The LFA-1 ligand ICAM-1 provides an important costimulatory signal for T cell receptor-mediated activation of resting T cells

Functional studies demonstrate that T cell activation often requires not only occupancy of the TCR but costimulatory interactions of other molecules, which remain largely undefined. We have tested the hypothesis that LFA-1 interaction with its ligand intercellular adhesion molecule 1 (CD54) (ICAM-1) is such a costimulatory interaction in a model system using biochemically purified ICAM-1 and TCR cross-linking by anti-CD3 mAb OKT3 immobilized on plastic. Resting T cells do not respond to OKT3 mAb immobilized on plastic. However ICAM-1 deposited on plastic together with the nonmitogenic immobilized OKT3 results in a potent activating stimulus. This costimulation cannot be readily accounted for by ICAM-1-mediated adhesion but is consistent with a role in signaling, which is observed in ICAM-1-mediated augmentation of activation induced by PMA/ionomycin. The ability of ICAM-1 to costimulate with immobilized CD3 contrasts with minimal costimulatory activity of cytokines IL-1 beta, IL-2, and IL-6. The proliferative response to co-immobilized OKT3 and ICAM-1 is dependent on the IL-2R, which is induced only in the presence of both OKT3 and ICAM-1. The present data demonstrate that LFA-1/ICAM-1 interaction is a potent costimulus for TCR-mediated activation; this observation, interpreted in light of previous reports, suggests that LFA-1/ICAM-1 is of major physiologic importance as a costimulatory signal.     

LFA-1-mediated costimulation of CD8+ T cell proliferation requires phosphatidylinositol 3-kinase activity

LFA-1 binding to ICAM-I provides a costimulatory signal for CD8(+) T cell activation that results in increased IL-2 mRNA levels and protein production to support proliferation. CD28 binding to its B7 ligands has the same effect, and the two costimulatory receptors activate some of the same intracellular signaling events, including up-regulation of phosphatidylinositol (PI) 3-kinase activity. However, costimulation by LFA-1 depends upon the activity of this enzyme, whereas costimulation by CD28 does not, as evidenced by differential effects of specific inhibitors of PI 3-kinase. When cells are costimulated with ICAM-1 in the presence of the inhibitors wortmannin or LY294002, proliferation is blocked, but increases in IL-2 mRNA levels and protein production are not. Costimulation also results in increased surface expression of CD25, which is essential for formation of an active IL-2R. This is blocked by the PI 3-kinase inhibitors when costimulation is via LFA-1 but not when it is via CD28. Finally, IL-2-driven proliferation is not blocked by the inhibitors once CD25 surface expression has increased. Thus, the PI 3-kinase-dependent step in CD8 T cell costimulation by LFA-1 is up-regulation of IL-2R expression. In contrast, CD28 engagement also increases IL-2R surface expression, but the up-regulation does not depend upon PI 3-kinase activity.     

Signaling pathways activated by leukocyte function-associated Ag-1-dependent costimulation

LFA-1 binding to ICAM-1 can enhance TCR-dependent proliferation of T cells, but it has been difficult to distinguish contributions from increased adhesion, and thus TCR occupancy, versus costimulatory signaling. Whether LFA-1 ligation results in generation of a unique costimulatory signal(s) distinct from those activated by the TCR has been unclear. Using purified ligands, it is shown that ICAM-1 and B7. 1 provide comparable costimulation for proliferation of CD8+ T cells, and that both ligands up-regulate the activities of phosphatidylinositol 3-kinase, sphingomyelinase, and c-Jun NH2-terminal kinase (JNK). These pathways are distinct from those activated by the TCR, and have previously been implicated in up-regulating IL-2 production in response to CD28-B7 interaction. Thus, under conditions in which ICAM-1 provides costimulation of proliferation, LFA-1 ligation activates some of the same signaling pathways as does CD28 ligation. LFA-1 and CD28 do not act identically, however, as indicated by differential sensitivity to inhibitors of phosphatidylinositol 3-kinase; LFA-1-dependent costimulation of proliferation is inhibited, while CD28-dependent costimulation is not. Given the broad distribution of class I and ICAMs on many cell types, the ability of LFA-1 to provide costimulatory signals has implications for where and how CD8+CTL may become activated in response to an antigenic challenge.     

