Enhanced response of T lymphocytes from Pgap3 knockout mouse: Insight into roles of fatty acid remodeling of GPI anchored proteins

Glycosylphosphatidylinositol (GPI) is a complex glycolipid that serves as a membrane anchor for many cell-surface proteins, such as Thy-1 and CD48. GPI-anchored proteins (GPI-APs) play important roles in many biological processes, such as signal transduction and cell-cell interaction, through their association with lipid rafts. Fatty acid remodeling of GPI-APs in the Golgi apparatus is required for their efficient association with lipid rafts, i.e., the unsaturated fatty acid at the sn-2 position of the PI moiety is exchanged for the saturated fatty acid by PGAP2 and PGAP3. To investigate the immunological role of the fatty acid remodeling of GPI-APs, we generated a Pgap3 knockout mouse. In this mouse, GPI-APs are expressed on the cell surface without fatty acid remodeling, and fail to associate with lipid rafts. Male Pgap3 knockout mice were born alive at a ratio lower than expected from Mendel's law, whereas the number of female mice followed Mendel's law. All mice exhibited growth retardation and abnormal reflexes such as limb grasping. We focused T cell function in these mice and found that T cell development in the absence of Pgap3 was normal. However, the response of T cells was enhanced in Pgap3 knockout mice in both in vitro and in vivo studies, including alloreactive response, antigen-specific immune response, and experimental autoimmune encephalomyelitis. Cross-linking of Thy-1 in wild-type cells inhibited the signal transduced by the T cell receptor (TCR), whereas cross-linking of Thy-1 in Pgap3 knockout cells enhanced the TCR signal. These results suggest that GPI-APs localized in lipid rafts may modulate signaling through the TCR.     

Activation of T lymphocytes through the Ly-6 pathway is defective in A strain mice

The Ly-6 alloantigens have been shown to participate in the process of T cell activation based on the ability of anti-Ly-6 mAb to induce IL-2 production and proliferation of T lymphocytes. In the present investigation we have demonstrated that peripheral T lymphocytes from A strain mice exhibited abnormally low proliferative responses after stimulation through Ly-6A/E and Ly-6C molecules when compared to responses of T cells from numerous other mouse strains. The abnormal activation of the Ly-6 pathway of A strain T cells was not due to ineffective FcR cross-linking of the anti-Ly-6 mAb, to inappropriate cellular expression of the Ly-6A/E alloantigen in A strain T cells, or to an active suppressive phenomenon. T lymphocytes from A strain mice proliferated normally when the cells were activated by mAb to Thy-1 or the CD3/TCR complex suggesting that A strain mice did not exhibit a generalized T cell activation defect. Cell separation studies of T cells and accessory cells demonstrated that this defect was quantitative, rather than qualitative, and that it was complex, residing at both the T cell and accessory cell levels. These results suggest that activation of T lymphocytes via the Ly-6 molecule may involve a signaling pathway and/or cell-cell interactions distinct from those required for optimal activation via CD3/TCR.     

Internalization of phosphatidylinositol-anchored lymphocyte proteins. I. Documentation and potential significance for T cell stimulation

