Short communicationHelper function of CD4+ lymphocytes in antiviral immunity in ginbuna crucian carp, Carassius auratus langsdorfii
Introduction
Mammalian CD4+ helper T cells (Th-cells) act as commanders-in-chief for antigen-specific immunity and facilitate the activation of B-cells and cytotoxic T-lymphocytes (CTLs). Identification of Th-cell-related genes, such as several cytokines and CD4, and cellular functional studies have suggested that Th-cell functions in teleosts are similar to those in mammals (Toda et al., 2011, Laing and Hansen, 2011, Secombes et al., 2011, Zou and Secombes, 2011, Kono and Korenaga, 2013). Therefore, it is believed that Th-cell function is well conserved among fish and mammals. However, there is no direct evidence to suggest that fish CD4+ Th-cells are actually involved in both humoral and cellular immunity during a secondary immune response.
For teleost fish, two types of CD4-like molecules, CD4 (CD4-1) and CD4L-2 (or CD4REL), have been identified in several fish species (Suetake et al., 2006, Laing et al., 2006, Dijkstra et al., 2006, Edholm et al., 2007, Buonocore et al., 2008, Nonaka et al., 2008). Like mammalian CD4 molecules, teleost CD4 consists of four extracellular Ig-like domains, whereas teleost CD4L-2 comprises only two or three extracellular Ig domains. Furthermore, a recent study showed that teleost CD4+ cells expressed Th-specific cytokines, suggesting that these CD4+ cells possess functions similar to those of mammalian Th-cells (Kono and Korenaga, 2013). Thus, it is likely that teleost CD4+ cells are equivalent to mammalian Th-cells.
Clonal ginbuna crucian carp have been used as a model for studying T cell functions in teleost fish (Nakanishi et al., 2011, Fischer et al., 2013, Somamoto et al., 2013b). In particular, this model has provided in vivo functional evidence for antigen-specific T cells. Transferring sensitized leukocytes that include CD8+ cells into allogeneic recipients induced a graft versus host reaction (GVHR), whereas leukocytes without CD8+ cells did not, suggesting that CTLs played essential roles during the induction of acute GVHR (Shibasaki et al., 2010). Furthermore, adoptive transfer studies using this clonal fish showed protective effects of lymphocytes against viral infection in vivo (Somamoto et al., 2002, Somamoto et al., 2013b). Recently, a monoclonal antibody (mAb) that recognized CD4 was successfully generated in clonal ginbuna crucian carp (Toda et al., 2011). Thus, this mAb can now be used to monitor CD4+ cells in cellular functional studies. This study aimed to analyze the facilitating functions of teleost CD4+ cells on cell-mediated and humoral immunity of fish.
Section snippets
Fish, virus and cell lines
Two clonal strains (S3n and OB1) of ginbuna crucian carp, Carassius auratus langsdorfii, were hatched at the National Fisheries University and reared at Kyushu University. They were maintained at a temperature of 25 °C and were daily fed commercial food pellets. CFS cells from the S3n strain of ginbuna crucian were used as syngeneic target cells (Hasegawa et al., 1997). Crucian carp hematopoietic necrosis virus (CHNV) was inoculated into CFS cells that were then grown in Eagle’s minimal
Donor cell populations
Unsorted PBLs expressed all T cell markers, including TCR-β, CD4, and CD4L-2 (or CD4REL), whereas CD4− cells did not express CD4 mRNA (Fig. 1). This indicated that CD4− donor cells isolated by MACS were free of CD4+ cells. CD4L-2 was expressed by unsorted cells, CD4+ cells, and CD4− cells, indicating that CD4L-2+ cells were included in both unsorted donor cells and CD4− donor cells. CD4− donor cells also expressed TCR-β, indicating that this fraction contained T cells other than CD4+ cells.
Acknowledgments
This research was supported in part by a Grant-in-Aid for Young Scientists (B) (Grant Number 22780176) and a Grant-in-Aid for Scientific Research (B) (Grant Number 21380126) from the Japan Society for the Promotion of Science (JSPS). The costs of publication were supported, in part, by a Research Grant for Young Investigators of the Faculty of Agriculture, Kyushu University. The authors would like to thank Enago (www.enago.jp) for the English language review.
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