Ligation of the T cell co-stimulatory receptor CD28 activates the serine-threonine protein kinase protein kinase B

The intracellular signaling pathways activated upon ligation of the co-stimulatory receptor CD28 remain relatively ill-defined, although CD28 ligation does result in the strong association with, and activation of, phosphatidylinositol (PI) 3-kinase. The downstream effector targets of the CD28-activated PI 3-kinase-dependent signaling pathway remain poorly defined, but recent evidence from other systems has shown that Akt/protein kinase B (PKB) is a major target of PI 3-kinase and have indicated that a major function of PKB is the regulation of cell survival events. Given the strong coupling of CD28 to PI 3-kinase and the known protective effects of both CD28 and PI 3-kinase against apoptosis in different cell models, we investigated the effects of CD28 on PKB activation. We demonstrate that ligation of CD28 by either anti-CD28 monoclonal antibodies or the natural ligand B7.1, results in the marked activation of PKB in both the leukemic T cell line Jurkat and freshly isolated human peripheral blood-derived normal T lymphocytes. Our data suggest therefore, that PKB may be an important intracellular signal involved in CD28 signal transduction and demonstrate CD28 coupling to downstream elements of a signaling cascade known to promote cell survival.     

Cytokine-mediated inhibition of apoptosis in non-transformed T cells and neutrophils can be dissociated from protein kinase B activation

In the absence of survival-inducing cytokines activated T cells and neutrophils enter apoptosis spontaneously. Phosphatidylinositol 3-kinase (PI3 K) activation and signaling through PKB/AKT have been widely linked to the inhibition of apoptosis by cytokines. Here we have investigated the role of PKB in the inhibition of spontaneous apoptosis of activated human CD4+ T cells and neutrophils. We used a range of cytokines known to induce survival and/or activation of PKB. We found activation of PKB in T cells treated with IL-2 and insulin, and neutrophils cultured with N-formyl-Met-Leu-Phe (fMLP), insulin or granulocyte-macrophage colony-stimulating factor. Insulin did not inhibit apoptosis in neutrophils or T cells and fMLP did not delay neutrophil apoptosis. Intriguingly, IFN-beta induced PI3 K-dependent survival in both cell types, but did not activate PKB. IL-2 mediated rescue of T cells from apoptosis but no induction of proliferation occurred in thepresence of LY294002, an inhibitor of PI3 K, which also blocked subsequent PKB activation. The main role of PI3 K in IL-2-mediated signaling may therefore be in the regulation of proliferation. These findings suggest that activation of PKB and inhibition of apoptosis can be dissociated in cytokine-mediated rescue of non-transformed CD4+ T cells and neutrophils.     

Mechanism of phosphatidylinositol 3-kinase-dependent increases in BAC1.2F5 macrophage-like cell density in response to M-CSF: Phosphatidylinositol 3-kinase inhibitors increase the rate of apoptosis rather than inhibit DNA synthesis

OBJECTIVE AND DESIGN: To determine the role of phosphatidylinositol 3-kinase (PI 3-kinase) in macrophagecolony stimulating factor (M-CSF)-induced macrophage proliferation. MATERIALS: The M-CSF-dependent BAC1.2F5 murine macrophage cell line was used. METHODS: PI 3-kinase activity, Protein kinase B activation, increased cell numbers, induction of DNA synthesis and apoptosis were measured in response to serum, M-CSF and PI 3-kinase inhibitors. RESULTS: Wortmannin or LY294002 inhibited M-CSF-stimulated increases in BAC1.2F5 cell density. Further analysis showed that inhibition of PI 3-kinase had an insignificant effect on DNA synthesis, but significantly induced apoptosis. Other co-factors in serum mediated cell survival and prevented programmed cell death, in a PI 3-kinase-dependent manner. Stimulation of BAC1.2F5 macrophages with M-CSF induced phosphorylation of PKB/Akt as detected by activation-specific antibodies. Activation of PKB/Akt correlated with PI 3-kinase activation, suggesting that the protection from apoptosis in these cells is mediated by PKB/Akt. CONCLUSIONS: These results indicate that the lack of increase in cell numbers when cells are stimulated with M-CSF in the presence of PI 3-kinase inhibitors is due to a preferential PI 3-kinase requirement for protection against apoptosis, rather than a requirement for PI 3-kinase activation during the proliferation signal.     

