A monoclonal antibody to beta 1 integrin (CD29) stimulates VLA- dependent adherence of leukocytes to human umbilical vein endothelial cells and matrix components

The leukocyte beta 1 integrin receptor very late activation antigen-4 (VLA-4) (alpha 4 beta 1, CD49d/CD29) binds to vascular cell adhesion molecule-1 (VCAM-1) expressed on cytokine-activated endothelium. A mAb designated 8A2 was identified that stimulated the binding of U937 cells to CHO cells transfected with VCAM-1 cDNA but not endothelial-leukocyte adhesion molecule or CD4 cDNA. mAb 8A2 also rapidly stimulated the adherence of peripheral blood lymphocytes (PBLs) to VCAM-1-transfected CHO cells or recombinant human tumor necrosis factor-treated human umbilical vein endothelial cells. mAb 8A2-stimulated binding of PBL was inhibited by mAbs to VLA-4 or VCAM-1. Surface expression of VLA-4 was not altered by mAb 8A2 treatment and monovalent Fab fragments of mAb 8A2 were active. Immunoprecipitation studies reveal that mAb 8A2 recognizes beta 1-subunit (CD29) of integrin receptors. In contrast to mAbs directed to VLA-4 alpha-subunit (alpha 4, CD49d), mAb 8A2 did not induce homotypic aggregation of PBL. Additionally, mAb 8A2 stimulated adherence of PBL and hematopoietic cell lines to purified matrix components laminin and fibronectin. This binding was blocked by mAbs to the VLA alpha-subunits alpha 6 (CD49f), or alpha 5 (CD49e) and alpha 4 (CD49d), respectively. We conclude that mAb 8A2 modulates the affinity of VLA-4 and other leukocyte beta 1 integrins, and should prove useful in studying the regulation of beta 1 integrin function.     

An alternative leukocyte homotypic adhesion mechanism, LFA-1/ICAM-1- independent, triggered through the human VLA-4 integrin

The VLA-4 (CD49d/CD29) integrin is the only member of the VLA family expressed by resting lymphoid cells that has been involved in cell-cell adhesive interactions. We here describe the triggering of homotypic cell aggregation of peripheral blood T lymphocytes and myelomonocytic cells by mAbs specific for certain epitopes of the human VLA alpha 4 subunit. This anti-VLA-4-induced cell adhesion is isotype and Fc independent. Similar to phorbol ester-induced homotypic adhesion, cell aggregation triggered through VLA-4 requires the presence of divalent cations, integrity of cytoskeleton and active metabolism. However, both adhesion phenomena differed at their kinetics and temperature requirements. Moreover, cell adhesion triggered through VLA-4 cannot be inhibited by cell preincubation with anti-LFA-1 alpha (CD11a), LFA-1 beta (CD18), or ICAM-1 (CD54) mAb as opposed to that mediated by phorbol esters, indicating that it is a LFA-1/ICAM-1 independent process. Antibodies specific for CD2 or LFA-3 (CD58) did not affect the VLA-4-mediated cell adhesion. The ability to inhibit this aggregation by other anti-VLA-4-specific antibodies recognizing epitopes on either the VLA alpha 4 (CD49d) or beta (CD29) chains suggests that VLA-4 is directly involved in the adhesion process. Furthermore, the simultaneous binding of a pair of aggregation-inducing mAbs specific for distinct antigenic sites on the alpha 4 chain resulted in the abrogation of cell aggregation. These results indicate that VLA-4-mediated aggregation may constitute a novel leukocyte adhesion pathway.     

Costimulation of proliferative responses of resting CD4+ T cells by the interaction of VLA-4 and VLA-5 with fibronectin or VLA-6 with laminin

