T cell responses to contact allergens.

T lymphocytes are instrumental in the prevention of infections. With their antigen-specific T cell receptor (TCR), these cells recognize short peptides in the peptide-binding groove on MHC molecules of antigen-presenting cells. However, conventional T cells can also recognize non-peptide antigens including carbohydrates, phosphate groups, organic chemicals, and metal ions. The molecular basis of the interaction of TCR with these structures in the context of MHC has been partly solved. Organic chemicals and carbohydrates are recognized when bound to MHC-associated peptides, whereas metal ions are recognized due to their ability to form non-covalent coordination bonds with MHC molecules, bound peptides, and TCR. Peptide-independent metal ion recognition has also been described.

Advanced tests for skin and respiratory sensitization assessment.

Sens-it-iv is an FP6 Integrated Project that finished in March 2011 after 66 months of activity, thanks to 12 million € of funding. The ultimate goal of the Sens-it-iv project was the development of a set of in vitro methods for the assessment of the skin and respiratory sensitization potential of chemicals and proteins. The level of development was intended to be at the point to enter the pre-validation phase. At the end of the project it can be concluded that the goal has been largely accomplished. Several advanced methods were evaluated extensively, and for some of them a detailed Standard Operating Procedure (SOP) was established. Other, less advanced methods also contributed to our understanding of the mechanisms driving sensitization. The present contribution, which has been prepared with the support of CAAT-Europe, represents a short summary of what was discussed during the 3-day end congress of the Sens-it-iv project in Brussels. It presents a list of methods that are ready for skin sensitization hazard assessment. Potency evaluation and the possibility of distinguishing skin from respiratory sensitizers are also well advanced.

Guiding principles for the implementation of non-animal safety assessment approaches for cosmetics: skin sensitisation.

Characterisation of skin sensitisation potential is a key endpoint for the safety assessment of cosmetic ingredients especially when significant dermal exposure to an ingredient is expected. At present the mouse local lymph node assay (LLNA) remains the 'gold standard' test method for this purpose however non-animal test methods are under development that aim to replace the need for new animal test data. COLIPA (the European Cosmetics Association) funds an extensive programme of skin sensitisation research, method development and method evaluation and helped coordinate the early evaluation of the three test methods currently undergoing pre-validation. In May 2010, a COLIPA scientific meeting was held to analyse to what extent skin sensitisation safety assessments for cosmetic ingredients can be made in the absence of animal data. In order to propose guiding principles for the application and further development of non-animal safety assessment strategies it was evaluated how and when non-animal test methods, predictions based on physico-chemical properties (including in silico tools), threshold concepts and weight-of-evidence based hazard characterisation could be used to enable safety decisions. Generation and assessment of potency information from alternative tools which at present is predominantly derived from the LLNA is considered the future key research area.

Novel fluorescence assay for tracking molecular and cellular allergen-protein interactions.

T cells recognizing nickel (Ni) are key mediators in human Ni allergy, which represents the most common form of human contact hypersensitivity. In contrast to well-characterized Ni-specific human T cell clones, molecular knowledge about the extra- and intracellular route(s) of antigen/allergen presentation and processing of Ni-specific epitopes is still fragmentary. Here, we demonstrate a new metal-specific fluorescent technique to detect and quantify metal ions, like Ni(2+), while they are associated with isolated metalloproteins. Moreover, utilizing the fluorescent metal sensor molecule Newport Green (NPG) a novel method has been developed, which permits the metal-specific detection of Ni(2+) binding to surface or intracellular structures of individual human antigen presenting cells by flow cytometry. We expect such metal-specific fluorescent analyses to contribute to a better basic understanding of molecular and cellular immune processes involved in Ni-specific T cell epitope generation and the pathogenesis of human nickel allergy.

Management of an integrated project (Sens-it-iv) to develop in vitro tests to assess sensitisation.

Sens-it-iv is an integrated project, funded by European Commission Framework Programme 6, the overall objective of which is to develop in vitro tests and test strategies to be used by the chemical, cosmetic and pharmaceutical industries to assess the risk for potential contact and respiratory sensitisers. Such tests, once formally validated and accepted, will permit the evaluation of the sensitising potential of existing and new chemical entities and the products of the European industries for classification and labelling, as required by the new EU REACH legislation on chemicals, or for the purpose of risk assessment as required by the 7th Amendment to the EU Cosmetics Directive. Sens-it-iv involves 28 partners, representing industries, universities and regulatory bodies, including various institutes in the EU Member States and different competencies, all with the common aim of achieving a final deliverable - increasing the safety of consumer products, whilst reducing animal experimentation. This paper provides an overview of the structure of the project and a detailed description of the organisation of its management.

