Abstract
The CD3 coreceptor is a master T cell surface marker, and genes encoding CD3ζ, γδ, and ε chains have been reported in several teleost fish. Here, a complete cDNA sequence of CD3ɛ chain was identified from a sea bass (Dicentrarchus labrax L.) gill transcriptome. Its basal expression was quantified in both lymphoid and non-lymphoid organs of sea bass juveniles with real-time qPCR analysis. After either in vitro stimulation of head kidney leukocytes with the T-cell mitogen phytohaemagglutinin or in vivo stimulation with an orally administered Vibrio anguillarum vaccine, CD3ε expression levels increased in head kidney leukocytes, confirming that CD3ε T cells may play important roles in fish systemic protection against pathogens. Further, three peptides were designed on the CD3ɛ cytoplasmic tail region and employed as immunogens for antibody production in rabbit. One antiserum so obtained, named RACD3/1, immunostained a band of the expected size in a western blot of a sea bass thymocyte lysate. The distribution of CD3ε+ lymphocyte population in the lymphoid organs and mucosal tissues was addressed in healthy fish by IHC. In decreasing percentage order, CD3ε+ lymphocytes were detected by flow cytometry in thymus, peripheral blood leukocytes, gills, head kidney, gut, and spleen. Finally, a significant in vivo enhancement of CD3ε+ T intestinal lymphocytes was found in fish fed on diets in which 100% fish meal was replaced by the microalgae Nannochloropsis sp. biomass. These results indicate that CD3ε+ T cells are involved in nutritional immune responses.
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This research was partially funded by the project ALGAFISH (PTDC/MAR-BIO/6233/2014-POCI-01-0145-FEDER016796), supported by FCT and co-financed by FEDER from COMPETE 2020 UID/Multi/04423/2019, and by the “Department of Excellence-2018” Program (Dipartimenti di Eccellenza) of the Italian Ministry of Education, University and Research, DIBAF-Department of University of Tuscia, Project “Landscape 4.0 – food, wellbeing and environment”.
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Animal manipulation during the diet experiments complied with the guidelines of the European Union Directive (2010/63/EU) and Portuguese legislation (DL113/2013) for animal treatments. The Organism for Animal Welfare of CIIMAR and the Portuguese Directorate-General for Food and Veterinary approved all the protocols performed in the present study (approval 0421/000/000/2017 DGAV). Fish handling was performed under ethylene glycol monophenyl ether anesthesia (0.3 mL/L; ref.: 8.07291.2500, Merck, Whitehouse Station, USA), and discomfort, stress, and pain to the experimental animals was avoided, as much as possible, along the experiment. For sampling, fish were euthanized by anesthetic overdose (ethylene glycol monophenyl ether; ref.: 8.07291.2500, Merck, Whitehouse Station, USA).
For the experiments performed in Italy, ethical approval by ethics committee was not required, as per the Explanatory note of the Italian Ministry of Health’s Directorate-General for Animal Health and Veterinary Medicinal Products (DGSAF) of 26 July 2017. Organs and tissues were dissected out following temporary immobilization with 1 mg/ml tricaine methane sulfonate (MS-222) (Sigma) in aquarium water, buffered with 0.6 g/L sodium bicarbonate. Anesthesia was followed by exsanguination or decapitation. All efforts were made to minimize suffering. The study was carried out in strict accordance to Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes and on National Legislative Decree 26 of 4 March 2014 “Attuazione della Direttiva 2010/63/UE sulla protezione degli animali utilizzati a fini scientifici.”
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Picchietti, S., Buonocore, F., Guerra, L. et al. Molecular and cellular characterization of European sea bass CD3ε+ T lymphocytes and their modulation by microalgal feed supplementation. Cell Tissue Res 384, 149–165 (2021). https://doi.org/10.1007/s00441-020-03347-x
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DOI: https://doi.org/10.1007/s00441-020-03347-x