Given prior evidence that adhesion molecules play critical roles in T cell recognition, it is important to identify new adhesion pathways and explore their role in T cell activation. Our studies of T cell proliferation complement concurrent studies of T cell adhesion; both demonstrate that resting CD4+ human T lymphocytes express the VLA integrins VLA-4, VLA-5, and VLA-6, and can use these receptors to interact with the extracellular matrix (ECM) proteins fibronectin (VLA-4 and VLA-5) and laminin (VLA-6). VLA-dependent interaction of resting human CD4+ T cells with fibronectin (FN) and laminin (LN) facilitates CD3-mediated T cell proliferation. Specifically, T cells do not proliferate in response to a wide range of concentrations of a CD3 mAb, OKT3, immobilized on plastic. However, coimmobilization with the CD3 mAb of FN or LN, but not other ECM proteins such as fibrinogen and collagen, consistently results in strong T cell proliferation. mAb blocking studies demonstrate that three VLA integrin receptor/ligand interactions mediate costimulation: VLA-4/FN, VLA-5/FN, and VLA-6/LN. VLA-5-dependent binding to FN but not costimulation by FN can be specifically blocked with peptides containing the RGD (arg-gly-asp) tripeptide sequence whereas VLA-4-dependent binding and costimulation can both be efficiently inhibited by a 12 amino acid peptide, LHGPEILDVPST (leu-his-gly-pro-glu-iso-leu-asp-val-pro-ser-thr), derived from the alternatively spliced IIICS region of FN. The costimulation provided by FN and LN in this system is stronger than and distinct from costimulatory signals provided by cytokines, such as IL-1 beta, IL-6,, and IL-7. These results suggest that, such as other adhesion molecules, T cell VLA integrins may also function in a dual capacity as adhesion and signalling molecules. In addition, they suggest that the interaction of T cells in vivo with ECM via VLA integrins plays a role not only in T cell migratory processes but may also influence Ag-specific T cell recognition.     

Human natural killer cells express VLA-4 and VLA-5, which mediate their adhesion to fibronectin.

Very late Ag (VLA)-3, VLA-4, and VLA-5, belonging to the beta-1 subfamily of integrins, have been recently identified as receptors for different binding regions of fibronectin (FN). We have detected VLA-4 and VLA-5, but not VLA-3, on fresh CD3-, CD16+, CD56+ human NK cells by flow cytometry and immunochemical analyses using mAb directed against beta-1, alpha-3, alpha-4, and alpha-5 subunits. Binding assays, performed on FN-coated plates, showed that NK cells specifically adhere to FN and their binding capacity is increased by MgCl2 but not by CaCl2. Using as inhibitory probes a polyclonal antibody against the beta-1 chain of the human FN receptor, the synthetic peptide GRGDSP, which is able to inhibit cellular adhesion mediated by VLA-5, the CS1 fragment, which contains the principal adhesion site in the IIICS domain recognized by VLA-4, and functional mAb directed against alpha-4 or alpha-5 subunits, we show that both VLA-4 and VLA-5 mediate the adhesion of human NK cells to FN. The expression of these integrin receptors may be relevant for NK interaction with extracellular matrix components and other cell types.     

Pseudomonas aeruginosa activates human mast cells to induce neutrophil transendothelial migration via mast cell-derived IL-1 alpha and beta

The mechanisms of neutrophil (PMN) recruitment to Pseudomonas aeruginosa infection remain incompletely defined. Mast cells (MC) involvement in this process has not been studied previously. In this study, we demonstrate that human cord blood-derived MC phagocytose P. aeruginosa and release mediators that activate HUVEC monolayers for supporting PMN transmigration. Pretreatment of supernatants from P. aeruginosa-MC cocultures with neutralizing anti-IL-1alpha plus anti-IL-1beta Abs, or IL-1R antagonist before addition to HUVEC for stimulation completely abrogated MC-induced PMN transmigration, while anti-TNF-alpha treatment had no effect. The expression of E-selectin and ICAM-1 on HUVEC, the latter a ligand for PMN CD11/CD18, was significantly up-regulated by P. aeruginosa-induced MC mediators. Pretreatment of human PMN with anti-CD18 mAb or pretreatment of HUVEC with a combination of three mAbs (against ICAM-1, ICAM-2, and E-selectin) inhibited by 85% the MC-dependent PMN transmigration. Moreover, P. aeruginosa-induced production of IL-1alpha and IL-1beta was down-regulated by IL-10 and dexamethasone. This study demonstrates for the first time that MC may mediate P. aeruginosa-induced PMN recruitment via production of IL-1alpha and beta. These findings have important implications for diseases involving P. aeruginosa infection and suggest novel targets for modulating P. aeruginosa-induced inflammation.     

Eosinophil transendothelial migration induced by cytokines. I. Role of endothelial and eosinophil adhesion molecules in IL-1 beta-induced transendothelial migration.

