Stimulation of murine T cells via the Ly-6C antigen: lack of proliferative response in aberrant T cells from lpr/lpr and gld/gld mice despite high Ly-6C antigen expression

Cross-linking of cell surface Ly-6C molecules with the 6C3 rat monoclonal antibody (MAb) followed by anti-rat immunoglobulin antibody acts in concert with phorbol myristate acetate (PMA) as a potent mitogenic stimulus for normal T cells. Specificity of this stimulation was demonstrated by its absence in T cells from NZB, NOD, or STb/J mice which lack the 6C3 determinant. In 6C3+ normal strains, the extent of 6C3-mediated stimulation varied, depending on the level of 6C3 antigen expression. Analysis of this stimulation in purified T cell subsets revealed that in Ly-6.1 strains (e.g., BALB/c, CBA/J), Lyt-2+ cells responded, but not L3T4+ cells, whereas in Ly-6.2 strains (e.g., C57BL/6, MRL-+/+), both subsets produced IL 2 and proliferated, although with different kinetics. Moreover, in adult MRL-+/+ mice, the minor Lyt-2-/L3T4- subset from the lymph nodes gave low responses to 6C3 cross-linking, whereas that from the thymus reacted strongly. Stimulation via Ly-6C therefore provides a pathway for differential activation of normal T cells. In contrast, the expanding population of Lyt-2-/L3T4- T cells from lpr/lpr or gld/gld mice did not proliferate in response to 6C3 antigen cross-linking plus PMA despite high levels of 6C3 antigen expression. Responsiveness of lpr/lpr T cells could not be restored with IL 1, IL 2, or both. These T cells also failed to be triggered by conjunction of PMA with either Thy-1 antigen cross-linking or concanavalin A. Moreover, they were not stimulated, in the presence of PMA, by doses of ionomycin that were optimal for normal T cells, but did respond to higher ionomycin concentrations (2 micrograms/ml), and this response was not altered by Ly-6C cross-linking. It is concluded that the Ly-6C pathway of T cell activation is not functional in the aberrant lpr/lpr (and gld/gld) T cells, and that this defect may reflect abnormalities of intracellular signaling.     

Divergent effects of cyclosporine A on interferon- and mitogen-induced Ly-6A antigen suggest autocrine regulation of Ly-6A expression in activated T cells

The murine Ly-6A cell surface antigen is normally present on a minor subset of mature T cells. This marker has been shown to become highly expressed on mitogen-activated T cells. We found that expression of Ly-6A is also markedly increased in resting T cells by incubation with IFN-alpha/beta or IFN-gamma. Here, we compared the effect of the immunosuppressant cyclosporine A (CsA) on Ly-6A induction by IFN and concanavalin A (Con A). The augmentation of Ly-6A expression produced by treatment of T cells with IFN-alpha/beta or IFN-gamma was found not to be affected by CsA concentrations up to 2 micrograms/ml. In contrast, at doses as low as 50 ng/ml, CsA prevented the enhancement of Ly-6A expression in Con A-treated T-cell cultures. Culture supernatant transfer experiments were performed to further explore this effect of CsA. It was found that supernatants from Con A-activated T cells enhanced Ly-6A expression in resting T cells. This activity could be neutralized with an anti-IFN-gamma monoclonal antibody. Supernatants from T cells treated with Con A in presence of CsA lacked Ly-6A-enhancing activity. Taken together, these data suggest that the inhibition by CsA of Ly-6A induction in Con A-treated T cells reflects the known inhibitory effects of the drug on IFN-gamma secretion. This may imply the existence in T cells of an autocrine circuit involving IFN-gamma and regulating Ly-6A expression.     

