Foxp3+ regulatory T cells are activated in spite of B7‐CD28 and CD40‐CD40L blockade

Costimulatory signals are required for priming and activation of naive T cells, while it is less clear how they contribute to induction of regulatory T (Treg)‐cell activity. We previously reported that the blockade of the B7‐CD28 and CD40L‐CD40 interaction efficiently suppresses allogeneic T‐cell activation in vivo. This was characterized by an initial rise in Foxp3⁺ cells, followed by depletion of host‐reactive T cells. To further investigate effects of costimulatory blockade on Treg cells, we used an in vitro model of allogeneic CD4⁺ cell activation. When CTLA‐4Ig and anti‐CD40L mAb (MR1) were added to the cultures, T‐cell proliferation and IL‐2 production were strongly reduced. However, Foxp3⁺ cells proliferated and acquired suppressive activity. They suppressed activation of syngeneic CD4⁺ cells much more efficiently than did freshly isolated Treg cells. CD4⁺ cells activated by allogeneic cells in the presence of MR1 and CTLA‐4Ig were hyporesponsive on restimulation, but their response was restored to that of naive CD4⁺ cells when Foxp3⁺ Treg cells were removed. We conclude that natural Treg cells are less dependent on B7‐CD28 or CD40‐CD40L costimulation compared with Foxp3^? T cells. Reduced costimulation therefore alters the balance between Teff and Treg‐cell activation in favor of Treg‐cell activity.     

Orai1 controls C5a‐induced neutrophil recruitment in inflammation

Stromal interaction molecule 1 (STIM1)‐dependent store operated calcium‐entry (SOCE) through Orai1‐mediated calcium (Ca²⁺) influx is considered a major pathway of Ca²⁺ signaling, serving T‐cell, mast cell, and platelet responses. Here, we show that Orai1 is critical for neutrophil function. Orai1‐deficient neutrophils present defects in fMLP and complement C5a‐induced Ca²⁺ influx and migration, although they respond normally to another chemoattractant, CXCL2. Up until now, no specific contribution of Orai1 independent from STIM1 or SOCE has been recognized in immune cells. Here, we observe that Orai1‐deficient neutrophils exhibit normal STIM1‐dependent SOCE and STIM1‐deficient neutrophils respond to fMLP and C5a efficiently. Despite substantial cytokine production, Orai1^?/? chimeric mice show impaired neutrophil recruitment in LPS‐induced peritonitis. Moreover, Orai1 deficiency results in profoundly defective C5a‐triggered neutrophil lung recruitment in hypersensitivity pneumonitis. Comparative evaluation of inflammation in Stim1^?/? chimeras reveals a distinct pathogenic contribution of STIM1, including its involvement in IgG‐induced C5a production. Our data establish Orai1 as key signal mediator of C5aR activation, contributing to inflammation by a STIM1‐independent pathway of Ca²⁺‐influx in neutrophils.     

CD4+ T‐cell activation is differentially modulated by bacteria‐primed dendritic cells,but is generally down‐regulated by n‐3 polyunsaturated fatty acids

Appropriate activation of CD4⁺ T cells is fundamental for efficient initiation and progression of acquired immune responses. Here, we showed that CD4⁺ T‐cell activation is dependent on changes in membrane n‐3 polyunsaturated fatty acids (PUFAs) and is dynamically regulated by the type of signals provided by dendritic cells (DCs). Upon interaction with DCs primed by different concentrations and species of gut bacteria, CD4⁺ T cells were activated according to the type of DC stimulus. The levels of CD80 were found to correlate to the levels of expression of CD28 and to the proliferation of CD4⁺ T cells, while the presence of CD40 and CD86 on DCs inversely affected inducible costimulator (ICOS) and cytotoxic T‐lymphocyte antigen‐4 (CTLA‐4) levels in CD4⁺ T cells. For all DC stimuli, cells high in n‐3 PUFAs showed reduced ability to respond to CD28 stimulation, to proliferate, and to express ICOS and CTLA‐4. Diminished T‐cell receptor (TCR) and CD28 signalling was found to be responsible for n‐3 PUFA effects. Thus, the dietary fatty acid composition influences the overall level of CD4⁺ T‐cell activation induced by DCs, while the priming effect of the DC stimuli modulates CD80, CD86 and CD40 levels, thereby affecting and shaping activation of acquired immunity by differential regulation of proliferation and costimulatory molecule expression in CD4⁺ T cells.     

