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.     

Acute Inhibition of Selected Membrane-Proximal Mouse T Cell Receptor Signaling by Mitochondrial Antagonists

T cells absorb nanometric membrane vesicles, prepared from plasma membrane of antigen presenting cells, via dual receptor/ligand interactions of T cell receptor (TCR) with cognate peptide/major histocompatibility complex (MHC) plus lymphocyte function-associated antigen 1 (LFA-1) with intercellular adhesion molecule 1. TCR-mediated signaling for LFA-1 activation is also required for the vesicle absorption. Exploiting those findings, we had established a high throughput screening (HTS) platform and screened a library for isolation of small molecules inhibiting the vesicle absorption. Follow-up studies confirmed that treatments (1 hour) with various mitochondrial antagonists, including a class of anti-diabetic drugs (i.e., Metformin and Phenformin), resulted in ubiquitous inhibition of the vesicle absorption without compromising viability of T cells. Further studies revealed that the mitochondrial drug treatments caused impairment of specific membrane-proximal TCR signaling event(s). Thus, activation of Akt and PLC-γ1 and entry of extracellular Ca²⁺ following TCR stimulation were attenuated while polymerization of monomeric actins upon TCR triggering progressed normally after the treatments. Dynamic F-actin rearrangement concurring with the vesicle absorption was also found to be impaired by the drug treatments, implying that the inhibition by the drug treatments of downstream signaling events (and the vesicle absorption) could result from lack of directional relocation of signaling and cell surface molecules. We also assessed the potential application of mitochondrial antagonists as immune modulators by probing effects of the long-term drug treatments (24 hours) on viability of resting primary T cells and cell cycle progression of antigen-stimulated T cells. This study unveils a novel regulatory mechanism for T cell immunity in response to environmental factors having effects on mitochondrial function.     

Attenuated T Cell Responses to a High-Potency Ligand In Vivo

αβ T cell receptor (TCR) recognition of foreign peptides bound to major histocompatibility complex (pMHC) molecules on the surface of antigen presenting cells is a key event in the initiation of adaptive cellular immunity. In vitro, high-affinity binding and/or long-lived interactions between TCRs and pMHC correlate with high-potency T cell activation. However, less is known about the influence of TCR/pMHC interaction parameters on T cell responses in vivo. We studied the influence of TCR/pMHC binding characteristics on in vivo T cell immunity by tracking CD4⁺ T cell activation, effector, and memory responses to immunization with peptides exhibiting a range of TCR/pMHC half-lives and in vitro T cell activation potencies. Contrary to predictions from in vitro studies, we found that optimal in vivo T cell responses occur to ligands with intermediate TCR/pMHC half-lives. The diminished in vivo responses we observed to the ligand exhibiting the longest TCR/pMHC half-life were associated with attenuation of intracellular signaling, expansion, and function over a broad range of time points. Our results reveal a level of control over T cell activation in vivo not recapitulated in in vitro assays and highlight the importance of considering in vivo efficacy of TCR ligands as part of vaccine design.     

The dependence for leukocyte function-associated antigen-1/ICAM-1 interactions in T cell activation cannot be overcome by expression of high density TCR ligand

The leukocyte-specific integrin, LFA-1, can enhance T cell activation. However, it is unclear whether the binding of LFA-1 to its ligand, ICAM-1, functions through intercellular adhesion alone, resulting in an augmentation of the TCR signal, or involves an additional LFA-1-mediated cellular signal transduction pathway. We have previously shown that naive CD4+ lymph node T cells, isolated from DO11.10 TCR transgenic mice, are activated by increasing doses of exogenous OVA peptide presented by transfectants expressing both class II and ICAM-1, but not by cells expressing class II alone. To determine whether LFA-1/ICAM-1 interactions were simply enhancing the presentation of low concentrations of specific MHC/peptide complexes generated from exogenously added peptide, we transfected cells with class II that is covalently coupled to peptide, alone or in combination with ICAM-1. These cells express 100-fold more specific class II/peptide complexes than can be loaded onto class II-positive cells at maximum concentrations of exogenous peptide. Despite this high density of TCR ligand, activation of naive CD4+ T cells still requires the coexpression of ICAM-1. LFA-1/ICAM-1 interactions are not required for effective conjugate formation and TCR engagement because presentation of class II/peptide complexes in the absence of ICAM-1 does induce up-regulation of CD25 and CD69. Thus, high numbers of engaged TCR cannot compensate for the lack of LFA-1/ICAM-1 interactions in the activation of naive CD4+ T cells.     

