Isolation and properties of a Lyt-2.1-negative mutant of a Lyt-2.1/Lyt-2.2 CTL line

Cytolytic activity and release of interleukin 2 (IL-2) were induced in Lyt-2-positive T-T cell hybrids by incubation with either concanavalin A or irradiated stimulator cells. Since hybrids of Lyt-2-positive class I-specific cytotoxic T lymphocytes (CTLs) with the fusable mouse thymoma cell line, BW5147, are invariably Lyt-2-negative, a derivative of BW5147 was produced by transfection which constitutively expresses surface Lyt-2.1. This cell line, 3B2, was fused with the H-2Ld-specific long term CTL line, 2C. Such hybrids expressed the transfected Lyt-2 gene but not the endogenous gene of the 2C fusion partner. That Lyt-2 plays a functional role in hybrids of 3B2 with 2C is shown by the observations that: 1) cytolysis by Lyt-2-positive hybrids was inhibited by Lyt-2-specific monoclonal antibody (mAb); 2) Lyt-2-positive but not Lyt-2-negative subclones of one such line develop specific cytotoxicity when incubated with stimulator cells; 3) Less IL-2 was released from Lyt-2-negative subclones incubated with stimulator cells than from Lyt-2-positive subclones; 4) Lyt-2-specific mAb inhibits release of IL-2 from Lyt-2-positive hybrids incubated with stimulator cells. All Lyt-2-positive hybrids expressed functional surface Lyt-3 encoded by the CTL fusion partner, demonstrating that expression of the Lyt-3 gene is not sensitive to the negative regulation which shuts off the endogenous Lyt-2 gene in hybrids of class I-specific CTLs with the 3B2 or BW5147 cell lines. The existence of inducible T-T cell hybrids expressing functional Lyt-2 and Lyt-3 provides a system for evaluation of the role(s) of Lyt-2 and Lyt-3 in the induction of function independent of cell growth.

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Differential susceptibility of mouse Lyt-2 and Lyt-3 genes to negative regulation

Hwang

Gottlieb

1993

Immunogenetics

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Hybrids of Lyt-2/Lyt-3-positive class I-specific cytotoxic T lymphocytes (CTLs) with the BW5147 thymoma cell line (Lyt-2/Lyt-3-negative) are known to be Lyt-2/Lyt-3-negative due to shutoff of transcription of the CTL's Lyt-2 gene. Hybrids of a constitutively Lyt-2-positive transfectant of BW5147 (3B2) with a long term CTL line, 2C, and with CTLs generated in a mixed leucocyte reaction (MLR) shut off the CTL's Lyt-2 gene as expected but express the CTL's Lyt-3 gene product as a heterodimer with the product of the transfected Lyt-2 gene. Thus the Lyt-3 gene is not subject to the same negative regulatory influences as the Lyt-2 gene. That expression of Lyt-2 is not necessary for Lyt-3 gene transcription to continue is demonstrated by the finding that hybrids of MLR-generated CTLs with either BW5147 (Lyt-2-negative) or 3B2 (Lyt-2-positive) cells express Lyt-3 RNA. Southern hybridization and structural analysis of DNA fragments generated using the polymerase chain reaction demonstrated that hybrids contain several species of Lyt-3 RNA, one of which lacks the exon encoding the extracellular V-like domain and appears to be the product of an alternatively-spliced RNA transcript.

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Isolation and properties of a Lyt-2.1-negative mutant of a Lyt-2.1/Lyt-2.2 CTL line

Cited by 3 publications

References 40 publications

Mutants of cultured mouse cells deficient in Ly-2 antigen

Mutants of cultured mouse cells deficient in Ly-2 antigen

Inducible functions in hybrids of a Lyt-2+ BW5147 transfectant and the 2C CTL line

Differential susceptibility of mouse Lyt-2 and Lyt-3 genes to negative regulation

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