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Background: Skin patch testing is still seen as the gold standard for the diagnosis of allergic hypersensitivity. For several metals and for patients with a suspected adverse reaction to their medical device implant material, patch testing can be unreliable. The current alternative to metal allergy patch testing is the in vitro lymphocyte proliferation test (LPT) using tritiated thymidine. This method is well-established but requires handling of radioactive material, often uses heat-inactivated allogenic human pooled serum and cannot determine T cell subsets. Objective: To develop a radioactive free LPT by using carboxyfluorescein succinimidyl ester (CFSE) and to evaluate the influence of serum source (heat-inactivated human pooled serum [HI HPS] vs autologous serum) on the sensitivity and specificity of the nickel-specific LPT. Methods: Peripheral blood mononuclear cells derived from nickel-allergic patients and healthy controls were collected, labelled with CFSE and cultured in medium containing 10% HI HPS or 10% autologous serum with or without additional T cell skewing cytokine cocktails (Th1: IL-7/IL-12, Th2: IL-7/IL-4 or Th17: IL-7/IL-23/IL-1β) in the absence or presence of NiSO4. The stimulation index (SI) was calculated as the ratio of divided cells, that is the percentage of CFSElow/neg CD3+CD4+ T-lymphocytes upon nickel stimulation compared to the percentage of CFSElow/neg CD3+CD4+ T-lymphocytes without antigen. These results were compared with the history of Ni allergy, patch test results and the MELISA test. Results: Autologous serum positively influenced Ni-specific proliferation while HI HPS negatively influenced Ni-specific proliferation. The test protocol analysing CD4+ cells and autologous serum without skewing cytokines scored the best diagnostic values (sensitivity 95%; specificity 93%; and overall accuracy 94%) compared to the parallel test using HI HPS (accuracy 60%). Cytokine supplements did not further improve the test protocol which used autologous serum. The protocol using HI HPS could be further improved by addition of the cytokine skewing cocktails. Conclusions: Here, we describe an optimized and highly accurate flow cytometric LPT which comprises of CFSE-labelled cells cultured in autologous serum (not heat inactivated) and without the presence of T cell skewing cytokines. Clinical relevance: The sensitivity and specificity of LPT is enhanced, compared to HI HPS, when autologous serum without skewing cytokines is used.

SummaryBackground Toll-like receptor 2 (TLR2) is a widely expressed pattern recognition receptor critical for innate immunity. TLR2 is also a key regulator of mucosal immunity implicated in the development of allergic disease. TLR2 activators are found in many common foods, but the role of TLR2 in oral tolerance and allergic sensitization to foods is not well understood. Objective The purpose of this study was to evaluate the impacts of TLR2 expression and TLR2 activation on oral tolerance to food antigens in a murine model. Methods Mice were fed ovalbumin (OVA) or peanut butter with or without the addition of low doses of TLR2 activators Pam3CSK4 or FSL-1. Oral tolerance was assessed by analysing antibody responses after a systemic antigen challenge. OVA-specific Tregs were assessed in the Peyer's patches, mesenteric lymph nodes, and spleen in wild-type and TLR2−/− mice. Low-dose Pam3CSK4 was also tested as an oral adjuvant. Results Oral tolerance was successfully induced in both wild-type and TLR2−/− recipient mice, with an associated regulatory T-cell response. Oral TLR2 activation, with low-dose Pam3CSK4 or FSL-1, during oral antigen exposure was found to alter oral tolerance and was associated with the development of substantial IgE and IgA responses to foods upon systemic challenge. Low-dose oral Pam3CSK4 treatment also selectively enhanced antigen-specific IgA responses to oral antigen exposure. Conclusions and Clinical Relevance TLR2 is not necessary for oral tolerance induction, but oral TLR2 activation modulates humoral IgE and IgA responses during tolerance development. Low-dose Pam3CSK4 is also an effective oral adjuvant that selectively enhances IgA production. These observations are pertinent to the optimization of oral allergen immunotherapy and oral vaccine development.