T cell receptor- and beta 1 integrin-mediated signals synergize to induce tyrosine phosphorylation of focal adhesion kinase (pp125FAK) in human T cells

The beta 1 subfamily of integrins is thought to play an important role in both the adhesion/migration and proliferation/differentiation of T cells. beta 1 integrins can provide T cell costimulation through interaction of very late antigen (VLA) 4 (VLA-4) (alpha 4 beta 1) and VLA-5 (alpha 5 beta 1) with the extracellular matrix protein fibronectin (FN), or by VLA-4 binding to its cell surface ligand, vascular cell adhesion molecule (VCAM) 1. The mechanism by which beta 1 integrin members transduce T cell-costimulatory signals is poorly understood. Studies in non-T cells have demonstrated regulation of the tyrosine focal adhesion kinase pp125FAK by beta 1 integrin engagement and, most recently, indicate a role for pp125FAK in linking integrin-mediated signal transduction to the Ras pathway (Schaller, M. D., and J. T. Parsons, 1994, Curr. Opin. Cell. Biol. 6: 705-710; Schlaepfer, D. D., S. K. Hanks, T. Hunter, and P. Van der Geer. 1994. Nature (Lond.), 372:786-790). Although pp125FAK kinase occurs in T cells, there are no reports on its regulation in this cell type. The studies described in this article characterize novel regulation of pp125FAK by the T cell receptor (TCR)-CD3 antigen complex and beta 1 integrins, and provide the first account, in any cell type, of integrin alpha 4 beta 1-mediated pp125FAK tyrosine phosphorylation. We demonstrate a rapid and sustained synergistic increase in tyrosine phosphorylation of human pp125FAK in Jurkat T cells after simultaneous (a) triggering of the TCR-CD3 complex, and (b) alpha 4 beta 1 and alpha 5 beta 1 integrin-mediated binding of these cells to immobilized FN or alpha 4 beta 1 integrin-mediated binding to immobilized VCAM-1. Studies with normal peripheral blood-derived CD4+ human T blasts confirm the synergistic action of a TCR-CD3 complex-mediated costimulus with a FN- or VCAM-1-dependent signal in the induction of T cell pp125FAK tyrosine phosphorylation. In vitro kinase assays performed on pp125FAK immunoprecipitates isolated from Jurkat cells and normal CD4+ T cells identified a coprecipitating 57-kD tyrosine-phosphorylated protein (pp57), distinct from pp59fyn or pp56lck. These results indicate, for the first time, the involvement of a specific kinase, pp125FAK, in alpha 4 beta 1- and alpha 5 beta 1-mediated T cell-costimulatory signaling pathways. In addition, the data demonstrate novel regulation of pp125FAK tyrosine phosphorylation by the TCR-CD3 complex.     

The role of B7-1 and LFA-3 in costimulation of CD8+ T cells

This study compares the ability of LFA-3 (CD58) and B7-1 (CD80) ligands to provide costimulatory signals for superantigen (SAg)-stimulated CD8+ and CD4+ T cells. We show that B7-1 and LFA-3 costimulation activate CD8+ T cells to proliferation, cytokine production (IL-2, TNF, and IFN-gamma), and cytotoxicity. A long-lasting proliferative response was observed after combined DR/B7-1/LFA-3 costimulation. Detailed analysis of SEA-activated CD8+ T cells revealed that maximal production of IFN-gamma was seen in LFA-3-costimulated cells, while production of IL-2 was mainly induced after B7-1 costimulation. A fivefold increase in the IFN-gamma production was observed when activated CD8+ T cells were costimulated with Chinese hamster ovary (CHO)-DR/LFA-3 cells compared with the secretion induced by CHO-DR/B7-1. In contrast, SEA-treated CD4+ T cells costimulated with B7-1 or LFA-3 gave rise to a similar production of IFN-gamma, suggesting a preferential function for the CD2/LFA-3 pathway in the regulation of IFN-gamma in CD8+ T cells. Moreover, the generation of CTL was supported similarly by B7-1 and LFA-3 costimulation, but not by CHO-DR cells. We conclude that ligation of the CD28 and CD2 receptors mediate distinct effect on CD8+ and CD4+ T cell effector functions.     

