Induction Of Contact Sensitivity Research Articles

Assessing the dermal compatibility of a new female incontinence product line

Context: We have developed a line of products designed to better meet the overall needs of women suffering from urinary incontinence. The products are more discrete and contain a unique odor neutralizing technology (ONT).Objective: This paper describes the overall skin compatibility program for this product line in which the new products were compared to negative controls and/or commercially marketed reference products with an established history of safe use.Materials and methods: Test products consisted of several product forms (light pads/pantiliners, moderate pads, briefs and taped diapers) with ONT and having various degrees of protection. Studies were conducted using standard protocols for 4-day and 21-day cumulative irritation, the Human Repeat Insult Patch Test (HRIPT), and the Behind-the-Knee (BTK) test for mechanical and chemical irritation. In one 4-day irritation study and one HRIPT, test subjects consisted of individuals with self-assessed sensitive skin. In addition, one 4-day study was conducted using normal skin sites, and sites compromised by tape stripping. Nonirritant controls were physiologic saline and/or current, commercially marketed incontinence products. All responses were evaluated by visual scoring of erythema. In addition, in the BTK, transepidermal water loss (TEWL) and adverse sensory effects collected from panelists’ daily diaries were also evaluated.Results and discussion: Two 4-day cumulative irritation studies and one 21-day study demonstrated that a wide range of product forms (liners, light and moderate pads, briefs and adult diapers) produced skin reactions similar to the nonirritant controls. The 4-day study conducted using sensitive skin subjects showed good skin compatibility, and the test products were comparable to the nonirritant controls. In the 4-day study with both normal and compromised skin sites, test products produced mean erythema scores similar to the nonirritant controls. Three HRIPT separate studies confirm that the products do not induce contact sensitization, including one study conducted on individuals with self-assessed sensitive skin. In the BTK, test and control products produced similar irritation, as assessed by erythema, TEWL and sensory effects.Conclusion: The results from the patch tests and mechanical irritation test demonstrate good skin compatibility of the new line of products with the unique ONT. In addition, the forms of the product (i.e. liner, pad or brief), were equally compatible with skin.

Down-regulation of Macrophage Immune Activity by Natural CD8+ Regulatory T Cells*

To evaluate the influence of natural regulatory CD8+ T cells on macrophages we investigated in vitro production of cytokines, reactive oxygen intermediates (ROIs) and expression of CD80 surface costimulatory molecules by macrophages (MF) of wild type (WT) B10PL and syngeneic knock-out (KO) strains, TCRalpha-/-, beta2m-/- and CD1d-/- mice. MF of TCR alpha-/- (CD4- and CD8-) and beta2m-/- (CD8-) animals produced higher levels ofTNF-a, IL-6, IL-12 and ROIs and showed increased expression of CD80 costimulatory molecules in comparison to MF of WT or CD1d-/- (NKT-) mice. When MF of these strains were conjugated with TNP hapten and injected i.v. into WT mice to test either induction of contact sensitivity (CS) or tolerance, only TNP-MF of TCRalpha-/- and beta2m-/- animals induced a significant CS reaction, while cells of WT and CD1d-/- strains were tolerogenic. MF of the tested strains can be classified functionally as resembling either proinflammatory (TCRalpha-/- and beta2m-/-mice) or immunosuppressive (WT and CD1d-/-) phenotypes. We suggest the presence of an in vivo regulatory loop in which innate CD8+ Treg cells control the transition between MF phenotypes and thus adjust the magnitude of the inflammatory response to strictly local requirements.

Tea Tree Oil

Tea tree oil is an increasingly popular ingredient in a variety of household and cosmetic products, including shampoos, massage oils, skin and nail creams, and laundry detergents. Known for its potential antiseptic properties, it has been shown to be active against a variety of bacteria, fungi, viruses, and mites. The oil is extracted from the leaves of the tea tree via steam distillation. This essential oil possesses a sharp camphoraceous odor followed by a menthol-like cooling sensation. Most commonly an ingredient in topical products, it is used at a concentration of 5% to 10%. Even at this concentration, it has been reported to induce contact sensitization and allergic contact dermatitis reactions. In 1999, tea tree oil was added to the North American Contact Dermatitis Group screening panel. The latest prevalence rates suggest that 1.4% of patients referred for patch testing had a positive reaction to tea tree oil.