F-actin flow drives affinity maturation and spatial organization of LFA-1 at the immunological synapse

Integrin-dependent interactions between T cells and antigen-presenting cells are vital for proper T cell activation, effector function, and memory. Regulation of integrin function occurs via conformational change, which modulates ligand affinity, and receptor clustering, which modulates valency. Here, we show that conformational intermediates of leukocyte functional antigen 1 (LFA-1) form a concentric array at the immunological synapse. Using an inhibitor cocktail to arrest F-actin dynamics, we show that organization of this array depends on F-actin flow and ligand mobility. Furthermore, F-actin flow is critical for maintaining the high affinity conformation of LFA-1, for increasing valency by recruiting LFA-1 to the immunological synapse, and ultimately for promoting intracellular cell adhesion molecule 1 (ICAM-1) binding. Finally, we show that F-actin forces are opposed by immobilized ICAM-1, which triggers LFA-1 activation through a combination of induced fit and tension-based mechanisms. Our data provide direct support for a model in which the T cell actin network generates mechanical forces that regulate LFA-1 activity at the immunological synapse.     

Interplay between TCR affinity and necessity of coreceptor ligation: high-affinity peptide-MHC/TCR interaction overcomes lack of CD8 engagement

CD8 engagement is believed to be a critical event in the activation of naive T cells. In this communication, we address the effects of peptide-MHC (pMHC)/TCR affinity on the necessity of CD8 engagement in T cell activation of primary naive cells. Using two peptides with different measured avidities for the same pMHC-TCR complex, we compared biochemical affinity of pMHC/TCR and the cell surface binding avidity of pMHC/TCR with and without CD8 engagement. We compared early signaling events and later functional activity of naive T cells in the same manner. Although early signaling events are altered, we find that high-affinity pMHC/TCR interactions can overcome the need for CD8 engagement for proliferation and CTL function. An integrated signal over time allows T cell activation with a high-affinity ligand in the absence of CD8 engagement.     

TCR Triggering by pMHC Ligands Tethered on Surfaces via Poly(Ethylene Glycol) Depends on Polymer Length

Antigen recognition by T cells relies on the interaction between T cell receptor (TCR) and peptide-major histocompatibility complex (pMHC) at the interface between the T cell and the antigen presenting cell (APC). The pMHC-TCR interaction is two-dimensional (2D), in that both the ligand and receptor are membrane-anchored and their movement is limited to 2D diffusion. The 2D nature of the interaction is critical for the ability of pMHC ligands to trigger TCR. The exact properties of the 2D pMHC-TCR interaction that enable TCR triggering, however, are not fully understood. Here, we altered the 2D pMHC-TCR interaction by tethering pMHC ligands to a rigid plastic surface with flexible poly(ethylene glycol) (PEG) polymers of different lengths, thereby gradually increasing the ligands’ range of motion in the third dimension. We found that pMHC ligands tethered by PEG linkers with long contour length were capable of activating T cells. Shorter PEG linkers, however, triggered TCR more efficiently. Molecular dynamics simulation suggested that shorter PEGs exhibit faster TCR binding on-rates and off-rates. Our findings indicate that TCR signaling can be triggered by surface-tethered pMHC ligands within a defined 3D range of motion, and that fast binding rates lead to higher TCR triggering efficiency. These observations are consistent with a model of TCR triggering that incorporates the dynamic interaction between T cell and antigen-presenting cell.     

SKAP1 protein PH domain determines RapL membrane localization and Rap1 protein complex formation for T cell receptor (TCR) activation of LFA-1

Although essential for T cell function, the identity of the T cell receptor (TCR) "inside-out" pathway for the activation of lymphocyte function-associated antigen 1 (LFA-1) is unclear. SKAP1 (SKAP-55) is the upstream regulator needed for TCR-induced RapL-Rap1 complex formation and LFA-1 activation. In this paper, we show that SKAP1 is needed for RapL binding to membranes in a manner dependent on the PH domain of SKAP1 and the PI3K pathway. A SKAP1 PH domain-inactivating mutation (i.e. R131M) markedly impaired RapL translocation to membranes for Rap1 and LFA-1 binding and the up-regulation of LFA-1-intercellular adhesion molecule 1 (ICAM-1) binding. Further, N-terminal myr-tagged SKAP1 for membrane binding facilitated constitutive RapL membrane and Rap1 binding and effectively substituted for PI3K and TCR ligation in the activation of LFA-1 in T cells.     