IL-1 beta promotes adhesiveness in human umbilical vein endothelial cells (HuVEC) for eosinophils through expression of adhesion molecules including intercellular adhesion molecules-1 (ICAM-1), E-selectin, and vascular cell adhesion molecule-1 (VCAM-1). Using an in vitro endothelial monolayer system, we examined whether IL-1 beta or TNF-alpha can promote eosinophil transendothelial migration. We also evaluated the contributions of ICAM-1, E-selectin, VCAM-1, leukocyte adhesion complex (CD11/18), and very late Ag-4 (CD11b/18) (VLA-4) in this process using blocking mAb, and determined the changes in expression of CD11b and L-selectin on eosinophils that had undergone transmigration. IL-1 beta and TNF-alpha treatment of HuVEC (4 h, 5 ng/ml) induced significant transendothelial migration of eosinophils (a 4.1 +/- 0.4-fold (IL-1 beta) and 2.0 +/- 0.9-fold (TNF-alpha) increase from the spontaneous value of 3.2 +/- 0.3%). Increased CD11b expression and shedding of L-selectin were observed on eosinophils following IL-1 beta-induced eosinophil transendothelial migration. Studies with mAb revealed that blockade of either ICAM-1 or CD11/18 inhibited transmigration, while antibodies against VCAM-1 and VLA-4 had no inhibitory effect. Among antibodies which block beta 2 integrins, anti-CD18 mAb had the best inhibitory effect (88% inhibition). The combined inhibitory effect of anti-CD11a mAb and anti-CD11b mAb was roughly equal to that of anti-CD18, although anti-CD11a (31% inhibition) and anti-CD11b (52% inhibition) were less effective individually. Anti-ICAM-1 by itself inhibited IL-1 beta-induced eosinophil transendothelial migration (24% inhibition) whereas neither anti-E-selectin nor anti-VCAM-1 were effective inhibitors. Interestingly, the combination of anti-E-selectin and anti-VCAM-1 with anti-ICAM-1 inhibited IL-1 beta-induced eosinophil transendothelial migration significantly better (53% inhibition) than anti-ICAM-1 alone. These results suggest that although the initial attachment of eosinophils to IL-1 beta-activated endothelial cells involves VCAM-1, E-selectin, and ICAM-1, the subsequent transendothelial migration process relies heavily on ICAM-1 and CD11/18. Finally, the changes that eosinophils have been observed to undergo during infiltration in vivo, namely increased expression of CD11/18 and shedding of L-selectin, appear to take place as a direct result of the interaction between eosinophils and endothelial cells.     

Functional evidence for three distinct and independently inhibitable adhesion activities mediated by the human integrin VLA-4. Correlation with distinct alpha 4 epitopes.

The human integrin VLA (very late activation antigens)-4 (CD49d/CD29), the leukocyte receptor for both the CS-1 region of plasma fibronectin (Fn) and the vascular cell surface adhesion molecule-1 (VCAM-1), also mediates homotypic aggregation upon triggering with specific anti-VLA-4 monoclonal antibody (mAb). Epitope mapping of this integrin on the human B-cell line Ramos, performed with a wide panel of anti-VLA-4 mAb by both cross-competitive cell binding and protease sensitivity assays, revealed the existence of three topographically distinct epitopes on the alpha 4 chain, referred to as epitopes A-C. By testing this panel of anti-VLA-4 mAb for inhibition of cell binding to both a 38-kDa Fn fragment containing CS-1 and to VCAM-1, as well as for induction and inhibition of VLA-4 mediated homotypic cell adhesion, we have found overlapping but different functional properties associated with each epitope. Anti-alpha 4 mAb recognizing epitope B inhibited cell attachment to both Fn and VCAM-1, whereas mAb against epitope A did not block VCAM-1 binding and only partially inhibited binding to Fn. In contrast, mAb directed to epitope C did not affect cell adhesion to either of the two VLA-4 ligands. All mAb directed to site A, as well as a subgroup of mAb recognizing epitope B (called B2), were able to induce cell aggregation, but this effect was not exerted by mAb specific to site C and by a subgroup against epitope B (called B1). Moreover, although anti-epitope C and anti-epitope B1 mAb did not trigger aggregation, those mAb blocked aggregation induced by anti-epitope A or B2 mAb. In addition, anti-epitope A mAb blocked B2-induced aggregation, and conversely, anti-epitope B2 mAb blocked A-induced aggregation. Further evidence for multiple VLA-4 functions is that anti-Fn and anti-VCAM-1 antibodies inhibited binding to Fn or to VCAM-1, respectively, but did not affect VLA-4-mediated aggregation. In summary, we have demonstrated that there are at least three different VLA-4-mediated adhesion functions, we have defined three distinct VLA-4 epitopes, and we have correlated these epitopes with the different functions of VLA-4.     