Differential costimulatory effects of adhesion molecules B7, ICAM-1, LFA-3, and VCAM-1 on resting and antigen-primed CD4+ T lymphocytes.

Optimal proliferation of T cells although initiated via ligation of the CD3/TCR complex requires additional stimulation resulting from adhesive interactions between costimulatory receptors (R) on T cells and their counter-R on APC. At least four distinct adhesion molecules (counter-R) present on APC, B7, ICAM-1 (CD54), LFA-3 (CD58), and VCAM-1 have been individually shown to costimulate T cell activation. Because some of these molecules may be expressed simultaneously on APC, it has been difficult to examine relative contributions of individual counter-R during the induction of T cell proliferation. We have produced soluble IgC gamma 1 fusion chimeras (receptor globulins or Rg) of B7, ICAM-1, LFA-3, and VCAM-1 and compared their relative abilities to costimulate proliferation of resting or Ag-primed CD4+ T cells. When co-immobilized with mAb directed at TCR alpha beta or CD3 but not CD2 or CD28, each Rg induced proliferation of both resting and Ag-primed CD4+ cells. In contrast, similarly co-immobilized CD7 Rg or ELAM-1 Rg were ineffective. Resting CD4+ T cells produced more IL-2, expressed significantly higher levels of IL-2R alpha, and proliferated more efficiently when costimulated with either ICAM-1 Rg or VCAM-1 Rg than with B7 Rg or LFA-3 Rg. CD4+ CD45RO+ memory T cells proliferated more vigorously in response to the costimulation by each of the four Rg than CD4+ CD45RA+ naive T cells. In contrast with the behavior of resting CD4+ T cells, proliferation of Ag-preactivated CD4+ T cells was most efficient when costimulated by B7 Rg. The costimulatory effect of LFA-3 Rg on Ag-primed CD4+ T cells was weaker than that of B7 Rg but was significantly greater than that of either ICAM-1 Rg or VCAM-1 Rg. These results suggest that resting and Ag-primed CD4+ T cells preferentially respond by proliferation to different costimulatory counter-R. ICAM-1 and VCAM-1 may be involved in the initiation of proliferation of Ag-responsive T cells, and B7 and LFA-3 may facilitate sustained proliferation of Ag-primed T cells. The cumulative costimulation by the above counter-R may facilitate optimal expression of various regulatory and effector functions of T 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.     

The role of CD11a/CD18-CD54 interactions in human T cell-dependent B cell activation.

The role of leukocyte function-associated Ag-1 (LFA-1, CD11a/CD18) and intercellular adhesion molecule 1 (ICAM-1, CD54) interactions in human T cell and B cell collaboration was examined by studying the effect of mAb to these determinants on B cell proliferation and differentiation stimulated by culturing resting B cells with CD4+ T cells activated with immobilized mAb to the CD3 molecular complex. In this model system, mAb to either the alpha (CD11a) or beta (CD18) chain of LFA-1 or ICAM-1 (CD54) inhibited B cell responses significantly. The mAb did not directly inhibit B cell function, inasmuch as T cell-independent activation induced by formalinized Staphylococcus aureus and IL-2 was not suppressed. Moreover, DNA synthesis and IL-2 production by immobilized anti-CD3-stimulated CD4+ T cells were not suppressed by the mAb to LFA-1 or ICAM-1. Although the mAb to LFA-1 inhibited enhancement of IL-2 production by co-culture of immobilized anti-CD3-stimulated CD4+ T cells with B cells, addition of exogenous IL-2 or supernatants of mitogen-activated T cells could not abrogate the inhibitory effects of the mAb to LFA-1 or ICAM-1 on B cell responses. Inhibition was most marked when the mAb were present during the initial 24 h in culture. Immobilized anti-CD3-stimulated LFA-1-negative CD4+ T cell clones from a child with leukocyte adhesion deficiency could induce B cell responses, which were inhibited by mAb to LFA-1 or ICAM-1. These results indicate that the interactions between LFA-1 and ICAM-1 play an important role in mediating the collaboration between activated CD4+ T cells and B cells necessary for the induction of B cell proliferation and differentiation, and for enhancement of IL-2 production by CD4+ T cells. Moreover, the data are consistent with a model of T cell-B cell collaboration in which interactions between LFA-1 on resting B cells and ICAM-1 on activated CD4+ T cells play a critical role in initial T cell-dependent B cell activation.     