Functional association of CD7 with phosphatidylinositol 3-kinase: interaction via a YEDM motif

Human CD7 is a 40 kDa protein expressed on thymocytes, earlyT, B, NK and myeloid lineage cells in bone marrow, and on matureT and NK cells. Previous studies suggested human CD7 may beInvolved in T and NK cell activation and/or adhesion, and thatCD7-mediated cell activation may be transduced via the lipidkinase phosphatldyllnositol 3-kinase (PI3-klnase), a heterodimerlccytosollc protein consisting of an 85 kDa adaptor subunit thatis coupled to a 110 kDa catalytic subunit. It has recently beenshown that a sequence motif present in the cytoplasmic tailof both human and mouse CD7 bound with high affinity to recombinantSH2 domains present in the p85 subunit of PI3-kinase. In thiswork, we used co-precipitation with anti-CD7 mAb 3A1 and recombinantp85 SH2-GST fusion proteins and peptide competition analysisto demonstrate that the cytoplasmic tall of CD7 interacts witha functional PI3-kJnase via the pTyr-X-X-Met motif. Furthermore,we show that cross-linking of CD7 markedly increased the amountof PI3-kinase activity associated with CD7. The Interactionof CD7 with the PI3-kinase signal transduction pathway providesa mechanism for the previously observed functional responsesattributed to CD7-mediated T and NK cell activation.     

Cellular stimulation via CD95 involves activation of phospho-inositide-3-kinase

 Several distinct intracellular pathways have been recently shown to be activated during CD95/Fas/APO-1-mediated apoptosis. Here, we demonstrate that CD95 ligation induces a rapid and transient tyrosine phosphorylation and activation of phosphoinositide-3-kinase (PI-3-K) in Jurkat T lymphocytes or CD95-sensitive glioma cells. Experiments using p56lck-deficient or p56lck-reconstituted Jurkat clones and the tyrosine kinase inhibitor herbimycin A revealed that tyrosine phosphorylation and activation of PI-3-K by CD95 depends on expression of Src-like tyrosine kinases, in particular p56lck. PI-3-K stimulation seems to be critical for CD95 receptor signalling since, first, inhibition of PI-3-K prevents CD95-mediated apoptosis and, second, CD95 receptor ligation fails to induce tyrosine phosphorylation or activation of PI-3-K in CD95-resistant glioma cells. Thus, PI-3-K activation may be an early signalling event during CD95-induced apoptosis, and failure to stimulate PI-3-K may predict tumor cell resistance to CD95-triggered apoptosis. Received: 14 May 1997 / Received after revision: 28 October 1997 / Accepted: 29 October 1997     

Down-regulation of LFA-1-mediated T cell adhesion induced by the HIV envelope glycoprotein gp160 requires phosphatidylinositol-3-kinase activity

Human immunodeficiency virus binds to CD4⁺ T lymphocyte by the interaction, in part, between its gp120 envelope glycoprotein and the CD4 molecule. We and others have reported that the lipid kinase phosphatidylinositol-3-kinase (PI3-kinase) is associated with the CD4-p56^lck complex and can be activated by various CD4 ligands. In a previous report we showed that the gp160 envelope down-regulates lymphocyte function-associated antigen-1 (LFA-1)-dependent adhesion between CD4⁺ T cells and B cells. This down-regulation was shown to be p56^lck-dependent. Here we investigate the role of PI3-kinase in the inhibition of adhesion induced by gp160 binding to CD4. We found that gp160 activates the PI3-kinase of HUT78 CD4⁺ T cell lines in a way dependent on CD4-p56^lck association, since no activation was detected when the interaction between CD4 and p56^lck was disrupted. It was also shown, using different inhibitors of the PI3-kinase (wortmannin, Ly294002 and antisense oligonucleotides), that this lipid kinase was necessary for the down-regulation of LFA-1-mediated adhesion induced by gp160. These results strongly suggest that PI3-kinase activation induced by gp160 leads to down-regulation of LFA-1-mediated T cell adhesion to B cells. Inhibition by gp160 of cytoskeleton rearrangement-dependent, anti-CD3-mediated T cell adhesion to B cells was blocked by neutralization of PI3-kinase activity, while inhibition of cytoskeleton rearrangement-independent, Mg²⁺-induced T cell adhesion was not. These results emphasize the role of PI3-kinase in the regulation of cytoskeleton structure. It is proposed that gp160 activates both p56^lck and PI3-kinase which lead to a cytoskeleton organization unfavorable for LFA-1 function.     