Subpopulations of mouse Lyt-2+ T cells defined by the expression of an Ly-6-linked antigen, B4B2

The production and characterization of a rat mu,kappa monoclonal anti-mouse T cell subset antibody, B4B2, is reported in this paper. B4B2 typing of lymphoid tissues of commonly used inbred mouse strains revealed two types of reactivity patterns. They can be characterized as C57BL/6-like (B6-like) or C3H/He-like (C3H-like). Among B6-like strains, B4B2 recognizes 5 to 10% of spleen cells, 30 to 50% of bone marrow cells, and less than 2 to 3% of thymocytes. In C3H-like strains, B4B2 reacts with less than 1% of spleen cells, 2 to 8% of bone marrow cells, and less than 1% of thymocytes. B4B2 recognizes a T cell subset differentiation antigen expressed by B6-like strains but not by C3H-like strains. Typing of BXH recombinant inbred strains showed linked expression of B4B2 and the Ly-6 antigen. The expression of B4B2 antigen appears to be under codominant control as the median fluorescence distribution of B4B2+ cells in C57BL/6 was approximately twice that of (C57BL/6xC3H)F1. B4B2 was shown to react with approximately 40 to 50% of Lyt-2+ T cells and less than 1% of L3T4+ T cells. No staining of resting or activated B cells by B4B2 was detected. The ratio of B4B2+:Lyt-2+ cells was similar for resting T cells and activated T cells obtained from mitogen-stimulated cultures or mixed lymphocyte cultures. In neonatal spleen, substantially more B4B2+ than Lyt-2+ cells were found. With increasing age, however, a rapid decline in B4B2+ cells and a corresponding increase of Lyt-2+ cells was observed. By approximately 1 mo of age, the relative proportion of these subsets had reversed so that Lyt-2+ cells became more numerous than B4B2+ cells.     

Retrovirus-induced changes in major histocompatibility complex antigen expression influence susceptibility to lysis by cytotoxic T lymphocytes

Retrovirus infection of murine fibroblasts was found to alter the expression of major histocompatibility complex (MHC) antigens. Fibroblasts infected with Moloney murine leukemia virus (M-MuLV) exhibited up to a 10-fold increase in cell surface expression of all three class I MHC antigens. Increases in MHC expression resulted in the increased susceptibility of M-MuLV-infected cells to lysis by allospecific cytotoxic T lymphocytes (CTL). M-MuLV appears to exert its effect at the genomic level, because mRNA specific for class I antigens, as well as beta 2-microglobulin, show a fourfold increase. Fibroblasts infected with the Moloney sarcoma virus (MSV):M-MuLV complex show no increase in MHC antigen expression or class I mRNA synthesis, suggesting that co-infection with MSV inhibits M-MuLV enhancement of MHC gene expression. Quantitative differences in class I antigen expression on virus-infected cells were also found to influence the susceptibility of infected cells to lysis by H-2-restricted, virus-specific CTL. Differential lysis of infected cells expressing varied levels of class I antigens by M-MuLV-specific bulk CTL populations and CTL clones suggests that individual clones may have different quantitative requirements for class I antigen expression. The MSV inhibition of MHC expression could be reversed by interferon-gamma. Treatment of MSV:M-MuLV-infected fibroblasts with interferon-gamma increased their susceptibility to lysis by both allogeneic and syngeneic CTL. The data suggest that interferon-gamma may function in the host's immune response to viral infections by enhancing MHC antigen expression, thereby increasing the susceptibility of virus-infected cells to lysis by H-2-restricted, virus-specific CTL.     

Functional tolerance of CD8+ T cells induced by muscle-specific antigen expression

Skeletal muscles account for more than 30% of the human body, yet mechanisms of immunological tolerance to this tissue remain mainly unexplored. To investigate the mechanisms of tolerance to muscle-specific proteins, we generated transgenic mice expressing the neo-autoantigen OVA exclusively in skeletal muscle (SM-OVA mice). SM-OVA mice were bred with OT-I or OT-II mice that possess a transgenic TCR specific for OVA peptides presented by MHC class I or class II, respectively. Tolerance to OVA did not involve clonal deletion, anergy or an increased regulatory T cell compartment. Rather, CD4+ T cell tolerance resulted from a mechanism of ignorance revealed by their response following OVA immunization. In marked contrast, CD8+ T cells exhibited a loss of OVA-specific cytotoxic activity associated with up-regulation of the immunoregulatory programmed death-1 molecule. Adoptive transfer experiments further showed that OVA expression in skeletal muscle was required to maintain this functional tolerance. These results establish a novel asymmetric model of immunological tolerance to muscle autoantigens involving Ag ignorance for CD4+ T cells, whereas muscle autoantigens recognized by CD8+ T cells results in blockade of their cytotoxic function. These observations may be helpful for understanding the breakage of tolerance in autoimmune muscle diseases.     