Several proteins that are anchored to the surface of T lymphocytes via a phosphatidylinositol (PI) moiety can initiate cell stimulation upon cross-linking. Inasmuch as these proteins do not traverse the plasma membrane, it is not clear how they are capable of signaling across the membrane. Herein we report two distinct sets of experiments that examine the consequence of cross-linking PI-anchored molecules on murine T cells. We first analyzed the fate of antibody cross-linked TAP (Ly-6A.2) and Thy-1 molecules on T-T hybrids. Using an assay to measure receptor-mediated endocytosis, an intracellular accumulation of 125I labeled anti-TAP and anti-Thy-1 mAb was documented that was specific and Ag dependent. The internalization of these molecules was confirmed by cytotoxicity assays using antibody-toxin conjugates, and electron microscopic studies. Although the PI-anchored proteins lack a cytoplasmic domain that is necessary for the internalization of many receptors, they nevertheless can be induced to enter the cell upon cross-linking. The rate of entry of cross-linked TAP and Thy-1 into cells was shown to be 10 and 2% per hour, respectively, which is considerably less than that observed for the transferrin receptor or TCR/CD3 complex. To assess whether the internalization of TAP and Thy-1 might be of importance in their ability to stimulate T cells, we attempted to cross-link these molecules under conditions where the mAb or its cross-linked complex can not enter the cell. We observed that anti-TAP and anti-Thy-1 mAb conjugated to a cell impermeant matrix fail to stimulate T cells. This loss of stimulatory activity was observed with multiple T-T hybridomas and mAb over a wide titration of antibody concentration and was independent of the mAb isotype. Results from experiments with anti-Ig cross-linking of the mAb-PI anchored protein complex suggested that the loss of T cell stimulation upon mAb immobilization is not simply due to an alteration in the degree of antibody cross-linking. These findings were generalized to three distinct PI-anchored proteins: TAP, Thy-1, and Ly6C on normal T cells. When the same cells were stimulated through the TCR/CD3 complex, only immobilized mAb are stimulatory. These results demonstrate a marked difference in the cross-linking requirements for stimulating T cells through PI-anchored molecules in contrast to the transmembrane TCR complex. Furthermore, these findings raise the possibility that molecular internalization of Ab-PI-anchored complexes may be necessary in signaling through these molecules.     

sgp-60, a signal-transducing glycoprotein concerned with T cell activation through the T cell receptor/CD3 complex

T cell activation depends not only on the expression of a TCR, but also on that of accessory molecules that function in cell-cell adhesion and/or signal transduction. The subject of this report is the biochemical and functional characterization of what appears to be a novel murine lymphocyte cell surface antigen, provisionally termed sgp-60. Extensive, higher-order cross-linking of this glycoprotein with an anti-sgp-60 mAb and a second-step antibody reagent results in the activation of resting CD4+ T cells in the presence of a second signal. Monovalent or bivalent engagement of sgp-60 by the anti-sgp-60 antibody results in profound and direct inhibition of anti-CD3- or Con A-driven T cell activation, whereas alternative T cell activation via the phosphatidylinositol-linked proteins Thy-1 and TAP/Ly-6A is not affected. These findings raise the possibility that the sgp-60 molecule may be specifically involved in signal transduction through the TCR/CD3 complex and thus point to an important physiologic role for this protein in CD4+ T cells.     

A regulatory role for the CD4 and CD8 molecules in T cell activation

The role of the CD4 and CD8 molecules in T cell activation is presently a matter of controversy. Although their role as associative binding elements to MHC class II or class I is well documented, their influence on the triggering process in unclear. Because antibodies to CD4 or CD8 block T cell activation in the absence of their respective ligands, a negative signaling by these molecules has been suggested. However, recent experimental evidence argues against a negative regulatory effect of these molecules, since, e.g., simultaneous cross-linking of TCR and CD4 leads to enhanced T cell activation. Therefore, a current model suggests that the association of TCR and CD4 in the membrane gives a positive signal essential for triggering. In this report we present evidence that this model is likely to be too simple. Anti-CD4 and CD8 antibodies inhibit alternative, nonreceptor pathways of T cell triggering via Tp103 and Tp44 in the absence of class II positive accessory or target cells. These antibodies also inhibit bypass activation of T cells by phorbol ester and calcium ionophore in an accessory cell-free system. Furthermore, if the CD4 or CD8 molecules are removed from the cell surface by antibody-induced modulation, the proliferative and cytotoxic response of T cell clones is enhanced. This enhancement is also observed if resting peripheral blood T cells are used as responder cells. These data show that the CD4 or CD8 molecules have a complex regulatory function in T cell activation beyond the requirement for co-cross-linking with the TCR.     

Induction of negative signal through CD2 during antigen-specific T cell activation.