The role of intercellular adhesion molecule-1/LFA-1 interactions in the generation of tumor-specific CD8+ T cell responses

The activation of naive CD4+ T cells requires both TCR engagement and a second costimulatory signal mediated by the interaction of CD28 with CD80/CD86 expressed on professional APC. However, the situation for naive CD8+ T cells is less clear. Although evidence indicates that induction of CD8+ T cell responses is also dependent on professional APC, the ability of some tumors, which do not express CD80/CD86, to induce CTL suggests that other pathways of costimulation exist for the activation of CD8+ T cells. We examined the ability of tumor cells expressing different levels of a tumor-specific Ag to directly prime CD8+ T cells. We demonstrate that CD8+ T cells are directly activated by tumor cells in a CD80/CD86-CD28 independent manner. In this system, costimulation requires ICAM-1/LFA-1 interaction. This results in the generation of CTL capable of inhibiting tumor growth in vivo, and maintaining long-term survival.     

The haemopoietic growth factors interleukin 3 and colony stimulating factor-1 stimulate proliferation but do not induce inositol lipid breakdown in murine bone-marrow-derived macrophages.

The haemopoietic growth factors interleukin 3 (IL-3) and colony stimulating factor-1 (CSF-1) stimulate the survival and proliferation of murine normal bone-marrow-derived macrophages. To establish whether these growth factors elicit their effects via the hydrolysis of phosphatidylinositol(4,5)bisphosphate [PtdIns(4,5)P2] to form the second messengers inositol (1,4,5)trisphosphate [Ins(1,4,5)P3] and diacylglycerol, macrophages were labelled with tracer quantities of [3H]inositol. Treatment of these cells with either IL-3 or CSF-1 did not alter the levels of PtdIns(4,5)P2 or Ins(1,4,5)P3. However, addition of the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) which does not stimulate proliferation in macrophages caused a marked and rapid increase in the levels of Ins(1,4,5)P3, inositol bisphosphate and inositol monophosphate, and a decrease in the amount of PtdIns(4,5)P2. FMLP also evoked a rapid increase in intracellular cytosolic Ca2+ levels, as measured with quin 2 the Ca2+-sensitive fluorescent probe, whereas IL-3 and CSF-1 had no such effect. These results suggest that FMLP stimulates the hydrolysis of PtdIns(4,5)P2 to form the second messenger Ins(1,4,5)P3 which acts to increase the levels of cytosolic Ca2+, and that IL-3- and CSF-1-stimulated proliferation in macrophages is not associated with the formation of PtdIns(4,5)P2-derived second messengers.     

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.     

Intercellular adhesion molecule 1 is critical for activation of CD28-deficient T cells

Presentation of Ag to T lymphocytes in the absence of the requisite costimulatory signals leads to an Ag-specific unresponsiveness termed anergy, whereas Ag presentation in conjunction with costimulation leads to clonal expansion. B7/CD28 signaling has been shown to provide this critical costimulatory signal and blockade of this pathway may inhibit in vitro and in vivo immune responses. Although T cells from CD28-deficient mice are lacking in a variety of responses, they nonetheless are capable of various primary and secondary responses without the induction of anergy expected in the absence of costimulation. This suggests that there may be alternative costimulatory pathways that can replace CD28 signaling under certain circumstances. In this paper, we show that ICAM-1becomes a dominant costimulatory molecule for CD28-deficient T cells. ICAM-1 costimulates anti-CD3-mediated T cell proliferation and IL-2 secretion in CD28-deficient murine T cells. Furthermore, splenocytes from ICAM-1-deficient mice could not activate CD28-deficient T cells and splenocytes lacking both ICAM and CD28 fail to proliferate in response to anti-CD3-induced T cell signals. This confirms that not only can ICAM-1 act as a CD28-independent costimulator, but it is the dominant, requisite costimulatory molecule for the activation of T cells in the absence of B7/CD28 costimulation.     

