IL-2-dependent murine T-cell lines and clones expressing gamma/delta T-cell antigen receptors. I. Functional and biochemical characterization.
We have developed two stable IL-2-dependent T-cell lines designated AKV-I and AKV-N from the enlarged spleens, respectively, of an AKV1 and an NFS mouse. Immunofluorescence staining with the appropriate monoclonal antibodies revealed that cells of the AKV-I cell line were alpha beta TCR-CD3+CD4-CD5-CD8+CD25+, whereas cells of the AKV-N cell line were alpha beta TCR-CD3+CD4-CD5+CD8-CD25+. A number of T-cell clones were developed from the AKV-I or AKV-N T-cell lines by limiting dilution and analysed by immunofluorescence. All clones tested were alpha beta TCR-CD3+CD4-CD25+. Certain T-cell clones expressed the CD5 antigen, whereas others expressed the CD8 antigen. The AKV-I cell line responded by proliferation to rIL2, rIL4, phorbol myristate acetate (PMA), PMA plus IL-4 and PMA plus PHA or Con A. In contrast, the AKV-N cell line did not respond to rIL-4 or rIL-4 plus PMA and exhibited only a modest proliferative response to PMA alone. Both AKV-I and AKV-N T-cell lines as well as a large number of T-cell clones examined were able to lyse cells of the PU5-IR murine cell line in the presence of the anti-CD3 (clone 145-2C11) MoAb, demonstrating their ability to mediate cytotoxicity in this system. Biochemical analysis of both AKV lines and a number of clones by immunoprecipitation with the anti-CD3 MoAb, followed by one-dimensional (either non-reducing or reducing) or two-dimensional (non-reducing/reducing) SDS-PAGE, revealed that the AKV lines and clones expressed a disulphide-linked dimer. Under non-reducing conditions, a band in the range of 75-85 kDa was observed and upon reduction it was resolved into two discrete polypeptide chains of 43-44 kDa and 48 kDa in certain AKV-I cells or 38 kDa and 42 kDa in certain AKV-N cells. In other T-cell clones or lines a broad band of 42-47 kDa was observed in AKV-I cells or 38-45 kDa in AKV-N cells. These results suggest the presence of different forms of disulphide-linked dimers on these cells. Northern blotting analysis using probes specific for the constant regions of the alpha-, beta-, gamma- and delta-chains of the T-cell antigen receptor revealed that all the AKV cell lines or clones tested expressed full-length alpha-, gamma- and delta-chain mRNA, whereas beta-chain mRNA was absent.(ABSTRACT TRUNCATED AT 250 WORDS)
Morkowski
J J
JJ
Department of Immunology, University of Texas, Houston.
Nanno
M
M
Chen
P F
PF
Itoh
K
K
Ioannides
C G
CG
Kruzel
E
E
Becker
F F
FF
Platsoucas
C D
CD
D000818:Animals; D015703:Antigens, CD / Q000032:analysis; D000945:Antigens, Differentiation, T-Lymphocyte / Q000276:immunology; D017252:CD3 Complex; D002460:Cell Line; D002999:Clone Cells; D003602:Cytotoxicity, Immunologic; D007376:Interleukin-2 / Q000096:biosynthesis / Q000494:pharmacology*; D008213:Lymphocyte Activation; D051379:Mice; D011233:Precipitin Tests; D012333:RNA, Messenger / Q000032:analysis; D011948:Receptors, Antigen, T-Cell / Q000276:immunology; D016692:Receptors, Antigen, T-Cell, gamma-delta / Q000032:analysis* / Q000235:genetics; D011994:Recombinant Proteins / Q000494:pharmacology; D013601:T-Lymphocytes / Q000276:immunology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D017252:CD3 Complex; D007376:Interleukin-2; D012333:RNA, Messenger; D011948:Receptors, Antigen, T-Cell; D016692:Receptors, Antigen, T-Cell, gamma-delta; D011994:Recombinant Proteins; D013755:Tetradecanoylphorbol Acetate