Multiple cytokine interactions regulate Ly-6E antigen expression: cooperative Ly-6E induction by IFNs, TNF, and IL-1 in a T cell lymphoma and in its induction-deficient variants.

The cell surface Ly-6E antigen, known to play a role in T cell activation, is up-regulated by IFNs. In the present study, we investigated the possible interactions between IFNs and other cytokines in this regulation. As a model system, we used the YAC T cell lymphoma, in which Ly-6E is normally absent but can be highly induced both at the mRNA and surface protein levels by IFN-gamma or IFN-alpha/beta. The combination of the two IFNs was found to result in markedly synergistic Ly-6E induction in this cell line. Moreover, mutants of YAC cells were isolated that did not respond to the Ly-6E-inducing action of IFN-gamma or IFN-alpha/beta alone but did respond to their combination. Such a synergistic interaction is consistent with the notion that the two IFN types utilize different intracellular mechanisms to induce Ly-6E expression. Ly-6E induction mediated by IFN-gamma or IFN-alpha/beta was also enhanced by cotreatment with TNF-alpha or IL-1 alpha, which by themselves had no detectable Ly-6E-inducing effect. These two cytokines similarly synergized with IFNs to trigger a response in several Ly-6E-induction-deficient mutants. However, their action could be dissociated in one mutant (B54) where the response to IFN-alpha/beta was enhanced by TNF-alpha, but not by IL-1 alpha. Altogether, these data indicate that Ly-6E antigen expression is regulated by the interaction of several inflammatory cytokines, which may provide a mechanism for the local modulation of T cell activation. The YAC cell mutants described here should facilitate further analysis of the molecular bases of Ly-6E regulation.     

Treatment of resting T lymphocytes with interferon-alpha/beta augments their proliferative response to activation signals delivered through their surface Ly-6 antigen

Although the exact significance of Ly-6 antigens is unknown, recent evidence suggests they may provide an important alternative pathway for murine T-cell activation. Thus, Shevach et al. (1986, Fed. Proc. 45, 1131) discovered that cross-linking of Ly-6 antigens on the cell surface acts in concert with phorbol myristate acetate to trigger mitogenesis in T cells. Previously, we reported that surface expression of Ly-6 antigens on T cells is markedly increased following exposure to interferon-alpha/beta (IFN-alpha/beta). The purpose of the present work was to determine the effect of IFN-induced Ly-6 enhancement on Ly-6-mediated T-cell stimulation. Purified T cells were incubated in vitro for 1-27 hr with various doses (10-10(4) units/ml) of IFN-alpha/beta. This was found to result in various degrees of augmentation of the proliferative responses of these T cells to stimulation through their Ly-6 antigen. Surprisingly, while maximal enhancement of Ly-6 expression occurred only after the longest pulses with the highest IFN concentrations, treatment with as little as 100 units IFN/ml for 12 hr was sufficient to induce a dramatic (25-fold) and nearly maximal enhancement of proliferation. This high sensitivity to IFN-alpha/beta of the Ly-6 pathway of T-cell activation led us to speculate that this pathway may play a role in the immunomodulatory activities of IFN-alpha/beta.     

Morphological changes of human neuroblastoma cells induced by human gamma interferon

The treatment of GOTO cells, originated from human neuroblastoma, with recombinant human interferon-gamma (rHuIFN-gamma) induced the morphological changes: the extension and bifurcation of neurites and the multinucleated giant cell formation. The treatment of KP-N-RT cells, originated from human neuroblastoma, with rHuIFN-gamma also induced the similar morphological changes. The treatment of these cells with natural HuIFN-gamma also induced the same morphological changes, but those with recombinant human leukocyte interferon (rHuIFN-alpha A), recombinant human fibroblast interferon (rHuIFN-beta) and recombinant murine interferon-gamma (rMuIFN-gamma) did not induce it. The rHuIFN-gamma and the rHuIFN-beta inhibited more strongly the growth of GOTO and KP-N-RT cells than the rHuIFN-alpha A. This suggests that the morphological changes of these neuroblastoma cells are not simply due to the cell growth inhibition, but due to the property which only the rHuIFN-gamma possesses.     