Engagement of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) induces transforming growth factor beta (TGF-beta) production by murine CD4(+) T cells

Evidence indicates that cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) may negatively regulate T cell activation, but the basis for the inhibitory effect remains unknown. We report here that cross-linking of CTLA-4 induces transforming growth factor beta (TGF-beta) production by murine CD4(+) T cells. CD4(+) T helper type 1 (Th1), Th2, and Th0 clones all secrete TGF-beta after antibody cross-linking of CTLA-4, indicating that induction of TGF-beta by CTLA-4 signaling represents a ubiquitous feature of murine CD4(+) T cells. Stimulation of the CD3-T cell antigen receptor complex does not independently induce TGF-beta, but is required for optimal CTLA-4-mediated TGF-beta production. The consequences of cross-linking of CTLA-4, together with CD3 and CD28, include inhibition of T cell proliferation and interleukin (IL)-2 secretion, as well as suppression of both interferon gamma (Th1) and IL-4 (Th2). Moreover, addition of anti-TGF-beta partially reverses this T cell suppression. When CTLA-4 was cross-linked in T cell populations from TGF-beta1 gene-deleted (TGF-beta1(-/-)) mice, the T cell responses were only suppressed 38% compared with 95% in wild-type mice. Our data demonstrate that engagement of CTLA-4 leads to CD4(+) T cell production of TGF-beta, which, in part, contributes to the downregulation of T cell activation. CTLA-4, through TGF-beta, may serve as a counterbalance for CD28 costimulation of IL-2 and CD4(+) T cell activation.     

Pyk2 is differentially regulated by beta1 integrin- and CD28-mediated co-stimulation in human CD4+ T lymphocytes

Beta1 integrins can provide T cell co-stimulation, but little is known concerning their downstream signaling pathways. We found that Pyk2, a focal adhesion kinase-related tyrosine kinase, is regulated by beta1 integrin signaling in human T cells. Stimulation of Jurkat T cells with the alpha4beta1 integrin ligand VCAM-1 results in Pyk2 tyrosine phosphorylation, and combined stimulation with VCAM-1 and anti-CD3 mAb induces rapid and sustained synergistic Pyk2 phosphorylation. Studies with mAb suggest that in synergistic CD3- and alpha4beta1 integrin-mediated Pyk2 tyrosine phosphorylation, a major contribution of CD3-derived signals is independent of their effects on regulating integrin adhesion. Analysis of resting human CD4+ T cells confirmed the ability of CD3-derived signals to synergize with beta1 integrin-dependent signals in the induction of Pyk2 tyrosine phosphorylation. In addition, although CD28-mediated co-stimulatory signals were able to synergize with CD3-mediated signals in inducing ERK and JNK activation and secretion of IL-2 in the primary T cells, they did not contribute to the induction of Pyk2 phosphorylation. Taken together, these results indicate a potential role for Pyk2 in T cell co-stimulation mediated specifically by beta1 integrins.     

Maturation of human neonatal CD4+ and CD8+ T lymphocytes into Th1/Th2 effectors

The increased susceptibility of neonates to infections has been ascribed to the immaturity of their immune system. More particularly, T cell-dependent responses were shown to be biased towards a Th2 phenotype. Our studies on the in vitro maturation of umbilical cord blood T cells suggest that the Th2 bias of neonatal response cannot be simply ascribed to intrinsic properties of neonatal T cells. Phenotypically, neonatal CD4+ T cells are more immature than their adult CD45RO-/RA+ naive counterparts and they contain a subset (10-20%) of CD45RO-/RA+ CD31- cells which is very low in adults and displays some unique functional features. The activation and maturation of neonatal CD4+ T cells is particularly dependent upon the strength of CD28-mediated cosignal which dictates not only the cytokine profile released upon primary activation but also the response to IL-12. Activation of adult as well as neonatal CD4+ T cells in the context of low CD28 costimulation yields to the production of low levels of only one cytokine, i.e. IL-2. In contrast, strong CD28 costimulation supports the production of high levels of type 1 (IL-2, IFN gamma and TNF beta) and low levels of type 2 (IL-4 and IL-13) cytokines by neonatal T cells. The low levels of naive T cell-derived IL-4 are sufficient to support their development into high IL-4/IL-5 producers by an autocrine pathway. The ability of IL-12 to prime neonatal CD4+ T cells for increased production of IL-4 (in addition to IFN gamma) is observed only when CD28 cosignal is minimal. Under optimal activation conditions (i.e. with anti-CD3/B7.1 or allogenic dendritic cells) the response and the maturation of neonatal and adult naive T cells are similar. Thus the Th2 bias of neonatal immune response cannot be simply ascribed to obvious intrinsic T cell defect but rather to particular conditions of Ag presentation at priming. Unlike CD4+ T cells, neonatal CD8+ T cells strictly require exogenous IL-4 to develop into IL-4/IL-5 producers. Most importantly, anti-CD3/B7-activated neonatal CD8 T cells coexpress CD4 as well as CCR5 and CXCR4 and are susceptible to HIV-1 infection in vitro.     