Lovastatin and phospholipase Cgamma regulate constitutive and protein kinase C dependent integrin mediated interactions of human T-cells with collagen

We previously reported that human interleukin (IL)-2 dependent T cell lines derived from very late antigen (VLA)-1(+) CD45RO(+) peripheral blood (PB) T-cells adhere constitutively to collagen type IV, whereas lines from VLA-1(-) PB lymphocytes (L) adhere weakly. Here we report that the latter are induced to adhere by phorbol 12-myristate 13-acetate (PMA). Both PMA dependent and constitutive adhesion, including that of a Herpes Virus Saimiri (HVS) infected CD4(+)VLA-1(+) clone (HVST) were inhibited by anti-VLA-1 monoclonal antibodies (mAb), by inhibitors of phospholipase C (PLC)gamma and by lovastatin but not by a MEK1 inhibitor, whereas only PMA induced adhesion was blocked by inhibition of protein-kinase (PK) C. Furthermore, lovastatin enhanced PLCgamma and anti VLA-1 mAb blockade, and its effect was not reversed by mevalonic acid (MVA). Lovastatin also inhibited interferon (IFN)gamma secretion by T cells triggered with anti-CD3 and in cells detaching from collagen IV. These results suggest new ways for functional modulation of activated T-cells interacting with collagen.     

Molecules involved in the adhesion and cytotoxicity of activated monocytes on endothelial cells.

Our study was designed to investigate the surface molecules involved in the adhesion and cytotoxicity of activated human monocytes on resting and IL-1-stimulated endothelial cells (EC). Monocytes, exposed to the prototypic activating stimuli IFN-gamma and LPS, showed increased binding to resting and IL-1-treated EC. Activated monocytes were cytotoxic for resting and IL-1-treated EC in a 24- to 48-h [3H]TdR release assay. Anti-CD18 mAb significantly inhibited binding of monocytes on EC: in particular they caused 59 and 22% inhibition of adhesion of activated monocytes to resting and IL-1-stimulated EC, respectively. Anti-VLA4 mAb had little or no effect on binding when used alone, but combined use with anti-CD18 revealed an important role for this adhesion pathway: in particular, VLA4-dependent adhesion accounted for 40% of the binding of activated monocytes on IL-1-treated EC. Anti-CD18 mAb caused similar inhibition (77 and 81%) of the cytotoxicity of activated monocytes on resting and IL-1-treated EC in spite of the fact that this pathway accounted for only 22% of binding to activated EC. Moreover, anti-VLA4 mAb, alone or in combination with anti-CD18, had no effect on cytotoxicity. These results suggest that adhesion of activated monocytes to activated EC involves the CD18- and VLA4-dependent pathways, but that the former is dominant for the expression of cytotoxicity. Thus, in the ensemble of adhesion molecules available for interaction between endothelium and activated monocytes, the hierarchy of their importance may vary for different functions.     

CD28-independent costimulation of T cells by OX40 ligand and CD70 on activated B cells.

OX40 and its ligand (OX40L) have been implicated in T cell-dependent humoral immune responses. To further characterize the role of OX40/OX40L in T-B cell interaction, we newly generated an anti-mouse OX40L mAb (RM134L) that can inhibit the costimulatory activity of OX40L transfectants for anti-CD3-stimulated T cell proliferation. Flow cytometric analyses using RM134L and an anti-mouse OX40 mAb indicated that OX40 was inducible on splenic T cells by stimulation with immobilized anti-CD3 mAb in a CD28-independent manner, while OX40L was not expressed on resting or activated T cells. OX40L was inducible on splenic B cells by stimulation with anti-IgM Ab plus anti-CD40 mAb, but not by either alone. These activated B cells exhibited a potent costimulatory activity for anti-CD3-stimulated T cell proliferation and IL-2 production. Anti-CD80 and anti-CD86 mAbs partially inhibited the costimulatory activity, and further inhibition was obtained by their combination with RM134L and/or anti-CD70 mAb. We also found the anti-IgM Ab- plus anti-CD40 mAb-stimulated B cells exhibited a potent costimulatory activity for proliferation of and IL-2 production by anti-CD3-stimulated CD28- T cells from CD28-deficient mice, which was substantially inhibited by RM134L and/or anti-CD70 mAb. These results indicated that OX40L and CD70 expressed on surface Ig- and CD40-stimulated B cells can provide CD28-independent costimulatory signals to T cells.     

