Defining lipids and T cell receptors involved in the intrinsic allergenicity of nut proteins
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LETTER TO THE EDITOR
Clinical and Translational Allergy Open Access
Defining lipids and T cell receptors involved in the intrinsic allergenicity of nut proteins Rui Wang1, Ashfaq Ghumra1, Stella Cochrane3* , Lucy Fairclough2, Richard Broughton4, Louise V. Michaelson4, Frederic Beaudoin4 and Marcos J. C. Alcocer1
To the Editor What makes a protein allergenic, and in particular a food allergen, has not yet been defined. Previously, using model systems, it has been shown plant lipids have an essential role in the allergenicity of the Brazil nut allergen Ber e 1 and NKT-like cells are involved in the sensitisation phase to nut proteins [1–3]. Further progressing investigation of these findings we hereby share details of work to improve and optimise protocols for isolation of lipid responsive human NKT-like cells, sequence and express lipid-binding T cell receptors (TCRs) and use these TCRs to screen Brazil nut lipid fractions. Primary NKT cells (CD3+, CD56+) from 4 allergic and 2 healthy human volunteers, were targeted by FACS. The NKT cells were challenged with lipids, active cells individually sorted and α/β and γ/δ TCR sequences amplified. The lipid-activated specific populations of TCRs were then identified, sequenced and cloned into expression constructs for use in the in-vitro system shown in Fig. 1. The CD3 + CD56 + lymphocytes were co-cultured with 5 µg/ml controls (without lipid or α-GalCer ) or 1 × 106 lipid-mixture loaded APC cells (MUTZ3) as previously described [4]. Single CD3 + CD56 + CD69 + high cells were isolated by FACS and TCR pairs sequenced and characterised as described [5, 6]. From this screening around 103 pairs of TCR DNA sequences were obtained that were analyzed and classified against human TCR sequence libraries using the IMGT/V-QUEST website. *Correspondence: [email protected] 3 Unilever Safety and Environmental Assurance Centre (SEAC), Colworth Science Park, MK44 1LQ Sharnbrook, UK Full list of author information is available at the end of the article
From the 103 pairs of sequences, some containing the Jα-33 marker for MAIT cells, three TCR pair sequences (1 α/β and 2 γ/δ) were identified within the nut group that were not present in the controls. These 3 TCR pairs were cloned into acceptor bidirectional plasmids as previously described [4], named as pMJA290, pMJA295 and pMJA297 and filed into Genbank as MK764035, MK764036, MK764037. In an attempt to detect the functions of these 3 TCRs, a plasmid (pMJA219) containing the α-GalCer responsive human TRAV10 and TRBV25 sequences was constructed. The α/β TCR sequences used in pMJA219 (TRAV10/TRBV25) design have been previously described as α-GalCer-specific using lipid loaded tetramers [4]. pMJA219 (TRAV10/TRBV25) containing a synthetic TCR sequence specific to α-GalCer was used as control. To investigate which lipid(s) were recognized by the TCR sequences from allergic patients, nut lipids were fractionated in three major classes: neutrolipids (NL), glycolipids (GL) and polar lipids (PL) as described [7]. These lipid classes
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