Monoclonal antibodies to CD2 and lymphocyte function-associated antigen 3 inhibit human thymic epithelial cell-dependent mature thymocyte activation

Recent study of human thymocyte-thymic epithelial (TE) cell interactions has demonstrated that thymocytes bind to TE cells, and a consequence of this binding is the provision of accessory cell signals by TE cells for phytohemagglutinin (PHA)-induced mature thymocyte activation. In this paper we report on studies of the molecules involved in TE cell-dependent mature thymocyte activation. TE-thymocyte interactions necessary for PHA-induced thymocyte activation were inhibited by monoclonal antibodies against the cluster of differentiation (CD)2 antigen on thymocytes and lymphocyte function-associated (LFA)-3 antigen on TE cells. Inhibition of TE accessory cell signals by antibodies against CD2 (alpha CD2) and LFA-3 (alpha LFA-3) antigens occurred early on during thymocyte activation and prevented thymocyte interleukin 2 receptor expression. Further, alpha CD2 and alpha LFA-3 inhibited PHA-induced thymocyte activation in whole thymic explant cultures suggesting a significant role of the CD2 and LFA-3 antigens in thymocyte activation when accessory cell signals for PHA-induced thymocyte triggering were delivered by cells within an intact thymic microenvironment.     

Human T cell clones with gamma/delta and alpha/beta receptors are differently stimulated by monoclonal antibodies to CD2

Requirements for stimulating autocrine proliferation of human T cell clones expressing either alpha/beta or gamma/delta antigen receptors via the "alternative" CD2 pathway have been examined using a large set of monoclonal antibodies (mAb). In the presence of autologous accessory cells (AC, B-lymphoblastoid cell lines) 2 of 13 single CD2 mAb (CLB-T11.1/1 and 6F10.3) stimulated proliferation of gamma/delta but not alpha/beta cells. Interleukin (IL) 1 or IL 6 did not substitute for AC in stimulating gamma/delta clones. Addition of CD28 mAb YTH 913.12 with the CD2 mAb did not result in stimulation of any alpha/beta clones. In the absence of AC, none of the CD2 mAb singly could stimulate any T cell clones, but pairs of mAb directed to different epitopes of CD2 (CLB-T11.1/1 + CLB- T11.2/1 or 6F10.3 + 39C1.5) stimulated both alpha/beta and gamma/delta clones. In both cases, stimulation was reduced by the presence of CD3 mAb. These results confirm that the established AC-independent alternative pathway of T cell activation, which requires binding of two separate epitopes of CD2, operates in both gamma/delta and alpha/beta T cells, and further suggest that an additional pathway initiated by binding of a single CD2 epitope in the presence of AC is exclusively operational in gamma/delta cells.     

ICAM-3 interacts with LFA-1 and regulates the LFA-1/ICAM-1 cell adhesion pathway

The interaction of lymphocyte function-associated antigen-1 (LFA-1) with its ligands mediates multiple cell adhesion processes of capital importance during immune responses. We have obtained three anti-ICAM-3 mAbs which recognize two different epitopes (A and B) on the intercellular adhesion molecule-3 (ICAM-3) as demonstrated by sequential immunoprecipitation and cross-competitive mAb-binding experiments. Immunoaffinity purified ICAM-3-coated surfaces were able to support T lymphoblast attachment upon cell stimulation with both phorbol esters and cross-linked CD3, as well as by mAb engagement of the LFA-1 molecule with the activating anti-LFA-1 NKI-L16 mAb. T cell adhesion to purified ICAM-3 was completely inhibited by cell pretreatment with mAbs to the LFA-1 alpha (CD11a) or the LFA-beta (CD18) integrin chains. Anti-ICAM-3 mAbs specific for epitope A, but not those specific for epitope B, were able to trigger T lymphoblast homotypic aggregation. ICAM-3-mediated cell aggregation was dependent on the LFA-1/ICAM-1 pathway as demonstrated by blocking experiments with mAbs specific for the LFA-1 and ICAM-1 molecules. Furthermore, immunofluorescence studies on ICAM-3-induced cell aggregates revealed that both LFA-1 and ICAM-1 were mainly located at intercellular boundaries. ICAM-3 was located at cellular uropods, which in small aggregates appeared to be implicated in cell-cell contacts, whereas in large aggregates it appeared to be excluded from cell-cell contact areas. Experiments of T cell adhesion to a chimeric ICAM-1-Fc molecule revealed that the proaggregatory anti-ICAM-3 HP2/19 mAb was able to increase T lymphoblast attachment to ICAM-1, suggesting that T cell aggregation induced by this mAb could be mediated by increasing the avidity of LFA-1 for ICAM-1. Moreover, the HP2/19 mAb was costimulatory with anti-CD3 mAb for T lymphocyte proliferation, indicating that enhancement of T cell activation could be involved in ICAM-3-mediated adhesive phenomena. Altogether, our results indicate that ICAM-3 has a regulatory role on the LFA-1/ICAM-1 pathway of intercellular adhesion.     