Antigen-driven HIV expansion in allergen-specific T cells.

In industrialized countries there is a high prevalence of allergy toward nickel ions. The exposure of affected individuals to nickel leads to a delayed-type hypersensitivity reaction, which is induced by antigen-specific CD4 and CD8 T cells. Beside this antigenic potential, immunomodulatory properties of nickel ions were described. To dissect the role of both mechanisms for HIV replication, we studied HIV expansion in PBMC of nickel-allergic and nonallergic donors. Nickel ions promote HIV replication in PBMC as efficiently as protein antigens. The nickel-mediated virus expansion strictly required the presence of nickel-specific T cells. Data obtained with nickel-specific CD4 T cell clones showed that antigen-mediated proliferation is an absolute prerequisite for HIV expansion. However, the previously suggested immunomodulatory properties of nickel ions do not seem to contribute to HIV expansion. As a widely distributed antigen with increasing numbers of allergic people, nickel may be an important and underestimated factor of HIV expansion in vivo.

Differences in pairing and cluster formation of T cell receptor alpha- and beta-chains in T cell clones and fusion hybridomas.

The questions of T cell receptor (TCR) clustering and preferential pairing of TCR alpha- and beta-chains are discussed controversially. We here describe the rare case of a non-pairing TCR alpha-TCR beta combination detected in the murine T cell hybridoma Hy-E6. Of its two TCR alpha-chains (Valpha3.2, Vbeta17) and one Vbeta16-chain only the Valpha17/Vbeta16 TCR is exposed on the surface, despite intracellular expression of Valpha3.2 protein. The lack of Valpha3.2/Vbeta16 pairing was confirmed by TCR transfections. Surprisingly, however, the parental T cell clone CTL-E6 expressed both alpha-chains on its plasma membrane. Different size distribution of TCR clusters in CTL-E6 versus Hy-E6 and transfectants as determined by Blue-Native gel electrophoresis indicated differences in the supra-molecular TCR assembly as one possible reason for this phenomenon. Our data further reveal that the nominal specificity of CTL-E6 for the fully agonistic trinitrophenyl (TNP) modified peptide M4L-TNP was specifically mediated by the trimeric Valpha3.2/Valpha17/Vbeta16 TCR of CTL-E6. In contrast, the Valpha17/Vbeta16 combination in Hy-E6 only conferred specificity for the cross-reactive partial agonist O4-TNP. Both specificities are H-2Kb-restricted and, hence, appear to be positively selected. The differences in TCR clustering in CTL and hybridoma might indicate differences in the reception and transmission of TCR-signals between these two cell types.

Subproteomic analysis of metal-interacting proteins in human B cells.

Metal-protein interactions are vitally important in all living organisms. Metalloproteins, including structural proteins and metabolic enzymes, participate in energy transfer and redox reactions or act as metallochaperones in metal trafficking. Among metal-associated diseases, T cell mediated allergy to nickel (Ni) represents the most common form of human contact hypersensitivity. With the aim to elucidate disease-underlying mechanisms such as Ni-specific T cell activation, we initiated a proteomic approach to identify Ni-interacting proteins in human B cells. As antigen presenting cells, B cells are capable of presenting MHC-associated Ni-epitopes to T cells, a prerequisite for hapten-specific T cell activation. Using metal-affinity enrichment, 2-DE and MS, 22 Ni-interacting proteins were identified. In addition to known Ni-binding molecules such as tubulin, actin or cullin-2, we unexpectedly discovered that at least nine of these 22 proteins belong to stress-inducible heat shock proteins or chaperonins. Enrichment was particularly effective for the hetero-oligomeric TRiC/CCT complex, which is involved in MHC class I processing. Blue Native/SDS electrophoresis analysis revealed that Ni-NTA-beads specifically retained the complete protein machinery, including the associated chaperonin substrate tubulin. The apparent Ni-affinity of heat shock proteins suggests a new function of these molecules in human Ni allergy, by linking innate and adaptive immune responses.

Malaria-specific transgenic CD4(+) T cells protect immunodeficient mice from lethal infection and demonstrate requirement for a protective threshold of antibody production for parasite clearance.

