Julia L. Greenstein

The role of the L3T4 molecule in mitogen and antigen-activated signal transduction

We investigated the role of the L3T4 molecule in mitogen and antigen-initiated signal transduction in the L3T4(+) murine T cell hybridoma, 3DT52.5.9 and an L3T4(-) variant, 3DT52.5.24. Both Concanavalin A (Con A) and specific antigen stimulated increases in cytosolic-free calcium ([Ca2+]i), phosphatidylinositol turnover, and interleukin-2 (IL-2) production in both cell lines. About 85% of the stimulated rise in [Ca2+]i was from an extracellular source. Anti-L3T4 monoclonal antibody (MAb) inhibited 90% of antigen- and 50% of Con A-stimulated increases in [Ca2+]i and IL-2 production but had no effect on the ability of either activation signal to stimulate phosphatidylinositol turnover in the parent L3T4(+) cells. Stimulus-response coupling in the L3T4(-) cells was unaffected by the MAb. The anti-L3T4-insensitive increase in [Ca2+]i induced by Con A was inhibited by EGTA, suggesting that this mitogen also stimulated an influx of Ca2+ via an additional transport mechanism distinct from that stimulated by antigen. The fact that anti-L3T4 antibodies inhibit antigen and Con A-stimulated Ca2+ transport and IL-2 production without affecting phosphatidylinositol turnover suggests that L3T4 may play a critical role in modulating the activation of the T cell receptor-associated Ca2+ transporter in T cell stimulus-response coupling.

Human Cytolytic T-Lymphocyte Clones and Their Function-Associated Cell Surface Molecules

Cytolytic T lymphocytes (CTLs) are important effectors in the recognition of viruses,1 allografts,2 and some tumors.3 A molecular understanding of the CTL-target cell interaction therefore may be relevant to the etiology and/or treatment of a variety of disease states. Tissue culture techniques, first reported by Gillis and Smith,4 have allowed the generation of long-term T-cell lines that retain function. A number of human long-term cytolytic T lymphocyte lines have been generated by continued stimulation of peripheral blood lymphocyte lines with “foreign” cells in the presence of the T-cell growth factor interleukin 2 (IL-2).5,6 We have used CTL lines and clones to define target antigens recognized by human allogeneic lymphocytes, to correlate lymphocyte phenotype with antigen specificity, and to generate monoclonal antibodies that block lymphocyte function. Our findings have provided new insights into the cell surface molecules involved in the CTL-target cell interaction. In this chapter we describe our methodologies for the generation and maintenance of CTL lines and clones and the use of the cells in concert with monoclonal antibodies to define and analyze function-associated cell surface molecules.

The mechanism of antigen presentation by dendritic cells and splenic adherent cells in the induction of an allogeneic cytotoxic T-lymphocyte response to H-2Kk liposomes

Induction of an allogeneic cytotoxic T-lymphocyte (CTL) response to purified alloantigen is partially dependent on uptake and processing of the class I alloantigen by antigen-presenting cells (APC) followed by recognition of the alloantigen and self Ia by helper T cells (TH). The activated TH provides the helper signal(s) to the alloantigen-specific CTL for proliferation and differentiation into an active effector CTL. The role of antigen processing and presentation of major histocompatibility complex alloantigens was examined and the ability of different types of APC to present purified H-2Kk liposomes was investigated. Splenic adherent cells (SAC), splenic dendritic cells (DC), and B-cell lymphoblastoid lines were all shown to be effective in the presentation of H-2Kk liposomes. The relative ability of these cells to serve as APC was determined to be DC greater than B-cell tumors greater than SAC. The role of processing of H-2Kk liposomes by SAC and DC was examined by investigating the effect of weak bases on pulsing of the APC. These experiments suggest that presentation of alloantigen by both SAC and DC involves a step which is sensitive to inhibition by weak bases. We examined whether the TH were activated by similar mechanisms when stimulated by the various APC. The functional involvement of the T-cell surface marker L3T4 was demonstrated in the induction of TH. In contrast, L3T4 was not involved in the subsequent generation of CTL since monoclonal antibody (MAb) specific for L3T4 was not effective in blocking CTL function in the presence of nonspecific T helper factor (THF). Similarly, Ia on the APC was shown to be involved in the stimulation of the TH pathway but not directly in the differentiation of the CTL. Thus, DC and B cells in addition to SAC can present H-2Kk to TH. The presentation of alloantigen by both cell types may involve an intracellular route as demonstrated by the blocking of the TH response by weak bases. Both Ia and L3T4 are required on the APC for induction of the TH response. The minimal requirements for activation of the CTL were H-2Kk liposomes and a source of THF.

Functional Analysis of Cd2, cd4, and cd8 in t‐Cell Activationa

T cells may be activated by the antigen-specific T-cell receptor (TCR)-CD3 complex upon interaction with allogeneic major histocompatibility complex (MHC) antigens or foreign antigens in association with syngeneic MHC molecules. In addition t o the TCR. several other cell-surface molecules are important for T-cell adhesion and activation. These molecules include lymphocyte-function associated antigen (LFA)-I. CD2 (TII, Leu 5, LFA-2)CD4 (T4, Leu 3 in the human, L3T4 in the mouse), and CD8 (T8, Leu 2 in the human, Lyt-2 in the mouse) on the T cell, and LFA-3 on the target or stimulator cell (TABLE 1) . LFA-1 is a heterodimer involved in antigen-independent conjugate formation between the T cell and target or stimulator cell, and may play a wider role in cell adhesion of lymphoid cells.’.? CD2 is a receptor on T cells whose natural ligand on the stimulator cell is LFA-3, a broadly distributed glycoprotein.1-4 Although the CD2/ LFA-3 interaction clearly functions in cell-cell adhesion, recent data suggest that it may play a role in T-cell activation. Functional studies and monoclonal antibody (mAb)-inhibition data indicate that the CD4 and CD8 glycoproteins appear to interact with nonpolymorphic regions of MHC class I1 and class I molecules. respectively. and may serve to enhance antigen-specific recognition.’ Much of our understanding about the cell-surface antigens CD2, CD4, and CD8 has been derived from the analysis of the functional effect of mAb directed against these molecules. In the last several years, it has become clear that the

Cell Surface Antigens Involved in Human Cytolytic T Lymphocyte-mediated Cytolysis

Cytotoxic T lymphocytes (CTL) play an important role in host resistance to viral infections1, some types of tumors2 and organ allografts.3 The molecular mechanisms involved in the human CTL response has been the subject of intense investigation. An important development in the study of the lytic mechanism has been the use of monoclonal reagents — both monoclonal antibodies (MAb) and monoclonal CTL. Monoclonal antibodies have been used to probe the surface of the CTL. Functionally relevant cell surface molecules have been identified by the ability of appropriate MAb to inhibit CTL cytolysis. Cloned CTL with known specificity and phenotype have enhanced the sensitivity of this technique.