Suppression of human lymphocyte chemotaxis and transendothelial migration by anti-LFA-1 antibody

The role of lymphocyte function-associated antigen 1 (LFA-1) in human T cell chemotaxis was investigated by using mAb specific to the beta-chain (TS1/18) (CD18) and alpha-chain (TS1/22) (CD11a) of LFA-1. T cell chemotaxis in response to IL-2 and to lymphocyte chemotactic factor (LCF) was markedly suppressed by the addition of TS1/18. TS1/22 was a less effective inhibitor than TS1/18 with only LCF stimulated responses showing significant inhibition when compared in seven different T cell preparations. Neither TS1/18 nor TS1/22 antibody inhibited random T cell migration. Control mAb to CD4 T cells failed to inhibit T cell random migration or chemotaxis. Additional studies to evaluate the adherence and migration of T cells through IL-1-stimulated human umbilical vein endothelial cell (HUVEC) monolayers showed that both TS1/22 and TS1/18 suppressed T cell migration through HUVEC, but failed to inhibit adherence of T cells to these cells. These studies indicate that LFA-1 plays a role in the migration of T cells through HUVEC and in the in vitro chemotactic response of T lymphocytes to IL-2 and LCF.     

Effects of interleukin-8 on suppression of human lymphocyte polarization and migration by anti-LFA-1 antibody.

We investigated the role of lymphocyte function-associated antigen 1 (LFA-1) in human T cell polarization and migration assay by using monoclonal antibody specific to beta chain (CD18) and alpha chain (CD11a). T cell polarization in response to fetal calf serum (FCS) and colchicine was suppressed by the addition of CD18 and CD11a antibodies. Furthermore, T cell migration in response to lymphocyte chemotactic factor (LCF) and casein was markedly depressed by the addition of CD18 and CD11a antibodies. Additional studies to evaluate effects of interleukin 8 (IL-8) on polarization and migration of T cells preincubated with CD18 or CD11a antibody showed that IL-8 restored the capability of migration of T cells, whereas did not restore polarization activity of such cells. These studies indicate that LFA-1 plays a role in the polarization and migration of T cells and that IL-8 may positively interfer with LFA-1-adhesion molecules.     

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.     

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.     

Functional CD2 mutants unable to bind to, or be stimulated by, LFA-3.

To define epitopes on the CD2 (T11, the T cell erythrocyte receptor) molecule that are necessary for interaction with lymphocyte function-associated Ag-3 (LFA-3), we have expressed the human wild-type CD2 cDNA and mutant CD2 cDNA in a murine Ag-specific T cell hybridoma that responds to human HLA-DR Ag. Here we have expressed mutations at amino acid 91 and 92 of CD2 in the T cell hybridoma. The mutated CD2 molecules were functional in that pairs of anti-CD2 mAb that continued to bind were able to stimulate IL-2 production by the hybridomas. However, CD2 mutants with either the 91 or 92 amino acid substitution had lost the ability to bind to or be activated by either SRBC, which bear an LFA-3 homologue, or by murine L cells expressing human LFA-3. Unlike hybridomas expressing the wild-type CD2 molecule, there was no enhanced response to Ag stimulation. Taken together, these data suggest that the mutated CD2 molecules were no longer able to bind to, or to utilize, LFA-3 for activation. We have previously demonstrated that a mutation at amino acid 51 of CD2 results in loss of binding to LFA-3. Whether these two regions of CD2, discrete and separable by amino acid sequence, form one or more binding sites for LFA-3 remains to be determined.     