Distinct effects of CD86-mediated costimulation on resting versus activated human CD4+ T cells

CD80 and CD86 are important in the initiation of T cell immunity. Although their costimulatory function has long been appreciated, it remains unclear whether the biological significance of the two B7 isoforms resides in their different patterns and kinetics of expression or whether differences exist in their function. We have addressed this issue using HLA‐DR1 transfectants co‐expressing CD80, CD86, or both molecules as stimulators for naïve, memory, and activated human CD4⁺ T cells. Both CD80 and CD86 efficiently costimulated alloresponses by unseparated peripheral blood CD4⁺ T cells; however, CD86 was substantially inferior in costimulating alloresponses by separated memory T cells, and completely incompetent in costimulating three human T cell clones. Furthermore, CD80/CD86 double transfectants stimulated lower responses by the clones than cells expressing CD80 alone. That CD86 was actively inhibitory rather than merely neutral was evidenced by the increase in response to the double CD80/CD86 APC when anti‐CD86 antibody was added. Furthermore, addition of anti‐CTLA‐4 Fab to cultures of HLA‐DR1 transfectants co‐expressing CD86, fully restored the proliferative response. These results indicate that CD80 and CD86 mediate distinct signals in previously activated T cells, and demonstrate that CTLA‐4 ligation may dominate the outcome of CD86‐mediated costimulation of activated CD4⁺ T cells.     

Unlike CD4+ T-cell help, CD28 costimulation is necessary for effective primary CD8+ T-cell influenza-specific immunity

The importance of costimulation on CD4⁺ T cells has been well documented. However, primary CTLs against many infections including influenza can be generated in the absence of CD4⁺ T‐cell help. The role of costimulation under such “helpless” circumstances is not fully elucidated. Here, we investigated such a role for CD28 using CTLA4Ig transgenic (Tg) mice. To ensure valid comparison across the genotypes, we showed that all mice had similar naïve precursor frequencies and similar peak viral loads. In the absence of help, viral clearance was significantly reduced in CTLA4Ig Tg mice compared with WT mice. CD44⁺BrdU⁺influenza‐specific CD8⁺ T cells were diminished in CTLA4Ig Tg mice at days 5 and 8 postinfection. Adoptive transfer of ovalbumin‐specific transgenic CD8⁺ T cells (OT‐I)‐I cells into WT or CTLA4Ig Tg mice revealed that loss of CD28 costimulation resulted in impairment in OT‐I cell division. As shown previously, neither viral clearance nor the generation of influenza‐specific CD8⁺ T cells was affected by the absence of CD4⁺ T cells alone. In contrast, both were markedly impaired by CD28 blockade of “helpless” CD8⁺ T cells. We suggest that direct CD28 costimulation of CD8⁺ T cells is more critical in their priming during primary influenza infection than previously appreciated.     

Food Allergen–Induced Mast Cell Degranulation is Dependent on PI3K‐Mediated Reactive Oxygen Species Production and Upregulation of Store‐Operated Calcium Channel Subunits

The importance of Ca²⁺ influx via store‐operated calcium channels (SOCs) leading to mast cell degranulation is well known in allergic disease. However, the underlying mechanisms are not fully understood. With food‐allergic rat model, the morphology of degranulated mast cell was analysed by toluidine blue stain and electron microscope. Ca²⁺ influx via SOCs was checked by Ca²⁺ imaging confocal microscope. Furthermore, the mRNA and protein expression of SOCs subunits were investigated using qPCR and Western blot. We found that ovalbumin (OVA) challenge significantly increased the levels of Th2 cytokines and OVA‐specific IgE in allergic animals. Parallel to mast cell activation, the levels of histamine in serum and supernatant of rat peritoneal lavage solution were remarkably increased after OVA treatment. Moreover, the Ca²⁺ entry through SOCs evoked by thapsigargin was increased in OVA‐challenged group. The mRNA and protein expressions of SOC subunits, stromal interaction molecule 1 (STIM1) and Orail (calcium‐release‐activated calcium channel protein 1), were dramatically elevated under food‐allergic condition. Administration of Ebselen, a scavenger of reactive oxygen species (ROS), significantly attenuated OVA sensitization‐induced intracellular Ca²⁺ rise and upregulation of SOCs subunit expressions. Intriguingly, pretreatment with PI3K‐specific inhibitor (Wortmannin) partially abolished the production of ROS and subsequent elevation of SOCs activity and their subunit expressions. Taken together, these results imply that enhancement of SOC‐mediated Ca²⁺ influx induces mast cell activation, contributing to the pathogenesis of OVA‐stimulated food allergy. PI3K‐dependent ROS generation involves in modulating the activity of SOCs by increasing the expressions of their subunit.     

