Molecular events associated with CD4-mediated Down-regulation of LFA-1-dependent adhesion.

We have previously shown that CD4 ligand binding inhibits LFA-1-dependent adhesion between CD4+ T cells and B cells in a p56(lck)- and phosphatidylinositol 3-kinase (PI3-kinase)-dependent manner. In this work, downstream events associated with adhesion inhibition have been investigated. By using HUT78 T cell lines, CD4 ligands were shown to induce a dissociation of LFA-1 from cytohesin, a cytoplasmic protein known to bind LFA-1 and to enhance the affinity/avidity of LFA-1 for its ligand ICAM-1. A dissociation of PI3-kinase from cytohesin is also observed. In parallel, we have found that CD4 ligand binding induced a redistribution of PI3-kinase and of the tyrosine phosphatase SHP-2 to the membrane and induced a transient formation of protein interactions including PI3-kinase; an adaptor protein, Gab2; SHP-2; and a SH2 domain-containing inositol phosphatase, SHIP. By using antisense oligonucleotides or transfection of transdominant mutants, down-regulation of adhesion was shown to require the Gab2/PI3-kinase association and the expression of SHIP and SHP-2. We therefore propose that CD4 ligands, by inducing these molecular associations, lead to sustained local high levels of D-3 phospholipids and possibly regulate the cytohesin/LFA-1 association.     

p56lck, LFA-1 and PI3K but not SHP-2 interact with GM1- or GM3-enriched microdomains in a CD4-p56lck association-dependent manner

We previously showed that the association of CD4 and G(M3) ganglioside induced by CD4 ligand binding was required for the down-regulation of adhesion and that aggregation of ganglioside-enriched domains was accompanied by transient co-localization of LFA-1 (lymphocyte function-associated antigen-1), PI3K (phosphoinositide 3-kinase) and CD4. We also showed that these proteins co-localized with the G(M1) ganglioside that partially co-localized with G(M3) in these domains. In the present study, we show that CD4-p56(lck) association in CD4 signalling is required for the redistribution of p56(lck), PI3K and LFA-1 in ganglioside-enriched domains, since ganglioside aggregation and recruitment of these proteins were not observed in a T-cell line (A201) expressing the mutant form of CD4 that does not bind p56(lck). In addition, we show that although these proteins associated in different ways with G(M1) and G(M3), all of the associations were dependent on CD4-p56(lck) association. Gangliosides could associate with these proteins that differ in affinity binding and could be modified following CD4 signalling. Our results suggest that through these associations, gangliosides transiently sequestrate these proteins and consequently inhibit LFA-1-dependent adhesion. Furthermore, while structural diversity of gangliosides may allow association with distinct proteins, we show that the tyrosine phosphatase SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase 2), also required for the down-regulation of LFA-1-dependent adhesion, transiently and partially co-localized with PI3K and p56(lck) in detergent-insoluble membranes without association with G(M1) or G(M3). We propose that CD4 ligation and binding with p56(lck) and their interaction with G(M3) and/or G(M1) gangliosides induce recruitment of distinct proteins important for CD4 signalling to form a multimolecular signalling complex.     

Cutting edge: LFA-1 integrin-dependent T cell adhesion is regulated by both ag specificity and sensitivity

Ab stimulation of the TCR rapidly enhances the functional activity of the LFA-1 integrin. Although TCR-mediated changes in LFA-1 activity are thought to promote T cell-APC interactions, the Ag specificity and sensitivity of TCR-mediated triggering of LFA-1 is not clear. We demonstrate that peptide/MHC (pMHC) tetramers rapidly enhance LFA-1-dependent adhesion of OT-I TCR transgenic CD8(+) T cells to purified ICAM-1. Inhibition of src family tyrosine kinase or PI3K activity blocked pMHC tetramer- and anti-CD3-stimulated adhesion. These effects are highly specific because partial agonist and antagonist pMHC tetramers are unable to stimulate OT-I T cell adhesion to ICAM-1. The Ag thresholds required for T cell adhesion to ICAM-1 resemble those of early T cell activation events, because optimal LFA-1 activation occurs at tetramer concentrations that fail to induce maximal T cell proliferation. Thus, TCR signaling to LFA-1 is highly Ag specific and sensitive to low concentrations of Ag.     