Blockade of B7-H1 on macrophages suppresses CD4+ T cell proliferation by augmenting IFN-gamma-induced nitric oxide production

PD-1 is an immunoinhibitory receptor that belongs to the CD28/CTLA-4 family. B7-H1 (PD-L1) and B7-DC (PD-L2), which belong to the B7 family, have been identified as ligands for PD-1. Paradoxically, it has been reported that both B7-H1 and B7-DC co-stimulate or inhibit T cell proliferation and cytokine production. To determine the role of B7-H1 and B7-DC in T cell-APC interactions, we examined the contribution of B7-H1 and B7-DC to CD4+ T cell activation by B cells, dendritic cells, and macrophages using anti-B7-H1, anti-B7-DC, and anti-PD-1 blocking mAbs. Anti-B7-H1 mAb and its Fab markedly inhibited the proliferation of anti-CD3-stimulated naive CD4+ T cells, but enhanced IL-2 and IFN-gamma production in the presence of macrophages. The inhibition of T cell proliferation by anti-B7-H1 mAb was abolished by neutralizing anti-IFN-gamma mAb. Coculture of CD4+ T cells and macrophages from IFN-gamma-deficient or wild-type mice showed that CD4+ T cell-derived IFN-gamma was mainly responsible for the inhibition of CD4+ T cell proliferation. Anti-B7-H1 mAb induced IFN-gamma-mediated production of NO by macrophages, and inducible NO synthase inhibitors abrogated the inhibition of CD4+ T cell proliferation by anti-B7-H1 mAb. These results indicated that the inhibition of T cell proliferation by anti-B7-H1 mAb was due to enhanced IFN-gamma production, which augmented NO production by macrophages, suggesting a critical role for B7-H1 on macrophages in regulating IFN-gamma production by naive CD4+ T cells and, hence, NO production by macrophages.     

ICAM-3 regulates lymphocyte morphology and integrin-mediated T cell interaction with endothelial cell and extracellular matrix ligands

Leukocyte activation is a complex process that involves multiple cross- regulated cell adhesion events. In this report, we investigated the role of intercellular adhesion molecule-3 (ICAM-3), the third identified ligand for the beta 2 integrin leukocyte function-associated antigen-1 (LFA-1), in the regulation of leukocyte adhesion to ICAM-1, vascular cell adhesion molecule-1 (VCAM-1), and the 38- and 80-kD fragments of fibronectin (FN40 and FN80). The activating anti-ICAM-3 HP2/19, but not other anti-ICAM-3 mAb, was able to enhance T lymphoblast adhesion to these proteins when combined with very low doses of anti-CD3 mAb, which were unable by themselves to induce this phenomenon. In contrast, anti-ICAM-1 mAb did not enhance T cell attachment to these substrata. T cell adhesion to ICAM-1, VCAM-1, FN40, and FN80 was specifically blocked by anti-LFA-1, anti-VLA alpha 4, and anti-VLA alpha 5 mAb, respectively. The activating anti-ICAM-3 HP2/19 was also able to specifically enhance the VLA-4- and VLA-5-mediated binding of leukemic T Jurkat cells to VCAM-1, FN40, and FN80, even in the absence of cooccupancy of the CD3-TcR complex. We also studied the localization of ICAM-3, LFA-1, and the VLA beta 1 integrin, by immunofluorescence microscopy, on cells interacting with ICAM-1, VCAM-1 and FN80. We found that the anti-ICAM-3 HP2/19 mAb specifically promoted a dramatic change on the morphology of T lymphoblasts when these cells were allowed to interact with those adhesion ligands. Under these conditions, it was observed that a large cell contact area from which an uropod-like structure (heading uropod) was projected toward the outer milieu. However, when T blasts were stimulated with other adhesion promoting agents as the activating anti-VLA beta 1 TS2/16 mAb or phorbol esters, this structure was not detected. The anti-ICAM-3 TP1/24 mAb was also unable to induce this phenomenon. Notably, a striking cell redistribution of ICAM-3 was induced specifically by the HP2/19 mAb, but not by the other anti-ICAM-3 mAb or the other adhesion promoting agents. Thus, ICAM-3 was almost exclusively concentrated in the most distal portion of the heading uropod whereas either LFA-1 or the VLA beta 1 integrin were uniformly distributed all over the large contact area. Moreover, this phenomenon was also observed when T cells were specifically stimulated with the HP2/19 mAb to interact with TNF alpha-activated endothelial cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Critical role of CD11a (LFA-1) in therapeutic efficacy of systemically transferred antitumor effector T cells