We have investigated the role of CD2 molecules in Ag-specific T cell activation by using a mouse model system in which the function of CD2 can be analyzed without the apparent influence of major accessory molecules, such as CD4 or LFA-1. Transfection of the CD2 gene into a CD2- T cell hybridoma confers the enhancement of IL-2 production upon Ag stimulation. Anti-CD2 mAb inhibits the Ag-specific response of the CD2-transfectant, not only to the level of CD2- cells but to the background. B cells, but not MHC class II-transfected L cells, serve as APC to induce the inhibition of Ag response. The complete abrogation of the response is observed only upon the stimulation through TCR with Ag in the presence of APC but not through either TCR-CD3 or other molecules such as Thy-1. Furthermore, the inhibition can also be observed when anti-CD2 mAb is immobilized on culture plates, suggesting that the inhibition of Ag response results from transducing the negative signal through the CD2 molecule. The experiments on cytoplasmic domain-deleted CD2-transfected T cells reveal that the cytoplasmic portion is responsible for the CD2-mediated abrogation of Ag responses. These results imply that CD2 has important roles in T cell responses not only as an activation and adhesion molecule but also as a regulatory molecule of Ag-specific responses through the TCR.     

CD2 can mediate TCR/CD3-independent T cell activation.

T lymphocytes can be activated clonotypically through TCR/CD3 complex or polyclonally via the CD2 molecule. Whether CD2-mediated activation is dependent on TCR/CD3 expression or signaling is controversial. We have re-explored this issue by using a series of CD2-transfected, TCR/CD3 surface membrane-negative human and mouse T cells. Our results clearly show that such T cells can be triggered for IL-2 secretion and increases in intracellular Ca2+ through the CD2 molecule in the absence of surface expression of TCR/CD3 complexes. These responses are only observed when cells express high levels of CD2 and there is a critical threshold of CD2 expression necessary for such activation in the absence of CD3. Concomitant expression of TCR/CD3 complex markedly lowers the level of CD2 required for activation via the latter pathway. These results provide a clear resolution of the controversy concerning the requirement for surface CD3 expression in T cell activation through CD2 and further suggest a possible role for CD2 in activation of TCR/CD3-negative cells.     

Selective regulation of TCR signaling pathways by the CD45 protein tyrosine phosphatase during thymocyte development

In CD45-deficient animals, there is a severe defect in thymocyte-positive selection, resulting in an absence of mature T cells and the accumulation of thymocytes at the DP stage of development. However, the signaling defect(s) responsible for the block in development of mature single-positive T cells is not well characterized. Previous studies have found that early signal transduction events in CD45-deficient cell lines and thymocytes are markedly diminished following stimulation with anti-CD3. Nevertheless, there are also situations in which T cell activation and TCR signaling events can be induced in the absence of CD45. For example, CD45-independent TCR signaling can be recovered upon simultaneous Ab cross-linking of CD3 and CD4 compared with cross-linking of CD3 alone. These data suggest that CD45 may differentially regulate TCR signaling events depending on the nature of the signal and/or on the differentiation state of the cell. In the current study, we have assessed the role of CD45 in regulating primary thymocyte activation following physiologic stimulation with peptide. Unlike CD3-mediated stimulation, peptide stimulation of CD45-deficient thymocytes induces diminished, but readily detectable TCR-mediated signaling events, such as phosphorylation of TCR-associated zeta, ZAP70, linker for activation of T cells, and Akt, and increased intracellular calcium concentration. In contrast, phosphorylation of ERK, which is essential for positive selection, is more severely affected in the absence of CD45. These data suggest that CD45 has a selective role in regulating different aspects of T cell activation.     