CD73 engagement promotes lymphocyte binding to endothelial cells via a lymphocyte function-associated antigen-1-dependent mechanism

CD73 is a GPI-anchored lymphocyte adhesion molecule possessing an ecto-5'-nucleotidase enzyme activity. In this work, we show that engagement of lymphocyte CD73 increases lymphocyte binding to cultured endothelial cells (EC) in an LFA-1-dependent fashion. Engagement of CD73 by an anti-CD73 mAb 4G4 increases the adhesion of lymphocytes to cultured EC by about 80% compared with that of lymphocytes treated with a negative control Ab, and the increased adhesion can be blocked by an anti-CD18 mAb. The CD73-regulated increase in lymphocyte adhesion is not due to a conformational change leading to high-affinity LFA-1 receptors as assayed using mAb 24 against an activation-induced epitope of the molecule. Instead, CD73 engagement induces clustering of LFA-1 that is inhibitable by calpeptin, indicating involvement of Ca(2+)-dependent activation of a calpain-like enzyme in this process. In conclusion, the results shown here demonstrate that CD73 regulates the avidity of LFA-1 by clustering. This indicates a previously undescribed role for CD73 in controlling the poorly characterized activation step in the multistep cascade of lymphocyte extravasation. Moreover, these results suggest that in physiological conditions the activation step may result in clustering of LFA-1 rather than in an affinity change of the molecule.     

CD98 induces LFA-1-mediated cell adhesion in lymphoid cells via activation of Rap1

CD98 is a multifunctional heterodimeric membrane protein involved in the regulation of cell adhesion as well as amino acid transport. We show that CD98 cross-linking persistently activates Rap1 GTPase in a LFA-1-dependent manner and induces LFA-1/ICAM-1-mediated cell adhesion in lymphocytes. Specific phosphatidylinositol-3-kinase (PI3K) inhibitors suppressed both LFA-1 activation and Rap1GTP generation, and abrogation of Rap1GTP by retroviral over-expression of a specific Rap1 GTPase activating protein, SPA-1, totally inhibited the LFA-1/ICAM-1-mediated cell adhesion. These results suggest that CD98 cross-linking activates LFA-1 via the PI3K signaling pathway and induces accumulation of Rap1GTP in a LFA-1-dependent manner, which in turn mediates the cytoskeleton-dependent cell adhesion process.     

Analysis of the role of leukocyte function-associated antigen-1 in activation of human influenza virus-specific T cell clones

The role of leukocyte function-associated Ag-1 (LFA-1) in intercellular adhesion is well documented. Previously, we demonstrated that the LFA-1 molecule (CD11a/CD18) can also regulate the induction of proliferation of peripheral blood T cells. In these studies, we observed opposite effects of antibodies against CD11a (LFA-1-alpha-chain) or CD18 (LFA-1-beta-chain). Here, we determined the effects of anti-CD11a and anti-CD18 mAb on proliferation of cloned influenza virus-specific T cells. Anti-CD18 mAb had similar inhibiting effects on the proliferative response of T cell clones induced by immobilized anti-CD3 mAb as it had on the response of peripheral blood T cells. In contrast to its costimulatory effect on resting peripheral blood T cells, anti-CD11a mAb did not increase the proliferation of cloned T cells. Similar differences in effects of anti-CD11a and anti-CD18 mAb were observed when proliferation of the T cell clones was induced by immobilized anti-TCR mAb. When proliferation was induced by influenza virus presented by monocytes as APC, both anti-CD11a and anti-CD18 mAb inhibited T cell proliferation. However, when EBV-transformed B cells were used as APC, neither anti-CD11a nor anti-CD18 mAb inhibited proliferation. These results demonstrate that the effects of antibodies against CD11a (LFA-1-alpha) or CD18 (LFA-1-beta) on T cell proliferation depend on 1) the stage of activation of the T cells, 2) the activation stimulus and its requirement for intercellular adhesion involving LFA-1, and 3) the type of cell used to present Ag.     