Growth inhibition of a colonic adenocarcinoma cell line (HT29) by T cells specific for mutant p21 ras

Interleukin-2 (IL-2)-activated killer cells, also referred to as lymphokine-activated killer (LAK) cells, are stimulated by tumor cells to express cytotoxic activity and to also secrete cytokines such as interferon (IFN) and tumor necrosis factor (TNF ). We previously reported that secretion of cytokines by IL-2-activated T cells (LAK-T cells) is dependent on the initial cross-linking of the T cell receptor (TCR)-CD3-molecular complex, but the cross-linking of accessory molecules, such as LFA-1, CD2, CD44 and CD45, on LAK-T cells can enhance this cytokine production. We have developed an approach involving interspecific gene transfer to define further the contributions of LFA-1 and CD2 to the activation of LAK-T cells. The genes for huICAM-1 (a ligand for LFA-1) and huLFA-3 (a ligand for CD2) were transfected singly and in combination into a null mouse melanoma background, and clonal populations of cells that stably express ICAM-1 and/or LFA-3 were derived. Expression of the introduced ICAM-1 and/or LFA-3 by transfected cells enhanced their ability to bind LAK-T cells; the LFA-1/ICAM-1-mediated binding was not further enhanced by activation with phorbol 12-myristate 13-acetate. ICAM-1- and/or LFA-3-transfected cells, in the presence of immobilized anti-CD3, exhibited a greater ability to stimulate IFN secretion by LAK-T cells compared to the untransfected parental lines. This experimental system, which allows ICAM-1/LFA-1 and CD2/LFA-3 interactions to occur on the LAK-T cell at a site distal from the anti-CD3 signal, extends our understanding of LAK-T cell activation by establishing that both LFA-1/ICAM-1 and CD2/LFA-3 can mediate co-stimulation via adhesion and signaling events.     

Quantifying the strength of ligand antagonism in TCR triggering

Triggering of the T cell receptor (TCR) may be antagonized by ligands that are slight variants of the immunogenic peptide. This paper proposes a mathematical model to quantify the strength of the antagonistic effect. The model is based on the kinetics of association and dissociation of TCR and peptide/major histocompatibility (pMHC) molecules, and incorporates TCR triggering according to a kinetic proofreading mechanism. Model analysis indicates that while the average lifetime of the TCR/pMHC complex is the basic determinant of the contribution to TCR triggering made by the ligand, the affinity of the ligand and its MHC presentation level are also important. However, these contributions depend on the kinetic limitation regime. There is a continuum of limitation regimes, at the extremes of which are found TCR limitation and MHC limitation. Both ligand affinity and TCR and pMHC densities determine whether TCR triggering is TCR limited or MHC limited. The changing importance of affinity and antigen presentation level under various kinetic limitation regimes may explain the respective roles of antagonistic and agonistic self peptides in thymic selection. Moreover, TCR down-regulation under TCR-limited conditions may allow the T cell to differentiate between the average lifetime of the TCR/pMHC complex and the presentation level of the ligand. A method for experimental differentiation between passive and active antagonistic effects is proposed which exploits the differences between TCR and MHC limitation.     

The CD8 T cell coreceptor exhibits disproportionate biological activity at extremely low binding affinities

T lymphocytes recognize peptides presented in the context of major histocompatibility complex (MHC) molecules on the surface of antigen presenting cells. Recognition specificity is determined by the alphabeta T cell receptor (TCR). The T lymphocyte surface glycoproteins CD8 and CD4 enhance T cell antigen recognition by binding to MHC class I and class II molecules, respectively. Biophysical measurements have determined that equilibrium binding of the TCR with natural agonist peptide-MHC (pMHC) complexes occurs with KD values of 1-50 microm. The pMHCI/CD8 and pMHCII/CD4 interactions are significantly weaker than this (KD >100 microm), and the relative roles of TCR/pMHC and pMHC/coreceptor affinity in T cell activation remain controversial. Here, we engineer mutations in the MHCI heavy chain and beta2-microglobulin that further reduce or abolish the pMHCI/CD8 interaction to probe the significance of pMHC/coreceptor affinity in T cell activation. We demonstrate that the pMHCI/CD8 coreceptor interaction retains the vast majority of its biological activity at affinities that are reduced by over 15-fold (KD > 2 mm). In contrast to previous reports, we observe that the weak interaction between HLA A68 and CD8, which falls within this spectrum of reduced affinities, retains substantial functional activity. These findings are discussed in the context of current concepts of coreceptor dependence and the mechanism by which TCR coreceptors facilitate T cell activation.     

