The hypervariable region 4 (HV4) and position 93 of the α chain modulate CD1d‐glycolipid binding of iNKT TCRs

Abstract

TCRs of invariant NKT (iNKT) cells bind α-galactosylceramide (αGC) loaded CD1d in a highly conserved fashion and show a characteristic TCR gene usage: An "invariant" α chain with a canonical AV14/AJ18 rearrangement in mice (AV24/AJ18 in humans) is paired with β chains containing characteristic Vβ segments. In the rat, a multimember AV14 gene family increases the variability within this system. This study characterizes CD1d binding of rat AV14 gene segments in TCR transductants as well as CD1d binding and iNKT TCR expression of expanded polyclonal F344 rat iNKT populations. It defines an important role of position 93 at the V-J transition for TCR avidity and species cross-reactivity of the rat iNKT TCR. Furthermore, for the first time we identified variability within the fourth hypervariable loop (HV4) of the α chain as a modulator of CD1d:αGC binding in rat and mouse. Additionally, we confirmed the importance of the CDR2β for CD1d:αGC binding, but also show that the CDR3β may even have opposite effects on binding depending on the pairing α chain. Altogether, we characterized naturally occurring sources of variability for the iNKT TCR and speculate that they rather level than increase the largely germline encoded differences of iNKT TCR ligand avidity.Keywords: CD1d r iNKT cells r Molecular modeling r Rat r T-cell receptors Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionType 1 NKT cells (invariant NKT (iNKT) cells) are a subset of αβ T cells characterized by the expression of a semi-invariant TCR. In contrast to conventional αβ T cells, iNKT cells are not MHCrestricted, but recognize CD1d, a nonpolymorphic MHC class Ilike molecule presenting lipid antigens [1,2]. The lineage-defining iNKT TCR contains an invariant α chain with a uniform AV14/AJ18 rearrangement in mice (AV24/AJ18 in human) and a limited number of β chain V segments (mostly BV8S2, BV7, and BV2 in mice Correspondence: Prof. Thomas Herrmann e-mail: herrmann-t@vim.uni-wuerzburg.de and the BV8S2 homologue BV11 in humans) [3][4][5][6][7]. This decreased variability within the TCR leads to a very restricted CDR repertoire with CDR3β as the only exception.The first characterized and one of the strongest antigens for iNKT cells is KRN7000 (α-galactosylceramide, αGC), the synthetic derivative of a compound isolated from the marine sponge Agelas mauritanus [3]. The rather fixed mode of interaction between the iNKT TCR and the CD1d:αGC complex does not only allow for direct identification of iNKT cells by CD1d oligomers loaded with αGC or its derivative PBS57, but also defines this lineage of αβ T cells. Crystallographic studies have shown that, for mouse and human, direct contact is mostly mediated by CDR1α, CDR3α, and CDR2β of the iNKT TCR, displaying the restricted usage of CDRs within this system [8,9]. Thereby, an alanine C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2015. 45: 2122-2133 Molecular immunology 2123 scanning mutagenesis study...

Function and expression of CD1d and invariant natural killer T‐cell receptor in the cotton rat (Sigmodon hispidus)

“…In some rearrangements glycine 93 or valine 92 were substituted by valine or alanine, respectively. This is analogous to substitutions that, in rat and mouse, respectively, were found to affect ligand binding . Furthermore, the cytokine production of splenocytes in response to typical iNKT cell antigens such as α GC and PBS57 indicates the existence of a typical iNKT cell population in this species.…”

“…a). The CDR1–3 region and the hypervariable region 4 (HV4) are based on published data and indicated in the alignment . The number of identical amino acids/nucleotides was divided by the total length of the alignment, so taking gaps into account, and the results were expressed as a percentage.…”

“…This is analogous to substitutions that, in rat and mouse, respectively, were found to affect ligand binding. 28,39 Furthermore, the cytokine production of splenocytes in response to typical iNKT cell antigens such as aGC and PBS57 indicates the existence of a typical iNKT cell population in this species. The levels of IFN-c were about 100-fold higher and for IL-4 twice as high as levels in F344 rats.…”

“…Fifty microlitres per well was used to coat wells of U‐bottom 96‐well suspension culture plates and plates were incubated at 4° overnight and afterwards washed three times with PBS. Then, 5 × 10 4 rat iNKT TCR‐expressing mouse T‐cell hybridoma cells, BW r/m CD28 EGN rAV14 S6 93A S65T CDR2+4 L14V, were added in RPMI‐1640 medium [Gibco, Grand Island, NY; supplemented with 10% FBS, 1 m m sodium pyruvate, 0·05 m m glutamine, 0·1 m m non‐essential amino acids, 5 m m β ‐mercaptoethanol, penicillin (100 U/ml), streptomycin (100 μg/ml)], and cultured for 22 hr at 37° in 5% CO 2 . Supernatants were analysed with a mouse interleukin‐2 (IL‐2) sandwich ELISA (BD OptEIA™ mouse IL‐2 ELISA Kit).…”

“…The CDR1-3 region and the hypervariable region 4 (HV4) are based on published data and indicated in the alignment. 28 The number of identical amino acids/nucleotides was divided by the total length of the alignment, so taking gaps into account, and the results were expressed as a percentage. Comparable to the sequence analysis of CD1d, the cotton rat AV14/AJ18 rearrangement shared an amino acid (nucleotide) homology of 80% (86%) with the predicted hamster rearrangement, 75% (83%) with rat, 74% (82%) with mouse and only 66% (75%) with the homologous human rearrangement (Fig.…”