Evidence for an accessory role of LFA-1 in lymphocyte-high endothelium interaction during homing

In a variety of lymphocyte interactions, lymphocyte function-associated antigen-1 (LFA-1) plays an important role as an accessory mechanism mediating cell adhesion. We tested the possibility that LFA-1 could also be involved in the specific binding of lymphocytes to high endothelial venules (HEV) during homing. Antibodies against LFA-1 but not against various other cell surface molecules (except the putative gp90 homing receptor defined by the MEL-14 antibody) were found to inhibit in vitro adherence of lymphocytes to HEV in frozen sections of lymph nodes. Binding of T cell lines to HEV was also inhibited by anti-LFA-1 antibody. Using sublines selected for differential expression of the MEL-14 antigen, MEL-14 high cells (which bind well to HEV) were less susceptible to inhibition by anti-LFA-1 than poor binders with low levels of the homing receptor, supporting the model of LFA-1 being an accessory mechanism strengthening weak interactions between cells. Parallel results were found in vivo where anti-LFA-1 antibodies reduced the migration of normal lymphocytes into lymph nodes and Peyer's patches by 40 to 60%. Localization in the lung, especially of activated lymphocytes, was also impaired, although to a lesser extent. These findings suggest that LFA-1 plays an accessory role in cellular interactions relevant for lymphocyte migration.     

Endothelial cell lymphocyte function-associated antigen-3 and an unidentified ligand act in concert to provide costimulation to human peripheral blood CD4+ T cells.

Our previous studies have demonstrated that cultured human endothelial cells (EC) provide costimulation to PHA-activated CD4+ T cells, measured as augmentation of IL-2 synthesis, through a cell contact-department pathway. Here we show that fixed and living EC provide comparable degrees of costimulation to CD4+ T cell populations, indicating that EC costimulation does not depend upon active metabolism. EC achieve these effects in part by utilizing lymphocyte function-associated antigen-3 (LFA-3) to interact with T cell CD2 as shown by observations that EC augmentation of IL-2 is partially (50-70%) blocked by eight of eight mAb tested which recognize LFA-3; that purified phosphatidylinositol-linked LFA-3 (PI-LFA-3) can also provide costimulation to CD4+ T cells; and that there is a delay of the EC effect on CD4+ T cells which express low levels of CD2 compared to those which express high levels of CD2. However, three lines of evidence suggest that EC also utilize at least one additional ligand. First, there is incomplete replacement of the EC effect by PI-LFA-3 such that the costimulatory ability of EC combined with PI-LFA-3 is additive at all concentrations of PI-LFA-3 tested. Second, costimulation by PI-LFA-3, but not by EC, is fully inhibited by anti-CD2 or anti-LFA-3 mAb. Finally, costimulation by PI-LFA-3, but not by EC, is completely suppressed by cyclosporine A. We have not formally identified the second ligand but it does not appear to be intercellular adhesion molecule-1, vascular cell adhesion molecule-1, CD44, or B7/BB1.     

Human resting B lymphocytes can serve as accessory cells for anti-CD2-induced T cell activation.

Although resting B cells are poor accessory cells for signals transmitted through the TCR/CD3 complex, we report that these B cells can support T cell proliferation when T cell activating signals are delivered through CD2. This was first suggested when leucine methyl ester treatment of PBMC abolished proliferation induced by anti-CD3, but not by the accessory cell-dependent anti-CD2 mAb combination, GT2 and OKT11. Then we demonstrated that unstimulated, resting B cells could support the proliferation of both CD4+ and CD8+ T cells. Aggregated IgG inhibited proliferation, suggesting that anti-CD2 mAb bound to T cells were cross-linked by attachment to B cell FcR. Two lines of evidence suggested that lymphocyte function-associated Ag-1/intercellular adhesion molecule-1 interaction was crucial for anti-CD2-induced proliferation. First, proliferation was blocked by mAb against these adhesion molecules. Second, intercellular adhesion molecule-1 expression rapidly increased on resting B cells after the addition of anti-CD2, but not anti-CD3. This was of interest because fixed monocytes, but not fixed B cells, were able to support the proliferative response. In contrast to lymphocyte function-associated Ag-1/intercellular adhesion molecule-1, CD28/B7 interaction was not required for anti-CD2-induced proliferation, although ligation of these molecules provided important costimulatory signals for stimulation by anti-CD3. Finally, neutralizing antibodies against IL-1 alpha, IL-1 beta, and IL-6 showed only modest inhibitory effects on T cell proliferation. The addition of IL-1 and/or IL-6 to T cells failed to substitute for accessory cells and were only partially effective with fixed B cells. Further evidence of a linkage between CD2 and CD45 isoforms was obtained. Anti-CD45RA, but not anti-CD45RO, potentiated anti-CD2-induced T cell proliferation. These studies have revealed a novel role for resting B cells as accessory cells and have documented costimulatory signals that are important for this effect. Because Ag-presentation by resting B cells to T cells generally leads to T cell nonresponsiveness, it is possible that this tolerogenic signal may be converted to an activation signal if there is concurrent perturbation of CD2 on T cells.     