Regulation of C-C chemokine production by murine T cells by CD28/B7 costimulation

C-C chemokines play an important role in recruitment of T lymphocytes to inflammatory sites. T lymphocytes secrete chemokines, but the activation requirements for chemokine production by T cells are uncertain. We studied the regulation of C-C chemokine production by CD28 costimulatory signals by murine T lymphocytes. Splenocytes from BALB/c mice cultured with anti-CD3 mAb expressed macrophage-inflammatory protein (MIP)-1alpha mRNA and secreted MIP-1alpha, which was inhibited by anti-B7-1 plus anti-B7-2 mAbs. MIP-1alpha production by Ag-stimulated T cells from DO.11.10 TCR transgenic mice was augmented by anti-CD28 mAb and increased compared with DO.11.10/CD28(-/-) cells. When T cell costimulation was provided by IL-2, MIP-1alpha was not enhanced. Studies with IL-2, IL-4, STAT4, and STAT6 knock-out mice suggested that chemokine production is controlled by pathways different from those regulating T cell differentiation. Thus, CD28 costimulation may amplify an immune response by stimulating T cell survival, proliferation, and production of chemokines that recruit T cells to inflammatory sites.     

Dissection of pathways implicated in integrin-mediated actin cytoskeleton assembly. Involvement of protein kinase C, Rho GTPase, and tyrosine phosphorylation

A panel of antibodies to the alphaIIbbeta3 integrin was used to promote adhesion of Chinese hamster ovary cells transfected with the alphaIIbbeta3 fibrinogen receptor. While some alphaIIbbeta3 antibodies were not able to induce p125 focal adhesion kinase (p125FAK) tyrosine phosphorylation, all the antibodies equally support cell adhesion but not spreading and assembly of actin stress fibers. Absence of stress fibers was also obtained by plating on antibodies directed to the hamster beta1 integrin. In contrast, cells plated on matrix proteins spread organizing actin stress fibers. Treatment with phorbol esters phorbol 12-myristate 13-acetate (PMA) induced cells to spread on antibodies-coated dishes but not to organize actin in stress fibers. The combination of PMA and cytotoxic necrotizing factor 1 (CNF1), a specific Rho activator, induced cell spreading and organization of stress fibers. PMA or the combination of PMA and CNF1 also increases tyrosine phosphorylation of p125FAK in response to antibodies that were otherwise unable to trigger this response. These data show that: 1) matrix proteins and antibodies differ in their ability to induce integrin-dependent actin cytoskeleton organization (while matrix induced stress fibers formation, antibodies did not); 2) p125FAK tyrosine phosphorylation is insufficient per se to trigger actin stress fibers formation since antibodies that activate p125FAK tyrosine phosphorylation did not lead to actin stress fibers assembly; and 3) the inability of anti-integrin antibodies to trigger stress fibers organization is overcome by concomitant activation of the protein kinase C (PKC) and Rho pathways; PKC activation leads to cell spreading and Rho activation is required to organize actin stress fibers.     