T cells are important in the immune response to malaria, both for their cytokines and their help for antibody production. To look at the relative importance of these roles, a T-cell receptor (TCR) transgenic mouse has been generated carrying a TCR specific for an epitope of the merozoite surface protein 1 (MSP-1) of the malaria parasite, Plasmodium chabaudi. In adoptive transfer experiments, malaria-specific CD4(+) T cells expand and produce interferon gamma (IFN-gamma) early in infection, but the population contracts quickly despite prolonged persistence of the parasite. MSP-1-specific CD4(+) cells can protect immunodeficient mice from lethal infection; however, the parasite is only completely cleared in the presence of B cells showing that T helper cells are critical. Levels of malaria-specific antibody and the speed of their production clearly correlate with the time of resolution of infection, indicating that a critical threshold of antibody production is required for parasite clearance. Furthermore, T cells specific for a shed portion of MSP-1 are able to provide help for antibody to the protective region, which remains bound to the infected erythrocyte, suggesting that MSP-1 has all of the components necessary for a good vaccine.

T cell receptor (TCR) interaction with haptens: metal ions as non-classical haptens.

Haptens are classified as low molecular chemicals with an intrinsic potential to covalently modify proteins, and many of them are strong inducers of contact hypersensitivity (CHS). CHS is T cell mediated, and hapten-specific T cells have been shown to interact with hapten-modified, MHC-associated peptides. However, the most common contact sensitizer in the industrialized world is nickel. In contrast to classical haptens, nickel ions do not form covalent bonds to proteins, but rather become caught in reversible coordination complexes. We here review work demonstrating that some T cells, indeed, may react to such Ni complexes on the MHC/peptide-surface absolutely comparable to other haptens. In other cases, Ni ions unlike classical haptens, may activate T cells by crosslinking their receptors to MHC molecules, independent of the nature of the associated peptide. Moreover, Ni-interacting proteins appear to make use of the reversibility of Ni-binding, and to mediate the transfer of Ni-ions to the receptor-MHC interphase. We have demonstrated such properties for human serum albumin (HSA) as well as for transferrin and identified numerous new Ni-binding proteins in human B-cell lines or dendritic cells by affinity purification and mass spectroscopy. These proteins include a notable number of known heat shock proteins and chaperones, implying that Ni may functionally interfere with these stress proteins.

Fas-mediated inhibition of CD4+ T cell priming results in dominance of type 1 CD8+ T cells in the immune response to the contact sensitizer trinitrophenyl.

One of the unusual properties of chemically reactive haptens is their capacity to simultaneously generate immunogenic determinants for hapten-specific CD8(+) and CD4(+) T cells. Surprisingly, however, a clear dominance of CD8(+) effector T cells is observed in murine contact hypersensitivity to various haptens and upon T cell priming with hapten-modified APCs in vitro. In this study we show that trinitrophenyl-specific CD8(+) T cells actively prevent CD4(+) T cell priming in vitro. This process requires cell-cell contact and is dependent on the expression of Fas on the CD4(+) T cells. Our results reveal an important Fas-dependent mechanism for the regulation of hapten-specific CD4(+) T cell responses by CD8(+) T cells, which causes the dominance of CD8(+) effector T cells and the active suppression of a CD4(+) T cell response. Moreover, our demonstration of reduced contact hypersensitivity to trinitrophenyl in the absence of Fas, but not of perforin and/or granzymes A and B, underlines the important role of Fas as a pathogenetic factor for contact hypersensitivity.

Drug interaction with T-cell receptors: T-cell receptor density determines degree of cross-reactivity.

BACKGROUND: Immune-mediated adverse reactions to drugs are often due to T-cell reactivity, and cross-reactivity is an important problem in pharmacotherapy. OBJECTIVE: We investigated whether chemical inert drugs can stimulate T cells through their T-cell receptor (TCR) and analyzed the cross-reactivities to related compounds. METHODS: We transfected human TCRs isolated from two drug-reactive T-cell clones (TCCs) by PCR into a TCR-negative mouse T-cell hybridoma. The TCCs were isolated from a patient with drug hypersensitivity to the antibacterial sulfonamide sulfamethoxazole (SMX). RESULTS: The transfectants reacted to SMX only in the presence of antigen-presenting cells (APCs). Glutaraldehyde-fixed APCs, however, were sufficient to elicit T-cell stimulation, indicating a processing-independent direct interaction of the drug with the TCR and MHC molecule. The transfected hybridomas secreted IL-2 in a drug dose-dependent manner, whereas the degree of reactivity was dependent on the level of TCR expression. One transfectant reacted not only to SMX but also to related sulfonamide compounds. Interestingly, high TCR expression increased cross-reactivity to other structurally related compounds. In addition, SMX-specific TCR cross-reacted only with sulfonamides bearing a sulfanilamide core structure but not with sulfonamides such as celecoxib, furosemide, or glibenclamide. CONCLUSIONS: These results demonstrate that the T-cell reactivity to drugs is solely determined by the TCR. Moreover, these results show that cross-reactivity of structurally similar compounds correlates with the density of the TCR. Stably transfected T-cell hybridomas may represent a powerful screening tool for cross-reactivity of newly generated sulfonamide-containing compounds such as celecoxib.