CD44 cross-linking induces integrin-mediated adhesion and transendothelial migration in breast cancer cell line by up-regulation of LFA-1 (alpha L beta2) and VLA-4 (alpha4beta1)

CD44, a widely expressed cell surface glycoprotein, plays a major role in cell-cell adhesion, cell-substrate interaction, lymphocyte homing, and tumor metastasis. For tumor metastasis to occur through the blood vessel and lymphatic vessel pathway, the tumor cells must first adhere to endothelial cells. Recent studies have shown that high expression of CD44 in certain types of tumors is associated with the hematogenic spread of cancer cells. However, the functional relevance of CD44 to tumor cell metastasis remains unknown. In this study, we investigated the mechanisms of CD44 cross-linking-induced adhesion and transendothelial migration of tumor cells using MDA-MB-435S breast cancer cell line. Breast cancer cells were found to express high levels of CD44. Using flow cytometric analysis and immunofluorescence staining, we demonstrated that cross-linking of CD44 resulted in a marked induction of the expression of lymphocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4) by exocytosis. These results were also observed with the Hs578T breast cancer cell line. Furthermore, LFA-1- and VLA-4-mediated adhesion and transendothelial cancer cell migration were also studied. Anti-LFA-1 mAb or anti-VLA-4 mAb alone had no effect on adhesion or transendothelial cancer cell migration, but were able to inhibit both of these functions when added together. This shows that CD44 cross-linking induces LFA-1 and VLA-4 expression in MDA-MB-435S cells and increases integrin-mediated adhesion to endothelial cells, resulting in the transendothelial migration of breast cancer cells. These observations provide direct evidence of a new function for CD44 that is involved in the induction of LFA-1 and VLA-4 expression by exocytosis in MDA-MB-435S cells. Because these induced integrins promote tumor cell migration into the target tissue, it may be possible to suppress this by pharmacological means, and thus potentially cause a reduction in invasive capability and metastasis.     

Role of LFA-1 and VLA-4 in the adhesion of cloned normal and LFA-1 (CD11/CD18)-deficient T cells to cultured endothelial cells. Indication for a new adhesion pathway.

Patients with the leukocyte adhesion deficiency (LAD) syndrome have a genetic defect in the common beta 2-chain (CD18) of the leukocyte integrins. This defect can result in the absence of cell surface expression of all three members of the leukocyte integrins. We investigated the capacity of T cell clones obtained from the blood of an LAD patient and of normal T cell clones to adhere to human umbilical vein endothelial cells (EC). Adhesion of the number of LAD T cells to unstimulated EC was approximately half of that of leukocyte function-associated antigen (LFA)-1+ T cells. Stimulation of EC with human rTNF-alpha resulted in an average 2- and 2.5-fold increase in adhesion of LFA-1+ and LFA-1- cells, respectively. This effect was maximal after 24 h and lasted for 48 to 72 h. The involvement of surface structures known to participate in cell adhesion (integrins, CD44) was tested by blocking studies with mAb directed against these structures. Adhesion of LFA-1+ T cells to unstimulated EC was inhibited (average inhibition of 58%) with mAb to CD11a or CD18. Considerably less inhibition of adhesion occurred with mAb to CD11a or CD18 (average inhibition, 20%) when LFA-1+ T cells were incubated with rTNF-alpha-stimulated EC. The adhesion of LFA-1- T cells to EC stimulated with rTNF-alpha, but not to unstimulated EC, was inhibited (average inhibition, 56%) by incubation with a mAb directed to very late antigen (VLA)-4 (CDw49d). In contrast to LAD T cell clones and the LFA-1+ T cell line Jurkat, mAb to VLA-4 did not inhibit adhesion of normal LFA-1+ T cell clones to EC, whether or not the EC had been stimulated with rTNF-alpha. We conclude that the adhesion molecule pair LFA-1/intercellular adhesion molecule (ICAM)-1 plays a major role in the adhesion of LFA-1+ T cell clones derived from normal individuals to unstimulated EC. Adhesion of LFA-1-T cells to TNF-alpha-stimulated EC is mediated by VLA-4/vascular cell adhesion molecule (VCAM)-1 interactions. Since we were unable to reduce significantly the adhesion of cultured normal LFA-1+ T cells to 24 h with TNF-alpha-stimulated endothelium with antibodies that block LFA-1/ICAM-1 or VLA-4/VCAM-1 interactions, and lectin adhesion molecule-1 and endothelial leukocyte adhesion molecule-1 appeared not to be implicated, other as yet undefined cell surface structures are likely to participate in T cell/EC interactions.     