Patterns of costimulation of T cell clones by cross-linking CD3, CD4/CD8, and class I MHC molecules

The ability of mAb to class I MHC molecules, CD3, or CD4/CD8 to stimulate human T cell clones alone or in combination was examined. Cross-linking each of these surface Ag with appropriate mAb and goat anti-mouse Ig (GaMIg) resulted in a unique pattern of increase in intracellular free calcium ([Ca2+]i) and different degrees of functional activation. Cross-linking class I MHC molecules provided the most effective stimulus of IL-2 production and proliferation. Cross-linking more than one surface Ag induced a compound calcium signal with characteristics of each individual response. Cross-linking CD3 + HLA-A,B,C caused a rapid and prolonged increase in [Ca2+]i and synergistically increased IL-2 production and proliferation of all clones. Cross-linking CD3 + CD4/CD8 also generated a compound calcium signal and increased IL-2 production and DNA synthesis. Purposeful inclusion of CD3 was not required for costimulation as cross-linking HLA-A,B,C + CD4/CD8 also increased [Ca2+]i, IL-2 production, and proliferation. Cross-linking three surface Ag, CD3 + HLA-A,B,C + CD4/CD8, resulted in the greatest initial and sustained [Ca2+]i, IL-2 production, and DNA synthesis. Although there was a tendency for the various stimuli to increase both [Ca2+]i and functional responsiveness, neither the magnitude nor duration of the increased [Ca2+]i correlated with the amount of IL-2 produced or the ultimate proliferative response. To determine whether costimulation required that the various surface molecules were cross-linked together, experiments were carried out using isotype specific secondary antibodies. Augmentation of [Ca2+]i and costimulation of functional responses were noted when class I MHC molecules were cross-linked and CD3 was bound, but not cross-linked. Similarly, costimulation through CD3 and CD4/CD8 was observed when CD4/CD8 was cross-linked and the CD3 complex was engaged by an anti-CD3 mAb which was not further cross-linked. In contrast, costimulation by class I MHC molecules and CD4/CD8 was only observed when these molecules were cross-linked together. These data demonstrate that cross-linking class I MHC determinants or CD4/CD8 provides a direct signal to T cell clones that can be enhanced when CD3 is independently engaged. The results also indicate that T cell clones can be stimulated without engaging CD3 by the combination of signals delivered via class I MHC molecules and CD4/CD8, but only when these determinants were cross-linked together. These studies have demonstrated that these cell surface molecules differ in their capacity to deliver activation signals to T cell clones and also exhibit unique patterns of positive cooperativity in signaling potential.     

Intracellular events involved in CD5-induced human T cell activation and proliferation.

In this report we describe a novel pathway of human T cell activation and proliferation involving the CD5 surface Ag. The CD5-specific Cris1 mAb induces by itself monocyte-dependent proliferation of PBMC. Among a panel of CD5-specific mAb (Leu1, OKT1, LO-CD5a, F101-1C5, and F145GF3), only the F145GF3 mAb shared this property with Cris1. The analysis of the biochemical pathway involved in this activation showed the lack of detectable hydrolysis of inositol phosphates or early increments in the intracellular cytosolic calcium concentration, after triggering cells with the mitogenic CD5 mAb. However, stimulation with CD5 induces activation of protein kinase C, as measured by phosphorylation of a specific peptide substrate (peptide GS), which can be inhibited by a pseudosubstrate peptide inhibitor. Stimulation with CD5 mAb induces also tyrosine kinase activity, with a substrate pattern that differs from that induced after triggering lymphocytes through the TCR-CD3 complex. On the other hand the IL-2/IL-2R pathway seems involved in the CD5-mediated proliferation of PBMC because anti-IL-2R-specific mAb inhibits CD5-induced proliferation, and stimulation with mitogenic CD5 mAb induces production of IL-2 and expression of IL-2R alpha and beta chains. Therefore, the triggering of the CD5 Ag can induce IL-2- and monocyte-dependent human T cell proliferation by a biochemical pathway that differs, at least in the first stages, from the one that mediates TCR-CD3 complex-induced T cell activation.     

