Antigen-dependent T lymphocytes (TcRß+) are primarily differentiated in the thymus rather than in other lymphoid tissues in sea bass (Dicentrarchus labrax, L.).

All jawed vertebrates share lymphocyte receptors that allow the recognition of pathogens and the discrimination between self and non-self antigens. The T cell transmembrane receptor (TcR) has a central role in the maturation and function of T lymphocytes in vertebrates via an important role in positive selection of the variable region of TcR αß/γδ chains. In this study, the TcRß transcript expression and TcRß(+) cell distribution during the ontogeny of the immune system of sea bass (Dicentrarchus labrax, L.) were analysed. RT-PCR analysis of larvae during early development demonstrated that the ß chain transcript is expressed by 19 days post-fertilisation (p.f.). RNA probes specific for the ß chain were synthesised and used for in situ hybridisation experiments on 30 day p.f. to 180 day old juvenile larvae. A parallel immunohistochemical study was performed using the anti-T cell monoclonal antibody DLT15 developed in our laboratory [Scapigliati et al., Fish Shellfish Immunol 1996; 6:383-401]. The first thymus anlage was detectable at 32-33 days p.f. (Corresponding to about 27 days post-hatch). DLT15(+) cells were detected at day 35 p.f. in the thymus whereas TcRß(+) cells were recognisable at day 38 p.f. in the thymus and at day 41 p.f. in the gut. TcRß(+) cells were observed in capillaries from 41 to 80 days p.f. At day 46 p.f., TcRß(+) cells were identified in the head kidney and were detected in the spleen 4 days later. The present results demonstrate that TcRß(+) cells can be differentiated first in the thymus and then in other organs/tissues, suggesting potential TcRß(+) cell colonisation from the thymus to the middle gut. Once the epithelial architecture of the thymus is completed with the formation of the cortical-medullary border (around 70-75 days p.f.), DLT15(+) cells or TcRß(+) cells are confined mainly to the cortex and cortical-medullary border. In particular, a large influx of TcRß(+) cells was observed at the cortical-medullary border from 72 to 90 days p.f., suggesting a role in positive selection for this thymic region during the ontogeny of the fish immune system. This study provides novel information about the primary differentiation and distribution of TcRß(+) cells in sea bass larvae and juveniles.

Majority of TcRbeta+ T-lymphocytes located in thymus and midgut of the bony fish, Dicentrarchus labrax (L.).

Real-time polymerase chain reaction (PCR) and in situ hybridization analyses were performed to investigate the occurrence and distribution of T-lymphocytes expressing TcRbeta in intestine and lymphoid tissues of the bony fish, Dicentrarchus labrax (sea bass). Immunohistochemistry with the monoclonal antibody DLT15 (pan-T-cell marker) was carried out to compare the cytology, distribution and number of T-cells and TcRbeta+ cells in the various sampled lymphoid organs. The highest TcRbeta expression was revealed by real-time PCR in the thymus, with high levels also being found in the gut. In the thymus, DLT15+ and TcRbeta+ cell populations were concentrated in the cortex and TcRbeta+ cells were notably reactive at the cortical-medullary border, suggesting a specialized role of this region in thymocyte selection. The density of DLT15+ T-cells increased from the anterior to posterior intestine, whereas TcRbeta+ lymphocytes were more numerous in the middle intestine compared with other segments. The existence, in fish thymus, of a medulla and a cortex comparable with those of mammals is revealed by this study. The concentration of TcRbeta+ cells in the sea bass midgut also strongly suggests a special role of this intestinal segment in antigen-specific cellular immunity. The large population of TcRbeta(-)/DLT15+ T-cells in the posterior gut can probably be ascribed to the TcRgammadelta phenotype fraction.

The ontogeny of mucosal immune cells in common carp (Cyprinus carpio L.).

The ontogeny of carp (Cyprinus carpio L.) immune cells was studied in mucosal organs (intestine, gills and skin) using the monoclonal antibodies WCL38 (intraepithelial lymphocytes), WCL15 (monocytes/macrophages) and WCI12 (B cells). In addition, recombination activating gene 1 expression was examined in the intestine with real time quantitative PCR and in situ hybridization to investigate extrathymic generation of lymphocytes. WCL38(+) intraepithelial lymphocytes (putative T cells) appeared in the intestine at 3 days post-fertilization (dpf), which is shortly after hatching but before feeding, implying an important function at early age. These lymphoid cells appear in the intestine before the observation of the first thymocytes at 3-4 dpf, and together with the expression of recombination activating gene 1 in the intestine, suggests that similar to mammals at least part of these cells are generated in the intestine. WCL15(+)monocytes/macrophages appeared in the lamina propria of the intestine at 7 dpf, but considerably later in the epithelium, while WCI12(+) (B) cells appeared in intestine and gills at 6-7 weeks. From these results it can be concluded that putative T cells occur much earlier than B cells, and that B cells appear much later in the mucosae than in other internal lymphoid organs (2 wpf).

Immunoglobulin protein and gene transcripts in ovarian follicles throughout oogenesis in the teleost Dicentrarchus labrax.

Transfer of immunoglobulins (IgM-like) from the female to the teleost embryo has been demonstrated but mechanisms of uptake into and storage within the eggs remain to be clarified. The monoclonal antibody DLIg3 against Dicentrarchus labrax Ig light chain revealed an active role of both follicle cells and oocytes in the Ig uptake. The primordial follicular cells showed DLIg3 immunoreactivity even at a pre-vitellogenetic stage. Early vitellogenetic oocytes (lipid vesicle stages) had DLIg3 staining of pore canals, plasmalemma and outer cortex and of their follicular cells. In protein yolk granule oocytes, DLIg3 staining was also detected within vesicles of the outer-mid cortex and juxtanuclear yolk granules; therefore, a centripetal transport of Ig throughout oocyte development is apparently carried out. Immunoelectron microscopy confirmed the presence of Ig within thecal and granulosa cells (and in the interposed basement membrane) of pre-vitellogenic and vitellogenic follicles. Thus, the transport of Ig to the egg apparently occurs also by transcytosis across the follicle cells. Igs were localised in the pore canals surrounding the microvilli and in vesicles of outer-mid cortex of vitellogenic oocytes. Reverse transcription/polymerase chain reaction with primers designed for the constant region of sea bass Ig light chain detected Ig mRNA in hydrated oocytes, a smaller content in released eggs and no signal in larvae at day two post-hatching. These findings show that a significant level of Ig gene transcription in the oocyte and/or a transfer of transcripts may also occur.