CD160-activating NK cell effector functions depend on the phosphatidylinositol 3-kinase recruitment

CD160 NK cell-activating receptor is a glycosyl-phosphatidylinositol-anchored molecule that, upon specific engagement, triggers both cytotoxicity and a unique cytokine production [IFN-gamma, tumor necrosis factor-alpha (TNF-alpha) and IL-6] through an undefined signaling pathway. In the current study, we have identified several signaling molecules recruited after mAb-specific CD160 engagement in freshly isolated human circulating NK cells. Using confocal microscopy, we found that CD160 engagement induces the recruitment and co-localization of phosphorylated molecules with redistributed, capped CD160 at the cell surface. We then demonstrated that phosphatidylinositol 3-kinase (PI3K) signaling molecule is required for CD160-mediated cytotoxicity and cytokine release. First, we observed by confocal microscopy that engagement of CD160 induces its polarization and co-localization with PI3K. Second, we showed that pharmacological inhibitors of PI3K abrogate both CD160-mediated cytotoxicity and IFN-gamma, TNF-alpha and IL-6 cytokine release. We further found that CD160 engagement induced marked phosphorylation of Akt, as evidenced by western blotting. We identified additional CD160-mediated signaling molecules recruited downstream and upstream of PI3K. Both induction of phosphorylated ERK molecules after CD160-specific engagement and prevention of CD160-induced cytokine release by MEK pharmacological inhibitor indicate that ERK downstream pathway is implicated. Similarly, we identified that Syk molecule upstream of PI3K is involved in the signaling cascade mediated by CD160 engagement. Two different Syk-specific inhibitors blocked CD160-mediated cytokine release, and CD160-specific engagement induced the enhancement of phosphorylated Syk proteins. These data demonstrate that PI3K is a crucial signaling element for both effector functions of the CD160 NK cell-activating receptor.     

Characterization of the interleukin-1beta-converting enzyme/ced-3-family protease, caspase-3/CPP32, in Hodgkin's disease: lack of caspase-3 expression in nodular lymphocyte predominance Hodgkin's disease.

Apoptosis (programmed cell death) serves an important role in the normal morphogenesis, immunoregulation, and homeostatic mechanisms in both normal and neoplastic cells. Caspase-3/CPP32, a member of the ICE/Ced-3-family of cysteine proteases, is an important downstream mediator of several complex proteolytic cascades that result in apoptosis in both hematopoietic and nonhematopoietic cells. Previous studies have demonstrated that caspase-3 is commonly expressed in classical Hodgkin's disease (CHD); however, the biological significance of its expression in Hodgkin's disease is unknown. In this report, the expression of caspase-3 in nodular lymphocyte predominance Hodgkin's disease (NLPHD) was evaluated by immunohistochemistry; in addition, we investigated the role of caspase-3 in CD95 (Fas)-mediated apoptosis in three CHD cell lines. Formalin-fixed, paraffin-embedded tissue sections from 11 cases of NLPHD were immunostained for caspase-3 using a polyclonal rabbit antibody that detects both the 32-kd zymogen and the 20-kd active subunit of the caspase-3 protease. Only 1/11 cases of NLPHD demonstrated caspase-3 immunopositivity in lymphocytic/histiocytic cells. Caspase-3 expression was also evaluated in three CHD cell lines, HS445, L428, and KMH2. Whereas caspase-3 expression was detected in HS445 and L428 cell lines, no expression was found in KMH2 cells by immunohistochemical staining. Treatment of HS445 and L428 cell lines for 72 hours with agonistic CD95 monoclonal antibody induced marked apoptosis that was significantly inhibited by pretreatment with the caspase-3 inhibitor, DEVD-FMK, as determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay and flow cytometric analysis of 7-amino-actinomycin D staining. In addition, a significant increase in caspase-3 activity as determined by an enzyme colorimetric assay was detected in HS445 and L428 cells after 48 hours of CD95 stimulation. In marked contrast, treatment of caspase-3-deficient KMH2 cells with anti-CD95 mAb did not demonstrate an increase in caspase-3 activity or induce apoptosis. These data demonstrate caspase-3 is important for CD95-mediated apoptosis in CHD cell lines. In addition, the majority of NLPHD cases examined in this study failed to express detectable levels of caspase-3, suggesting these tumor cells may be resistant to apoptotic stimuli dependent on caspase-3 activity. Furthermore, these data suggest the differential expression of caspase-3 noted between NLPHD and CHD may provide additional evidence that each is a unique disease entity.     

