Edward T.H. Yeh

TAP transcription and phosphatidylinositol linkage mutants are defective in activation through the T cell receptor

TAP is a phosphatidylinositol-anchored membrane protein that is involved in murine T lymphocyte activation. To determine the relationship between TAP and T cell receptor/CD3-mediated activation, we derived TAP expression mutants of a T-T hybridoma. Two phenotypically distinct classes of mutants were obtained. The first has a selective defect in the transcription of TAP, while the second has a defect in the biosynthesis of phosphatidylinositol-protein linkages. Both mutations affect antigen-stimulated, T cell receptor-mediated activation of the T-T hybrid. These variants have intact immune effector gene programs, as they are responsive to pharmacologic agents that mimic receptor signals. These findings support a role for phosphatidylinositol-linked cell-surface glycoproteins in physiologic T cell activation. Consistent with this interpretation, we observed similar defects in T cell responsiveness after enzymatic removal of phosphatidylinositol-linked proteins from normal T lymphocytes.

Structural characterization of the TAP molecule: A phosphatidylinositol-linked glycoprotein distinct from the T cell receptor/T3 complex and Thy-1

Here we characterize the T-cell-activating protein (TAP), an Ly-6 gene product involved in T cell activation, as a glycoprotein with a molecular weight of 10-12 kd under nonreducing conditions and 15-18 kd under reducing ones. Two of the three bands that are precipitated from metabolically labeled cells are expressed on the cell surface and can be recovered from the supernatants of cells treated with a phosphatidylinositol-specific phospholipase C. Thus TAP appears to be attached to the cell membrane via this lipid. Precisely the same anchorage is observed for the activating Thy-1 molecule, and is therefore of particular interest as a potentially novel linkage involved in membrane signal transduction.

Dietary marine lipids suppress continuous expression of interleukin-1β gene transcription1

Abstractn-3 Polyunsaturated fatty acids abundant in marine lipids suppress certain inflammatory and immune reactions, and dietary marine lipid supplements have antiinflammatory effects in experimental and human autoimmune disease. Previous work by other investigators demonstrated that dietary marine lipid supplements suppressed production of cytokines from stimulated human peripheral blood mononuclear cellsex vivo. The present study further documents the ability of n-3 fatty acids to inhibit cytokine formation, and in part defines the mechanism of the inhibition of production of interleukin-1β (IL-1β) by dietary n-3 fatty acid. Female BALB/c mice were each fed a fatfree balanced diet to which was added either a refined fish oil (FO) preparation as a source of n-3 fatty acid, or beef tallow (BT), which consisted primarily of saturated and monoenoic fatty acids. After ingesting the experimental diets for periods ranging from 3 to 12 wk, spleen cell preparations were stimulatedex vivo with either lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA), and prolL-1β mRNA (Il-1β mRNA) was measured by northern analysis. Levels of IL-1β mRNA in both LPS-and PMA-stimulated cells from BT-fed mice were elevated to a greater extent than in cells from FO-fed mice, at most concentrations of LPS and PMA. Stability of LPS-stimulated mRNA levels after actinomycin D was similar for BT and FO groups, indicating that lower levels of IL-1 mRNA with FO groups was related to suppressed IL-1 gene transcription and not due to accelerated transcript degradation. Nuclear run-on transcription assays revealed a more transient expression of the IL-1β gene in LPS-stimulated spleen cells from FO-fed mice compared to cells from BT-fed mice. We conclude that dietary marine lipids reduce transient expression of the IL-1β gene in stimulated splenic monocytic cells. Preliminary results from nuclear run-on transcription assays indicate that n-3 fatty acids may not change the initial rate of gene transcription but may promote more rapid shutting down of transcription of this gene after induction than do alternative lipids.

Defective signal transduction in CD4-CD8- T cells of lpr mice

Mice homozygous for the lpr gene develop a lymphoproliferative disorder due to expansion of a subset of CD4-CD8- T cells. Triggering of the T-cell receptor in these lpr T cells does not lead to translocation of protein kinase C or phosphorylation of CD3, interleukin-2 production, or proliferation, whereas a combination of phorbol ester and calcium ionophore does. Stimulation with concanavalin A or anti-CD3 induces phosphoinositide hydrolysis. The rise in inositol bisphosphate, inositol triphosphate, and inositol tetrakisphosphate, identified by HPLC, is similar in +/+ and lpr T cells. The concentration of cytoplasmic free calcium ([Ca2+]i), however, under basal and stimulated conditions is significantly lower in lpr T cells. The lower basal [Ca2+]i may explain why induction of proliferation with phorbol ester and calcium ionophore requires a higher concentration of ionophore in these cells than in normal T cells. The lower [Ca2+]i obtained on stimulation may contribute to the activation defect of CD4-CD8- lpr T cells.