Antibody blockade of Thy-1 (CD90) impairs mouse cytotoxic T lymphocyte induction by anti-CD3 monoclonal antibody

Thy-1 (CD90) expressed by mouse T cells is known to have signal transducing properties, but the ability of Thy-1 to enhance cytotoxic T lymphocyte (CTL) development is not well understood. Here we show that stimulation of mouse T cells with monoclonal antibodies (mAb) to CD3, CD28 and Thy-1 (clone G7), which were coimmobilized on polystyrene microbeads, resulted in a greater proliferative response than stimulation with only anti-CD3 and anti-CD28 mAb, indicating that Thy-1 cross-linking enhanced T cell receptor/CD28-driven T cell activation. Consistent with this finding, Thy-1 blockade with a soluble nonactivating anti-Thy-1 mAb (clone 30-H12) inhibited anti-CD3-induced proliferation of CD4+ and CD8+ T cells, and the induction of cytotoxic effector cells in a dose-dependent fashion. Interleukin-2 synthesis and CD25 expression were also impaired by Thy-1 blockade. The inhibitory effect involved a defect at or before the level of protein kinase C activation because the addition of phorbol ester ablated the anti-Thy-1-mediated inhibition of anti-CD3-induced T cell activation. The CTL that were induced in the presence of blocking anti-Thy-1 mAb adhered to target cells but showed reduced expression of granzyme B and perforin. In contrast, Fas ligand expression and function was not affected by Thy-1 blockade. We conclude that Thy-1 signalling promotes the in vitro generation of CTL that kill in a granule-dependent fashion.     

CD22 is a functional ligand for SH2 domain-containing protein-tyrosine phosphatase-1 in primary T cells

The intracellular Src homology 2 (SH2) domain-containing protein-tyrosine phosphatase (SHP-1) has been characterized as a negative regulator of T cell function, contributing to the definition of T cell receptor signaling thresholds in developing and peripheral mouse T lymphocytes. The activation of SHP-1 is achieved through the engagement of its tandem SH2 domains by tyrosine-phosphorylated proteins; however, the identity of the activating ligand(s) for SHP-1, within mouse primary T cells, is presently unresolved. The identification of SHP-1 ligand(s) in primary T cells would provide crucial insight into the molecular mechanisms by which SHP-1 contributes to in vivo thresholds for T cell activation. Here we present a combination of biochemical and yeast genetic analyses indicating CD22 to be a T cell ligand for the SHP-1 SH2 domains. Based on these observations we have confirmed that CD22 is indeed expressed on mouse primary T cells and capable of associating with SHP-1. Significantly, CD22-deficient T cells demonstrate enhanced proliferation in response to anti-CD3 or allogeneic stimulation. Furthermore, the co-engagement of CD3 and CD22 results in a raising of TCR signaling thresholds hence demonstrating a previously unsuspected functional role for CD22 in primary T cells.     

pp59fyn mutant mice display differential signaling in thymocytes and peripheral T cells.

We have generated mutant mice that do not express pp59fyn, a nonreceptor protein tyrosine kinase related to pp60src, by homologous recombination in embryonic stem cells. fyn- mice did not display an overt phenotype. Because fyn is associated with the T cell receptor (TCR), thymocyte and T cell signaling was analyzed in the mutant background. Cross-linking of TCR-CD3 in thymocytes led to markedly reduced calcium fluxes and abrogated proliferation, whereas mature splenic T cells retained largely normal proliferation despite depressed calcium movements and IL-2 production. Similarly, proliferation induced by Thy-1 cross-linking was reduced in thymocytes but not in splenic T cells. fyn- thymocytes were impaired at a late stage of maturation and showed limited clonal deletion to the Mls-1a self-super-antigen but not to staphylococcal enterotoxin A. These results implicate fyn as a critical component in TCR signaling in thymocytes and, potentially, in the process that determines T cell repertoire in the adult mouse.     

CD38 is associated with lipid rafts and upon receptor stimulation leads to Akt/protein kinase B and Erk activation in the absence of the CD3-zeta immune receptor tyrosine-based activation motifs.