Role of CD80, CD86, and CTLA4 on mouse CD4(+) T lymphocytes in enhancing cell-cycle progression and survival after activation with PMA and ionomycin

Cell surface interactions between the T cell costimulatory receptors, CD28 and cytotoxic T-lymphocyte antigen-4 (CTLA4), with their cognate ligands, CD80 and CD86, on antigen-presenting cells play an important role in T cell activation. Although CD80 and CD86 are induced on T cells after activation, not much is known about their role in modulating T cell function. We show that CD80, CD86, and CTLA4 are induced on purified CD4(+) T cells after in vitro activation with phorbol 12-myristate 13-acetate (PMA) and ionomycin, and they play an essential role for proliferation and survival. Blockade of CTLA4-CD80/CD86 interactions greatly reduces PMA and ionomycin-mediated mouse CD4(+) T cell activation. The three key features of this inhibition of activation are: First, late events in T cell activation (after 18 h) are affected; second, these cells do not undergo anergy; and third, CD4(+)CD25(+) regulatory T cells are not responsible. Activation of T cells with PMA and ionomycin together with CTLA4-CD80/CD86 blockade results in decreased induction of CD25 and Bcl-X(L), reduced interleukin (IL)-2, and enhanced transforming growth factor-beta (TGF-beta) production. Furthermore, extended CTLA4-CD80/CD86 blockade results in decreased cell-cycle progression and enhanced apoptosis in a large proportion of cells. This inhibition of T cell proliferation can be rescued completely with anti-CD28 or IL-2 and partially with TGF-beta antagonists. This study reveals a functional role for CD80, CD86, and CTLA4 on CD4(+) T lymphocytes and sheds light on the mechanisms by which these molecules enhance activation and survival with PMA and ionomycin.     

CD28-B7-mediated T cell costimulation in chronic cardiac allograft rejection: differential role of B7-1 in initiation versus progression of graft arteriosclerosis

Provision of adequate T cell costimulation is critical for the development of acute and chronic allograft rejection. We have previously reported that early blockade of CD28-B7 T cell costimulation prevents the development of graft arteriosclerosis, in the LEW into F344 rat cardiac transplant model. In this study, we used the same model to examine the requirement for CD28-B7-mediated T cell costimulation in the progression of established chronic rejection and examined the individual roles of B7-1 (CD80) and B7-2 (CD86) costimulatory molecules. Late blockade of CD28-B7 T cell costimulation by the fusion protein CTLA4Ig, which binds both CD80 and CD86, attenuated the development of transplant arteriosclerosis, mononuclear cell infiltration, and parenchymal fibrosis in this model. Selective blockade of CD80 using the mutant fusion protein Y100F was as effective as CTLA4Ig in this regard. In contrast to CTLA4Ig, blockade of CD80 alone by Y100F was ineffective at preventing early graft loss and prolonging graft survival when given early after transplantation. This study is the first to demonstrate that late blockade of CD28-B7 T cell costimulation interrupts chronic cardiac allograft rejection, and it indicates the importance of continued T cell activation in this process. This study further defines functional differences between CD80 and CD86 costimulatory molecules in vivo.     

ICAM‐1 and B7‐1 provide similar but distinct costimulation for CD8+ T cells,while CD4+ T cells are poorly costimulated by ICAM‐1

The function of purified ICAM‐1 in costimulating CD4⁺ and CD8⁺ T cell responses has been directly compared to that of B7‐1 in a model system that minimizes contributions of other receptor‐ligand interactions. While B7‐1 costimulates both subsets of T cells, ICAM‐1 is much more effective in the costimulation of CD8⁺ cells. ICAM‐1 also synergizes with B7‐1 for the induction of IL‐2 production in CD8⁺ but not CD4⁺ T cells. These differences are not explained by differences in LFA‐1 receptor expression on the two subsets of T cells. The CD8⁺ T cell response to ICAM‐1 costimulation is associated with increased proliferation and IL‐2 production at levels similar to those seen with B7‐1 costimulation, but clonal expansion in response to ICAM‐1 is not as great due to decreased cell survival. ICAM‐1‐mediated costimulation is effective for both naive and memory CT8⁺ T cells, is independent of CD28 engagement, and does not appear to be due solely to effects on adhesion. These results suggest that ICAM‐1‐dependent, B7‐independent costimulation may be important in initiating a CTL response to class I antigen presented by cells that are not professional APC.     

