Evidence for CD8-independent T cell maturation in transgenic mice.

Double-negative (CD4-/CD8-) T cells expressing the alpha/beta transgenic TCR from the 2C cell line (anti-H-2Ld) were examined in the periphery of animals whose MHC type produces positive, negative, or no selection for differentiation of the TCR on single positive (CD8+) cells. Regardless of the selection haplotype the CD4-/CD8- cells are capable of activation by anticlonotypic mAb indicating that negative selection does not inactivate the "forbidden" TCR. Rather, the lack of response to H-2Ld in the negative haplotype is likely to absence of CD8 required to produce a functional response to H-2Ld. The similarity of surface phenotype and functional activity of these CD4-/CD8- cells maturing in different haplotypes suggests they may arise by an alternative lineage which, unlike the dominant TCR alpha/beta pathway, does not require coexpression of CD4/CD8.

Cytotoxic T lymphocyte-induced loss of target cell adhesion and lysis involve common and separate signaling pathways.

Recently, we demonstrated that an early event in the CTL-target cell (TC) interaction is loss of TC adherence to substrate. This loss of adhesion is Ag-specific, but distinct from the lytic event because it can ensue in nominally Ca2+-free medium. In this study, we examine further the mechanism of CTL-induced loss of adhesion, concentrating mainly on the signal transduction pathway. Based on the differential sensitivity of CTL to extracellular Ca2+, protein kinase C activation/depletion and inhibition by anti-Lyt-2 (CD8) or anti-CTL receptor (TCR) reagents, we demonstrate that CTL-induced loss of adhesion can be initiated through multiple activation pathways. Although CTL-mediated lysis is restricted to a Ca2+ and protein kinase C-dependent signaling mechanism, CTL-induced loss of adhesion is initiated in the presence or absence of extracellular Ca2+ or functional protein kinase C activity. Furthermore, although under physiologic conditions, anti-CD8 or anti-TCR reagents strongly block both CTL activities, under non-lytic conditions, they fail to inhibit the ability of CTL to promote loss of adhesion. These findings implicate the participation of additional CTL-TC ligand interactions resulting in loss of adhesion, and thus, provide further evidence to support the hypothesis that CTL-induced loss of adhesion can be initiated through multiple triggering pathways.

Phosphorylation of a 15- to 17-kDa protein correlated with lytic function in cytotoxic T lymphocytes.

CTL are activated to lyse their targets through the interaction of the CTL-R and the appropriate Ag on the surface of the target cell. Experiments with tumor-promoting phorbol esters have suggested that the activation and translocation of protein kinase C (PKC) to the CTL membrane may be important in the activation process. We have studied the functional role of PKC in lytic signal transduction by correlating the phosphorylation of a set of CTL membrane proteins bound by the lectin Con A with lytic function in CTL clones. The data obtained indicate that the phosphorylation of a 15- to 17-kDa polypeptide in this subset is associated with the translocation of PKC to the membrane and the stimulation of lytic function. This suggests that the 15- to 17-kDa protein may be a physiologically relevant substrate for PKC translocated to the membrane as a result of Ag-specific perturbation of the CTL-R.

Antigen as a positive and negative regulator of proliferation in cytotoxic lymphocytes. A model for the differential regulation of proliferation and lytic activity.

The goal of these experiments has been to capitalize on the functionally distinct responses of heteroclitic CTL to cross-reactive Ag to explore the role of Ag in regulating CTL proliferation and lytic function. The experiments here have examined one of these clones in detail but several such clones appear similar. The principal findings were: 1) proliferation and lysis may be optimally stimulated by quantitatively different interactions, 2) Ag can have negative as well as positive effects on the proliferative potential of the responding cell, and 3) both positive and negative effects on lysis can be mimicked by agents which are similar to the second messengers produced by stimulating the TCR.

Loss of adhesion. A novel and distinct effect of the cytotoxic T lymphocyte-target interaction.

The ability of adherent target cells to remain attached to their substratum is lost as an early consequence of interaction with cytotoxic T lymphocytes. This phenomenon is shown to be a distinct event in the lytic process induced by cytotoxic T lymphocytes and does not occur during lysis by antibody plus complement. Thus loss of adhesion represents a previously unrecognized form of immune damage which may have important implications in both normal and pathologic responses.

Antagonistic effects of phorbol esters on lymphocyte activation. Evidence that protein kinase C provides an early signal associated with lytic function.

There is increasing evidence that protein kinase C plays a role in the transduction of an activation signal in lymphocytes. The bulk of this evidence is based on pharmacological experiments involving the tumor promoter phorbol myristate acetate (PMA) as a protein kinase C agonist. However, in cytotoxic T lymphocytes, PMA has been shown to both stimulate and inhibit lytic function. By examining the effects of a series of phorbol esters on protein kinase C activity in lymphocytes, we will demonstrate that these antagonistic effects of PMA on cytotoxic T lymphocyte function are related to multiple effects of PMA on protein kinase C activity.