Interleukin-3, but not granulocyte-macrophage colony-stimulating factor and interleukin-5, inhibits apoptosis of human basophils through phosphatidylinositol 3-kinase: requirement of NF-kappaB-dependent and -independent pathways

Basophils are key effector cells of allergic reactions. Although proinflammatory cytokines, such as interleukin (IL)-3, granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-5, inhibit eosinophil apoptosis in vitro, little is known about basophil apoptosis, and the signalling mechanisms required for basophil survival remain undefined. To address this issue, we used a novel negative-selection system to isolate human basophils to a purity of > 95%, and evaluated apoptosis by morphology using light and transmission electron microscopy, and by annexin-V binding and propidium iodide incorporation using flow cytometry. In this study, we demonstrated that the spontaneous rate of apoptotic basophils was higher than that of eosinophils as, at 24 hr, 57.6 +/- 4.7% of basophils underwent apoptosis compared with 39.5 +/- 3.8% of eosinophils. In addition, basophil cell death was significantly inhibited when cultured with IL-3 for 48 hr (84.6 +/- 4.9% vehicle-treated cells versus 40.9 +/- 3.9% IL-3-treated cells). IL-3 also up-regulated basophil CD69 surface expression. The effects of IL-3 on apoptosis and CD69 surface expression of human basophils were completely blocked by LY294002 (LY), a potent inhibitor of phosphatidylinositol 3-kinase (PI3-K), but only partially inhibited by lactacystin, a proteasome inhibitor that prevents degradation of IkappaB and NF-kappaB translocation. These observations reveal the novel finding that IL-3 prevents basophil apoptosis through the activation of PI3-K, which is only partially NF-kappaB dependent. As basophils are active participants in allergic reactions and IL-3 is one of the abundant proinflammatory cytokines in secretions from allergic tissue, we suggest that IL-3-mediated inhibition of basophil apoptosis may exacerbate the inflammation associated with allergic disorders.     

Fas/CD95-mediated apoptosis of type II cells is blocked by Toxoplasma gondii primarily via interference with the mitochondrial amplification loop

The intracellular protozoan Toxoplasma gondii induces persistent infections in various hosts and is an important opportunistic pathogen of humans with immature or deficient immune responses. The ability to survive intracellularly largely depends on the blocking of different proapoptotic signaling cascades of its host cell. Fas/CD95 triggers an apoptotic cascade that is crucial for immunity and the outcome of infectious diseases. We have determined the mechanism by which T. gondii counteracts death receptor-mediated cell death in type II cells that transduce Fas/CD95 ligation via caspase 8-mediated activation of the mitochondrial amplification loop. The results showed that infection with T. gondii significantly reduced Fas/CD95-triggered apoptosis in HeLa cells by inhibiting the activities of initiator caspases 8 and 9 and effector caspase 3/7. Parasitic infection dose dependently diminished cleavage of caspase 8, the BH3-only protein Bid, and the downstream caspases 9 and 3. Importantly, interference with Fas/CD95-triggered caspase 8 and caspase 3/7 activities after parasitic infection was largely dependent on the presence of caspase 9. Within the mitochondrial amplification loop, T. gondii significantly inhibited the Fas/CD95-triggered release of cytochrome c into the host cell cytosol. These results indicate that T. gondii inhibits Fas/CD95-mediated apoptosis in type II cells primarily by decreasing the apoptogenic function of mitochondria.     