Sensitization to thiourea derivatives among Finnish patients with suspected contact dermatitis

Thiourea derivatives in rubber products may induce contact sensitization and allergic contact dermatitis. Sensitization is most often from neoprene rubber, but the multitude of possible sensitizing products has remained poorly characterized. The aim of this study was to collect information on the occurrence of thiourea-related contact allergy and to show novel sources of sensitization. A mixture of dibutyl-, diethyl-, and diphenylthiourea was included in patch test baseline series in five Finnish dermatology clinics during 2002-2007. In addition, an extended series of rubber chemicals was tested in patients with suspected rubber allergy. Sources of sensitization to thioureas were analysed in sensitized patients. Thiourea mix yielded positive patch test reactions in 59 of 15,100 patients (0.39%); 33/59 patients were also tested with individual rubber chemicals. Diethylthiourea was positive in 24/33, diphenylthiourea in 5, and dibutylthiourea in 1 patient. The most common sources of sensitization included various neoprene-containing orthopaedic braces, sports equipment, and foot wear. The sources of sensitization to thiourea chemicals were detected in most cases. These sources comprise a heterogenous group of products extending from orthopaedic materials to sports equipment.

Contact sensitization to 4,4′‐diaminodiphenylmethane and to isocyanates among general dermatology patients

Diisocyanates and 4,4'-diaminodiphenylmethane (MDA) are industrial sensitizers. Occupational asthma is a risk among workers exposed to diisocyanates. Exposure may also lead to contact sensitization and allergic contact dermatitis. The aim of this study was to determine the occurrence of contact sensitization to MDA and to diisocyanates among general dermatology patients. Patch testing with MDA was carried out in 1595 patients. Diphenylmethane-4,4'-diisocyanate (MDI) and toluene-2,4-diisocyanate (TDI) were tested in 1023 patients and isophorone diisocyanate (IPDI) and 1,6-hexamethylene diisocyanate (HDI) in 433 patients. The clinical data and sources of exposure are analysed. MDA reactions were seen in 17 (1.1%) patients and MDI reactions in 4 patients. Six MDA-positive patients reacted to p-phenylenediamine and two to epoxy chemicals. 5/10 of the TDI reactions were seen concurrently with reactions to MDI, MDA, HDI, or to IPDI. IPDI reactions were seen in eight patients and HDI reactions in two patients. Possible sources of exposure were traced in most patients, although the association with the current dermatitis was not apparent in all cases. (Di)isocyanates may induce contact sensitization with or without allergic contact dermatitis.

Absence of contact sensitization to Aloe vera (L.) Burm. f.

Aloe vera has been used as a cosmetic and medical remedy since ancient times and has gained increasing popularity in recent years. Despite its widespread use, reports of allergic reactions are rare. We patch tested 702 consecutive patients with an oily extract from the leaves, Aloe pulvis from the entire plant and concentrated Aloe vera gel. A specially designed questionnaire was used for the use of Aloe vera, reasons and location of application, adverse reactions, occupation, hobbies and atopy. None of the subjects showed any reaction to one of the preparations. 2 components of the plant have to be distinguished: the bark of the leaves contains anthrachinones with pro-peristaltic and potential antibiotic and anticancer properties. Constraints have been imposed due to their considerable toxic potential. Today, mostly the Aloe gel from the center of the leaves is processed. It almost exclusively consists of carbohydrates to which also many medical effects have been attributed. Carbohydrates are not likely to induce contact sensitization, which might explain the outcome of our study. However, this does not justify unrestrained promotion of Aloe products, as scientific studies investigating the claims on its constitutional effects are few in number, and the majority of them have been unable to diminish the intuitive scepticism against miracle cures, like Aloe seems to be.

Mobilisation of specific T cells from lymph nodes in contact sensitivity requires substance P

Capsaicin-mediated depletion of neuropeptides in the skin was previously shown to abolish a dinitrocholorobenzene (DNCB)-induced contact sensitivity (CS) response. To understand the basis for this disruption, we explored whether nerve fibres innervating the draining lymph node (LN) could be involved. As expected, removal of the draining LN after DNCB sensitisation abolished the CS response. Furthermore, the CS response could be abolished by destroying the nerve fibres in the draining LN and could be restored by providing the LN with the neuropeptide substance P. The size of the CS response restored by substance P was dose dependent. The response was also inhibited by exposing the lymph node to a neurokinin-1 receptor antagonist which blocks binding of substance P. The results suggest that an afferent signal from the skin via the sympathetic arm of the central nervous system evokes an efferent signal to the LN which combines to regulate the CS response. The efferent signal may serve to control or release from the LN primed effector lymphocytes into the circulation.