CD4+ T cells from IRF-1-deficient mice exhibit altered patterns of cytokine expression and cell subset homeostasis

The cyclic hexapeptide CWLDVC (TBC 772) is an antagonist of alpha4 integrins and a potent inhibitor of lymphocyte interactions with fibronectin, vascular cell adhesion molecule-1, and muscosal vascular addressin cell adhesion molecule-1 (MAdCAM-1). As such, peptide TBC 772 effectively inhibits the activation of freshly isolated human T lymphocytes stimulated with purified vascular cell adhesion molecule-1 coimmobilized with anti-CD3 mAb. The influence of peptide binding on distinct sites of the alpha4beta1 complex was determined by flow cytometry and cellular adhesion assays employing a panel of mAbs. Binding of the alpha4-specific mAb L25 and the beta1-specific mAb 33B6 was not altered by the peptide; however, binding of mAb 19H8, which is specific for a combinatorial epitope of alpha4beta1, was dramatically inhibited. Treatment of lymphocytes with the peptide caused an increase in a ligand-induced epitope on beta1 integrin defined by mAb 15/7. In T cell activation studies using coimmobilized anti-CD3 mAb and the anti-integrin mAbs, the peptide had broader inhibitory activity, suppressing costimulation induced by all the integrin mAbs. The peptide was not generally toxic and was integrin selective in its suppressive activity, as coactivation by ligation of CD3 in conjunction with CD28 or CD26 was not affected. These results suggest that the antagonist peptide CWLDVC can effectively neutralize integrin coactivation systems by a mechanism independent of competitive binding.     

Induction of T cell adhesion to extracellular matrix or endothelial cell ligands by soluble or matrix-bound interleukin-7

The putative effects of interleukin (IL)-7, operating in the context of extracellular matrix (ECM), on the adhesion of human T cells were examined. Recombinant human, IL-7 was found to bind ECM or fibronectin (FN) with IC50 values of 10-100 nM. Nanogram amounts of both soluble and, especially, FN- or ECM-bound IL-7, which differentially affected the morphologies of FN-adherent T cells, induced the adhesion of resting CD4+ and CD8+ T cells in dose-dependent and beta 1 integrin-dependent manners. Under static and flow conditions, soluble IL-7 also induced the binding of unstimulated T cells to vascular cell adhesion molecule-1, suggesting that this cytokine can also modulate integrin binding to endothelial cell ligands. The effects of affinity modulation by IL-7 of FN-specific beta 1 integrins depend on the presence of soluble FN, which inhibited T cell adhesion to FN induced by FN-bound IL-7 or by an integrin-specific affinity-modulating monoclonal antibody, but not by soluble IL-7 or phorbol 12-myristate 13-acetate. These findings provide an example of a major ECM integrin ligand, FN, which is capable of modulating its adhesive interactions with specific immune cells by associating with and presenting a cytokine in a bio-active state.     

T cell receptor-mediated tyrosine phosphorylation of Cas-L, a 105-kDa Crk-associated substrate-related protein, and its association of Crk and C3G

Cas-L (pp105), a Crk-associated substrate (p130(Cas))-related protein, was first identified as a 105-kDa protein that is tyrosine-phosphorylated following beta1 integrin cross-linking in T cells. Cas-L contains possible multiple binding sites for the Src homology (SH) 2 domains of various signaling molecules, and appears to be involved in signal transduction through phosphorylated tyrosine-mediated protein-protein interaction. Since Cas-L is preferentially expressed in lymphocytes, it is conceivable that Cas-L plays an important role in lymphocyte-specific signals. Here, we show the involvement of Cas-L in the T cell receptor (TCR)/CD3 signaling pathway. Cas-L is transiently phosphorylated following CD3 cross-linking, and tyrosine-phosphorylated Cas-L binds to Crk and C3G. Furthermore, a Cas-L mutant that lacks the SH3 domain, the binding site for focal adhesion kinase (FAK), is also tyrosine-phosphorylated upon CD3 cross-linking, but not upon beta1 integrin crosslinking, suggesting that FAK is not involved in CD3-dependent Cas-L phosphorylation. Taken together, the present study indicates a novel signaling pathway mediated by tyrosine-phosphorylated Cas-L upon the TCR/CD3 stimulation.     

