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

Abstract

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.

MHC Class Ib-Restricted CD8 T Cells Differ in Dependence on CD4 T Cell Help and CD28 Costimulation over the Course of Mouse Polyomavirus Infection

“…A Q9:VP2.139-specific CD8 T cell hybridoma (C3K) was derived by fusing the C3-8 clone (described in ref (11)) with a CD8-transfected BW5147 fusion partner (described in ref (19)). Generation of replication-defective retroviral producer cell lines was performed as described in ref (20).…”

Interaction of the Nuclear Matrix-associated Region (MAR)-Binding Proteins, SATB1 and CDP/Cux, with a MAR Element (L2a) in an Upstream Regulatory Region of the Mouse CD8a Gene

“…Little is known concerning the regulation of the mouse CD8a and CD8b genes, but some insights have come from studies of T-T cell hybrids produced by fusing cytotoxic T lymphocytes with the BW5147 thymic lymphoma. Such hybridomas are invariably CD8-negative (26) due to shut-off of CD8a gene transcription (27), but interestingly the CD8b gene, which is located 36 kb upstream of the CD8a gene (28) (see Fig. 1), continues to be transcribed (27,29).…”

“…Such hybridomas are invariably CD8-negative (26) due to shut-off of CD8a gene transcription (27), but interestingly the CD8b gene, which is located 36 kb upstream of the CD8a gene (28) (see Fig. 1), continues to be transcribed (27,29). Working on the hypothesis that the shut-off of CD8a gene transcription in T-T cell hybridomas was due to cis-acting DNA elements upstream of the CD8a gene, stable clonal transfectants of BW5147 were produced using constructs of the CD8a gene containing varying amounts of 5Ј-flanking DNA and surface expression was monitored by flow microfluorometry.…”

Phenotypic Changes in Growth-Arrested T Cell Hybrids: A Possible Avenue to Produce Functional T Cell Hybridoma

“…On the other hand, BW5147 cells produced IL-2 by themselves when stimulated with calcium ionophore and phorbol ester mimicking the activation signals delivered when TCRs are engaged with cognate antigen (31). Furthermore, even cytolytic BW5147 hybridomas produced by fusion with cytotoxic T cells (CTLs) secreted IL-2 (32, 33) although CTLs usually had no ability to produce IL-2. Thus, the ability of hybridomas to produce IL-2 is most likely attributed to a property of the parental BW5147 lymphoma.…”