Clonal growth of carp (Cyprinus carpio) T cells in vitro: Long-term proliferation of Th2-like cells

Carp kidney leukocytes co-cultured with a supporting cell layer resulted in proliferation of polyclonal CD4⁺ αβT cells as described previously. These bulk-cultured T cells expressed transcripts for both T helper 1 cells (Th1) master regulator (T-bet) and T helper 2 cells (Th2) master regulator (GATA-3). To identify the Th subsets in bulk-cultured T cells, single cells were picked up from the bulk culture, proliferated, and characterized. The majority of the clones displayed characteristics consistent with CD4⁺ αβT cell identity. These clones expressed both TCRα and TCRβ, but could not produce a TCRγδ heterodimer since they typically only expressed either TCRγ or TCRδ. These clones also expressed the TCR co-receptor genes CD4-1 or CD4-2, whereas they did not express CD8α or CD8β. In addition, GATA-3 was expressed whereas T-bet was not. Among these clones, one clone (KoThL5) continued to proliferate on the supporting cells and was successively transferred for more than 10 months and 90-100 passages. To characterize the KoThL5 cells by their cytokine production profile, they were stimulated with PHA and investigated by real-time RT-PCR. mRNA expression of Th2-related cytokine (IL-4/13B) was only enhanced in KoThL5 cells whereas both Th1-related cytokine (IFNγ) and Th2-related cytokines (IL-4/13A andIL-4/13B) were significantly enhanced in bulk-cultured T cells. Taken together, KoThL5 cells share some features with mammalian Th2 cells.This is the first study to describe in vitro cultures of teleost cell with Th2-like features. The KoThL5 cell line has considerable potential for addressing questions concerning the properties of teleost Th2 cells.