Contact hypersensitivity to methyl red in female Balb/c mice

Rationale Methyl red is a commonly used azo dye in laboratory assays, textiles, and other commercial products but little is known about its sensitization properties in humans. These studies were conducted to evaluate the sensitizing potential of methyl red using murine models of hypersensitivity. Methods For the local lymph node assay, female BALB/c mice were dosed for 3 days with vehicle (dimethyl sulfoxide), positive control, or increasing concentrations of methyl red (0.05-1.0%) on the dorsal surface of each ear. Following injection of [3H]-thymidine, draining lymph nodes were radioassayed on day 6. In the phenotypic analysis assay, mice were similarly exposed for 4 days. On day 10 post-exposure, serum was collected for total serum IgE analysis and lymph nodes were excised to evaluate B220+ and IgE+B220+ cell populations. Results Exposures up to 1.0% methyl red resulted in a dose dependent increase in lymphocyte proliferation reaching statistical significance ( p<0.05) at 0.5% and a calculated EC3 value (concentration required to induce a significant response) of 50 μg/cm2. Phenotypic analysis demonstrated a significant increase in lymph node B220+ cells ( p<0.01) although no change in serum IgE levels was observed following exposure to methyl red. Conclusions These studies suggest the potential for methyl red to induce contact sensitization in humans.

Pre-clinical methods for detecting the hypersensitivity potential of pharmaceuticals: regulatory considerations

Immune-based systemic hypersensitivities account for a significant number of adverse drug reactions. There appear to be no adequate nonclinical models to predict systemic hypersensitivity to small molecular weight drugs. Although there are very good methods for detecting drugs that can induce contact sensitization, these have not been successfully adapted for prediction of systemic hypersensitivity. Several factors have made the development of adequate models difficult. The term systemic hypersensitivity encompases many discrete immunopathologies. Each type of immunopathology presumably is the result of a specific cluster of immunologic and biochemical phenomena. Certainly other factors, such as genetic predisposition, metabolic idiosyncrasies, and concomitant diseases, further complicate the problem. Therefore, it may be difficult to find common mechanisms upon which to construct adequate models to predict specific types of systemic hypersensitivity reactions. There is some reason to hope, however, that adequate methods could be developed for at least identifying drugs that have the potential to produce signs indicative of a general hazard for immune-based reactions.

Induction and Localization of Cutaneous Interleukin-1β mRNA during Contact Sensitization

Chemical allergens that induce contact sensitivity cause changes in levels of epidermal cytokines. In mice one of the earliest epidermal cytokines to be upregulated following sensitization is interleukin-1β (IL-1β). The present study investigated the kinetics and in situ localization of induced IL-1β expression in mouse skin following topical exposure to the contact allergen oxazolone. Mice were exposed topically to 1% oxazolone, with control mice exposed to vehicle (acetone:olive oil 4:1) alone, and at various times thereafter skin was excised for IL-1β mRNA and protein determination by in situ hybridization and enzyme-linked immunosorbant assay (ELISA), respectively. IL-1β mRNA was found to be expressed constitutively at low levels in skin from naı̈ve (untreated) and vehicle-treated mice, with mRNA localized in some hair follicles and sebaceous glands; no IL-1β mRNA was detected in the epidermis of control animals. Following topical exposure of mice to oxazolone for 5–15 min, upregulation of IL-1β mRNA was observed in the epidermis, dermis, hair follicles, and sebaceous glands; at 90 min and beyond the pattern of IL-1β mRNA expression declined toward control. Analysis of whole skin homogenates by ELISA demonstrated cutaneous IL-1β protein to be present constitutively in both vehicle-treated and naı̈ve mice. Following exposure to oxazolone, cutaneous IL-1β protein expression was elevated at 30 min, decreased at 1 h, and fell below the limit of detection of the assay at 2 h before returning to constitutive levels at 4 and 24 h. IL-1β protein levels in vehicle-treated mice, naı̈ve mice, and mice treated with the respiratory allergen trimellitic anhydride were unchanged over this time period. The present study demonstrated that IL-1β mRNA expression was upregulated rapidly and transiently in well-defined regions of mouse epidermis and dermis during contact sensitization, and was succeeded by an elevation in IL-1β protein. This early highly localized upregulation of IL-1β lends further support to the hypothesis that this cytokine plays a key role in the initial stages of skin sensitization. Such information will enhance our understanding of the molecular processes involved in allergic contact dermatitis and may provide a mechanistic basis for designing refined animal and in vitro alternatives to existing models of skin sensitization.