Rapid CD40-mediated rescue from CD95-induced apoptosis requires TNFR-associated factor-6 and PI3K

The activation molecule CD40 and the death receptor CD95/Fas play important roles in regulating B cells so that effective antimicrobial immunity occurs without autoimmunity. CD40 signaling increases CD95 expression, sensitizing cells to apoptosis, but sustained CD40 signals rescue B cells from CD95 killing. Here we describe a mechanism of early CD40-mediated rescue from CD95-induced apoptosis in B cells. Maximal rescue was achieved when CD40 signals were given within 1-2 h of initiating CD95 apoptosis. CD40 signaling did not block association of Fas-associated death domain-containing protein with CD95, but decreased CD95-induced activation of caspases 3 and 8. Rapid CD40 rescue did not require NF-kappaB activation and was independent of de novo protein synthesis, but was dependent upon active PI3 K. Signaling via a CD40 mutant that does not bind TNFR-associated factor (TRAF)1, TRAF2, and TRAF3 rescued B cells from CD95-induced apoptosis. TRAF1/2/3-independent rescue was confirmed in B cell lines made deficient in these TRAF molecules by gene targeting. In contrast, CD40 rescue was completely abrogated in TRAF6-deficient B cells, which showed reduced activation of Akt in response to CD40 engagement. These results reveal a new rapid mechanism to balance B cell activation and apoptosis.     

Distinct patterns of cleavage and translocation of cell cycle control proteins in CD95-induced and p53-induced apoptosis

Apoptotic cell death induced by p53 occurs at a late G1 cell cycle checkpoint termed the restriction (R) point, and it has been proposed that p53-induced apoptosis causes upregulation of CD95. However, as cells with defective in CD95 signaling pathway are still sensitive to p53-induced apoptosis, CD95 cannot be the sole factor resulting in apoptosis. In addition, unlike p53-induced apoptosis, the relationship between CD95-mediated apoptosis and the cell cycle is not clearly understood. It would therefore be worth investigating whether CD95-mediated cell death is pertinent with p53-induced apoptosis in view of cell cycle related molecules. In this report, biochemical analysis showed that etoposide-induced apoptosis caused the induction and the nuclear translocation of effector molecules involved in G1 cell cycle checkpoint. However, there was no such translocation in the case of CD95-mediated death. Thus, although both types of apoptosis involved caspase activation, the cell cycle related proteins responded differently. This argues against the idea that p53-induced apoptosis occurs through the induction of CD95/CD95L expression.     

CD72-mediated B cell activation involves recruitment of CD19 and activation of phosphatidylinositol 3-kinase

Occupancy of the B cell glycoprotein, CD72 results in syk-independent activation of phospholipase-C γ and calcium mobilization. The cytoplasmic tail of CD72 does not contain an immunoreceptor tyrosine-based activation motif to directly transduce signals into the B lymphocyte. Hence, we investigated whether other coreceptors such as CD19 and its associated phosphatidylinositol 3-kinase (PI 3-K) were involved in CD72 signaling. Two specific inhibitors of PI 3-K inhibited CD72-stimulated B cell proliferation in a dose-dependent manner. Activation of B lymphocytes via CD72 resulted in recruitment and activation of PI 3-K, which was mediated by CD19. Accordingly, CD72 ligation induced CD19 tyrosine phosphorylation. Thus, lipid products generated as a result of PI 3-K activation may have an important function in CD72-mediated B lymphocyte activation. The kinetics of CD19 tyrosine phosphorylation induced by CD72 ligation were strikingly different from those seen following B cell antigen receptor (BCR) stimulation. A transient increase in the tyrosine phosphorylation of the complement receptors, CD21 and CD35 was observed in BCR- but not CD72-stimulated cells. Co-cross-linking of CD72 and CD19 failed to induce syk tyrosine phosphorylation suggesting that even under these conditions, CD72 signaling was independent of syk activation. A transient and stimulation-dependent physical association between CD19 and CD72 was observed in CD72-ligated cells. These observations suggest a mechanism by which CD72 can recruit CD19 and influence activation of CD19-associated PI 3-K, which appears to be critical for CD72-mediated B cell activation.     

