Clonal growth of carp (Cyprinus carpio) T cells in vitro.
Carp kidney leukocytes co-cultured with a supporting cell layer resulted in the rapid proliferation of various types of leukocytes including immature leukocytes. Expressions of marker genes for multiple blood cell lineages were observed in the primary culture. However, after several passages, the proliferating cells expressed only T cell and macrophage marker genes. Further RT-PCR analysis revealed that the proliferating cells expressed TCR constant regions (Cα, Cβ, Cγ, Cδ), CD3γ/δ and CD4 (CD4L-1), but did not express CD8α and CD8β. Additionally, in situ hybridization analysis showed that the majority of proliferating cells expressed Cα, Cβ, Cγ, Cδ and CD4. Moreover, 5'-RACE sequences of TCR variable regions (Vα, Vβ, Vγ, Vδ) revealed that the proliferating cells contained a polyclonal T cell repertoire, and most of the Vα and Vβ sequences were functional, but the Vγ and Vδ sequences were non-functional with frame shifts and stop codons. Taken together, these results indicate that the proliferating cells after serial passages predominantly contained CD4+ CD8- αβT cells that simultaneously co-expressed non-functional γδTCR. To obtain CD4+ αβT cell (helper T cell) clones, single cells were picked up from the bulk culture, seeded into each well of 96-well plates and cultured in the presence of supporting cells and conditioned media. T cell colonies formed from single cells after 2-3 weeks. These colony cells expressed Cα, Cβ, Cδ and CD4, and weakly expressed Cγ, but did not express CD8α, CD8β and CD4L-2. Taken together, these results indicate that these clonal T cells resemble a subpopulation of mammalian CD4+ helper T cells.
Yamaguchi
Takuya
T
Laboratory of Fish Pathology, Department of Veterinary Medicine, Nihon University, Kameino 1866, Fujisawa, Kanagawa 252-0880, Japan.
Katakura
Fumihiko
F
Shitanda
Satoshi
S
Niida
Yoshimitsu
Y
Toda
Hideaki
H
Ohtani
Maki
M
Yabu
Takeshi
T
Suetake
Hiroaki
H
Moritomo
Tadaaki
T
Nakanishi
Teruyuki
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015704:CD4 Antigens / Q000235:genetics; D015496:CD4-Positive T-Lymphocytes; D016827:CD8 Antigens / Q000235:genetics; D002347:Carps / Q000276:immunology*; D049109:Cell Proliferation; D002478:Cells, Cultured; D002999:Clone Cells / Q000166:cytology / Q000276:immunology; D018920:Coculture Techniques; D015870:Gene Expression; D020869:Gene Expression Profiling; D019672:Genes, T-Cell Receptor; D017403:In Situ Hybridization; D008264:Macrophages / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D008969:Molecular Sequence Data; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000737:chemistry / Q000235:genetics; D016692:Receptors, Antigen, T-Cell, gamma-delta / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013601:T-Lymphocytes / Q000166:cytology* / Q000276:immunology* / Q000378:metabolism
D015704:CD4 Antigens; D016827:CD8 Antigens; C431698:CD8alpha antigen; C477867:CD8beta antigen; D016693:Receptors, Antigen, T-Cell, alpha-beta; D016692:Receptors, Antigen, T-Cell, gamma-delta