Sequential changes in MHC antigen expression induced by the v-Ki-ras oncogene

A series of early-passage cell lines were transformed with the v-Ki-ras oncogene with the aim of examining the effect of an activatedras gene on the ability of these cells to express major histocompatibility complex (MHC) antigens. These cell lines were found to undergo multiple phenotypic changes upon transformation and subsequent proliferation. At early passage, the predominant effect ofras was an increased ability to express class II antigens when induced with interferon (IFN). For class I antigens, maximum levels of expression induced with IFN were largely unaffected, however, decreased sensitivity to induction with this lymphokine was noted. With subsequent in vitro or in vivo passage, both class I and class II antigen inducibility was attenuated. The latter phenotypic change was found to be transferable by coculture, implicating a soluble IFN antagonist. Conditioned media fromras-transformed cells treated to activate their latent transforming growth factor (TGF) content mediated similar changes in MHC antigen inducibility, suggesting that TGF\ may be involved in modulating MHC antigen expression inras-transformed cells.     

Phenotypic changes of adult porcine mesenchymal stem cells induced by prolonged passaging in culture

The in vitro culture of porcine bone marrow-derived mesenchymal stem cells (MSCs) was used for the investigation of adult stem cell biology. Isolated porcine MSCs possessed the ability to proliferate extensively in an antioxidants-rich medium containing 5% fetal bovine serum (FBS). Greater than 40 serial MSC passages and 100 cell population doublings have been recorded for some MSC batches. Early and late passage MSCs were defined here as those cultures receiving less than 5 trypsin passages and more than 15 trypsin passages, respectively. Consistent with their robust ability to proliferate, both the early and late passage MSCs expressed the cell-cycle promoting enzyme p34cdc2 kinase. Late MSCs, however, exhibited certain features reminiscent of cellular aging such as actin accumulation, reduced substrate adherence, and increased activity of lysosomal acid beta-galactosidase. Early MSCs retained the multipotentiality capable of chondrogenic, osteogenic, and adipogenic differentiation upon induction in vitro. In contrast, late MSCs were only capable of adipogenic differentiation, which was greatly enhanced at the expense of the osteochondrogenic potential. Along with these changes in multipotentiality, late MSCs expressed decreased levels of the bone morphogenic protein (BMP-7) and reduced activity of alkaline phosphatase. Late MSCs also exhibited attenuated synthesis of the hematopoietic cytokines granulocyte colony-stimulating factor (G-CSF), leukemia inhibitory factor (LIF), and stem cell factor (SCF). The long-term porcine MSC culture, thus, provides a model system to study the molecular interplay between multiple MSC differentiation cascades in the context of cellular aging.     

Down-regulation of IL-2 production by activation of T cells through Ly-6A/E

Ly-6A/E molecules are expressed on the surface of T cells and have been shown to function in activation by the capacity of anti-Ly-6A/E mAb to induce T cell hybridomas or normal T cells to produce IL-2. Recent evidence suggests that activation through Ly-6A/E may be linked to the TCR signaling pathway. To further investigate the relationship between Ly-6- and TCR-induced T cell activation, we have examined whether an anti-Ly-6A/E mAb (D7) modulates TCR signaling in vitro. We now report that mAb D7 specifically inhibited IL-2 production by T cells also activated through TCR. Such inhibition was noted for normal T cells stimulated by soluble anti-CD3 or alloantigen and for T hybridomas stimulated by soluble anti-CD3. The ability of D7 to inhibit IL-2 production by T hybridomas was dependent on the nature of the TCR activating signal because IL-2 production was not inhibited when T hybridomas were stimulated with Ag or immobilized anti-CD3. Inhibition of IL-2 production by D7 apparently required cross-linking of the mAb because D7 F(ab')2 fragments were not effective for inhibition of IL-2 production. Similar to its ability to enhance anti-Ly-6A/E-induced activation of T and B cells, IFN-gamma enhanced the D7-induced inhibition of IL-2 production by alloantigen-activated normal T cells. These data further support the notion that Ly-6 and TCR signaling pathways are interrelated.     