T lymphocytes can be activated via the T cell receptor (TCR) or by triggering through a number of other cell surface structures, including the CD38 co-receptor molecule. Here, we show that in TCR+ T cells that express a CD3-zeta lacking the cytoplasmic domain, cross-linking with CD38- or CD3-specific monoclonal antibodies induces tyrosine phosphorylation of CD3-epsilon, zeta-associated protein-70, linker for activation of T cells, and Shc. Moreover, in these cells, anti-CD38 or anti-CD3 stimulation leads to protein kinase B/Akt and Erk activation, suggesting that the CD3-zeta-immunoreceptor tyrosine-based activation motifs are not required for CD38 signaling in T cells. Interestingly, in unstimulated T cells, lipid rafts are highly enriched in CD38, including the T cells lacking the cytoplasmic tail of CD3-zeta. Moreover, CD38 clustering by extensive cross-linking with an anti-CD38 monoclonal antibody and a secondary antibody leads to an increased resistance of CD38 to detergent solubilization, suggesting that CD38 is constitutively associated with membrane rafts. Consistent with this, cholesterol depletion with methyl-beta-cyclodextrin substantially reduces CD38-mediated Akt activation while enhancing CD38-mediated Erk activation. CD38/raft association may improve the signaling capabilities of CD38 via formation of protein/lipid domains to which signaling-competent molecules, such as immunoreceptor tyrosine-based activation motif-bearing CD3 molecules and protein-tyrosine kinases, are recruited.     

TCR complex-activated CD8 adhesion function by human T cells

The CD8 receptor plays a central role in the recognition and elimination of virally infected and malignant cells by cytolytic CD8(+) T cells. In conjunction with the TCR, the CD8 coreceptor binds Ag-specific class I MHC (MHC-I) molecules expressed by target cells, initiating signaling events that result in T cell activation. Whether CD8 can further function as an adhesion molecule for non-Ag MHC-I is currently unclear in humans. In this study, we show that in human CD8(+) T cells, TCR complex signaling activates CD8 adhesion molecule function, resulting in a CD8 interaction with MHC-I that is sufficient to maintain firm T cell adhesion under shear conditions. Secondly, we found that while CD8 adhesive function was triggered by TCR complex activation in differentiated cells, including in vitro generated CTL and ex vivo effector/memory phenotype CD8(+) T cells, naive CD8(+) T cells were incapable of activated CD8 adhesion. Lastly, we examine the kinetics of, and signaling for, activated CD8 adhesion in humans and identify notable differences from the equivalent CD8 function in mouse. Activated CD8 adhesion induced by TCR signaling may contribute to the more rapid and robust elimination of pathogen-infected cells by differentiated CD8(+) T cells.     

Itk/Emt/Tsk activation in response to CD3 cross-linking in Jurkat T cells requires ZAP-70 and Lat and is independent of membrane recruitment.

The Tec family tyrosine kinase, Itk has been implicated in T cell antigen receptor (TCR) signaling, yet little is known about Itk regulation. Here, we investigate the role of the tyrosine kinase ZAP-70 in regulating Itk. Whereas Itk was activated in Jurkat T cells in response to CD3 cross-linking, Itk activation was defective in the ZAP-70-deficient P116 Jurkat T cell line. Itk responsiveness to TCR engagement was restored in P116 cells stably transfected with ZAP-70 cDNA. ZAP-70 itself could not directly phosphorylate the Itk kinase domain, indicating an indirect regulation of Itk activity. No role was found for ZAP-70 in regulating Itk recruitment to the plasma membrane, an event that has been suggested to be rate-limiting for the activation of Tec family kinases. Indeed, Itk was found to be constitutively targeted to the membrane fraction in both Jurkat and P116 cells. Lat, a prominent in vivo substrate of ZAP-70 that mediates assembly of multimolecular signaling complexes at the plasma membrane of T cells was also found to be required for TCR-stimulated Itk activation. Itk could not be activated by CD3 cross-linking in a Lat-negative cell line, unless Lat expression was restored. Lat and Itk were observed to co-associate in response to CD3 cross-linking in Jurkat T cells, but not in P116 T cells. The Lat-Itk association correlated with Lat tyrosine phosphorylation, which was deficient in the P116 T cells. These data suggest that ZAP-70 and Lat play important, probably sequential, roles in regulating the activation of Itk following TCR engagement.     

