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Immunoglobulin T (IgT) is one of the key effector molecules of jawed vertebrate's adaptive immune system, and in this work we describe the quantitative distribution of IgT-expressing and IgT-producing cells in tissues of the European seabass Dicentrarchus labrax by using mRNA riboprobes and a specific anti-IgT antibody. A polyclonal antiserum (pAb) was prepared by immunizing rabbits with three synthetic peptides deduced from the full length IgT cDNA sequence and located in a surface-exposed CH3 domain of IgT constant region. The obtained antiserum, named RAIgT1, was able to recognize by ELISA immunization antigens and IgT from intestinal mucus and serum. In western blots of head kidney leukocytes lysates the antiserum recognized a 180 kDa polypeptide in non-reducing, and a 75 kDa peptide in reducing conditions. Interestingly, the RAIgT1 pAb crossreacted intensely in western blots with rainbow trout IgT purified from mucus and serum. Antisense mRNA IgT oligonucleotide sequences were employed in in situ hybridization to detect IgT-expressing cells in sections from lymphoid tissues, and positive cells were observed in head kidney, spleen, intestine and gills. By employing RAIgT1 in quantitative immunohistochemistry, the highest number of IgT-producing cells was observed in the gills (9.5 ± 0.7%), followed by intestine (8.4 ± 1.2%), head kidney (6.2 ± 1.4%), and spleen (4.1 ± 0.7%). Interestingly, the number of IgT-B cells showed a regionalization in the intestine, increasing from the proximal to the terminal part. By immunofluorescence and flow cytometry of live leukocytes, the percentages of RAIgT1 stained cells were 34 ± 11% in the intestine, 22 ± 5% in head kidney, 16 ± 7% in spleen, and 9 ± 5% in gills. At the fluorescence microscope, live cells from these tissues showed a typical membrane-associated positivity and a lymphocytic morphology, and no IgT/IgM double positive cells were detected. Immunoreactive cells have been purified from head kidney using magnetic beads, and IgT-enriched cells showed by RT-PCR an enhanced expression of the IgT gene, whereas IgT-depleted cells had an highest expression of IgM and TRβ genes. These data describe for the first time a quantitative panel of IgT-expressing and IgT-immunoreactive cells in tissues of a teleost fish species.

Cellular and molecular data have evidenced a gut-associated lymphoid tissue in a variety of teleost species, abundantly containing T cells, whose origin, selection and functions are still unclear. This study reports CD4, CD8-α, MHCI-α, MHCII-β, rag-1 and TCR-β gene transcription along the intestine (anterior, middle and posterior segments) and in the thymus of one year-old Dicentrarchus labrax (L.). Real-time PCR findings depicted a main role of the thymus in T cell development, but also rag-1 and CD8-α transcripts are detected in the intestine, having significant expression in the posterior segment. In the whole intestine TCR-β and CD8-α exceeded CD4 transcripts. RNA ISH confirmed these data and detailed that mucosal CD8-α+ cells were especially numerous in the epithelium and in aggregates in the lamina propria. Regional differences in T cell-specific gene expressions are first described in the intestine of a bony fish. High non-specific cytotoxic activity against xenogeneic and allogeneic cells was found in lymphocytes purified from the intestinal mucosa, providing further insight into their local defence roles. L'articolo è disponibile sul sito dell'editore http://www.sciencedirect.com/

To investigate mucosal immunity, from a whole transcriptome of sea bass gills we identified and successively cloned complete cDNA sequences of IgT and IgD heavy chains. These sequences have been employed to prepare riboprobes for ISH, and to identify conserved regions of sea bass IgT/IgD in order to select sequences from which synthetic peptides have been prepared. The peptides have been used as immunogens in rabbits to obtain antisera against IgT (RaIgT1) and IgD (RaIgD1) and by employing either riboprobes and antisera, the pattern of IgT/IgD expressing tissues and the presence of IgT/IgD immunoreactive cells has been investigated. The results by qPCR showed that the IgT gene is mainly expressed in muscle, intestine and gills, and the IgD gene in head kidney. By ISH, the presence of IgT-expressing cells has been detected in the intestine, with their number increasing toward the posterior segment, and in the gills. These data have been confirmed by IHC employing the rabbit antiserum RaIgT1, and extended by revealing the presence of IgTimmunoreactive cells in head kidney and spleen, as well as intense reactivity in intestinal mucus. As expected, in mucosal tissues IgT-immunoreactive cells outnumbered IgM-immunoreactive cells. By IIF and flow cytometry with RaIgT1 we determined the number of IgT-positive cells in organs and tissues, being around 32 ± 12 % in head kidney leukocytes, 14 ± 6 % in GIALT and 23 ± 10 % in spleen leukocytes. At the microscope the IIF-positive cells had the morphology of lymphocytes. By western blotting of head kidney lysates, RaIgT1 stained a 180 kDa band in non-reducing gels and a 74 kDa band in reducing gels and, in addition, RaIgT1 showed cross-reaction with partially purified rainbow trout IgT. Leukocytes from the head kidney were enriched in RaIgT1-positive cells by magnetic sorting, and PCR analysis showed an enrichment of IgT and depletion of IgM expression, respectively, in RaIgT1-enriched cells. These data suggest we obtained an antibody against IgT cells of sea bass. Preliminary data employing the RaIgD1 showed by IHC the presence of immunoreactive cells in gills, spleen, head kidney, intestine, and work is in progress to highlights the characteristics of sea bass IgD expressing cells. Acknowledgements. The work was financially supported by EU-6FP project Targetfish, n°311993.