The major histocompatibility complex-restricted antigen receptor on T cells: the genetics of expression of an allotype

The monoclonal antibody KJ16-133 binds an allelic determinant expressed on the antigen-specific, major histocompatibility complex (MHC)-restricted receptors on approximately 20% of T cells in most mouse strains. The locus controlling the presence or absence of the determinant mapped 9.8 +/- 2.2 centimorgans from the Igk/Ly-2 locus on chromosome 6 in mice, and may be the beta-chain locus. Other genetic loci were identified that controlled the frequency of cells that expressed the allele in positive mice. One of these was the MHC itself, which may control expression of the beta-chain allele by controlling T cell repertoire. The identity of the other, as yet unmapped locus is unknown. KJ16-133 was used to show that T cell receptor gene products are expressed in a manner consistent with allelic exclusion.     

T cell-mediated cytotoxicity induced by Listeria monocytogenes. III. Phenotypic characteristics of mediator T cells

Cytotoxic thymus-derived lymphocytes (CTL) generated in vitro by restimulating rat cells with Listeria antigen- (LMA) pulsed syngeneic accessory cells were characterized in respect to their surface membrane markers. LM-dependent CTL were devoid of detectable surface immunoglobulin (Ig) and receptors for the Fc region of rabbit IgG. Experiments with monoclonal antibodies to rat T cell markers revealed that these cytotoxic cells have the phenotypic profile W3/13+, W3/25-, MRC OX 8+. LM-dependent CTL also bind the monoclonal antibody, MRC OX 3, which recognizes an Ia-antigen-like determinant on rat cells. Although LM-dependent CTL lack the W3/25 marker, their generation depends on the cooperative interplay of W3/25+ and W3/25- T cells.     

Astrocytes as antigen-presenting cells. I. Induction of Ia antigen expression on astrocytes by T cells via immune interferon and its effect on antigen presentation

Ia antigens seem to control immune responses on at least two levels. First, they influence the antigen recognition repertoire of the T cells. Second, their variable expression on certain antigen-presenting cells is a powerful regulatory mechanism for the local immune reaction. This is particularly important in the central nervous system (CNS) in which no Ia antigens are normally expressed. Recent experiments in this context have shown that astrocytes are able to express Ia antigens during interaction with T cells, and that they function as antigen-presenting cells. The Ia-inducing activity is produced by activated T cells, and can be replaced by immune interferon (IFN-gamma). In this study we report on the functional and kinetic relationship between Ia antigen expression on astrocytes and the immune-specific activation of T cells by astrocytes. Normal resting astrocytes were found to be negative for Ia antigens by immunofluorescence and by biochemical criteria. Moreover, they are only able to stimulate T cells after they have been induced to express Ia antigens by a signal from the T cells, which is probably mediated by IFN-gamma. In conclusion, the immune-specific interaction between astrocytes and T lymphocytes is a sensitively controlled system that might be pivotal to the development of immune responses in the brain. Malfunction of the system could be an important factor in the pathogenesis of aberrant immune reactions in the CNS, e.g., in multiple sclerosis.     

Phenotypic and functional alterations of dendritic cells induced by human herpesvirus 6 infection

Human herpesvirus 6 (HHV-6) has a tropism for T lymphocytes and monocytes/macrophages, suggesting that HHV-6 infection affects the immunosurveillance system. In the present study, we investigated the HHV-6-induced phenotypic and functional alterations of dendritic cells (DCs), which are professional antigen-presenting cells. HHV-6 infection of monocyte-derived immature DCs appeared to induce the up-regulation of CD80, CD83, CD86, and HLA class I and class II molecules, suggesting that HHV-6 infection induces the maturation of DCs. In addition, the antigen capture capacity of DCs was found to decrease following infection with HHV-6. In contrast to up-regulation of mature-DC-associated surface molecules on HHV-6-infected DCs, their capacity for presentation of alloantigens and exogenous virus antigens to T lymphocytes decreased significantly from that of uninfected DCs. In contrast, there appeared to be no reduction in the capacity for presentation of an HLA class II-binding peptide to the peptide-specific CD4(+) T lymphocytes. These data indicate that HHV-6 infection induces phenotypic alterations and impairs the antigen presentation capacity of DCs. The present data also suggest that the dysfunction of HHV-6-infected DCs is attributable mainly to impairment of the antigen capture and intracellular antigen-processing pathways.     