Stimulator cell-dependent requirement for CD2- and LFA-1-mediated adhesions in T lymphocyte activation by superantigenic toxins.

The staphylococcal enterotoxins and related microbial T cell mitogens stimulate T cells by cross-linking variable parts of the T cell receptor (TCR) with MHC class II molecules on accessory or target cells. We have used cloned human T cells and defined tumor cells as accessory cells (AC) to study the requirements for T cell activation by these toxins. On AC expressing high levels of CD54 (intercellular adhesion molecule-1, ICAM-1) and CD58 (lymphocyte function-associated antigen-3, LFA-3), mAb to CD2 were relatively ineffective in inhibiting the response to the toxins and antibodies to the lymphocyte function-associated antigen-1 (LFA-1) did not inhibit at all. If added together, however, these mAb inhibited the response completely. Similar results were obtained using antibodies to the target structures of CD2 and LFA-1. In contrast, on cells expressing low levels of LFA-3, mAb to LFA-1 but not to CD2 were strongly inhibitory. The same pattern of inhibition was found when these same cells were used as presenters of specific antigen to the T cells. These data show that adhesions via CD2 or LFA-1 are alternatively required for the stimulation of the T cells by superantigenic toxins and demonstrate another similarity between T cell stimulation by superantigens and by specific antigen recognition.     

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.     

Cross-reactivity in T-cell antigen recognition

The molecular interactions between the T-cell receptor (TCR) and peptide-MHC (pMHC) have been elucidated in recent years. Nevertheless, the fact that binding of only slightly different ligands by a TCR, or ligation of the same pMHC at different developmental stages of the T cell, can have opposing consequences, continues to pose intellectual challenges. Kinetic proofreading models, which have at their core the dissociation rates of pMHC from the TCR, are best suited to account for these observations. However, T cells can be triggered by peptides with often minimal homology to the primary immunogenic peptide. This cross-reactivity of the TCR is manifest at several levels, from positive selection of immature thymocytes to homeostasis and antigen-cross- reactive immune responses of mature peripheral T cells. The implications of the high cross-reactivity of T-cell antigen recognition for self-tolerance and T-cell memory are discussed.     

Different T cell receptor affinity thresholds and CD8 coreceptor dependence govern cytotoxic T lymphocyte activation and tetramer binding properties

T cells have evolved a unique system of ligand recognition involving an antigen T cell receptor (TCR) and a coreceptor that integrate stimuli provided by the engagement of peptide-major histocompatibility complex (pMHC) antigens. Here, we use altered pMHC class I (pMHCI) molecules with impaired CD8 binding (CD8-null) to quantify the contribution of coreceptor extracellular binding to (i) the engagement of soluble tetrameric pMHCI molecules, (ii) the kinetics of TCR/pMHCI interactions on live cytotoxic T lymphocytes (CTLs), and (iii) the activation of CTLs by cell-surface antigenic determinants. Our data indicate that the CD8 coreceptor substantially enhances binding efficiency at suboptimal TCR/pMHCI affinities through effects on both association and dissociation rates. Interestingly, coreceptor requirements for efficient tetramer labeling of CTLs or for CTL activation by determinants displayed on the cell surface operated in different TCR/pMHCI affinity ranges. Wild-type and CD8-null pMHCI tetramers required monomeric affinities for cognate TCRs of KD < approximately 80 microM and approximately 35 microM, respectively, to label human CTLs at 37 degrees C. In contrast, activation by cellular pMHCI molecules was strictly dependent on CD8 binding only for TCR/pMHCI interactions with KD values >200 microM. Altogether, our data provide information on the binding interplay between CD8 and the TCR and support a model of CTL activation in which the extent of coreceptor dependence is inversely correlated to TCR/pMHCI affinity. In addition, the results reported here define the range of TCR/pMHCI affinities required for the detection of antigen-specific CTLs by flow cytometry.     

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.     

The T cell receptor triggering apparatus is composed of monovalent or monomeric proteins

Understanding the component stoichiometry of the T cell antigen receptor (TCR) triggering apparatus is essential for building realistic models of signal initiation. Recent studies suggesting that the TCR and other signaling-associated proteins are preclustered on resting T cells relied on measurements of the behavior of membrane proteins at interfaces with functionalized glass surfaces. Using fluorescence recovery after photobleaching, we show that, compared with the apical surface, the mobility of TCRs is significantly reduced at Jurkat T cell/glass interfaces, in a signaling-sensitive manner. Using two biophysical approaches that mitigate these effects, bioluminescence resonance energy transfer and two-color coincidence detection microscopy, we show that, within the uncertainty of the methods, the membrane components of the TCR triggering apparatus, i.e. the TCR complex, MHC molecules, CD4/Lck and CD45, are exclusively monovalent or monomeric in human T cell lines, implying that TCR triggering depends only on the kinetics of TCR/pMHC interactions. These analyses also showed that constraining proteins to two dimensions at the cell surface greatly enhances random interactions versus those between the membrane and the cytoplasm. Simulations of TCR-pMHC complex formation based on these findings suggest how unclustered TCR triggering-associated proteins might nevertheless be capable of generating complex signaling outputs via the differential recruitment of cytosolic effectors to the cell membrane.     