The lymphocyte function-associated antigen (LFA)-1 and CD2/LFA-3 pathways of antigen-independent human T cell adhesion

Human cytotoxic T lymphocyte clones form conjugates with both antigen-positive and antigen-negative lymphoblastoid cells. Conjugates with antigen-negative targets form as rapidly, and are almost as frequent, as those with antigen-positive targets; both types are strong. Monoclonal antibodies against lymphocyte function-associated antigen (LFA)-1, CD2, and LFA-3 (or their Fab fragments) each consistently inhibit conjugate formation, but only partially; mixes of alpha LFA-1 with either CD2 monoclonal antibodies or alpha LFA-3 cause complete inhibition. Our previous studies have demonstrated two distinct pathways of antigen-independent conjugate (AIC) formation, one involving LFA-1 and the other involving CD2/LFA-3. The present studies showing supra-additive inhibition with mixes of Fab indicate that at least a major fraction of the conjugates involve T cells which utilize both pathways. Preincubation studies (and restricted expression for CD2) demonstrate that in the CD2/LFA-3 pathway, CD2 is critical on the effector and LFA-3 on the target and that in the LFA-1 pathway, LFA-1 is critical on the effector. Analysis of conjugate formation by primary allosensitized T cells confirms the critical findings made with T cell clones. Among a panel of antigen-negative "target" cell lines tested, there is wide variation in the number of AIC formed with cytotoxic T lymphocyte clones; this variation correlates partially with differences in level of expression of LFA-3. Both pathways of adhesion are utilized in AIC formation with all five targets tested, but there was variation between targets in the relative contribution by each pathway. Studies of inhibition of lysis (rather than conjugate formation) support the relevance of the two-pathway model to the lytic process as a whole. These studies demonstrate the general involvement of two pathways of adhesion in human T cell interactions: one involving T cell LFA-1 and the other involving T cell CD2 binding to target cell LFA-3.     

Relative contribution of CD2 and LFA-1 to murine T and natural killer cell functions.

We have examined the functional property of murine CD2 as an intercellular adhesion molecule by using five anti-murine CD2 mAb which were classified into two groups according to their mutual competition in binding to cell surface CD2. Hamster fibroblasts transfected with murine CD2 cDNA exhibited increased conjugate formation with a murine mastocytoma P815 which expresses the putative murine LFA-3 mRNA detected by cross-hybridization with human LFA-3 cDNA under conditions of low stringency. This increase in conjugate formation was abrogated by both groups of anti-CD2 mAb, although some differences in the extent of inhibition were observed at lower concentrations of the mAb. We then examined the involvement of CD2 in several murine T cell responses by using these mAb to abrogate CD2-mediated cellular interactions. Anti-CD2 mAb significantly inhibited mitogenic T cell responses induced by suboptimal doses of Con A and PHA. In the allogenic MLR response and in the Ag response of two KLH/I-Ak-specific Th cell clones, the inhibitory effect of anti-CD2 mAb was also greatest under suboptimal conditions, i.e., with lesser doses of the Ag. These results indicate that the contribution of CD2 as an accessory molecule is variable, depending on the Ag dose used for stimulation, and they suggest that CD2 is involved in the Ag response of murine T cells under the physiologic conditions where only a limited amount of Ag is available. We next examined the contribution of CD2 to MHC-restricted cytotoxicity by CTL and to MHC-unrestricted cytotoxicity by NK and lymphokine-activated killer cells. Only a marginal inhibition by anti-CD2 mAb alone was observed. Anti-lymphocyte function-associated Ag (LFA)-1 mAb alone exhibited greater inhibitory effects than anti-CD2 mAb in all of the cases tested. In most cases, however, substantial levels of cytotoxicity remained, even in the presence of both anti-CD2 and anti-LFA-1 mAb. These results indicate a minor contribution of CD2, as compared with LFA-1, to cytotoxicity by murine CTL, NK cells, and lymphokine-activated killer cells, and they reveal the presence of undefined cellular interaction pathways other than those mediated by CD2 and LFA-1.     