Ligation of HLA class II molecules promotes sensitivity to CD95 (Fas antigen,APO-1)-mediated apoptosis

CD95 (Fas antigen/APO-1) is up-regulated in activated lymphocytes, and monoclonal antibody (mAb) to CD95 induces apoptosis. HLA class II molecules play a key role in antigen presentation, ligation of which induces signal transduction. We examined 18 lymphoid cell lines (15 B cell and 3 T cell lines) to investigate the effects of ligation of HLA class II molecules on CD95-mediated apoptosis. All of the five immature B cell lines were sensitive to anti-CD95 mAb, and ligation of HLA class II molecules promoted CD95-mediated apoptosis. In seven B-blastoid cell lines, two Burkitt lines were resistant to anti-CD95 mAb in spite of high expression of CD95. In three of five non-Burkitt B-blastoid lines, CD95-mediated apoptosis was augmented by treatment with anti-HLA class II mAb, while the other two lines lacking CD95 were resistant to anti-CD95 mAb. Three plasmacytic cell lines showed CD95-mediated apoptosis, but enhancement by anti-HLA class II mAb was slight in one cell line and was not observed in the other two lines. Out of three HLA class II antigen-positive T cell lines, CD95-mediated apoptosis was observed to some degree in one cell line but was not promoted by the treatment with anti-HLA class II mAb, and the other two cell lines were resistant to anti-CD95 mAb. Ligation of HLA class II molecules did not alter CD95 expression in the five cell lines examined, except Su-DHL-4 originated from a follicular lymphoma, which showed slight up-regulation. Taken together, ligation of HLA class II molecules apparently promotes CD95-mediated apoptosis in immature B cells and non-Burkitt B blasts. These findings highlight the role of HLA class II molecules in CD95-mediated apoptosis, which may facilitate rapid clearance of functionally useless cells from the immune system and might be involved in negative selection of B cells.     

Ligation of CD28 receptor by B7 induces formation of D-3 phosphoinositides in T lymphocytes independently of T cell receptor/CD3 activation

The co-stimulatory role of B7/CD28 interactions is important in promoting T cell activation. Very little is known about the intracellular events that follow CD28 engagement although recent evidence has implicated coupling of CD28 to a protein tyrosine kinase signal transduction pathway. In this study we have investigated the putative role of D-3 phosphoinositides as mediators of CD28 receptor signaling, since phosphoinositide (PI) 3-kinase, the enzyme responsible for D-3 phosphoinositide formation, is a known substrate for protein tyrosine kinases associated with certain T cell surface receptors such as CD4 and interleukin-2 receptor. The lipid products of PI 3-kinase activity have been suggested to play a role in mitogenic signaling and growth regulation in other cells. Chinese hamster ovary cells (CHO) previously transfected with B7 cDNA, induced time-dependent elevation above basal levels of phosphatidylinositol(3,4)-bisphosphate (PtdIns(3,4)P₂) and PtdIns(3,4,5)P₃, while parental CHO cells that did not express B7 had no effect on these lipids. Moreover, the elevation of these same lipids by CD3 ligation was potentiated in an additive manner by CHO-B7+ but not by CHO-B7^? cells. CHO-B7⁺ and CHO-B7^? cells did not activate phospholipase C as evidenced by their inability to modulate basal or CD3-induced changes in the levels of phosphatidic acid or D-4 and D-5 phosphoinositides. These data imply that PI 3-kinase but not phospholipase C, may be an important signal transduction molecule with respect to CD28-mediated co-stimulation and T cell activation following ligation by B7.     