STIM1 knock-down decreases the affinity of obinutuzumab for CD20 by altering CD20 localization to Triton-soluble membrane

Obinutuzumab is thought to exert its effects through its high antibody-dependent cellular cytotoxicity (ADCC) via glyco-engineering of the Fc region. In addition, obinutuzumab causes direct binding-induced cell death (DCD) only by specifically binding to its target CD20, a Ca²⁺ channel. However, the specific features of CD20 related to obinutuzumab binding-induction of cell death are not clearly understood. In this study, we evaluated the relationship between the Ca²⁺ channel features of CD20 as a store-operated Ca²⁺ channel (SOC) and obinutuzumab binding-induced cell death. Ca²⁺ channel function and biochemical analysis revealed that CD20 is an Orai1- and stromal interaction molecule (STIM1)-dependent Ca²⁺ pore. However, binding of obinutuzumab on CD20 did not have any effect on Ca²⁺ influx activity of CD20; the direct cell death rate mediated by obinutuzumab binding was almost equivalent with or without the extracellular Ca²⁺ condition. Given the apparent interaction between STIM1 and CD20, we observed Triton-X solubilized obinutuzumab-bound CD20 accompanied by STIM1. Subsequently, obinutuzumab binding and cell death were decreased by STIM1 knock-down in Ramos B cells. Thus, STIM1 directly contributes to cell death by increasing the affinity of cells for obinutuzumab by transferring CD20 to the Triton-soluble membrane region.     

Combination of nifedipine and subtherapeutic dose of cyclosporin additively suppresses mononuclear cells activation of patients with rheumatoid arthritis and normal individuals via Ca(2+) -calcineurin-nuclear factor of activated T cells pathway

Abnormal Ca²⁺‐mediated signalling contributes to the pathogenesis of rheumatoid arthritis (RA). However, the potential implication of calcium channel blocker in RA remained unknown. We hypothesized that nifedipine, an L‐type calcium channel blocker, combined with a calcineurin inhibitor, could suppress T cell activation via targeting different level of the Ca²⁺ signalling pathway. The percentage of activated T cells and the apoptotic rate of mononuclear cells (MNCs) was measured by flow cytometry. The MNC viability, cytokine production, cytosolic Ca²⁺ level and activity of the nuclear factor of activated T cells (NFAT) were measured by enzyme‐linked immunosorbent assay (ELISA). The NFAT‐regulated gene expression, including interleukin (IL)‐2, interferon (IFN)‐γ and granulocyte–macrophage colony‐stimulating factor (GM‐CSF), was measured by real‐time polymerase chain reaction (PCR). We found that the percentage of activated T cells in anti‐CD3 + anti‐CD28‐activated MNC was higher in RA patients. High doses of nifedipine (50 µM) increased MNCs apoptosis, inhibited T cell activation and decreased T helper type 2 (Th1) (IFN‐γ)/Th2 (IL‐10) cytokine production in both groups. The Ca²⁺ influx was lower in anti‐CD3 + anti‐CD28‐activated MNC from RA patients than healthy volunteers and suppressed by nifedipine. When combined with a subtherapeutic dose (50 ng/ml) of cyclosporin, 1 µM nifedipine suppressed the percentage of activated T cells in both groups. Moreover, this combination suppressed more IFN‐γ secretion and NFAT‐regulated gene (GM‐CSF and IFN‐γ) expression in RA‐MNCs than normal MNCs via decreasing the activity of NFATc1. In conclusion, we found that L‐type Ca²⁺ channel blockers and subtherapeutic doses of cyclosporin act additively to suppress the Ca²⁺‐calcineurin‐NFAT signalling pathway, leading to inhibition of T cell activity. We propose that this combination may become a potential treatment of RA.     