Phenotypic and functional analysis of gamma delta T cell receptor-positive murine dendritic epidermal clones

Thy-1+ dendritic cells isolated from the epidermis of normal mice (dEC)3 bear the gamma delta TCR associated with the CD3 complex. We have analyzed the effects of antibodies directed against the TCR complex, Ly-6C, and Thy-1, as well as pharmacologic agents which have been shown to activate T cells without engagement of the TCR complex, on levels of intracellular free calcium, activation of protein kinase C, cytolysis, IL-2R expression, and secretion of lymphokines by dEC clones. We have found that the dEC cells express a fully functional TCR complex which can function to transmit signals upon perturbation leading to an increase in IL-2R expression, release of lymphokines, and cytolytic activity. These results indicate that the gamma delta TCR+ dEC are capable of responding to activation signals in the same manner as mature alpha beta TCR+ cells and suggests that they may play a functional role in the skin.     

The glycophosphatidylinositol-anchored Thy-1 molecule interacts with the p60fyn protein tyrosine kinase in T cells.

Stimulation of murine T cells by engagement of the multi-component T cell antigen receptor or by cross-linking the Thy-1 molecule leads to a similar response characterized by lymphocyte activation and lymphokine production. The early biochemical events induced by engaging these molecules also are similar and begin with activation of a tyrosine kinase pathway and tyrosine phosphorylation of a comparable set of substrates. Previous work demonstrates that the protein tyrosine kinase p60fyn is associated with the antigen receptor and therefore it may participate in the tyrosine phosphorylations that are observed with antigen receptor signaling. In this study we demonstrate that the Thy-1 molecule is also associated with p60fyn in a murine T cell hybridoma and in murine thymocytes. The interaction is independent of antigen receptor expression. Thy-1 is a member of the class of molecules anchored to the plasma membrane by a glycophosphatidylinositol (GPI) group. The association of Thy-1 with p60fyn is dependent on the GPI linkage, since cleavage of the GPI anchor disrupts the interaction. The association of Thy-1 and p60fyn suggests a means by which Thy-1 cross-linking leads to tyrosine phosphorylation and T cell activation.     

Involvement of hematopoietic progenitor kinase 1 in T cell receptor signaling

Hematopoietic progenitor kinase 1 (HPK1), a mammalian Ste20-related serine/threonine protein kinase, is a hematopoietic-specific upstream activator of the c-Jun N-terminal kinase. Here, we provide evidence to demonstrate the involvement of HPK1 in T cell receptor (TCR) signaling. HPK1 was activated and tyrosine-phosphorylated with similar kinetics following TCR/CD3 or pervanadate stimulation. Co-expression of protein-tyrosine kinases, Lck and Zap70, with HPK1 led to HPK1 activation and tyrosine phosphorylation in transfected mammalian cells. Upon TCR/CD3 stimulation, HPK1 formed inducible complexes with the adapters Nck and Crk with different kinetics, whereas it constitutively interacted with the adapters Grb2 and CrkL in Jurkat T cells. Interestingly, HPK1 also inducibly associated with linker for activation of T cells (LAT) through its proline-rich motif and translocated into glycolipid-enriched microdomains (also called lipid rafts) following TCR/CD3 stimulation, suggesting a critical role for LAT in the regulation of HPK1. Together, these results identify HPK1 as a new component of TCR signaling. T cell-specific signaling molecules Lck, Zap70, and LAT play roles in the regulation of HPK1 during TCR signaling. Differential complex formation between HPK1 and adapters highlights the possible involvement of HPK1 in multiple signaling pathways in T cells.     

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.