A novel function for the Tec family tyrosine kinase Itk in activation of beta 1 integrins by the T-cell receptor

Stimulation of T cells via the CD3--T-cell receptor (TCR) complex results in rapid increases in beta 1 integrin-mediated adhesion via poorly defined intracellular signaling events. We demonstrate that TCR-mediated activation of beta 1 integrins requires activation of the Tec family tyrosine kinase Itk and phosphatidylinositol 3-kinase (PI 3-K)-dependent recruitment of Itk to detergent-insoluble glycosphingolipid-enriched microdomains (DIGs) via binding of the pleckstrin homology domain of Itk to the PI 3-K product PI(3,4,5)-P(3). Activation of PI 3-K and the src family kinase Lck, via stimulation of the CD4 co-receptor, can initiate beta 1 integrin activation that is dependent on Itk function. Targeting of Itk specifically to DIGs, coupled with CD4 stimulation, can also activate beta 1 integrin function independently of TCR stimulation. Changes in beta 1 integrin function mediated by TCR activation of Itk are also accompanied by Itk-dependent modulation of the actin cytoskeleton. Thus, TCR-mediated activation of beta 1 integrins involves membrane relocalization and activation of Itk via coordinate action of PI 3-K and a src family tyrosine kinase.     

Induction of tyrosine phosphorylation during ICAM-3 and LFA-1-mediated intercellular adhesion, and its regulation by the CD45 tyrosine phosphatase

Intercellular adhesion molecule (ICAM)-3, a recently described counter- receptor for the lymphocyte function-associated antigen (LFA)-1 integrin, appears to play an important role in the initial phase of immune response. We have previously described the involvement of ICAM-3 in the regulation of LFA-1/ICAM-1-dependent cell-cell interaction of T lymphoblasts. In this study, we further investigated the functional role of ICAM-3 in other leukocyte cell-cell interactions as well as the molecular mechanisms regulating these processes. We have found that ICAM-3 is also able to mediate LFA-1/ICAM-1-independent cell aggregation of the leukemic JM T cell line and the LFA-1/CD18-deficient HAFSA B cell line. The ICAM-3-induced cell aggregation of JM and HAFSA cells was not affected by the addition of blocking mAb specific for a number of cell adhesion molecules such as CD1 1a/CD18, ICAM-1 (CD54), CD2, LFA-3 (CD58), very late antigen alpha 4 (CD49d), and very late antigen beta 1 (CD29). Interestingly, some mAb against the leukocyte tyrosine phosphatase CD45 were able to inhibit this interaction. Moreover, they also prevented the aggregation induced on JM T cells by the proaggregatory anti-LFA-1 alpha NKI-L16 mAb. In addition, inhibitors of tyrosine kinase activity also abolished ICAM-3 and LFA-1- mediated cell aggregation. The induction of tyrosine phosphorylation through ICAM-3 and LFA-1 antigens was studied by immunofluorescence, and it was found that tyrosine-phosphorylated proteins were preferentially located at intercellular boundaries upon the induction of cell aggregation by either anti-ICAM-3 or anti-LFA-1 alpha mAb. Western blot analysis revealed that the engagement of ICAM-3 or LFA-1 with activating mAb enhanced tyrosine phosphorylation of polypeptides of 125, 70, and 38 kD on JM cells. This phenomenon was inhibited by preincubation of JM cells with those anti-CD45 mAb that prevented cell aggregation. Altogether these results indicate that CD45 tyrosine phosphatase plays a relevant role in the regulation of both intracellular signaling and cell adhesion induced through ICAM-3 and beta 2 integrins.     

Superantigen-induced T cell:B cell conjugation is mediated by LFA-1 and requires signaling through Lck, but not ZAP-70.