Lysis of human keratinocytes by allogeneic HLA class I-specific cytotoxic T cells. Keratinocyte ICAM-1 (CD54) and T cell LFA-1 (CD11a/CD18) mediate enhanced lysis of IFN-gamma-treated keratinocytes.

Exposure of human KC to IFN-gamma increases their susceptibility to lysis by CTL. The mechanism of this enhanced lysis was investigated by analyzing interactions of IFN-gamma-treated and nontreated cultured KC with allogeneic class I-specific CTL clones. rIFN-gamma treatment augmented KC lysis in a time- and dose-dependent manner. Increased lysis of IFN-KC was detected after only 2 h of IFN-gamma treatment and was maximal by 12 h. Enhanced lysis of IFN-KC was Ag-specific, inasmuch as nonantigenic IFN-KC were not lysed either directly or as bystanders during the lysis of antigenic KC. Parallel immunofluorescence and cytotoxicity assays of KC treated with IFN-gamma for various intervals revealed a direct correlation between the degree of increased KC lysis and levels of cell surface ICAM-1 (CD54), but not of specific alloantigen or beta 2-microglobulin. Lysis of nontreated KC was blocked by mAb against class I or CD3, but not by mAb against ICAM-1 or LFA-1. In contrast, lysis of IFN-KC was partially inhibited by anti-ICAM-1 or anti-LFA-1 mAb, but resisted inhibition by anti-class I mAb except in the presence of anti-ICAM-1. These results indicate that both ICAM-1/LFA-1 and Ag/CD3-TcR interactions are important for Ag-specific lysis of IFN-KC, whereas lysis of nontreated KC depends on Ag/CD3-TcR but not ICAM-1/LFA-1 interactions. Equivalent inhibition of IFN-KC lysis by mAb against ICAM-1 or LFA-1 suggests that ICAM-1 is the only LFA-1 ligand involved in enhanced IFN-KC lysis. Furthermore, enhanced CTL lysis of KC after short-term IFN-gamma treatment can be explained solely on the basis of ICAM-1 induction, because all of the increase in specific lysis associated with IFN-gamma treatment could be blocked by mAb that block ICAM-1/LFA-1 interactions.     

Defective CD8+ T cell activation and cytolytic function in the absence of LFA-1 cannot be restored by increased TCR signaling.

Signaling through the TCR as well as engagement of costimulatory molecules are required for efficient T cell activation and progression into differentiated effector cells. The beta2 integrin LFA-1 (CD11a/CD18) has been implicated in TCR costimulation as well as in cell-cell adhesion function, but its exact role is still ambiguous. The present study focuses on the requirement for LFA-1 in CD8+ T cell activation and effector function using LFA-1-deficient cells expressing the 2C transgenic TCR as a model system. The lack of LFA-1 expression in 2C T cells resulted in severely diminished proliferative response toward allogeneic BALB/c splenocytes. Increase in TCR signaling alone by pulsing stimulators with high affinity peptides, p2Ca or QL9, had minimal effects in restoring proliferation. Addition of exogenous IL-2, however, enhanced the effect of peptide pulsing on proliferation of LFA-1-deficient 2C T cells. LFA-1-deficient 2C CTLs generated from alloantigen stimulation exhibited a defective cytotoxic activity when tested on a variety of target cells. Cytolysis could be improved, but not fully rectified by peptide pulsing of target cells. Thus, in the 2C TCR model, LFA-1 has a requisite role for optimal CD8+ T cell activation and effector function, which cannot be overcome by increasing peptide/MHC density on either the APCs or target cells, respectively.     