Abnormal T cells from MRL/Mp-lpr/lpr mice do not respond to anti-T-cell-receptor antibody or tumor promoter and calcium lonophore A23187 despite the presence of the T-cell-receptor complex and protein kinase c

Abnormal T cells from autoimmune MRL/Mp-lpr/lpr mice express T-cell-receptor complexes on their surfaces. These receptors are nonfunctional, since monoclonal anti-T-cell-receptor antibody-conjugated beads, which normally activate receptor-bearing T cells, were unable to activate these abnormal T cells. In addition, these abnormal T cells are unresponsive to the synergistic effect of phorbolmyristate acetate (PMA) and calcium ionophore A23187, as quantitated by proliferation, interleukin-2(IL-2) production, and the expression of IL-2 receptors. The failure of abnormal T cells to respond to PMA is not due to the absence of PMA receptors since Scatchard plot analysis reveals that there are 1-1.5 X 10(5) PMA-binding sites/cell with a Kd of 6-10 nM on these abnormal T cells. Similar to normal T lymphocytes, protein kinase c activity can be readily detected in the cytosolic fraction of these abnormal T cells. More importantly, in vitro culture of the abnormal T cells with PMA results in translocation of protein kinase c activity from the cytosolic fraction to the membrane fraction. From these experiments we concluded that the defect in the signal-transducing machinery in MRL/Mp-lpr/lpr T cells is not due to the lack of PMA receptors or the absence of protein kinase c activity, but may result from events which occur after the activation and translocation of protein kinase c. However, whether defects in response to a physiological stimulus (i.e., anti-receptor antibody) could occur in a step prior to protein kinase c activation remains to be determined.

SHP-1 regulates hematopoietic stem cell quiescence by coordinating TGF-β signaling

Cell cycle quiescence is critical for hematopoietic stem cell (HSC) maintenance. TGF-&bgr; signaling in bone marrow niche has been identified in regulating HSC quiescence; however, the intrinsic regulatory mechanisms remain unclear. This study reports that Shp-1 knockout HSCs have attenuated quiescence and impaired long-term self-renewal. SHP-1–activated HSCs are surrounded by megakaryocytes, which regulate HSC quiescence by producing TGF-&bgr;1. Mechanistically, SHP-1 interacts with the immunoreceptor tyrosine-based inhibition motif on TGF-&bgr; receptor 1 and is critical for TGF-&bgr; signaling activation in HSCs. Functionally, Shp-1 knockout HSCs do not respond to TGF-&bgr;–enforced HSC quiescence regulation, both in vitro and in vivo. Therefore, we identify TGF-&bgr;–SHP-1 as a novel intrinsic regulatory mechanism for HSC quiescence maintenance.

Selection of T cell receptor expression mutants through the functionally linked Ly-6A

Ly-6A is a glycosyl-phosphatidylinositol (GPI)-anchored molecule that participates in murine T cell activation. Activation of T cell hybridomas with anti-Ly-6A monoclonal antibody (mAb) leads to production of interleukin-2 (IL-2), but also to a paradoxical growth inhibition, which was used to select for signaling mutants. Fifteen subclones derived from two independent mutageneses and anti-Ly-6A selection were characterized. Thirteen subclones responded poorly or not at all to soluble anti-Ly-6A mAb. Although the selective pressure was exerted through Ly-6A, only one mutant did not express the Ly-6A antigen. Interestingly, 10 of the 15 subclones expressed either nondetectable or a very low level of T cell receptor/CD3 complex (TCR/CD3). Preferential expansion of TCR/CD3 expression mutants following anti-Ly-6A selection further established functional linkage between Ly-6A and TCR/CD3 complex. The mechanism of the functional coupling was investigated by analyzing the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2), one of the early events in T cell activation. We showed that PIP2 was not hydrolyzed in response to anti-Ly-6A in TCR/CD3-negative mutants. Aluminum fluoride, which activates G protein directly, did induce PIP2 hydrolysis in these cells. These data suggest that activation signals originated from Ly-6A must be transmitted first to TCR/CD3 complex, which then couples to the G protein/phospholipase C system. A similar requirement also applies to the Thy-1 protein and lectin receptors. Thus, the TCR/CD3 complex plays a central role in the integration and transmission of activation signals that originated from several T cell surface molecules.