Use of Humanized Rat Basophilic Leukemia Reporter Cell Lines as a Diagnostic Tool for Detection of Allergen-Specific IgE in Allergic Patients: Time for a Reappraisal?
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The interaction between allergens and specific IgE is at the heart of the allergic response and as such lies at the center of techniques used for diagnosis of allergic sensitization. Although serological tests are available, in vivo tests such as double-blind placebo-controlled food challenges (DBPCFC) and skin prick test (SPT) associated to the patients’ clinical history are still the main guides to clinicians in many practices around the world. More recently, complex protein arrays and basophil activation tests, requiring only small amounts of whole blood, have been developed and refined, but are yet to enter clinical practice. Similarly, the use of rat basophilic leukemia (RBL) cell lines for detection of allergen-specific IgE has been made possible by stable transfection of the human FcεRI α chain into this cell line more than 20 years ago, but has not found widespread acceptance among clinicians. Here, we review the perceived limitations of diagnostic applications of humanized RBL systems. Furthermore, we illustrate how the introduction of reporter genes into humanized RBL cells is able to overcome most of these limitations, and has the potential to become a new powerful tool to complement the armamentarium of allergists. A demonstration of the usefulness of humanized RBL reporter systems for elucidation of complex IgE sensitization patterns against wheat proteins and a section on the use of fluorescence-based reporter systems in combination with allergen arrays close the review.
KeywordsRBL RS-ATL8 NFAT-DsRed IgE Allergic sensitization Allergy diagnosis
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Conflict of Interest
Drs. Falcone, Alcocer, Okamoto-Uchida, and Nakamura declare no conflicts of interest.
Human and Animal Rights and Informed Consent
This article does not contain any studies with human or animal subjects performed by any of the authors.
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- 2.•Fleischer DM, Burks AW. Pitfalls in food allergy diagnosis: serum IgE testing. J Pediatr. 2015;166:8–10. Up-to-date editorial summarising the key problems regarding the difficulties of diagnosing food allergy, in particular due to the gap between sensitization (the occurrence of sIgE in patient blood) and the presence or absence of clinical symptoms.CrossRefPubMedGoogle Scholar
- 3.Cox L, Williams B, Sicherer S, Oppenheimer J, Sher L, Hamilton R, et al. Pearls and pitfalls of allergy diagnostic testing: report from the American College of Allergy, Asthma and Immunology/American Academy of Allergy, Asthma and Immunology Specific IgE Test Task Force. Ann Allergy Asthma Immunol. 2008;101:580–92.CrossRefPubMedGoogle Scholar
- 4.Blackley CH. Experimental researches on the causes and nature of catarrhus æstivus (hay-fever or hay-asthma). 1873. Available from: doi: 10.1097/00000441-187413300-00026
- 5.••Santos AF, James LK, Bahnson HT, Shamji MH, Couto-Francisco NC, Islam S, et al. IgG4 inhibits peanut-induced basophil and mast cell activation in peanut-tolerant children sensitized to peanut major allergens. J Allergy Clin Immunol. 2015;135:1249–56. This work clearly demonstrates the benefits of using in vitro cellular readouts when studying sensitization patterns. Levels of allergen-specific IgE were only in part able to explain the observed differences in clinical reactivity between allergic and sensitized, but tolerant patients. Depletion experiments successfully demonstrated the importance of IgG4 in plasma samples of peanut-sensitized tolerant patients as a blocking antibody.PubMedCentralCrossRefPubMedGoogle Scholar
- 11.Bernard H, Guillon B, Drumare M-F, Paty E, Dreskin SC, Wal J-M, et al. Allergenicity of peanut component Ara h 2: contribution of conformational versus linear hydroxyproline-containing epitopes. J Allergy Clin Immunol. 2015;135:1267–74.e8.Google Scholar
- 17.Taudou G, Varin-Blank N, Jouin H, Marchand F, Weyer A, Blank U. Expression of the alpha chain of human Fc epsilon RI in transfected rat basophilic leukemia cells: functional activation after sensitization with human mite-specific IgE. Int Arch Allergy Immunol. 1993;100:344–50.CrossRefPubMedGoogle Scholar
- 18.Wang X, Cato P, Lin H-C, Li T, Wan D, Alcocer MJC, et al. Optimisation and use of humanised RBL NF-AT-GFP and NF-AT-DsRed reporter cell lines suitable for high-throughput scale detection of allergic sensitisation in array format and identification of the ECM-integrin interaction as critical factor. Mol Biotechnol. 2013;56:136–46.PubMedCentralCrossRefGoogle Scholar
- 20.••Nakamura R, Uchida Y, Higuchi M, Nakamura R, Tsuge I, Urisu A, et al. A convenient and sensitive allergy test: IgE crosslinking-induced luciferase expression in cultured mast cells. Allergy. 2010;65:1266–73. This was the first developed humanized RBL reporter system obtained on an SX-38 RBL background, called RS-ATL8. The inclusion of a luciferase reporter strongly boosted sensitivity, allowing higher serum dilutions to be used, thus avoiding issues of cytotoxicity which had marred the use of humanized RBL cells in the past.PubMedCentralCrossRefPubMedGoogle Scholar
- 25.Ladics GS, van Bilsen JHM, Brouwer HMH, Vogel L, Vieths S, Knippels LMJ. Assessment of three human FcepsilonRI-transfected RBL cell-lines for identifying IgE induced degranulation utilizing peanut-allergic patient sera and peanut protein extract. Regul Toxicol Pharmacol. 2008;51:288–94.CrossRefPubMedGoogle Scholar
- 40.•Wan D, Ludolf F, Alanine DGW, Stretton O, Ali Ali E, Al-Barwary N, et al. Use of humanised rat basophilic leukaemia cell line RS-ATL8 for the assessment of allergenicity of Schistosoma mansoni proteins. PLoS Negl Trop Dis. 2014;8, e3124. This is the first demonstration of the potential use of EXiLE for identification of novel allergens, in this case from the trematode Schistosoma mansoni . It is suggested that this could become an important tool for assessment of safety of anti-helminth vaccine candidates.Google Scholar
- 42.•Nakamura R, Nakamura R, Sakai S, Adachi R, Hachisuka A, Urisu A, et al. Tissue transglutaminase generates deamidated epitopes on gluten, increasing reactivity with hydrolyzed wheat protein-sensitized IgE. J Allergy Clin Immunol. 2013;132:1436–8. This is a good example of how the use of a humanized RBL reporter system played a key role in elucidating the role of tissue transglutaminase in generating neoepitopes in gluten, resulting in clinical reactions to ingestion of gluten by patients with negative serum gluten-specific IgE levels, but sensitized to hydrolysed wheat protein in a cosmetic product.CrossRefPubMedGoogle Scholar
- 51.Marti E, Wang X, Jambari NN, Rhyner C, Olzhausen J, Pérez-Barea JJ, et al. Novel in vitro diagnosis of equine allergies using a protein array and mathematical modelling approach: a proof of concept using insect bite hypersensitivity. Vet Immunol Immunopathol. 2015.Google Scholar