The formation of a conjugate between a T cell and an APC requires the activation of integrins on the T cell surface and remodeling of cytoskeletal elements at the cell-cell contact site via inside-out signaling. The early events in this signaling pathway are not well understood, and may differ from the events involved in adhesion to immobilized ligands. We find that conjugate formation between Jurkat T cells and EBV-B cells presenting superantigen is mediated by LFA-1 and absolutely requires Lck. Mutations in the Lck kinase, Src homology 2 or 3 domains, or the myristoylation site all inhibit conjugation to background levels, and adhesion cannot be restored by the expression of Fyn. However, ZAP-70-deficient cells conjugate normally, indicating that Lck is required for LFA-1-dependent adhesion via other downstream pathways. Several drugs that inhibit T cell adhesion to ICAM-1 immobilized on plastic, including inhibitors of mitogen-activated protein/extracellular signal-related kinase kinase, phosphatidylinositol-3 kinase, and calpain, do not inhibit conjugation. Inhibitors of phospholipase C and protein kinase C block conjugation of both wild-type and ZAP-70-deficient cells, suggesting that a phospholipase C that does not depend on ZAP-70 for its activation is involved. These results are not restricted to Jurkat T cells; Ag-specific primary T cell blasts behave similarly. Although the way in which Lck signals to enhance LFA-1-dependent adhesion is not clear, we find that cells lacking functional Lck fail to recruit F-actin and LFA-1 to the T cell:APC contact site, whereas ZAP-70-deficient cells show a milder phenotype characterized by disorganized actin and LFA-1 at the contact site.     

Integrin leukocyte function-associated antigen-1-mediated cell binding can be activated by clustering of membrane rafts

The leukocyte function-associated antigen-1 (LFA-1) integrin (CD11a/CD18) is an important adhesion molecule for lymphocyte migration and the initiation of an immune response. At the cell surface, LFA-1 activity can be regulated by divalent cations that enhance receptor affinity but also by membrane clustering induced by treatment of cells with substances such as phorbol esters. Membrane clustering leads to increased LFA-1 avidity. We report here that LFA-1-mediated binding of mouse thymocytes or activated T lymphocytes to intercellular adhesion molecule 1 can be rapidly induced by clustering of membrane rafts using antibodies to the glycosylphophatidylinositol-anchored molecule CD24 or cholera toxin (CTx). CD24 and CD18 were found to co-localize in rafts and cross-linking with CTx lead to enhanced LFA-1 clustering. We observed that disruption of raft integrity by lowering the membrane cholesterol content abolished the CTx and the phorbol 12-myristate 13-acetate-induced LFA-1 binding but left the ability to activate LFA-1 with Mg(2+)/EGTA unimpaired. In contrast to activation with Mg(2+)/EGTA, activation via raft clustering was dependent on PI3-kinase, required cytoskeletal mobility, and was accompanied by Tyr phosphorylation of a 18-kDa protein. Our results support the notion that rafts as preformed adhesion platforms could be important for the rapid regulation of lymphocyte adhesion.     

CD18 activation epitopes induced by leukocyte activation.

The cell surface adhesion molecule LFA-1 coordinates leukocyte trafficking and is a costimulatory molecule for T cell activation. We developed a panel of mAbs that recognize activation epitopes on the CD18 subunit, and show that stimulation of T lymphocytes appears to be accompanied by a conformational change in a subpopulation of LFA-1 that does not require ligand binding. Activation epitope up-regulation requires divalent cations, is sensitive to cellular signal transduction events, and correlates with cell adhesion. In addition, the stimulated appearance of these activation epitopes is absent in cell lines from patients with leukocyte adhesion deficiency-1/variant that has previously been shown to be defective in LFA-1 activation. Thus, these activation epitope Abs can be used to dissect signal transmission to CD18. Evidence suggests that these CD18 activation epitopes are induced early in cellular activation and are independent of actin rearrangement necessary for avid adhesion. We have also determined that function-blocking CD18 Abs inhibit the induction of activation epitopes. One activation epitope Ab binds to a site on CD18 distinct from that of the blocking Abs, indicating that the blocking Abs suppress a conformational change in LFA-1. We also find that these neoepitopes are present on rLFA-1 with high affinity for ICAM-1 and their binding is modulated in parallel with the affinity of LFA-1 for ICAM-1. Collectively, these neoepitope Abs identify a subpopulation of LFA-1 most likely with high affinity for ICAM-1 and necessary for LFA-1 function.     