Metal-protein complex-mediated transport and delivery of Ni2+ to TCR/MHC contact sites in nickel-specific human T cell activation.

Nickel allergy clearly involves the activation of HLA-restricted, skin-homing, Ni-specific T cells by professional APCs. Nevertheless, knowledge concerning the molecular details of metal-protein interactions underlying the transport and delivery of metal ions to APC during the early sensitization phase and their interactions with HLA and TCRs is still fragmentary. This study investigates the role of human serum albumin (HSA), a known shuttling molecule for Ni(2+) and an often-disregarded, major component of skin, in these processes. We show that Ni-saturated HSA complexes (HSA-Ni) induce and activate Ni-specific human T cells as potently as Ni salt solutions when present at equimolar concentrations classically used for in vitro T cell stimulation. However, neither HSA itself nor its Ni-binding N-terminal peptide are involved in determining the specificity of antigenic determinants. In fact, HSA could be replaced by xenogeneic albumins exhibiting sufficient affinity for Ni(2+) as determined by surface plasmon resonance (Biacore technology) or atomic absorption spectroscopy. Moreover, despite rapid internalization of HSA-Ni by APC, it was not processed into HLA-associated epitopes recognizable by Ni-specific T cells. In contrast, the presence of HSA-Ni in the vicinity of transient contacts between TCR and APC-exposed HLA molecules appeared to facilitate a specific transfer of Ni(2+) from HSA to high-affinity coordination sites created at the TCR/HLA-interface.

T-cell-mediated autoimmunity: novel techniques to characterize autoreactive T-cell receptors.

Histological samples of autopsy or biopsy tissue provide the best available evidence that autoreactive T cells are involved in the immunopathogenesis of many autoimmune diseases. However, morphology alone does not provide information on the antigen-specific T-cell receptor (TCR) of these cells, let alone on their antigen specificity. In this review article we discuss a number of emerging possibilities for identifying TCR sequences directly from biopsy tissue. We also review the methods for expressing presumably autoreactive TCR molecules and speculate on how the expressed TCR might be used to identify target antigens. Such information should eventually provide new insights into disease pathogenesis which lead to better therapies.

T cell receptor transfection shows non-HLA-restricted recognition of nickel by CD8+ human T cells to be mediated by alphabeta T cell receptors.

CD8+ T cells have been assigned a prominent role in allergic contact dermatitis, including nickel allergy; however, human nickel-reactive T cells of the CD8+ phenotype have largely escaped detailed investigation. Here we characterize two quite unusual nickel-specific cytotoxic T cell clones isolated from the peripheral blood of two nickel-sensitized patients. These clones mediate nickel-specific cytolysis of many human cell lines, independent of the expression of HLA class I, CD1, or HLA class II molecules. Lysis is mediated by the alphabeta T cell receptors and involves the perforin, but not the Fas/Fas ligand pathway. Both antigen receptors lack sequence homology to each other as well as to typical natural killer T cell receptors. A transfectant expressing the rearranged alphabeta T cell receptor derived from one of the T cell clones unequivocally demonstrates that the T cell receptor itself is necessary and sufficient to confer HLA-independent nickel specificity. The independent isolation of these clones from two individuals points to an important role of such cells in the pathology of nickel contact dermatitis.

A new type of metal recognition by human T cells: contact residues for peptide-independent bridging of T cell receptor and major histocompatibility complex by nickel.