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.     

Occupancy of lymphocyte LFA-1 by surface-immobilized ICAM-1 is critical for TCR- but not for chemokine-triggered LFA-1 conversion to an open headpiece high-affinity state

Lymphocyte arrest and spreading on ICAM-1-expressing APCs require activation of lymphocyte LFA-1 by TCR signals, but the conformational switches of this integrin during these critical processes are still elusive. Using Ab probes that distinguish between different LFA-1 conformations, we found that, unlike strong chemokine signals, potent TCR stimuli were insufficient to trigger LFA-1 extension or headpiece opening in primary human lymphocytes. Nevertheless, LFA-1 in these TCR-stimulated T cells became highly adhesive to both anchored and mobile surface-bound ICAM-1, although it failed to bind soluble ICAM-1 with measurable affinity. Rapid rearrangement of LFA-1 by immobilized ICAM-1 switched the integrin to an open headpiece conformation within numerous scattered submicron focal dots that did not readily collapse into a peripheral LFA-1 ring. Headpiece-activated LFA-1 microclusters were enriched with talin but were devoid of TCR and CD45. Notably, LFA-1 activation by TCR signals as well as subsequent T cell spreading on ICAM-1 took place independently of cytosolic Ca(2+). In contrast to LFA-1-activating chemokine signals, TCR activation of LFA-1 readily took place in the absence of external shear forces. LFA-1 activation by TCR signals also did not require internal myosin II forces but depended on intact actin cytoskeleton. Our results suggest that potent TCR signals fail to trigger LFA-1 headpiece activation unless the integrin first gets stabilized by surface-bound ICAM-1 within evenly scattered actin-dependent LFA-1 focal dots, the quantal units of TCR-stimulated T cell arrest and spreading on ICAM-1.     

Costimulation of T cell receptor/CD3-mediated activation of resting human CD4+ T cells by leukocyte function-associated antigen-1 ligand intercellular cell adhesion molecule-1 involves prolonged inositol phospholipid hydrolysis and sustained increase of intracellular Ca2+ levels.

Activation of resting human CD4+ T cells mediated by mAb ligation of the TCR/CD3 complex requires costimulatory signals to result in proliferation; these can be provided by intercellular cell adhesion molecule-1 (ICAM-1, CD54) a natural ligand of leukocyte function-associated Ag-1 (LFA-1, CD11a/CD18). We analyzed early signaling events involved in T cell activation to determine the contribution by the LFA-1/ICAM-1 interaction. We studied in detail the hydrolysis of phosphatidylinositol(4,5)bisphosphate and intracellular levels of free Ca2+ during stimulation with beads coated with the CD3 mAb OKT3 alone or in combination with purified ICAM-1 protein. Our investigations show no response to LFA-1/ICAM-1 alone, but that costimulation by LFA-1/CAM-1 interaction induces prolonged inositol phospholipid hydrolysis (up to 4 h), resulting in generation of both inositol(1,4,5)phosphate3 and inositol(1,3,4,5)phosphate4 and their derivatives. Based on studies with cycloheximide, this costimulatory effect of prolonged inositol phospholipid hydrolysis appears dependent in part on de novo protein synthesis. A sustained increase in intracellular levels of free Ca2+ level is also observed after LFA-1/ICAM-1 costimulation, which is at least partly dependent on extracellular sources of Ca2+. Kinetic studies indicate that costimulation requires a minimal period of 4 h of LFA-1/ICAM-1 interaction to provide maximal costimulation for OKT3-mediated T cell proliferation. Thus, the necessary costimulation required for OKT3-mediated proliferation in this model system may be provided by an extended LFA-1/ICAM-1 interaction that in combination with OKT3 mAb leads to signal-transducing events, resulting in prolonged phospholipase C activation and phosphatidylinositol(4,5)bisphosphate hydrolysis, and a sustained increase in intracellular levels of free Ca2+.