The systemic adoptive transfer of activated T cells, derived from tumor-draining lymph nodes (LNs), mediates the regression of established tumors. In this study, the requirement of cell adhesion molecules, CD11a/CD18 (LFA-1), CD54 (ICAM-1), CD49d/CD29 (VLA-4), and CD106 (VCAM-1), for T cell infiltration into tumors and antitumor function was investigated. Administration of anti-CD11a mAb completely abrogated the efficacy of adoptive immunotherapy for both intracranial and pulmonary metastatic MCA 205 fibrosarcomas. In contrast, adoptive immunotherapy was effective in animals treated with anti-CD49d mAb, anti-CD106 mAb, anti-CD54 mAb, or in CD54 knockout recipients. Trafficking of transferred cells to the intracranial tumor was not affected by any of the mAb. However, the tumor-specific secretion of IFN-gamma by activated LN T cells was suppressed by anti-CD11a mAb or anti-CD54 mAb. To account for the different effects of CD11a and CD54 blockade in vivo, an additional CD11a/CD18 ligand, CD102 (ICAM-2), was demonstrated on tumor-associated macrophages but not on tumor cells. These results show that CD11a mediates a critical function in interactions between effector T cells, tumor cells, and host accessory cells in situ leading to tumor regression.     

Fibronectin promotes proliferation of naive and memory T cells by signaling through both the VLA-4 and VLA-5 integrin molecules.

The capacity of purified fibronectin to costimulate human T cell DNA synthesis was examined. Low concentrations of immobilized fibronectin, but not soluble fibronectin, augmented anti-CD3-induced proliferation of highly purified human T cells. In the absence of anti-CD3 stimulation, immobilized fibronectin did not induce T cell proliferation alone or in the presence of IL-2 or phorbol dibutyrate. Although fibronectin is present in high concentrations in the serum, immobilized fibronectin was able to costimulate T cell proliferation when cells were cultured in serum-containing medium. Immobilized collagen type I did not enhance anti-CD3 stimulated T cell responses, whereas gelatin (denatured collagen) and laminin were able to enhance anti-CD3 stimulated T cell responses modestly. The effects of gelatin, however, appeared to be indirect, because it could not enhance responses in medium devoid of fibronectin. Immobilized fibronectin enhanced anti-CD3 induced proliferation of both CD45RA dim and CD45RA bright subsets within both the CD4+ and CD8+ subpopulations of T cells, although cells with the CD45RA dim phenotype were costimulated by lower concentrations of immobilized fibronectin. Enhancement of anti-CD3 induced proliferation by immobilized fibronectin was completely inhibited by a mAb to CD29, the integrin beta 1-chain (4B4) and not by a variety of other mAb. In contrast to its effects on proliferation, 4B4 only partially blocked T cell binding to anti-CD3 and fibronectin-coated macrowells. These findings suggested that the interaction between fibronectin and its receptor transduced a signal to the T cell and did not merely stabilize the interaction between anti-CD3 and the CD3 complex. Further experiments confirmed this observation. Thus fibronectin could enhance anti-CD3 responses when it was immobilized to a separate surface. The augmentation of anti-CD3 stimulated proliferation induced by immobilized fibronectin was also inhibited partially by mAb to either VLA-4 or VLA-5 and completely by a combination of the two mAb. The mAb to VLA-4 not only blocked the capacity of immobilized fibronectin to enhance anti-CD3-induced T cell proliferation but also directly costimulated T cell responses. Thus, at least two fibronectin receptors are involved in fibronectin-mediated costimulation of T cell proliferation. These studies indicate that signals are transduced through the fibronectin receptors, VLA-4 and VLA-5, that augment T cell responses and therefore implicate the extracellular matrix protein fibronectin as an important influence regulating T cell responsiveness in vivo.     

