUC1 (mucin)-derived peptides O-glycosylated with a Tn epitope (GalNAc) using HLA-A*0201 single chain (HHD)-transfected cell lines and transgenic mice. For part of the compounds Tn moiety did not interfere with the HLA-A*0201 binding. Moreover, part of the glycopeptides elicited effective cytotoxic responses, indicating recognition of the glycopeptide-HLA-A*0201 complex by the T cell receptor (TCR) and subsequent cytotoxic T lymphocyte (CTL) activation. The CTLs exhibited a substantial degree of cross-reactivity against target cells loaded with glycosylated and non-glycosylated forms of the same peptide. The studied (glyco)peptides showed cellular immunogenicity in both MUC1-HHD and HHD mice and induced effective lysis of (glyco)peptide-loaded target cells in CTL assays. However, the elicited CTLs did not induce selective lysis of human MUC1-expressing murine cell lines. Moreover, immunization with (glyco)peptide-loaded dendritic cells (DCs) did not induce significant immunotherapeutic effects. We conclude that Tn glycosylated MUC1-derived peptides can be presented by MHC class I molecules, and may be recognized by specific TCR molecules resulting in cytotoxic immune responses. However, the studied glycopeptides did not offer significant benefit as targets for cytotoxic immune response due apparently to (a) cross-reactivity of the elicited CTLs against the glycosylated and non-glycosylated forms of the same peptide and (b) low abundance of glycopeptides on tumour target cells.     

T‐cell receptors: Tugging on the anchor for a tighter hold on the tumor‐associated peptide

Although it has been shown that human tumor‐associated, HLA anchor residue modified “heteroclitic” peptides may induce stronger immune responses than wild‐type peptides in cancer vaccine trials, it has also been shown that some T cells primed with these heteroclitic peptides subsequently fail to recognize the natural, tumor‐expressed peptide efficiently. This may provide a molecular reason for why clinical trials of these peptides have been thus far unsuccessful. In this issue of the European Journal of Immunology, Madura et al. [Eur. J. Immunol. 2015. 45: 584–591] highlight a novel twist on T‐cell receptor (TCR) recognition of HLA–peptide complexes. Tumor‐associated peptides often lack canonical anchor residues, which can be substituted for the optimal residue to improve their antigenicity. T‐cell cross‐reactivity between the natural and modified (heteroclitic) peptides is essential for this approach to work and depends on whether the anchor residue substitution influences peptide conformation. The Melan‐A/MART‐1_26‐35 peptide epitope is an example where T cells can make this distinction, with the natural peptide stimulating higher affinity CD8⁺ T cells than the heteroclitic peptide, despite the heteroclitic peptide's more stable association with HLA‐A2. The molecular basis for peptide discrimination is identified through the structure of the TCR bound to the natural peptide; TCR engagement of the natural peptide “lifts” its amino‐terminus partly away from the HLA peptide binding groove, forming a higher affinity interface with the TCR than is formed with the anchor residue “optimized” heteroclitic peptide, which cannot be “pulled” from the HLA groove.     

T-cell reactivity to insulin peptide A1-12 in children with recently diagnosed type 1 diabetes or multiple beta-cell autoantibodies

Insulin-specific immune responses appear early in preclinical type 1 diabetes (T1D), and bovine insulin in cow's milk-based infant formulas has been suggested to be of importance in induction of the primary response to insulin in humans. To characterize insulin-specific T-cell reactivity we studied T-cell responses to 10 insulin peptides derived from bovine (BI) and human insulin (HI) in 42 children with recently diagnosed T1D, 47 children with multiple autoantibodies and 111 autoantibody-negative control children with risk-associated HLA alleles. Proliferation responses detected in antigen-stimulated peripheral blood mononuclear cells did not differ between the three groups when the comparison was performed without considering HLA genotypes. However, significant differences were observed, when children with the high-risk genotype HLA (DRB1*03)-DQA1*05-DQB1*02/DRB1*0401-DQA1*03-DQB1*0302 were analyzed separately. The responses to the peptides including amino acids A1-12 derived from B1 and H1 were significantly higher in children with T1D (P=0.008, P=0.004, for B1 and H1, respectively) and in children with diabetes-associated autoantibodies (P=0.002 and P=0.001, respectively) than in control children. Positive responses (stimulation indices SI>/=3) were seen more frequently in T1D children than in controls (4/7 vs 2/19; P=0.03 and 4/7 vs 1/19; P=0.01 for B1 and H1, respectively). T-cell response to the insulin peptide A1-12 is enhanced in clinical and preclinical T1D associated with the high-risk HLA-genotype emphasizing the importance of this epitope.     

