Mary C. Wacholtz

Accessory Cell Signals Involved in T‐Cell Activation

The physiologic recognition of antigen by T lymphocytes involves an interaction of the T-cell receptor (TCR) with fragments of antigen bound to either class I or class II major histocompatibility complex (MHC) molecules on the surface of an antigen-presenting cell (APC) (Buus et al. 1986, 1987, Babbitt et al. 1985, Ohashi et al. 1985). It is felt that activation signals generated as a result of recognition of the antigen-MHC complex by the TCR are transmitted via CD3, a multimolecular complex expressed in association with the TCR on the surface of all mature T cells. The CD3 moleeular complex has a number of features characteristic of signaling molecules, including polypeptide members with substantial intracytoplasmic domains containing amino acid residues that are phosphorylated after stimulation (Samelson et al. 1985a, 1985b). That CD3 serves to convey signals to the T-ee!l upon antigen recognition is derived from the observation that the TCR is structurally, albeit not covalently, associated with the CD3 molecular complex (Brenner et al. 1987), whereas the structure of the TCR itself has little in common with other known signaling molecules (Hedrick et al. 1984, Sim & Augustin 1985). Moreover, CD3 and the TCR are coordinately expressed on T cells (Weiss & Stobo 1984). In addition, monoclonal antibodies (mAb) to CD3 trigger T-cell activation {Chang et al. 1981, Van-Wauwe et al. 1980). Finally, expression of CD3 is required for the activation of T cells stimulated via a number of different surface molecules (Bockenstedt et al. 1988, Bamezai et al. 1988, Gunter et al. 1987, Fleischer et al. 1988, Geppert et al. 1989). These results all support the conclusion that the CD3 molecular complex plays a eentral role in the transduction of signals from a variety of receptors including the TCR that culminate in T-cell activation after antigen recognition.

Association of anticardiolipin antibodies and pregnancy loss in women with systemic lupus erythematosus

OBJECTIVE To analyze the pregnancy history in relation to the presence or absence of anticardiolipin antibodies in women who had been diagnosed with systemic lupus erythematosus (SLE). DESIGN One-hundred twenty-five women of reproductive age who were diagnosed with SLE and attended the Lupus Clinic at Parkland Memorial Hospital or Southwestern Medical Center were selected for this study. A retrospective review of patient histories, including anticardiolipin antibody test results and pregnancy histories, was conducted. Women who had therapeutic pregnancy terminations were excluded from this study. A chi 2 analysis was used to evaluate the significance of the data. RESULTS In women with SLE of childbearing age with anticardiolipin antibodies, a 39% pregnancy loss rate occurred, compared with an 11% loss rate in anticardiolipin antibody-negative women. In women with at least two pregnancies who had anticardiolipin antibodies, 27% experienced two or more losses, whereas only 3% of antibody-negative women had recurrent pregnancy loss. CONCLUSION We conclude that women with SLE and the presence of anticardiolipin antibodies are at increased risk for pregnancy loss.

Characterization of the 3',5'-cyclic adenosine monophosphate-mediated regulation of IL2 production by T cells and Jurkat cells

The effect of cyclic AMP-elevating agents on mitogen-stimulated IL2 production was examined. Prostaglandin E2 (PGE2) inhibited IL2 production by human peripheral blood T cells stimulated with PHA. In contrast, PGE2 did not inhibit PHA-stimulated IL2 production by the human leukemic T cell line. Jurkat, and often slightly enhanced IL2 production by those cells. Other cyclic adenosine monophosphate (cAMP) elevating agents (forskolin, isoproterenol, and the cAMP analogue, dibutyryl cAMP) also inhibited lectin-stimulated IL2 production by T cells, but could not inhibit IL2 production by Jurkat cells. Of the cAMP-elevating agents examined, only cholera toxin (CT) inhibited IL2 production by both Jurkat cells and peripheral blood T cells. Although phorbol myristate acetate (PMA) greatly enhanced PHA-stimulated IL2 production by Jurkat cells. CT remained markedly inhibitory. The combination of PMA and the calcium ionophore, ionomycin, also induced IL2 production by Jurkat cells, and this was similarly suppressed by CT, suggesting that a step after initial second messenger generation was inhibited. A prolonged increase in intracellular cAMP levels was induced by CT in both T cells and Jurkat cells, but the maximal level and the length of elevation achieved in T cells were much less than those observed in Jurkat cells. In contrast, PGE2 caused only a modest and transient increase in intracellular cAMP levels in Jurkat cells compared to that noted with T cells. PGE2 induced a more marked and sustained increase in cAMP levels in Jurkat cells treated with isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor. Moreover, in the presence of IBMX, PGE2 caused a marked inhibition of IL2 production by PHA-stimulated Jurkat cells. Differences in the capacity of PGE2 to induce cAMP could not be explained by disparities in the level of cAMP phosphodiesterase activity as this was comparable in Jurkat cells and in T cells. Thus, these observations indicate that IL2 production by both peripheral T cells and Jurkat cells can be modulated by cAMP-elevating agents. The data suggest that the diminished capacity of PGE2 to inhibit IL2 production by Jurkat cells reflects both a diminished capacity of PGE2 to induce increases in cAMP levels in these cells and an increase in the threshold of cAMP required to inhibit Jurkat cells.