Risk assessment and immunotoxicology

In general toxicity testing, maximal acceptable concentrations are derived from no-observed adverse effect levels (NOAEL) in rodents. Risk assessment then considers safety factors for the interspecies difference, and intraspecies variability. This approach can be used for assessing maximal acceptable concentrations for chemicals inducing direct immunotoxicity, resulting in e.g. reduced resistance to infections. As for predictive testing of chemicals in terms of sensitization, laboratory animal data are mostly used for risk assessment as well. Generally, the assessment of risk for chemicals that induce contact sensitivity is limited to hazard identification, and risk management is restricted to labeling. An alternative type of evaluation of the risk of adverse effects due to exposure to immunotoxic chemicals may be the so called parallellogram approach. In this parallellogram there are four cornerstones, one of which is the health effect of exposure to a chemical, assessed as an endpoint (e.g. infection model) in experimental animals, and another the quantitative prediction of this endpoint in humans. The other cornerstones are assays of parameters that are relevant to the mechanism of the adverse effect in experimental animals and humans, and are used for species comparison. Species comparisons between the animal species used for hazard identification and humans are crucial for extrapolation of animal data to the human situation. This approach can be used to provide relevant information on the dose-response relationship in humans. In concert with information on actual exposure, such data can then be used for the characterization of risk for adverse health effects in humans. Such approaches have been used for chemicals that exert direct immunotoxic activity (bis(tri-n-butyltin)oxide (TBTO)), and may hold promise for the risk evaluation of chemicals that exert skin sensitizing properties.

Susceptibility to Effects of UVB Irradiation on Induction of Contact Sensitivity, Relevance of Number and Function of Langerhans Cells and Epidermal Macrophages

Sensitization on skin exposed to acute low-dose UVB irradiation separates normal humans into two phenotypically distinct groups: One group, following sensitization on UVB-irradiated skin, develops contact sensitivity, designated UVB resistant (UVB-R) and the second group, following sensitization on UVB-irradiated skin, fails to develop contact sensitivity, designated UVB susceptible (UVB-S). To investigate whether UVB susceptibility in humans in related to antigen-presenting activity in the skin we studied the effect of UVB irradiation on the number and function of the epidermal antigen-presenting cells in volunteers identified as UVB-R and UVB-S. Single cell suspensions of epidermal cells from control skin and skin exposed to 3 minimal erythema doses (MED) of UVB 3 days previously were stained for Langerhans cells (CD1a+HLA-DR+) and epidermal macrophages (CD1a-HLA-DR+). The UVB exposure of the skin significantly decreased the percentage of Langerhans cells (UVB-R: n = 7, P < 0.02, UVB-S: n = 6, P < 0.03) and increased the percentage of epidermal macrophages (UVB-R: n = 7, P < 0.03, UVB-S: n = 6, P < 0.03) however to the same degree in both the UVB-R and the UVB-S group. To study the effect on Langerhans cell alloreactivity, epidermal cells were harvested immediately after UVB irradiation. However, in both UVB-R and UVB-S subjects the Langerhans cell alloreactivity was blocked to the same degree immediately after UVB irradiation compared to nonirradiated epidermal cells. To determine the effect of UVB irradiation on epidermal macrophages, epidermal cells were harvested 3 days after UVB irradiation. Irradiated epidermal cells from both UVB-R and UVB-S subjects demonstrated a strong antigen-presenting capacity compared to epidermal cells from control skin leading to activation of T cells that mainly secrete interferon (IFN)-gamma and not interleukin (IL)-4. In conclusion we found that UVB susceptibility was not correlated with the number of Langerhans cells or epidermal macrophages in the skin at the same time of sensitization. Neither was it correlated with the capacity of Langerhans cells nor UVB-induced epidermal macrophages to activate T cells in vitro.

Susceptibility to Effects of UVB Irradiation on Induction of Contact Sensitivity, Relevance of Number and Function of Langerhans Cells and Epidermal Macrophages

Susceptibility to Effects of UVB Irradiation on Induction of Contact Sensitivity, Relevance of Number and Function of Langerhans Cells and Epidermal Macrophages

Antigen-presenting cell function of epidermal cells activated by hapten application.