Interaction of CD2 with its ligand, LFA-3, in human T cell proliferation

Recently, it has been demonstrated that lymphocyte function-associated Ag (LFA-3) is a natural ligand for CD2 and that this receptor-ligand interaction functions in cell-cell adhesion. In this report, we demonstrate that LFA-3 plays a role in T cell activation. L cells were transfected with human genomic DNA and sorted for expression of LFA-3. We demonstrate that LFA-3+ L cells, together with anti-CD3 mAb or with suboptimal doses of PHA, stimulate proliferation of human peripheral blood T cells. Furthermore, thymocyte proliferation was induced by LFA-3+ L cells and suboptimal doses of PHA. Proliferation was inhibited by mAb directed against either CD2 or LFA-3. Stimulation of thymocytes by the combination of PHA and LFA-3+ L cells resulted in the increased expression of the IL-2R, as well as of the surface Ag 4F2, transferrin receptor, and HLA-DR. These data support the conclusion that LFA-3 plays a role in CD2-dependent T cell activation. LFA-3 is widely distributed and is expressed on all APC and target cells. Thus, the ability of the CD2/LFA-3 interaction to costimulate with an anti-CD3 mAb suggests that the CD2/LFA-3 interaction may be involved not only in an Ag-independent alternate pathway of T cell activation but also in Ag-specific T cell activation.     

H-Ras signals to cytoskeletal machinery in induction of integrin-mediated adhesion of T cells

The adhesive function of integrins is regulated through cytoplasmic signaling. The present study was performed to investigate the relevance of cytoplasmic signaling and cytoskeletal assembly to integrin-mediated adhesion induced by chemokines. Adhesion of T cells induced by chemokines macrophage inflammatory protein (MIP)-1alpha and MIP-1beta was inhibited by pertussis toxin, wortmannin, and cytochalasin B, suggesting that both G protein-sensitive phosphatidylinositol (PI) 3-kinase activation and cytoskeletal assemblies are involved. The chemokine-induced T cell adhesion could be mimicked by expression of small G proteins, fully activated H-RasV12, or H-RasV12Y40C mutant, which selectively binds to PI 3-kinase, in T cells, inducing activated form of LFA-1alpha and LFA-1-dependent adhesion to ICAM-1. H-Ras expression also induced F-actin polymerization which colocalized with profilin in T cells. Adult T cell leukemia (ATL) cells spontaneously adhered to ICAM-1, which depended on endogenous MIP-1alpha and MIP-1beta through activation of G protein-sensitive PI 3-kinase. H-Ras signal pathway, leading to PI 3-kinase activation, also induced active configuration of LFA-1 and LFA-1-mediated adhesion of ATL cells, whereas expression of a dominant-negative H-Ras mutant failed to do. Profilin-dependent spontaneous polymerization of F-actin in ATL cells was reduced by PI 3-kinase inhibitors. In this paper we propose that H-Ras-mediated activation of PI 3-kinase can be involved in induction of LFA-1-dependent adhesion of T cells, which is relevant to chemokine-mediated signaling, and that profilin may form an important link between chemokine- and/or H-Ras-mediated signals and F-actin polymerization, which results in triggering of LFA-1 on T cells or leukemic T cells.     

Human endothelial-cell specific molecule-1 binds directly to the integrin CD11a/CD18 (LFA-1) and blocks binding to intercellular adhesion molecule-1.

ICAMs are ligands for LFA-1, a major integrin of mononuclear cells involved in the immune and inflammatory processes. We previously showed that endothelial cell specific molecule-1 (ESM-1) is a proteoglycan secreted by endothelial cells under the control of inflammatory cytokines. Here, we demonstrate that ESM-1 binds directly to LFA-1 onto the cell surface of human blood lymphocytes, monocytes, and Jurkat cells. The binding of ESM-1 was equally dependent on Ca(2+), Mg(2+), or Mn(2+) divalent ions, which are specific, saturable, and sensitive to temperature. An anti-CD11a mAb or PMA induced a transient increase in binding, peaking 5 min after activation. Direct binding of ESM-1 to LFA-1 integrin was demonstrated by specific coimmunoprecipitation by CD11a and CD18 mAbs. A cell-free system using a Biacore biosensor confirmed that ESM-1 and LFA-1 dynamically interacted in real time with high affinity (K(d) = 18.7 nM). ESM-1 consistently inhibited the specific binding of soluble ICAM-1 to Jurkat cells in a dose-dependent manner. These results suggest that ESM-1 and ICAM-1 interact with LFA-1 on binding sites very close to but distinct from the I domain of CD11a. Through this mechanism, ESM-1 could be implicated in the regulation of the LFA-1/ICAM-1 pathway and may therefore influence both the recruitment of circulating lymphocytes to inflammatory sites and LFA-1-dependent leukocyte adhesion and activation.     