Novel T‐cell epitopes on Schistosoma japonicum SjP40 protein and their preventive effect on allergic asthma in mice

Allergic asthma is a chronic inflammatory disease mediated by Th2 cell immune responses. Currently, immunotherapies based on immune deviation are attractive, preventive, and therapeutic strategies for asthma. Many studies have shown that intracellular bacterial infections such as mycobacteria and their components can suppress asthmatic reactions by enhancing Th1 responses, while helminth infections and their proteins can inhibit allergic asthma via immune regulation. However, some helminth proteins such as SmP40, the major egg antigen of Schistosoma mansoni, are found as Th1 type antigens. Using a panel of overlapping peptides, we identified T‐cell epitopes on SjP40 protein of Schistosoma japonicum, which can induce Th1 cytokine and inhibit the production of Th2 cytokines and airway inflammation in a mouse model of allergic asthma. These results reveal a novel form of immune protective mechanism, which may play an important role in the modulating effect of helminth infection on allergic asthmatic reactions.     

Identification of the HLA-A24 peptide epitope within cytomegalovirus protein pp65 recognized by CMV-specific cytotoxic T lymphocytes.

Among cytomegalovirus (CMV) tegument proteins, phosphoprotein 65 (pp65) has been identified as the important target antigen of the cytotoxic T lymphocyte (CTL) response against the virus. We synthesized seven CMV-pp65-derived peptides carrying an HLA-A24-binding motif, and investigated the ability of these peptides to induce CMV-specific CTL. We identified one nonamer peptide (pp65113-121; VYALPLKML) able to bind HLA-A24 and induce CTL responses in vitro in peripheral blood mononuclear cells (PBMC) from CMV-seropositive individuals. The peptide-specific CTLs generated were capable of recognizing pp65 expressed on CMV-infected fibroblasts as well as pp65113-121 peptide bound to the surface of C1R-A*2402 cells in an HLA-A24-restricted manner. The pp65113-121 peptide thus might be considered a synthetic peptide vaccine in HLA-A24-positive individuals.     

CD4+ T cells from type 1 diabetic and healthy subjects exhibit different thresholds of activation to a naturally processed proinsulin epitope

Recent studies suggest that insulin is a primary autoantigen for type 1 diabetes. Several studies have identified preproinsulin (PPI) 76-90 as an immunodominant CD4+ T cell epitope. We developed a class II tetramer reagent using a modified PPI peptide with a lysine to serine substitution at position 88 (PPI 78-90(88S)) that has high binding affinity to DRA1*0101/DRB1*0401 (DR0401). Using this tetramer, positive responses were observed from both DR0401 healthy and type 1 diabetic subjects when T cells were stimulated with the PPI 78-90(88S) peptide. Seventy percent of these T cells proliferated in response to both the wild type PPI 76-90 and PPI 78-90(88S) peptides. However, when T cells were stimulated with wild type peptide and assayed with DR0401/PPI 78-90(88S), positive responses were only detected in the diabetic group but not in healthy subjects. When highly purified CD4+CD25-CD45RA+ T cells were stimulated with PPI 78-90(88S) peptide in the absence of antigen-presenting cells, T cells from the diabetic group were able to respond to peptide stimulation, while T cells from healthy subjects were not. These data suggest that T cells from type 1 diabetic subjects have a lower threshold of activation in response to PPI peptide stimulation as compared to healthy subjects.