Exposure to haptens initiates a series of immune and inflammatory reactions that cause migration of epidermal Langerhans cells (LC) to draining lymph nodes, antigen processing, and presentation to T cells. In the present study, the antigen-presenting cell (APC) function of epidermal cells (EC) following hapten application was determined using a cell transfer system. This function of EC in inducing contact sensitivity (CS) in the recipient mice appeared as early as 6 h after hapten painting, and reached its maximum at 24 h. The amount of hapten on EC did not correlate with the function, i.e. the amount retained on the cells was greatest immediately after hapten painting and decreased over time. Several experiments were performed to identify the cell type responsible for the APC function. Through immunomagnetic bead separation, the APC function was detected in Ia- EC, as well as in unfractionated EC from hapten-painted mice. A purified population of Ia+ cells (LC) induced CS with much less efficiency than unseparated cells. Depletion of LC by anti-Ia monoclonal antibody (mAb) and complement-mediated lysis did not impair the APC function, whereas it was reduced by the depletion of Thy-1+ cells by anti-Thy-1 mAb and complement-mediated lysis. Moreover, adherent cells that were harvested from a 48-h culture of EC obtained from hapten-painted skin, and were free of contaminating LC and gamma omega T cells, had a strong capacity to induce CS. These findings indicate that keratinocytes (KC) acquire APC function as well as LC, with hapten application. Phenotypically increased expression of intercellular adhesion molecule-1 (ICAM-1) and Thy-1 on EC was observed following hapten application, whereas expression of Ia and B7/BB1 was unaltered. The APC function of EC from hapten-painted skin was dependent on ICAM-1 and Thy-1 expression, as the mAbs for these molecules reduced the capacity to induce CS. These results suggest that hapten application induces not only LC but also KC to mature functionally and become potent APCs, and that these KC exert the APC function complementarily at local sites following the migration of LC with potent APC function to the draining lymph node.

PRODUCTION AND IN SITU LOCALIZATION OF CUTANEOUS TUMOUR NECROSIS FACTOR α (TNF-α) AND INTERLEUKIN 6 (IL-6) FOLLOWING SKIN SENSITIZATION

The induction of contact sensitization and other cutaneous immune responses is dependent upon the activity of epidermal cytokines. One such, keratinocyte-derived tumour necrosis factor α (TNF-α), is thought to provide the stimulus for the migration of Langerhans cells from the epidermis and their accumulation as immunocompetent dendritic cells in draining lymph nodes. In these investigations we have examined the stimulation be allergen of cutaneous TNF-α production and the induced epidermal expression of mRNA for TNF-α. Topical exposure of mice to oxazolone, a skin-sensitizing chemical, resulted in cutaneous TNF-α protein production that was maximal 2-h following treatment and then declined markedly. The same treatment resulted in highly localized and transient expression of epidermal TNF-α mRNA as judged by in situ hybridization. Epidermal mRNA for TNF-α was apparent 10 min following exposure to oxazolone, but was no longer detectable at 20 min. A similar pattern of TNF-α mRNA expression in the epidermis was provoked by intradermal exposure to interleukin 1β, a cytokine shown previously to induce TNF-α. Such rigorous regulation of temporal and spatial expression was shown not to be a characteristic of all epidermal cytokines induced by chemical allergen. Exposure to oxazolone under the same conditions resulted in a more widespread and more persistent expression of epidermal mRNA for interleukin 6. These data demonstrate that during skin sensitization the induced expression of epidermal TNF-α is finely controlled in space and time. It is proposed that such regulation facilitates the initiation of cutaneous immune responses while preventing excessive inflammation that would result from more persistent TNF-α production.

Susceptibility to Effects of UVB Irradiation on Induction of Contact Sensitivity, Relevance of Number and Function of Langerhans Cells and Epidermal Macrophages

Susceptibility to Effects of UVB Irradiation on Induction of Contact Sensitivity, Relevance of Number and Function of Langerhans Cells and Epidermal Macrophages

Induction of contact sensitization to monotertiary butyl hydroquinone

Induction of contact sensitization to monotertiary butyl hydroquinone

Draining Lymph Node Cells of Contact-Sensitized Mice Induced Suppression of Contact Sensitivity