Mobilization and homing of peripheral blood progenitors is related to reversible downregulation of alpha4 beta1 integrin expression and function

Despite the wide use of mobilized peripheral blood (PB) progenitor cells (PBPC) for clinical transplantation the mechanism(s) underlying their mobilization and subsequent engraftment are still unknown. We compared the adhesive phenotype of CD34(+) colony-forming cells (CFC) in bone marrow (BM) and PB of normal donors before and after administration of granulocyte colony-stimulating factor (G-CSF) for 5 d. G-CSF-mobilized PB CFC cells adhered significantly less to BM stroma, fibronectin, and to the alpha4 beta1 binding fibronectin peptide, CS1, because of decreased expression of the alpha4 integrin. Since incubation of BM CD34(+) cells for 4 d with G-CSF at concentrations found in serum of G-CSF- treated individuals did not affect alpha4-dependent adhesion, G-CSF may not be directly responsible for the decreased alpha4-mediated adhesion of PB CFC. Culture of G-CSF-mobilized PB CD34(+) cells with cytokines at concentrations found in BM stromal cultures upregulated alpha4 expression and restored adhesion of mobilized PB CFC to stroma, fibronectin, and CS1. Adhesion of cultured, mobilized PB CFC to stroma and CS1 could not be further upregulated by the beta1 activating antibody, 8A2. This indicates acquisition of a maximally activated alpha4 beta1 integrin once PB CFC have been removed from the in vivo mobilizing milieu. Thus, decreased alpha4 expression on CD34(+) CFC in PB may be responsible for the aberrant circulation of mobilized PB CD34(+) cells. Reexpression of a maximally activated alpha4 beta1 integrin on mobilized PB CFC removed from the mobilizing in vivo milieu may contribute to the early engraftment of mobilized PBPC.     

T cell activation modulates retrovirus-mediated gene expression

Important considerations for T lymphocyte-based gene therapy include efficient gene delivery and expression in primary, human T cells. In this study, retrovirus-mediated gene transfer and the fate of proviral gene expression were evaluated in human T cells activated using (1) immobilized anti-CD3 monoclonal antibody (MAb) plus interleukin 2, or (2) cis costimulation using beads carrying coimmobilized anti-CD3 and anti-CD28 MAbs. By cross-linking the CD3 and CD28 receptors, these MAbs mimic in vivo signaling events, leading to cytokine production and proliferation. A modified human interleukin 1beta (IL-1beta) cDNA inserted into the MFG retroviral vector served as an indicator gene. Retroviral transduction frequencies were similar for T lymphocytes activated by the respective methods. However, early after MAb stimulation and virus exposure, proviral gene expression was greater at the RNA and protein levels in optimized anti-CD3/anti-CD28 bead-activated T cells, corresponding with augmented endogenous cytokine responses and mitogenesis. Proviral gene expression was not regulated by extrinsic cell factors present in activated T cell supernatants. Regardless of the MAb stimulation method, proviral IL-1beta expression declined in later T cell cultures concomitant with a decrease in cellular cytokines. Restimulation by either method reinduced both T cell activity and vector expression. Our finding that proviral gene regulation is downmodulated in the absence of T cell signaling events has implications for clinical strategies using retrovirus-modified T cells.     

CD9-mediated costimulation of TCR-triggered naive T cells leads to activation followed by apoptosis

The induction of full activation or death in TCR-triggered T cells depends largely on whether appropriate costimulatory signals are provided. In this study, we show that the costimulation of CD9 on naive T cells during TCR stimulation results in transient, albeit potent, activation followed by apoptosis, rather than full activation. Anti-CD9 mAb synergized with suboptimal doses of anti-CD3 mAb in inducing T cell activation. [3H]TdR incorporation determined 2 days after CD9 costimulation was as potent as that induced by CD28 costimulation. In contrast to progressive T cell proliferation induced by CD28 costimulation, CD9 costimulation led to the induction of apoptosis of once-activated T cells. Although IL-2R expression was induced significantly earlier and to a greater degree after CD9 costimulation than after CD28 costimulation, CD9 costimulation only transiently produced a small amount of IL-2 and induced apparently low levels of bcl-xL compared with those observed in CD28 costimulation. Addition of rIL-2 to cultures of CD9 costimulation induced strikingly enhanced expression of bcl proteins, especially of bcl-xL, and protected TCR-stimulated T cells from apoptosis. These data indicate that CD9-mediated costimulation of TCR-triggered naive T cells leads to activation followed by apoptosis as the result of failure to generate a positive signal for sufficient levels of IL-2 production.     