Inhibition of CD95 (Fas/Apo1)-mediated apoptosis by vaccinia virus WR

Stimulation of the CD95 (Apo-1/Fas) molecule either by the CD95 ligand or by monoclonal antibodies induces programmed cell death by apoptosis in a variety of cell lines and primary cells. In this study we observed that infection of B lymphoblast and T lymphoblast cell lines with vaccinia virus strain WR and recombinant vaccinia WR constructs, but not strain Copenhagen, rendered cells refractory to CD95-mediated apoptosis. In particular, vaccinia virus infection suppressed anti-CD95 antibody-induced membrane disintegration, apoptotic nuclear morphology of cells, and DNA fragmentation. Inhibition of apoptosis was not mediated by CD95 down-regulation or reduced binding of anti-CD95 antibody to infected cells, and occurred at a time point when cellular metabolism was not yet affected by the lytic vaccinia virus infection. Vaccinia virus (WR)-infected cells were resistant to CD95 ligand–CD95-mediated lysis by CD4⁺ and CD8⁺, T lymphocytes. Because cytolysis mediated by CD95 is one of two major mechanisms used by cytotoxic T lymphocytes to kill target cells, inhibition of CD95-mediated apoptosis may constitute a novel immune escape mechanism for this virus. Additionally, this mechanism may contribute to the higher pathogenicity of vaccinia virus strain WR compared with strain Copenhagen.     

CD4-induced down-regulation of T cell adhesion to B cells is associated with localization of phosphatidyl inositol 3-kinase and LFA-1 in distinct membrane domains

We have previously shown that binding of anti-CD4 antibody inhibit LFA-1-dependent adhesion between CD4+ T cells and B cells in a p56(lck) and a PI3-kinase-dependent manner. In this work, we investigated with two different T cell lines (Jurkat and A201) whether CD4 binding could alter interactions of the proteins putatively involved in this adhesion regulatory pathway. Anti-CD4 binding was shown to induce a transient association between PI3-kinase and LFA-1, which took place in different regions of the plasma membrane. It was detected in detergent soluble membrane but also in detergent insoluble membrane consisting in raft microdomains, composed of GM1 and/or GM3 gangliosides. These results show that anti-CD4 Ab could modify the interaction between LFA-1 and signaling molecules, such as PI3-kinase and induce, in part, their recruitment in raft domains. By using specific inhibitors, raft integrity and CD4 association with GM3 were found necessary for observing the CD4-dependent inhibition of LFA-1-mediated adhesion. These results strongly suggest that these molecular rearrangements in the membrane are necessary to induce down-regulation of LFA-1-mediated adhesion.     

Inhibition of CD28-mediated T cell costimulation by the phosphoinositide 3-kinase inhibitor wortmannin

T lymphocyte activation requires at least two signals, one via the antigen-specific T cell receptor and a second via the surface molecule CD28 which provides signals critical to interleukin-2 (IL-2) production and T cell proliferation. We have previously shown (Ward S. G., Westwick J., Hall N. and Sansom D. M. Eur. J. Immunol. 1993. 23: 2572) that CD28 stimulates phosphoinositide (PI) 3-kinase activity, indicating that D-3 phosphoinositides may act as mediators of CD28-induced T cell costimulation. Here, we report that immunoprecipitation of CD28 molecules from Jurkat cells stimulated with the CD28-ligand B7, results in a ligand-dependent association of CD28 with PI 3-kinase. This association correlates with the appearance of PI 3-kinase enzymatic activity in CD28 immunoprecipitates and the formation of D-3 phosphoinositides. Consistent with the hypothesis that D-3 phosphoinositides are important mediators of CD28 signaling, treatment of T cells with the PI 3-kinase inhibitor wortmannin, inhibited both T cell proliferation and production of IL-2, but not the response of T cells to exogenous IL-2. Hence, abrogation of PI 3-kinase activity by wortmannin, appears sufficient to disrupt the costimulatory pathway utilized by CD28, indicating a central role for this enzyme in the CD28 signaling pathway.