The application of hapten to the skin of mice can induce contact sensitivity (CS). It has also been well established that draining lymph node (DLN) cells can induce CS to the hapten used for skin painting when injected into naive mice. This is true for DLN cells recovered about 24 h after skin painting with hapten. It is unclear, however, whether DLN cells recovered shortly after hapten application have the same ability. By using an adoptive transfer assay system, we examined the ability of DLN cells recovered from mice at various times after skin painting with hapten to induce CS. DLN cells harvested 18-24 h after the application of fluorescein isothiocyanate (FITC) or 2,4-dinitrofluorobenzene (DNFB) induced strong CS when injected into naive mice. DLN cells harvested 3-6 h after the application of FITC or DNFB induced either only weak or no CS but induced suppression of the subsequent immunization to the two haptens. The suppression was hapten-specific, MHC restricted, and associated with the appearance of splenic suppressor T lymphocytes. Analyses with antibodies and ultraviolet (UV) B radiation demonstrated that suppression-inducing cells in DLNs were Ia+, Thy-1(-), and functionally UV-sensitive. These data suggest that epicutaneous sensitization with hapten first induces immunologically specific suppressor activity in the draining lymph nodes, whereas immunogenic activity becomes predominant later.

A re-appraisal of the skin-sensitizing activity of 2,4-dinitrothiocyanobenzene

A debate continues regarding the immunological properties of 2,4-dinitrothiocyanobenzene (DNTB). In some investigations this chemical was shown not to cause skin sensitization when applied topically but to induce instead hyporesponsiveness or immunological tolerance. In other studies DNTB was found to pause skin sensitization, but not tolerance. However, this chemical continues to be used to discriminate between the properties of skin sensitizing and non-sensitizing chemicals. This study demonstrates that topical exposure of mice to DNTB induces skin sensitization in mice and that this is associated with the accumulation of dendritic cells in draining lymph nodes and the stimulation of lymph node cell proliferation; the latter responses being of equivalent magnitude to those stimulated by 2,4-dinitrochlorobenzene (DNCB), a chemical known to cause contact sensitization. Moreover, exposure of mice to DNTB, as with exposure to DNCB, resulted in the development of a cytokine secretion pattern by draining lymph node cells (LNC) characteristic of contact allergens. Thus, DNTB and DNCB each induced the production by LNC of high levels of interferon-y, but little or no interleukin 4 or interleukin 10. Finally, DNTB was shown in the guinea pig maximization test to behave as an extreme skin sensitizer. These results confirm that DNTB should not be regarded as a universal tolerogen and that it possesses a significant potential to induce contact sensitization. The use of this chemical as a presumptive non-sensitizer and/or tolerogen for the evaluation of the selectivity of new predictive test methods for the identification of contact allergens is therefore considered to be inappropriate.

Carrier-independent hapten recognition and promiscuous MHC restriction by CD4 T cells induced by trinitrophenylated peptides.

The elucidation of mechanisms underlying the recognition of haptens by class II MHC-restricted T cells is instrumental for the understanding of chemical- and drug-induced allergies. We have previously demonstrated that trinitrophenyl (TNP) peptides represent dominant antigenic epitopes for CD8+ and CD4+ mouse T cells triggered by chemically TNP-modified APC. Here, we report the characterization of TNP-specific, CD4+ mouse T cell lines and hybridomas that were induced in vivo and in vitro by defined hapten-conjugated peptides. These peptides, which we had previously shown to induce contact sensitivity to picryl chloride in vivo regardless of sequence homologies to mouse proteins, were found to activate carrier-independent TNP-specific T cells in vitro. We interpret these findings to support our view that carrier-independent T cells, reactive to particularly repetitive hapten epitopes, may play a crucial role in allergies to chemicals and drugs. In addition to carrier independence, one of our hybridomas (IT-H6/A11) exhibited a striking promiscuity of MHC restriction. Although absolutely dependent in its TNP reactivity on the presence of MHC class II molecules, the IT H6/A11 hybridoma completely ignored class II polymorphism and even reacted to TNP peptides presented on human DR molecules. Regarding hapten allergies in humans with a heterozygous situation for three types of class II molecules (DR, DP, and DQ), such promiscuous MHC restriction should lead to the presentation of even higher epitope densities to the respective T cell clones. Hybridoma IT-H6/A11, reacting to TNP independent of carrier peptide and of MHC haplotype, also allowed for an unusually systematic study of the minimal requirements for TNP recognition. Despite an almost complete ignorance of amino acid side chains on the carrier peptide, our data indicate a clearly position-specific interaction of hapten and TCR.