Porphyromonas gingivalis fimbriae use beta2 integrin (CD11/CD18) on mouse peritoneal macrophages as a cellular receptor, and the CD18 beta chain plays a functional role in fimbrial signaling

In this study, we demonstrate that Porphyromonas gingivalis fimbriae use molecules of beta2 integrin (CD11/CD18) on mouse peritoneal macrophages as cellular receptors and also show that the beta chain (CD18) may play a functional role in signalling for the fimbria-induced expression of interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) genes in the cells. Using a binding assay with 125I-labeled fimbriae, we observed that fimbrial binding to the macrophages was inhibited by treatment with CD11a, CD11b, CD11c, or CD18 antibody but not by that with CD29 antibody. Western blot assays showed that the fimbriae bound to molecules of beta2 integrin (CD11/CD18) on the macrophages. Furthermore, Northern blot analyses showed that the fimbria-induced expression of IL-1beta and TNF-alpha genes in the cells was inhibited strongly by CD18 antibody treatment and slightly by CD11a, CD11b, or CD11c antibody treatment. Interestingly, intracellular adhesion molecule 1 (ICAM-1), a ligand of CD11/CD18, inhibited fimbrial binding to the cells in a dose-dependent manner. In addition, ICAM-1 clearly inhibited the fimbria-induced expression of IL-1beta and TNF-alpha genes in the cells. However, such inhibitory action was not observed with laminin treatment. These results suggest the importance of beta2 integrin (CD11/CD18) as a cellular receptor of P. gingivalis fimbriae in the initiation stage of the pathogenic mechanism of the organism in periodontal disease.     

Interleukin-15 induces adhesion receptor redistribution in T lymphocytes

Chemotactic factors such as cytokines and chemokines direct the migration of leukocytes into inflammatory sites. Chemokines play a role regulating both the expression and adhesive properties of leukocyte integrins. We have recently described an additional function of chemokines in the induction of cell polarization and adhesion receptor redistribution during the initial step of leukocyte locomotion. We herein report that interleukin (IL)-15, a newly described cytokine with chemotactic properties, is able to induce uropod formation on T lymphoblasts to which intercellular adhesion molecule (ICAM)-3, a leukocyte-restricted counter-receptor for the lymphocyte function-associated antigen (LFA)-1 integrin, is redistributed. Other adhesion molecules, such as ICAM-1, ICAM-2, CD43 and CD44, also redistributed to the uropod, although in a lower proportion of the cells. The induction of uropod formation by IL-15 was observed on T lymphoblasts adhering to the integrin ligands fibronectin, vascular cell adhesion molecule (VCAM)-1 and ICAM-1, but not to bovine serum albumin or poly-L-lysine. The effect of IL-15 was dose dependent and specifically inhibited by a monoclonal antibody (mAb) against this cytokine. Blocking experiments with anti-IL-2 receptor beta chain mAb showed an inhibitory effect on IL-15-mediated redistribution of ICAM-3, whereas no effect was observed in the presence of anti-IL-2 receptor alpha chain mAb. The uropod induced by IL-15 is enriched in many different adhesion receptors and, being well exposed to the external milieu, is likely to modulate the adhesive properties of lymphocytes.     

Studies using antigen-presenting cells lacking expression of both B7-1 (CD80) and B7-2 (CD86) show distinct requirements for B7 molecules during priming versus restimulation of Th2 but not Th1 cytokine production

The differentiation of CD4+ T cells into a Th1 vs Th2 phenotype profoundly influences the outcome of autoimmune and infectious diseases. B7 costimulation has been shown to affect the production of both Th1 and Th2 cytokines, depending on the system studied. There is, consequently, great interest in manipulating the B7 costimulatory signal for therapeutic purposes. To optimally manipulate this key immunoregulatory pathway, the contribution of B7 costimulation to cytokine production requires further clarification. We have compared the B7 requirement for cytokine production by naive vs previously activated T cells using DO11.10 TCR transgenic CD4+ T cells and splenic APCs from mice lacking B7 expression. Our data indicate that induction of IL-4 production and Th2 differentiation by naive T cells is highly dependent on B7 molecules, whereas IL-4 production by previously activated T cells is B7 independent. The predominant contribution of B7-mediated signals to Th1 cytokine production by both naive and primed T cells is upon IL-2 production (and expansion) rather than IFN-gamma (effector cytokine) production. Thus, our studies demonstrate that the antigenic experience of a T cell at the time of B7 blockade may determine whether blockade predominantly affects T cell expansion, differentiation, or effector cytokine production. These differential effects of B7 costimulation on IL-2 vs IFN-gamma production and on IL-4 production by naive vs primed T cells have important implications for understanding how B7:CD28/CTLA4 blockade can be effectively used to manipulate cytokine production in vivo.     

