Characterization of CD3γ/δ+ cells in grass carp (Ctenopharyngodon idella)

CD3 is an essential component of the TCR-CD3 complex which plays a key role in adaptive immunity. Non-mammalian CD3 complex consists of CD3 gamma/delta, CD3 epsilon and CD3 zeta subunits. In this study, homologues of CD3 gamma/delta and CD3 epsilon (termed CiCD3 gamma/delta and CiCD3 epsilon) have been identified in grass carp (Ctenopharyngodon idella). Like their counterparts from other vertebrates, the CiCD3 gamma/delta and CiCD3 epsilon are clustered in the same locus in the genome and encode proteins which are structurally conserved, comprising a signal peptide, an extracellular domain, a transmembrane domain and a cytoplasmic tail containing two ITAM motifs. Sequence analyses identified two novel conserved motifs in the cytoplasmic tail of CiCD3 gamma/delta and CiCD3 epsilon, one is composed of an arginine and lysine motif (RK or RR) at the C terminus of CiCD3 gamma/delta and a proline rich domain (PxxPxP/Q) located at the N terminus of ITAM motifs of CiCD3 epsilon. Both genes were highly expressed at the mRNA level in the spleen and gills of healthy fish and could be modulated by infection of Flavobacterium columnare and grass carp reovirus. A monoclonal antibody against the CiCD3 gamma/delta (GC38T) was produced and showed good reactivity with the native molecule in Western blotting analysis and flow cytometry. The CiCD3 gamma/delta(+) cells were analysed in the primary leucocytes, accounting for 5.5% of lymphocytes isolated from spleen, 4.5% from head kidney and 2.8% from peripheral blood. The CiCD3 gamma/delta(+) cells were localized in the gills and head kidney by fluorescent confocal microscopy.

Automated summary

CD3 is a crucial component of the TCR-CD3 complex in adaptive immunity, with its homologues CiCD3γ/δ and CiCD3ε identified in grass carp and shown to be highly expressed in healthy fish and modulated by infections. A monoclonal antibody against CiCD3γ/δ was successfully produced, which showed good reactivity in Western blotting and flow cytometry analysis. The CiCD3γ/δ(+) cells were found to be localized in the gills and head kidney, accounting for a small percentage of lymphocytes in different organs.