We have used antisense oligodeoxynucleotides corresponding to genes encoding the variable (V) region of the T-cell receptor (TCR) a and j3 chains (V. and Vp) to control TCR expression in T-cell hybridomas. Two hybridomas, A1.1 and B1.1, recognize a synthetic polypeptide antigen designated poly 18 {poly[Glu-Tyr-Lys-(Glu-Tyr-Ala)51} together with I-Ad. We have found that TCR function (production of lymphokines in response to antigen) and T3 expression were removed after protease treatment of the cells and were fully recovered 48 hr later. However, when antisense oligodeoxynucleotides corresponding to the appropriate TCR V genes were present after protease treatment, little or no recovery of TCR function or T3 expression was observed. This effect was specific for the TCR V genes utilized by the T cell: antisense oligodeoxynucleotides corresponding to the TCR V regions of AM.1 had no effect on TCR expression in B1.1 and vice versa. Thus, antisense oligodeoxynucleotides can be used to temporarily block expression of a TCR gene in a T-cell hybridoma. This technique was then applied to a paradoxical phenomenon in Al.1 cells. We had observed previously that Al.1 releases an antigen-specific immunoregulatory activity that shows the same antigenic fine specificity as is displayed by the TCR of AL.L. We now report that antisense oligodeoxynucleotides corresponding to the AM.1 V. gene blocked the production of this soluble antigen-specific activity by the cell. Antisense oligodeoxynucleotides corresponding to All Vp, on the other hand, had no effect on the production of this antigen-specific activity. We discuss these observations in the context of recent findings on the nature of T cell-derived antigen-specific regulatory factors.T lymphocytes specifically recognize foreign antigen together with self major histocompatibility complex (MHC) molecules through the cell-surface T-cell receptor (TCR) complex. This complex is composed of the TCR a and p chains, which are responsible for antigen and MHC specificity (1), and the T3 molecules, which may be responsible for transducing the membrane signal (2). a Chain, p chain, and T3 are only expressed as a complex; in the absence ofany one component, cell-surface expression does not occur (2).A helper T-cell hybridoma (A1.1) has been described (3) that expresses TCR a and 8 molecules specific for a synthetic polypeptide designated poly 18 {poly[Glu-Tyr-Lys-(Glu-TyrAla)5]} and, in the presence of specific antigen and I-Ad, releases lymphokines. This T-cell hybridoma also constitutively produces a poly 18-specific cell-free factor involved in antigen-specific induction of suppression, which we call "suppressor-inducer factor" (4). Analysis has revealed (4) that the factor produced by Al. 1 displayed the same antigenic fine specificity exhibited by the TCR on the Al.1 cell surface.Further, an anti-TCR antiserum was found to bind the antigen-specific factor (5). These results led us to speculate that at least some of the genes encoding the TCR a and 83 chains may be responsibl...

We have previously described an antigenspecific I-Ad-restricted T-cell hybridoma, AM.l, that constitutively releases an antigen-specific immunoregulatory activity into supernatants. Using retrovirally mediated gene transfer, we have found that transfer of the T-cell receptor a chain (TCRa) gene from Al.l to a number of other T-cell hybridomas effectively transferred the ability to produce the activity.

No abstract

No abstract

Conventional and thiophosphonate-derivatized oligonucleotides were employed to specifically regulate functional gene expression in murine T-cell hybridomas. For example, induction of apoptotic cell death following activation of T-cell hybridomas was examined using antisense oligonucleotides corresponding to several protooncogenes. We found that antisense oligodeoxynucleotides corresponding to c-myc inhibited both the characteristic DNA fragmentation and the loss of cell viability following activation without affecting production of lymphokines. Functional antisense oligonucleotides corresponding to c-fos had no effect in this system. These results demonstrate the use of antisense oligonucleotides to regulate function in T-cell hybridomas and provide valuable insights into the molecular bases of this biological phenomenon. Antisense oligonucleotides were also used to study another problem, the relation of T-cell-derived antigen-specific immunoregulatory factors to the T-cell receptor (TCR). Because the translation start of each TCR gene usually varies from one T cell to another, antisense oligonucleotides corresponding to the TCR V alpha or V beta of different cells were shown to act in a cell-specific manner. Furthermore, this method was used to demonstrate that a soluble antigen-specific regulatory activity produced by one of the T-cell lines depends on expression of the specific TCRa, an observation that has since been confirmed by gene transfer experiments. Expression of the CD3-TCR complex on the cell surface was also blocked by antisense oligonucleotides corresponding to CD3 gamma and CD3 zeta; however, neither these nor TCR V beta antisense oligonucleotides had any effect on production of the soluble regulatory activity.