Species Specific Differences of CD1d Oligomer Loading In Vitro.

Daniel Paletta,Thomas Herrmann,Lisa Starick,Alina Suzann Fichtner,Paul B Savage,Steven A Porcelli

doi:10.1371/journal.pone.0143449

Abstract

CD1d molecules are MHC class I-like molecules that present glycolipids to iNKT cells. The highly conserved interaction between CD1d:α-Galactosylceramide (αGC) complexes and the iNKT TCR not only defines this population of αβ T cells but can also be used for its direct identification. Therefore, CD1d oligomers are a widely used tool for iNKT cell related investigations. To this end, the lipid chains of the antigen have to be inserted into the hydrophobic pockets of the CD1d binding cleft, often with help of surfactants. In this study, we investigated the influence of different surfactants (Triton X-100, Tween 20, Tyloxapol) on in vitro loading of CD1d molecules derived from four different species (human, mouse, rat and cotton rat) with αGC and derivatives carrying modifications of the acyl-chain (DB01-1, PBS44) and a 6-acetamido-6-deoxy-addition at the galactosyl head group (PBS57). We also compared rat CD1d dimers with tetramers and staining of an iNKT TCR transductant was used as readout for loading efficacy. The results underlined the importance of CD1d loading efficacy for proper analysis of iNKT TCR binding and demonstrated the necessity to adjust loading conditions for each oligomer/glycolipid combination. The efficient usage of surfactants as a tool for CD1d loading was revealed to be species-specific and depending on the origin of the CD1d producing cells. Additional variation of surfactant-dependent loading efficacy between tested glycolipids was influenced by the acyl-chain length and the modification of the galactosyl head group with PBS57 showing the least dependence on surfactants and the lowest degree of species-dependent differences.

Highlights

CD1 molecules belong to a non-polymorphic family of non-classical MHC class I molecules (MHC Ib molecules) [1, 2]
This allows insertion of hydrophobic chains and lipid antigen presentation. In this way CD1d of most species is able to present antigens to Type 1 NKT cells, a “non-conventional” αβ T cell subpopulation [11,12,13,14,15]. This distinct population can be defined by the highly conserved interaction between its semi-invariant iNKT TCR (Vα14/Jα18 paired with Vβ8.2 in mice and Vα24/ Jα18 paired with Vβ11 in human) and the cerebroside α-Galactosylceramide presented by CD1d [16,17,18,19,20]
Included in the analysis were CD1d dimers of cotton rat which have recently been generated in our laboratory [36]

Summary

Introduction

CD1 molecules belong to a non-polymorphic family of non-classical MHC class I molecules (MHC Ib molecules) [1, 2]. The α1 and α2 domains of CD1d form an antigen binding cleft substantially different from that of MHC class I molecules: Accessible through a single narrow portal (F’ portal), it is located between both helices where two deep hydrophobic pockets (A’ and F’) are formed beneath the surface of the CD1d molecule [8,9,10]. This allows insertion of hydrophobic chains and lipid antigen presentation. During the binding of iNKT TCRs to CD1d molecules presenting αGC, the acyl and sphingosine chains are inserted into the A’ and F’ pockets of CD1d, respectively, while the polar head group protrudes above the antigen binding cleft to make direct contacts with the iNKT TCR [11,12,13,14]

Methods

Results

Conclusion