Role of CD11a/CD18-CD54 interactions in suppression of human B cell responsiveness by CD4+ suppressor T cells.

The role of leukocyte function-associated Ag-1 (LFA-1, CD11a/CD18) and intercellular adhesion molecule 1 (ICAM-1, CD54) interactions in the suppression of human B cell function by immobilized anti-CD3-activated CD4+ T cells was examined by studying the effects of mAb to these determinants. The suppressive activity was assessed by the effects of CD4+ T cells without mitomycin C treatment activated by immobilized anti-CD3 for 72 hr on the differentiation into Ig-secreting cells of B cells activated for 72 hr with immobilized anti-CD3-stimulated CD4+ T cells that had been treated with mitomycin C (T4 mito). Suppression was not observed when activated CD4+ T cells and B cells were separated by filter membranes, indicating that the suppression requires the direct interactions between anti-CD3-activated CD4+ T cells and activated B cells. In this model system, mAb to either the alpha (CD11a) or beta (CD18) chain of LFA-1 or ICAM-1 (CD54) reversed the suppression of B cell function by suppressor CD4+ T cells significantly. Reversal of suppression of B cell function was most marked when activated B cells were treated with mAb to ICAM-1 and suppressor CD4+ T cells were treated with mAb to LFA-1, but not vice versa. Studies using fluorescence-activated cell sorter revealed marked increase of expression of ICAM-1 on B cells after 72 hr of activation with immobilized anti-CD3-stimulated T4 mito. These results indicate that the interactions between LFA-1 and ICAM-1 play an important role in mediating the suppressive activity of anti-CD3-activated CD4+ T cells to B cells. Moreover, the data are consistent with a model of T-cell-mediated B cell suppression in which interactions between LFA-1 on suppressor T cells and ICAM-1 on activated B cells play a central role in the suppression of B cell function.     

Gonadotropin releasing hormone stimulates the formation of inositol phosphates in rat anterior pituitary tissue.

The production of inositol phosphates in response to gonadotropin releasing hormone (GnRH) was studied in rat anterior pituitary tissue preincubated with [3H]inositol. Prelabelled paired hemipituitaries from prepubertal female rats were incubated in the presence or absence of GnRH in medium containing 10 mM-Li+ X Li+, which inhibits myo-inositol-1-phosphatase, greatly amplified the stimulation of inositol phosphate production by GnRH (10(-7) M) to 159, 198 and 313% of paired control values for inositol 1-phosphate, inositol bisphosphate and inositol trisphosphate respectively after 20 min. The percentage distribution of [3H]inositol within the phosphoinositides was 91.3, 6.3 and 2.4 for phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate respectively and was unaffected by GnRH. The stimulation of inositol trisphosphate production by GnRH was evident after 5 min incubation, was dose-dependent with a half-maximal effect around 11 nM, and was not inhibited by removal of extracellular Ca2+. Elevation of cytosolic Ca2+ by membrane depolarization with 50 mM-K+ had no significant effect on inositol phosphate production. These findings are consistent with the hypothesis that GnRH action in the anterior pituitary involves the hydrolysis of phosphatidylinositol 4,5-bisphosphate. The resulting elevation of inositol trisphosphate may in turn lead to intracellular Ca2+ mobilization and subsequent stimulation of gonadotropin secretion.     

Intercellular adhesion molecule-2, a second counter-receptor for CD11a/CD18 (leukocyte function-associated antigen-1), provides a costimulatory signal for T-cell receptor-initiated activation of human T cells.