Western blotting analysis of heat shock proteins of Rickettsiales and other eubacteria

Promyelocytic leukemia HL-60 cells promoted by PMA to differentiate along the monocyte pathway adhere to tissue culture plates. To explore the regulation of adhesion molecules in cells promoted to differentiate, the expression and secretion of osteopontin (OPN) and expression of associated cell surface receptors, CD44 and integrin subunits alpha(v), beta3, beta1, were examined. Results were as follows: 1) PMA induced OPN mRNA and OPN secretion into media; 2) untreated cells expressed beta1 and CD44 mRNA, and PMA induced alpha(v), and beta3 mRNA and increased beta1 and CD44 mRNA expression; 3) PMA increased levels of alpha(v), beta3, beta1 and CD44 protein on the cell surface; and 4) retinoic acid, which promotes granulocytic differentiation of HL-60 cells, did not affect OPN, alpha(v), beta3, beta1, or CD44 mRNA or protein expression. These data suggest that induction of OPN and associated receptors may play a role during monocytic differentiation of HL-60 cells.     

Focal adhesion kinase-related fakB is regulated by the integrin LFA-1 and interacts with the SH3 domain of phospholipase C gamma 1

Signal transduction through integrin molecules expressed on platelets and nonlymphoid cells involves activation of the intracellular focal adhesion kinase ppI25FAK (FAK) to phosphorylate substrate proteins on tyrosine residues. Similar mechanisms are also functional in T-lymphocytes through the beta 1-integrin VLA-4. A putative FAK-related phosphoprotein (fakB) was identified that is responsive to intracellular signals induced through ligation of antigen receptors on both T- and B-lymphocytes, and whose induced tyrosine phosphorylation is augmented by TCR costimulation through the adhesion/costimulatory receptors CD2 and CD4. In this report, fakB is shown to respond to extracellular signals through the beta 2-integrin LFA-1 in the absence of primary signals through the TCR. Protein-protein complex formation was observed involving an association between fakB, phospholipase C gamma 1 (PLC gamma 1), and the tyrosine phosphoprotein pp35-36. Evidence is provided here that fakB interacts with PLC gamma 1 through its SH3 domain. The association between fakB and PLC gamma 1 does not appear to require T-cell activation, whereas the induced tyrosine phosphorylation of the protein complex components occurs following engagement of LFA-1. These data indicate that the beta2-integrin LFA-1 expressed on T-lymphocytes stimulates a novel, FAK-related molecule that may function in the interplay between adhesion receptors and intracellular signaling enzymes responsible for downstream second messenger generation.     

Differential effects of CD28 engagement and IL-12 on T cell activation by altered peptide ligands

To futher our understanding of the mechanisms underlying the diverse effects of altered peptide ligands (APL) on T cell activation, we used a population of nonactivated spleen cells from mice that expressed a transgenic TCR specific for myelin basic protein Ac1-11 and peptide analogues that display either enhanced or decreased affinities for TCR/MHC to address the question whether APL-induced signaling through the TCR can regulate the capability of APC to activate T cells. We demonstrate that weak agonists APL are poor inducers of all aspects of the activation of both the responder T cells and the APC. Enhancement of the antigenic signal by augmenting the binding of the weak agonists to MHC reversed their defective activating capacity. Enhancement of costimulation by engagement of CD28 only resulted in augmentation of the capacity of the weak agonist APL to induce proliferation and IL-2/IL-3 production, but not CD40L or IL-12Rbeta2 chain expression on T cells, CD80/CD86 expression on APC, IL-12 secretion, or IFN-gamma production. Exogenous IL-12 promoted IFN-gamma production in the presence of the weak agonists. These studies demonstrate that there is a critical threshold of antigenic signal required for full activation of the T cell-APC interactions needed for the differentiation of Th1 cells. The provision of excess costimulation can overcome some of the defects in T cell activation by weak agonists, but is insufficient to induce a sufficient level of CD40L expression needed for engagement of CD40 on APC with subsequent IL-12 production and induction of IL-12Rbeta2 chain expression.