Plasticity of Ly-6C(hi) myeloid cells in T cell regulation

CD11b(+)Ly-6C(hi) cells, including inflammatory monocytes (IMCs) and inflammatory dendritic cells (IDCs), are important in infectious, autoimmune, and tumor models. However, their role in T cell regulation is controversial. In this article, we show that T cell regulation by IMCs and IDCs is determined by their activation state and is plastic during an immune response. Nonactivated IMCs and IDCs function as APCs, but activated IMCs and IDCs suppress T cells through NO production. Suppressive IMCs are induced by IFN-γ, GM-CSF, TNF-α, and CD154 derived from activated T cells during their interaction. In experimental autoimmune encephalomyelitis, CD11b(+)Ly-6C(hi) cells in the CNS are increasingly activated from disease onset to peak and switch their function from Ag presentation to T cell suppression. Furthermore, transfer of activated IMCs or IDCs enhances T cell apoptosis in the CNS and suppresses experimental autoimmune encephalomyelitis. These data highlight the interplay between innate and adaptive immunity: immunization leads to the expansion of Ly-6C(hi) myeloid cells initially promoting T cell function. As T cells become highly activated in the target tissue, they induce activation and NO production in Ly-6C(hi) myeloid cells, which in turn suppress T cells and lead to the contraction of local immune response.     

Xenogeneic monoclonal antibody to an Ly-6-linked murine cell surface antigen: differential reactivity with T cell subpopulations and bone marrow cells

The patterns of cellular and strain reactivity of a monoclonal antibody (6C3 MAb) derived from the fusion of SP2/0 cells with splenocytes from rats immunized against MRL/MpJ-lpr/lpr T cells were characterized by using flow cytofluorometry (FCF) analysis. This MAb was found to stain 70 to 90% of T cells of mice with the lpr/lpr genotype and 20 to 60% of T cells of congenic +/+ strains. Dual-parameter FCF analysis of Lyt-2 vs 6C3 expression revealed the existence of several Lyt-2- and Lyt-2+ T cell subsets, one of which (Lyt-2- bright 6C3+) was expanded in lpr/lpr-bearing mice. The 6C3 MAb stained only 2 to 5% normal thymocytes but reacted with 40 to 50% bone marrow (BM) cells. A strain survey demonstrated the expression of the 6C3 antigen on peripheral T cells (and BM cells) of all strains examined, with the exception of NOD, NZB/B1NJ, and ST/bJ. Interestingly, in the positive strains, two types of 6C3 staining patterns of T cells were observed: bimodal or trimodal. Study of BXH and CXB recombinant inbred (RI) strains demonstrated that the bimodal and trimodal 6C3 patterns are associated with the Ly-6.1 and Ly-6.2 phenotypes, respectively. Linkage of 6C3 expression with the Ly-6 locus was confirmed by using the congenic C3H.B6-Ly-6b strain. Moreover, the 6C3 staining of T cells in Ly-6.2 strains was reduced by preincubation with the H9/25 and SK-142-446 MAb, which are known to recognize Ly-6.2-associated antigens. Therefore, the 6C3 MAb appears to detect a frame-work determinant on an Ly-6-linked antigen that is absent from T cells of NOD, NZB, and ST/bJ mice. Analysis of (NZB x C58) NX8 RI strains demonstrated a correlation between the lack of 6C3 expression on T cells and unresponsiveness in autologous mixed lymphocyte reaction (a property of NZB/B1NJ mice). The 6C3 MAb should prove useful for further genetic and biochemical analysis of the Ly-6 locus and its product(s), and for the delineation of functional subsets of T cells and BM cells in normal and lpr/lpr-bearing mice.     

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.