Activation of T cells often requires both activation signals delivered by ligation of the TCR and those resulting from costimulatory interactions between certain T cell surface accessory molecules and their respective counter-receptors on APC. CD11a/CD18 complex on T cells modulate the activation of T cells by interacting with its counter-receptors intracellular adhesion molecule (ICAM-1) (CD54) and/or ICAM-2 on the surface of APC. The costimulatory ability of ICAM-1 has been demonstrated. Using a soluble ICAM-2 Ig fusion protein (receptor globulin, Rg) we demonstrate the costimulatory effect of ICAM-2 during the activation of CD4+ T cells. When coimmobilized with anti-TCR-1 mAb ICAM-2 Rg induced vigorous proliferative response of CD4+ T cells. This costimulatory effect of ICAM-2 was dependent on its coimmobilization with mAb directed at the CD3/TCR complex but not those directed at CD2 or CD28. Both resting as well as Ag-primed CD4+ T cells responded to the costimulatory effects of ICAM-2. The addition of mAb directed at the CD11a or CD18 molecules almost completely inhibited the responses to ICAM-2 Rg. These results are consistent with the role of CD11a/CD18 complex as a receptor for ICAM-2 mediating its costimulatory effects. Stimulation of T cells with coimmobilized anti-TCR-1 and ICAM-2 resulted in the induction of IL-2R (CD25), and anti-Tac (CD25) mAb inhibited this response suggesting the contribution of endogenously synthesized IL-2 during this stimulation. These results demonstrate that like its homologue ICAM-1, ICAM-2 also exerts a strong costimulatory effect during the TCR-initiated activation of T cells. The costimulatory effects generated by the CD11a/CD18:ICAM-2 interaction may be critical during the initiation of T cell activation by ICAM-1low APC.     

Signaling from LFA-1 contributes signal transduction through CD2 alternative pathway in T cell activation.

LFA-1, a member of the integrin family of molecules, is involved in mediating cellular adhesion in all phases of the immune response, playing a role in the interaction of helper T cells as well as in killing of target cells by both cytotoxic T cells and natural killer cells. We have developed a monoclonal antibody, anti-HVS6B6, which recognizes a functionally unique epitope of the LFA-1 molecule. Although this mAb itself was not mitogenic against T cells, it induced a strong proliferative response when added to T cells with submitogenic concentrations of anti-CD2 (anti-T11(2) and anti-T11(3)) mAbs. In contrast, other anti-LFA-1 mAbs (CD11a and CD18) suppressed this anti-CD2 mAb-induced T cell proliferation. Kinetic studies showed that anti-HVS6B6 acts on an early event in CD2-mediated T cell activation. Although T11(3)-epitope expression induced by anti-T11(2) mAb was not affected by treatment of cells with anti-HVS6B6, both Ca2+ influx and phosphatidylinositol turnover induced by anti-CD2 mAbs were markedly enhanced by the pretreatment of T cells with anti-HVS6B6 mAb. These results indicate that the LFA-1 mediating signal contributes to a very early phase of signal transduction during CD2-mediated T cell activation.     

Beta2-integrin, LFA-1, and TCR/CD3 synergistically induce tyrosine phosphorylation of focal adhesion kinase (pp125(FAK)) in PHA-activated T cells

Complete T cell activation requires not only a first signal via TCR/CD3 engagement but also a costimulatory signal through accessory receptors such as CD2, CD28, or integrins. Focal adhesion kinase, pp125(FAK) (FAK), was previously shown to be localized in focal adhesions in fibroblasts and to be involved in integrin-mediated cellular activation. Although signaling through beta1- or beta3-integrins induces tyrosine phosphorylation of FAK, there has been no evidence that activation of T cells through the beta2-integrin, LFA-1, involves FAK. We report here that crosslinking of LFA-1 induces tyrosine phosphorylation of FAK in PHA-activated T cells. Moreover, cocrosslinking with anti-LFA-1 mAb and suboptimal concentration of anti-CD3 mAb markedly increases tyrosine phosphorylation of FAK in an antibody-concentration-dependent and time-kinetics-dependent manner compared with stimulation through CD3 alone, which correlates well with enhanced proliferation of PHA-activated T cells. Furthermore, LFA-1beta costimulation with CD3 induces tyrosine phosphorylation of Syk associated with FAK. These results indicate, for the first time, that signals mediated by LFA-1 can regulate FAK, suggesting that LFA-1-mediated T cell costimulation may be involved in T cell activation at least partially through FAK.