Dual role of H-Ras in regulation of lymphocyte function antigen-1 activity by stromal cell-derived factor-1alpha: implications for leukocyte transmigration

We investigated the role of H-Ras in chemokine-induced integrin regulation in leukocytes. Stimulation of Jurkat T cells with the CXC chemokine stromal cell-derived factor-1alpha (SDF-1alpha) resulted in a rapid increase in the phosphorylation, i.e., activation of extracellular signal receptor-activated kinase (ERK) but not c-Jun NH(2)-terminal kinase or p38 kinase, and phosphorylation of Akt, reflecting phosphatidylinositol 3-kinase (PI3-K) activation. Phosphorylation of ERK in Jurkat cells was enhanced and attenuated by expression of dominant active (D12) or inactive (N17) forms of H-Ras, respectively, while N17 H-Ras abrogated SDF-1alpha-induced Akt phosphorylation. SDF-1alpha triggered a transient regulation of adhesion to intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 mediated by lymphocyte function antigen-1 (LFA-1) and very late antigen-4 (VLA-4), respectively, and a rapid increase in LFA-1 binding to soluble ICAM-1.Ig, which was inhibited by D12 but not N17 H-Ras. Both D12 and N17 H-Ras abrogated the regulation of LFA-1 but not VLA-4 avidity, and impaired LFA-1-mediated transendothelial chemotaxis but not VLA-4-dependent transmigration induced by SDF-1alpha. Analysis of the mutant Jurkat J19 clone revealed LFA-1 with constitutively high affinity and reduced ERK phosphorylation, which were partially restored by expression of active H-Ras. Inhibition of PI3-K blocked the up-regulation of Jurkat cell adhesion to ICAM-1 by SDF-1alpha, whereas inhibition of mitogen-activated protein kinase kinase impaired the subsequent down-regulation and blocking both pathways abrogated LFA-1 regulation. Our data suggest that inhibition of initial PI3-K activation by inactive H-Ras or sustained activation of an inhibitory ERK pathway by active H-Ras prevail to abolish LFA-1 regulation and transendothelial migration induced by SDF-1alpha in leukocytes, establishing a complex and bimodal involvement of H-Ras.     

Modulation of antigen presentation and peptide-MHC-specific, LFA-1-dependent T cell-macrophage adhesion

Incubation of peritoneal macrophages in vitro before fixation increased their ability to present exogenous peptides to 3A9 T hybridoma cells. The enhanced level of presentation correlated with a greatly increased, peptide-specific adhesion of 3A9 cells to the macrophages, whereas peptide-independent adhesion was minimal and essentially unaltered. 3A9 cells exhibited rapid peptide-specific adhesion (plateau by 5 to 10 min) and deadhesion (complete reversal by 5 min). Peptide-specific adhesion was blocked by anti-I-Ak and anti-LFA-1. Interaction of T cell receptors and CD-4 with peptide-I-Ak complexes appeared to provide little direct contribution to the avidity of T cell-macrophage adhesion, but activated a LFA-1-mediated adhesion mechanism. In addition, anti-T cell receptor, anti-CD3, and anti-CD4 antibodies themselves activated LFA-1-dependent adhesion in the absence of peptide. Unlike the peptide-induced adhesion, this adhesion was similar for macrophages whether or not they were incubated in vitro before fixation. We conclude that the different macrophage populations supported LFA-1-mediated adhesion equally. Therefore, the enhancement of T cell stimulation observed after in vitro incubation of macrophages was due to increased peptide presentation and consequently increased triggering of LFA-1-mediated adhesion. Mechanisms may exist to regulate the effectiveness with which peptide-class II MHC complexes are displayed for T cell recognition.     