Assessment of a positive selection technique using an avidin column to isolate human peripheral blood T cell subsets

The current studies were designed to assess a new technique for positively selecting human T cells from whole peripheral blood mononuclear cells using the minimal amount of monoclonal antibody required to bind the T cell to an avidin column indirectly via a biotin-conjugated secondary antibody. Positive selection of T cells has previously been avoided because the saturating amounts of antibodies required for other isolation procedures can lead to aberrant results in assays of T cell activation and function. The avidin column technique for obtaining purified T cell subsets was compared to a multi-step procedure that included negative selection panning. The positive selection technique was easily performed within 4 h whereas the negative selection technique required a minimum of 12 h to complete. The avidin column technique proved to be a rapid and simple method for isolating T cell subsets of high purity and normal functional capabilities. Since minimal amounts of monoclonal antibodies were used for the purification protocol, no consistent inhibitory or stimulatory effect of the residual antibody was noted in assays of activation and proliferation of positively selected T cells compared to T cells isolated by negative selection panning.

Comparison of the capacity of murine and human class I MHC molecules to stimulate T cell activation.

The mechanism underlying the apparent differences in the capacity of murine and human class I MHC molecules to function as signal transducing structures in T cells was examined. Cross-linking murine class I MHC molecules on splenic T cells did not stimulate an increase in intracellular calcium ([Ca2+]i) and failed to induce proliferation in the presence of IL-2 or PMA. In contrast, modest proliferation was induced by cross-linking class I MHC molecules on murine peripheral blood T cells or human class I MHC molecules on murine transgenic spleen cells, but only when costimulated with PMA. Moreover, cross-linking murine class I MHC molecules or the human HLA-B27 molecule on T cell lines generated from transgenic murine splenic T cells stimulated only modest proliferation in the presence of PMA, but not IL-2. On the other hand, cross-linking murine class I MHC molecules expressed by the human T cell leukemic line, Jurkat, transfected with genes for these molecules, generated a prompt increase in [Ca2+]i, and stimulated IL-2 production in the presence of PMA. The results demonstrate that both murine and human class I MHC molecules have the capacity to function as signal transducing structures, but that murine T cells are much less responsive to this signal.

Modulatory effect of aggregating the CD3 molecular complex on T cell activation

The role of cross-linking the TCR/CD3 complex in the induction of T cell activation was examined using human peripheral blood T cells and the Jurkat leukemic T cell line. IL-2 production was induced from these cells by pulsing them with mAb to CD3 and costimulating with phorbol myristate acetate (PMA). Cross-linking the anti-CD3 mAb with soluble goat anti-mouse immunoglobulin (GaMIg) markedly inhibited IL-2 production by these cells. Soluble GaMIg did not induce a generalized inhibition of IL-2 production as it was required for responses induced by mAb to class I MHC molecules. In addition, cross-linking anti-CD3 mAb with GaMIg did not inhibit IL-2 production induced by PMA and ionomycin. Inhibition of IL-2 production induced by soluble GaMIg reflected diminished accumulation of mRNA for IL-2. By contrast, immobilized GaMIg was a potent stimulus for IL-2 production by T cells pulsed with anti-CD3 mAb and costimulated with PMA. Cross-linking anti-CD3 with soluble GaMIg induced enhanced aggregation of the ligated molecules, but it did not alter the profile of the change in intracellular calcium induced. To determine whether cross-linking of mAb played a role in inducing IL-2 production as well as in limiting responsiveness, F(ab) fragments were employed. F(ab) fragments of anti-CD3 mAb failed to induce IL-2 production by PMA costimulated Jurkat cells. However, cross-linking of anti-CD3 F(ab)-pulsed Jurkat cells with low concentrations of soluble GaMIg induced IL-2 production in the presence of PMA, whereas higher concentrations suppressed responses. The data indicate that induction of IL-2 production requires aggregation of the TCR/CD3 complex, whereas excessive cross-linking diminishes the induction of IL-2 production. Moreover, the results indicate that various biologic activities of the CD3 molecular complex, including aggregation, signaling capability, and the ability to induce IL-2 gene transcription, are differentially affected by cross-linking.

Lymphocyte Function-Associated Antigen-1 (LFA-1) Is a Signaling Molecule for Human T Lymphocytes

The CD18 family of integrin molecules defines a group of structurally related glycoproteins found on hematopoietic cells (1). The different members of this group (LFA-1, Mac-1, p150,95) all share a common s chain (95 kD; CD18), but have different α chains (LFA-1–180 kD, CD11a; Mac-1–165kD, CD11b; p150,95–150kD, CD11c). In a number of different circumstances, these proteins have been found to play an important role in cell to cell interactions and adhesion (2). Consistent with this, the LFA-1 molecule has been found to play a role in a number of the functional activities of lymphocytes. Much of the understanding of the involvement of LFA-1 molecules in lymphocyte function has been defined by use of anti-LFA-1 monoclonal antibodies (MAbs) and includes mediation of the adhesion of the target and effector cells in cytotoxic T lymphocyte and NK cell mediated killing. LFA-1 also plays a role in the induction of T cell proliferation by accessory cell-dependent stimuli by facilitating the induction of required physical interactions between responding T cells and accessory cells. Similarly, LFA-1 plays a role in the development of cell to cell contact required for some T-cell dependent B cell responses (2,3).