Direct stimulation of human T cells via TLR5 and TLR7/8: flagellin and R-848 up-regulate proliferation and IFN-gamma production by memory CD4+ T cells

TLRs are involved in innate cell activation by conserved structures expressed by microorganisms. Human T cells express the mRNA encoding most of TLRs. Therefore, we tested whether some TLR ligands may modulate the function of highly purified human CD4+ T lymphocytes. We report that, in the absence of APCs, flagellin (a TLR5 ligand) and R-848 (a TLR7/8 ligand) synergized with suboptimal concentrations of TCR-dependent (anti-CD3 mAb) or -independent stimuli (anti-CD2 mAbs or IL-2) to up-regulate proliferation and IFN-gamma, IL-8, and IL-10 but not IL-4 production by human CD4+ T cells. No effect of poly(I:C) and LPS, ligands for TLR3 and TLR4, respectively, was detected. We also observed that CD4+CD45RO+ memory T cell responses to TLR ligands were more potent than those observed with CD4+CD45RA+ naive T cells. Moreover, among the memory T cells, CCR7- effector cells were more sensitive to TLR ligands than CCR7+ central memory cells. These data demonstrate for the first time a direct effect of TLR5 and TLR7/8 ligands on human T cells, and highlight an innate arm in T cell functions. They also suggest that some components from invading microorganisms may directly stimulate effector memory T cells located in tissues by up-regulating cytokine and chemokine production.     

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.     

Adhesion of T and B lymphocytes to extracellular matrix and endothelial cells can be regulated through the beta subunit of VLA

Investigating the regulation of very late antigen (VLA)-mediated functions, we found that TS2/16, a mAb directed against the beta chain of the VLA group of integrins, can induce binding of resting peripheral blood lymphocytes, cloned T lymphocytes, and Epstein Barr virus-transformed B cells to extracellular matrix components, fibronectin, laminin, and collagen, but not to fibrinogen. The antibody stimulates VLA-4-, VLA-5-, and VLA-6-mediated binding. Furthermore, it induces VLA-4-mediated binding to vascular cell adhesion molecule-1 expressed by rTNF-alpha-stimulated endothelial cells, but it does not stimulate homotypic aggregation of cells as described for a number of anti-VLA-4 alpha antibodies (Bednarczyk, J.L., and B. W. McIntyre. 1990. J. Immunol. 144: 777-784; Campanero, M. R., R. Pulido, M. A. Ursa, M. Rodríguez-Moya, M. O. de Landázuri, and F. Sánchez-Madrid. 1990. J. Cell Biol. 110:2157-2165). Therefore, the stimulating activity of this anti-beta 1 antibody clearly contrasts with that of the anti-VLA-4 alpha antibodies, which induce homotypic cell aggregation, but not binding of cells to extracellular matrix components or endothelial cells, indicating that TS2/16 may generate different signals. The observation that also F(ab')2 or Fab fragments of this anti-beta 1 antibody stimulate binding to extracellular matrix components and endothelial cells excludes the possibility that binding requires receptor crosslinking, or is Fc receptor mediated. Induction of this adhesion is cation and energy dependent and requires an intact cytoskeleton. Although changes in the conformation of VLA integrins induced by this antibody may regulate their functional activity, the dependence on metabolic energy indicates that intracellular processes may also play a role.     

CD4 ligation promotes the IL-4-independent development of IL-4-producing clones from naive CD4(+) T cells.

The signals that trigger IL-4-independent IL-4 synthesis by conventional CD4(+) T cells are not yet defined. In this study, we show that coactivation with anti-CD4 mAb can stimulate single naive CD4(+) T cells to form IL-4-producing clones in the absence of APC and exogenous IL-4, independently of effects on proliferation. When single CD4(+) lymph node cells from C57BL/6 mice were cultured with immobilized anti-CD3epsilon mAb and IL-2, 65-85% formed clones over 12-14 days. Coimmobilization of mAb to CD4, CD11a, and/or CD28 increased the size of these clones but each exerted different effects on their cytokine profiles. Most clones produced IFN-gamma and/or IL-3 regardless of the coactivating mAb. However, whereas 0-6% of clones obtained with mAb to CD11a or CD28 produced IL-4, 10-40% of those coactivated with anti-CD4 mAb were IL-4 producers. A similar response was observed among CD4(+) cells from BALB/c mice. Most IL-4-producing clones were derived from CD4(+) cells of naive (CD44(low) or CD62L(high)) phenotype and the great majority coproduced IFN-gamma and IL-3. The effect of anti-CD4 mAb on IL-4 synthesis could be dissociated from effects on clone size since anti-CD4 and anti-CD11a mAb stimulated formation of clones of similar size which differed markedly in IL-4 production. Engagement of CD3 and CD4 in the presence of IL-2 is therefore sufficient to induce a substantial proportion of naive CD4(+) T cells to form IL-4-producing clones in the absence of other exogenous signals, including IL-4 itself.     