In spite of high frequencies of metal allergies, the structural basis for major histocompatibility complex (MHC)-restricted metal recognition is among the unanswered questions in the field of T cell activation. For the human T cell clone SE9, we have identified potential Ni contact sites in the T cell receptor (TCR) and the restricting human histocompatibility leukocyte antigen (HLA)-DR structure. The specificity of this HLA-DR-promiscuous VA22/VB17+ TCR is primarily harbored in its alpha chain. Ni reactivity is neither dependent on protein processing in antigen-presenting cells nor affected by the nature of HLA-DR-associated peptides. However, SE9 activation by Ni crucially depends on Tyr29 in CDR1alpha, an N-nucleotide-encoded Tyr94 in CDR3alpha, and a conserved His81 in the HLA-DR beta chain. These data indicate that labile, nonactivating complexes between the SE9 TCR and most HLA-DR/peptide conjugates might supply sterically optimized coordination sites for Ni ions, three of which were identified in this study. In such complexes Ni may effectively bridge the TCR alpha chain to His81 of most DR molecules. Thus, in analogy to superantigens, Ni may directly link TCR and MHC in a peptide-independent manner. However, unlike superantigens, Ni requires idiotypic, i.e., CDR3alpha-determined TCR amino acids. This new type of TCR-MHC linkage might explain the high frequency of Ni-reactive T cells in the human population.

A high frequency of allergen-specific CD8+ Tc1 cells is associated with the murine immune response to the contact sensitizer trinitrophenyl.

Chemical haptens induce a variety of allergic immune reactions by induction of hapten-specific T cells. Contact sensitizers such as the hapten trinitrochlorobenzene (TNCB) elicit an allergic response, which is confined to the area of antigen exposure. Despite this localized allergic response, we show here that the trinitrophenyl (TNP)-specific immune response is characterized by a rapid induction of CD8+ Tc1 type cytotoxic effector cells already after a single allergen contact which can be detected in all secondary lymphoid organs tested. We furthermore demonstrate that the rapid induction of CD8+ Tc1 effector cells correlates with an unusually high frequency of polyclonal TNP-specific CD8+ effector T cells with specificities for a variety of MHC class I binding TNP-peptides carrying the hapten in different positions. These data suggest that allergies to chemical haptens may in part be due to an unusually high frequency of polyclonal, allergen-specific effector cells which are detected in all secondary lymphoid organs.

Components of the ligand for a Ni++ reactive human T cell clone.

The major histocompatibility complex (MHC) restriction element for a human Ni(2+) reactive T cell, ANi-2.3, was identified as DR52c. A series of experiments established that the functional ligand for this T cell was a preformed complex of Ni(2+) bound to the combination of DR52c and a specific peptide that was generated in human and mouse B cells, but not in fibroblasts nor other antigen processing-deficient cells. In addition, ANi-2.3 recognition of this complex was dependent on His81 of the MHC beta chain, suggesting a role for this amino acid in Ni(2+) binding to MHC. We propose a general model for Ni(2+) recognition in which betaHis81 and two amino acids from the NH(2)-terminal part of the MHC bound peptide coordinate Ni(2+) which then interacts with some portion of the Valpha CDR1 or CDR2 region.

An autoreactive gamma delta TCR derived from a polymyositis lesion.

To investigate the role of gammadelta T cells in human autoimmune disease we expressed and characterized a gammadelta TCR from an autoimmune tissue lesion. The TCR was first identified in a rare form of polymyositis characterized by a monoclonal infiltrate of gammadelta T cells which invaded and destroyed skeletal muscle fibers. The Vgamma1.3-Jgamma1-Cgamma1/Vdelta2-Jdelta3 TCR cDNA of the original muscle invasive gammadelta T cell clone was reconstructed from unrelated cDNA and transfected into the mouse hybridoma BW58alpha(-)beta(-). Appropriate anti-human gammadelta TCR Abs stimulated the TCR transfectants to produce IL-2, thus demonstrating that the human gammadelta TCR functionally interacted with murine signaling components. The transfected Vgamma1.3/Vdelta2 TCR recognized a cytosolic protein expressed in cultured human myoblasts and TE671 rhabdomyosarcoma cells. The Ag was recognized in the absence of presenting cells. Using a panel of control gammadelta TCR transfectants with defined exchanges in different positions of both TCR chains, we showed that the gammadelta TCR recognized its Ag in a TCR complementarity-determining region 3-dependent way. To our knowledge, this is the first example of a molecularly defined gammadelta TCR directly derived from an autoimmune tissue lesion. The strategy used in this study may be applicable to other autoimmune diseases.