Lymphocyte function-associated antigen-1-mediated T cell adhesion is impaired by low molecular weight phosphotyrosine phosphatase-dependent inhibition of FAK activity

Protein tyrosine phosphorylation is one of the earliest signaling events detected in response to lymphocyte function-associated antigen-1 (LFA-1) engagement during lymphocyte adhesion. In particular, the focal adhesion kinase p125FAK, involved in the modulation and rearrangement of the actin cytoskeleton, seems to be a crucial mediator of LFA-1 signaling. Herein, we investigate the role of a FAK tyrosine phosphatase, namely low molecular weight phosphotyrosine phosphatase (LMW-PTP), in the modulation of LFA-1-mediated T cell adhesion. Overexpression of LMW-PTP in Jurkat cells revealed an impairment of LFA-1-dependent cell-cell adhesion upon T cell receptor (TCR) stimulation. Moreover, in these conditions LMW-PTP causes FAK dephosphorylation, thus preventing the activation of FAK downstream pathways. Our results also demonstrated that, upon antigen stimulation, LMW-PTP-dependent FAK inhibition is associated to a strong reduction of LFA-1 and TCR co-clustering toward a single region of T cell surface, thus causing an impairment of receptor activity by preventing changes in their avidity state. Because co-localization of both LFA-1 and TCR is an essential event during encounters of T cells with antigen-presenting cells and immunological synapse (IS) formation, we suggest an intriguing role of LMW-PTP in IS establishment and stabilization through the negative control of FAK activity and, in turn, of cell surface receptor redistribution.     

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.     

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.     

The role of accessory molecules in T-helper activation induced by antigen, lectin, or CD3 antibodies

We examined the role of L3T4 and LFA-1 molecules in T-helper-cell activation, under conditions where the physical stability of T helper-accessory cell interactions was not an issue. T-helper hybridomas were activated by accessory cells coated either with concanavalin A (Con A) or with CD3 antibodies. Activation of the T helper cells was measured by microtubule-organizing center (MTOC) reorientation as an early activation event, and by interleukin-2 (IL-2) production as an indication of a fully matured response. Both parameters were strongly blocked by L3T4 and LFA-1 antibodies in the case of Con A activation. In the case of stimulation with accessory cell-bound CD3 antibody, activation was blocked by LFA-1 but not L3T4 antibody. These results support the notion that L3T4 and LFA-1 molecules play more than a simple adhesion role in T-cell activation. The differential effect of L3T4 antibody in the case of Con A activation vs CD3 activation is consistent with the possibility that L3T4 and the alpha/beta portions of the T-cell receptor must interact during antigen- and lectin-stimulated T-cell activation.     

The accessory function of murine intercellular adhesion molecule-1 in T lymphocyte activation. Contributions of adhesion and co-activation.

These studies demonstrate that the murine intercellular adhesion molecule-1 (ICAM-1) performs at least two roles in enhancing T cell activation. These two roles are evident in both of our experimental systems: with ICAM-1 expressed on the surface of transfected fibroblast cells, and with purified ICAM-1 immobilized on plastic. First, as has been documented by many investigators, ICAM-1 mediates adhesion between ICAM-1- and lymphocyte function-associated Ag-1 (LFA-1)-bearing cells. This adhesive interaction occurs even in the absence of T cell stimulation, although it is increased by addition of phorbol ester and calcium ionophore. Although ICAM-1 expression does markedly increase intercellular adhesion, the increase is significantly less than the improvement ICAM-1 expression makes in the Ag-presenting ability of MHC class II-transfected fibroblast cells. We have investigated whether this difference is due to LFA-1-mediated signaling, and we present data that demonstrates that although ICAM-1 does not deliver costimulatory signals required for T cell activation, the interaction of LFA-1 with ICAM-1 does synergize with TCR-transduced signals. This synergy is observed for ICAM-1 on live and on chemically fixed accessory cells, and for purified ICAM-1 molecules, but in all cases occurs only when the ICAM-1 and the TCR ligands are on the same surface. Finally, when the ICAM-1 is present on the surface of accessory cells, it enhances T cell activation by changing the Ag dose-dependence of the T cell, but when ICAM-1 and CD3 mAb are co-immobilized, ICAM-1 increases the peak response of the T cell without affecting the dose dependence of the response.