Regulation of the VLA integrin-ligand interactions through the beta 1 subunit

Integrins from the very late activation antigen (VLA) subfamily are involved in cellular attachment to extracellular matrix (ECM) proteins and in intercellular adhesions. It is known that the interaction of integrin proteins with their ligands can be regulated during cellular activation. We have investigated the regulation of different VLA-mediated adhesive interactions through the common beta 1 chain. We have found that certain anti-beta 1 antibodies strongly enhance binding of myelomonocytic U-937 cells to fibronectin. This beta 1-mediated regulatory effect involved both VLA-4 and VLA-5 fibronectin receptors. Moreover, anti-beta 1 mAb also induced VLA-4-mediated binding to a recombinant soluble form of its endothelial cell ligand VCAM-1. Non-activated peripheral blood T lymphocytes, unable to mediate VLA-4 interactions with fibronectin or VCAM-1, acquired the ability to bind these ligands in the presence of anti-beta 1 mAb. The anti-beta 1-mediated changes in the affinities of beta 1 integrin for their ligands were comparable to those triggered by different lymphocyte activation agents such as anti-CD3 mAb or phorbol ester. Adhesion of melanoma cells to other ECM proteins such as laminin or collagen as well as that of alpha 2-transfected K-562 cells to collagen, was also strongly enhanced by anti-beta 1 mAb. These beta 1-mediated regulatory effects on different VLA-ligand interactions do not involve changes in cell surface membrane expression of different VLA heterodimers. The anti-beta 1-mediated functional effects required an active metabolism, cytoskeleton integrity and the existence of physiological levels of intracellular calcium as well as a functional Na+/H+ antiporter. Beta 1 antibodies not only increased cell attachment but also promoted spreading and cytoplasmic extension of endothelial cells on plates coated with either fibronectin, collagen, or laminin as well as induced the rapid appearance of microspikes in U-937 cells on fibronectin. Moreover, both beta 1 integrin and the cytoskeletal protein talin colocalized in the anti-beta 1 induced microspikes. These results emphasize the central role of the common beta 1 chain in regulating different adhesive functions mediated by VLA integrins as well as cellular morphology.     

Requirement for VLA-4 and VLA-5 integrins in lymphoma cells binding to and migration beneath stromal cells in culture

Physical interaction between human lymphomas and murine bone marrow derived stromal cells were studied. Nalm-6 pre-B cells adhered to BMS2 stromal cells and subsequently migrated beneath them, while Ramos Burkitt lymphoma cells, adhered but did not migrate. Four mAbs were established against Nalm-6 cells, which were able to block initial adhesion of Nalm-6 cells. Two of them were directed against the alpha 4 chain of VLA-4, and other two recognized the beta 1 chain of VLA integrins. Therefore, the initial adhesion of Ramos and Nalm-6 cells to BMS2 was largely mediated by the VLA-4 integrin expressed on lymphocytes. The corresponding ligand on stromal cells appears to be VCAM-1, because antibodies against murine VCAM-1 blocked the adhesion. However, antibodies against the alpha chain of VLA-4 were not capable of blocking subsequent migration beneath stromal cells. In contrast, antibodies against the beta chain of VLA integrins blocked the migration beneath stromal cells as well as the initial adhesion. Because a common beta chain can be shared among integrins, the role of other VLA integrins in Nalm-6 cells migration was investigated. VLA-5 and VLA-6 as well as VLA-4 were expressed on Nalm-6 cells, but not on Ramos cells. Additional blocking experiments revealed that VLA-4 and VLA-5 are likely to work in concert to mediate the migration of Nalm-6 cells beneath stromal cells. Thus, particular VLA integrins appear to be responsible not only for lymphocyte adhesion but also for migration with respect to stromal cells. These findings may have implications for cell-cell interactions and directed migration of lymphocytes in bone marrow and other tissues.