Diversity, molecular characterization and expression of T cell receptor γ in a teleost fish, the sea bass (Dicentrarchus labrax, L)

Abstract

Two lineages of T cells, expressing either the αβ T cell receptor (TR) or the γδ TR, exist in Gnathostomes. The latter type of T cells account for 1-10 % of T cells in blood and up to 30 % in the small intestine. They may recognize unconventional antigens (phosphorylated microbial metabolites, lipid antigens) without the need of major histocompatibility class I (MH1) or class II (MH2) presentation. In this work we have described cloning and structural characterization of TR -chain (TRG) from the teleost Dicentrarchus labrax. Further, by means of quantitative PCR analysis, we analyzed TRG expression levels both in poly I:C stimulated leukocytes in vitro, and following infection with betanodavirus in vivo. Two full length cDNAs relative to TRG, with the highest peptide and nucleotide identity with Japanese flounder, were identified. A multiple alignment analysis showed the conservation of peptides fundamental for TRG biological functions, and of the FGXG motif in the FR4 region, typical of most TR and immunoglobulin light chains. A 3D structure consisting of two domains mainly folded as beta strands with a sandwich architecture for each domain was also reported. TRG CDR3 of 8-18 AA in length and diversity in the TRG rearrangements expressed in thymus and intestine for a given V/C combination were evidenced by junction length spectratyping. TRG mRNA expression levels were high in basal conditions both in thymus and intestine, while in kidney and gut leukocytes they were up-regulated after in vitro stimulation by poly I:C. Finally, in juveniles the TRG expression levels were up-regulated in the head kidney and down-regulated in intestine after in vivo infection with betanodavirus. Overall, in this study the involvement of TRG-bearing T cells during viral stimulation was described for the first time, leading to new insights for the identification of T cell subsets in fish.

Figure 1. IMGT Colliers de Perles of the two identified sea bass TRG sequences.

Panel a) Vdomain (V-GAMMA) and C- domain (C-GAMMA) of Diclab1, Panel b) V- domain (V-GAMMA) and C- domain (C-GAMMA) of Diclab2. Amino acid positions are according to the IMGT unique numbering for V domain and C domain. Anchor positions are shown as squares. In FR-IMGT, the hydrophobic amino acids (hydropathy index with positive value) and tryptophan (W) found at a given position in more than 50% of sequences are displayed with a blue background color.

Figure 2. Alignment of the predicted sea bass TRγ chain amino acid sequences with other known TRγ molecules.

The position of the framework (FR) and CDR regions for the V-DOMAINs following the IMGT numbering are indicated above the sequences. Conserved amino acid residues are evidenced. Accession numbers: Dicentrarchus labrax clone 1 FR745889; Dicentrarchus labrax clone 2 FR751038; Paralichthys olivaceus AB076073; Ictalurus punctatus DQ435303; Ginglymostoma cirratum FJ513785; Cyprinus carpio ABD04398; Danio rerio AY973921; Oryctolagus cuniculus RABTCRG; Mus musculus MUSTCRGZZX; Homo sapiens S01895; Xenopus laevis AF440821; Gallus gallus GGU22666.

Figure 3. Spectratyping analysis of the γ TR diversity in gut and thymus of healthy fish.

CDR3 length distribution profiles of γ transcripts for selected Vγ-Cγ combinations in naive thymus and gut. Typical results from the same fish are shown. Y-axis: fluorescence arbitrary units, x-axis: CDR3 size, with values indicated for the smallest and longest CDR3 amplified in each profile.

Figure 4. 3D model of sea bass TRγ chain.

Panel a). 3D model of sea bass Diclab1. The backbone ribbon and secondary structure topology are shown: yellow arrows represent β-strands and red cylinders represent α-helices. The Cys residue are evidenced along the sequence. Panel b). Amino acid residues putatively involved in the interactions with TRδ chain in both Diclab1 and Diclab2 sea bass TRγ sequences are represented as space-fill balls. The basic and acidic amino acid residues have been indicated in blue and red, respectively. Arrows and labels indicate the charged amino acids of the evidenced areas in the two sea bass TRγ sequences.

Figure 5. TRG expression analysis.

Panel a) and b). Stimulation with poly I:C. TRγ mRNA levels expressed as a ratio relative to rRNA 18 levels in the same samples after real-time PCR analysis of head kidney and gut leukocytes stimulated with PBS (control) and with 20 µg/ml of poly I:C for 6 and 24 h and normalised against the non-stimulated 0 h control. Data are expressed as the mean ± SD; one asterisk indicates when p<0.05 with respect to the time 0 control; two asterisks indicate when p<0.01 with respect to the time 0 control and three asterisks indicates when p<0.001 with respect to the time 0 control. Panel c) and d). Infection with betanodavirus. TRγ mRNA levels as a ratio relative to rRNA 18 levels in the same samples after real-time PCR analysis of head kidney and gut leukocytes in control fish and in infected fish at different sampling times. Data are expressed as indicated above for panel a) and b).