T cell-specific adapter protein inhibits T cell activation by modulating Lck activity

We previously reported the isolation of a cDNA encoding a T cell-specific adapter protein (TSAd). Its amino acid sequence contains an SH2 domain, tyrosines in protein binding motifs, and proline-rich regions. In this report we show that expression of TSAd is induced in normal peripheral blood T cells stimulated with anti-CD3 mAbs or anti-CD3 plus anti-CD28 mAbs. Overexpression of TSAd in Jurkat T cells interfered with TCR-mediated signaling by down-modulating anti-CD3/PMA-induced IL-2 promoter activity and anti-CD3 induced Ca2+ mobilization. The TCR-induced tyrosine phosphorylation of phospholipase C-gamma1, SH2-domain-containing leukocyte-specific phosphoprotein of 76kDa, and linker for activation of T cells was also reduced. Furthermore, TSAd inhibited Zap-70 recruitment to the CD3zeta-chains in a dose-dependent manner. Consistent with this, Lck kinase activity was reduced 3- to 4-fold in COS-7 cells transfected with both TSAd and Lck, indicating a regulatory effect of TSAd on Lck. In conclusion, our data strongly suggest an inhibitory role for TSAd in proximal T cell activation.     

HIV envelope gp120 activates LFA-1 on CD4 T-lymphocytes and increases cell susceptibility to LFA-1-targeting leukotoxin (LtxA)

The cellular adhesion molecule LFA-1 and its ICAM-1 ligand play an important role in promoting HIV-1 infectivity and transmission. These molecules are present on the envelope of HIV-1 virions and are integral components of the HIV virological synapse. However, cellular activation is required to convert LFA-1 to the active conformation that has high affinity binding for ICAM-1. This study evaluates whether such activation can be induced by HIV itself. The data show that HIV-1 gp120 was sufficient to trigger LFA-1 activation in fully quiescent naïve CD4 T cells in a CD4-dependent manner, and these CD4 T cells became more susceptible to killing by LtxA, a bacterial leukotoxin that preferentially targets leukocytes expressing high levels of the active LFA-1. Moreover, virus p24-expressing CD4 T cells in the peripheral blood of HIV-infected subjects were found to have higher levels of surface LFA-1, and LtxA treatment led to significant reduction of the viral DNA burden. These results demonstrate for the first time the ability of HIV to directly induce LFA-1 activation on CD4 T cells. Although LFA-1 activation may enhance HIV infectivity and transmission, it also renders the cells more susceptible to an LFA-1-targeting bacterial toxin, which may be harnessed as a novel therapeutic strategy to deplete virus reservoir in HIV-infected individuals.     

Inhibition of CD4+ T cell activation and adhesion by peptides derived from the gp160.

It has been previously demonstrated that the HIV envelope glycoprotein gp160 can inhibit the activation of T cells triggered by phytohemagglutinin, anti-CD3 antibody and Ag, caused in part by the modulation of the expression of CD4. In this study, we show that gp160 is also able to inhibit the Ag-independent adhesion of CD4+ T cells to B cells as anti-CD4 antibodies do. In addition, synthetic peptides (14 to 21 mer) derived from the gp160 sequence and analogous to the putative binding site of gp160 to CD4 (residues 418-460), and also covering residues 460 to 474 inhibit the capacity of both CD4+ T cell proliferation induced by tuberculin and anti-CD3 antibody and adhesion. This was not associated with inhibition of Ca2+ flux in T cell activation. These inhibitory activities are specific because a) CD4+ T cells but not CD8+ T cells are susceptible to their effects, and b) soluble CD4 neutralizes the inhibitory activities. Peptides are, however, about 100- to 1000-fold less potent inhibitors than the native gp160. In addition, they do not induce CD4 modulation. It is thought therefore that at least part of the gp160 inhibitory activity is not secondary to CD4 modulation but may rely either upon steric hindrance of CD4-MHC class II interaction, of CD4/CD3 TCR complex interaction, or upon negative signaling through binding to CD4. The latter hypothesis is suggested by the inhibition by gp160, gp160-derived peptides, and anti-CD4 antibodies of the Ag-independent adhesion of CD4+ T cells. This adhesion process has been previously shown to be mediated by the LFA-1 and CD2 molecules and not by the TCR/CD3 complex and by CD4. Together, these results support the role of part of the 418-460 region of gp160 as a binding site to CD4, and suggest that binding of part of this region to CD4 can alter T cell proliferation and adhesion. It is proposed that these effects are mainly mediated by negative signaling through CD4.