CTLA4-CD80/CD86 interactions on primary mouse CD4+ T cells integrate signal-strength information to modulate activation with Concanavalin A

The mechanisms by which concanavalin A (Con A), a lectin, activates T cells are poorly studied. A low dose of Con A is stimulatory for T cells, whereas a high dose of Con A results in suppression of proliferation and enhanced T cell death. The expression and functional roles of costimulatory receptors, CD28 and cytotoxic T-lymphocyte antigen 4 (CTLA4), and their ligands, CD80 and CD86, on primary mouse CD4(+) T cells after activation with different doses of Con A were studied. CTLA4-CD80/CD86 interactions in this T:T cell activation model demonstrate distinct outcomes depending on the dose of Con A. CTLA4-CD80/CD86 interactions inhibit CD4(+) T cell cycling and survival after activation with a suppressive dose of Con A by increasing oxidative stress and decreasing levels of BclX(L). The enhanced CD4(+) T cell death with a suppressive dose of Con A is dependent on excess H(2)O(2) and nitric oxide but is independent of Fas and caspase activity. It is surprising that the increased proliferation of CD4(+) T cells with a suppressive dose of Con A on blocking CTLA4-CD80/CD86 interactions is largely interleukin (IL)-2-independent but is cyclosporine A-sensitive. On activation with a stimulatory dose of Con A, CTLA4-CD80/CD86 interactions enhance T cell activation and survival by reducing the production of reactive oxygen species, increasing IL-2 and BclX(L) levels. Here IL-10 but not transforming growth factor-beta plays a functional role. In summary, CTLA4-CD80/CD86 interactions on T cells integrate signal strength, based on the dose of Con A, to enhance or inhibit primary mouse CD4(+) T cell cycling and survival.     

CD28 days later: Resurrecting costimulation for CD8+ memory T cells

Rapid activation and proliferative expansion of specific CD8⁺ memory T (CD8⁺T_M) cells upon antigen re‐encounter is a critical component of the adaptive immune response that confers enhanced immune protection. In this context, however, the requirements for costimulation in general, and CD28 signaling in particular, remain incompletely defined. In the current issue of the European Journal of Immunology, Fröhlich et al. [Eur. J. Immunol. 2016. 46: 1644‐1655] provide definitive evidence that optimal elaboration of CD8⁺T_M‐cell recall responses is indeed contingent on CD28 expressed by these cells. Here, we discuss the “CD28 costimulation paradigm” in its historical context and highlight some of the unresolved complexities pertaining to CD28‐dependent interactions that shape CD8⁺ T‐cell phenotypes, functionalities, and recall reactivity.     

CTLA4‐Ig inhibits allergic airway inflammation by a novel CD28‐independent,nitric oxide synthase‐dependent mechanism

The T‐cell response to antigen depends on coordinate signaling between costimulatory and inhibitory receptors. The altered function of either may underlie the pathophysiology of autoimmune and/or chronic inflammatory diseases and manipulation of these pathways is an important emerging area of therapeutics. We report here that the immunosuppressant drug CTLA4‐Ig inhibits the effector phase of allergic airway inflammation through a CD28‐independent, nitric oxide synthase (NOS)‐dependent mechanism. Using mice deficient in both B‐ and T‐lymphocyte attenuator (BTLA) and CD28, we demonstrate that simultaneous deficiency of an inhibitory receptor can rescue the in vivo but not the in vitro CD28‐deficient phenotype. Furthermore, we demonstrate that inflammation in CD28/BTLA‐double‐deficient mice is suppressed by CTLA4‐Ig. This suppression is reversed by treatment with the NOS inhibitor, N⁶‐methyl‐L ‐arginine acetate (L‐NMMA). In addition, CTLA4‐Ig is ineffective at inhibiting inflammation in NOS2‐deficient mice when given at the effector phase. Thus, CD28 and BTLA coordinately regulate the in vivo response to inhaled allergen, and CTLA4‐Ig binding to B7‐proteins inhibits the effector phase of inflammation by a CD28‐independent, NOS‐dependent mechanism.     

Cell‐intrinsic and ‐extrinsic control of Treg‐cell homeostasis and function revealed by induced CD28 deletion

While the requirement for CD28 and its ligands for the generation and function of “natural” (n)Treg cells is well established, it has not been possible yet to investigate cell‐intrinsic effects after interrupted CD28 expression. Here, we demonstrate a selective loss of Treg cells after disruption of the CD28 gene. The decline in Treg‐cell number was accompanied by reduced homeostatic proliferation, probably due to lack of costimulation during self‐antigen recognition, and by impaired Treg‐cell function including downregulation of CTLA‐4. The decline in Treg‐cell number was unaffected by thymectomy or by the presence of CD28 expressing T cells within the same animal, indicating that impairment of peripheral homeostasis and function of nTreg cells by CD28 deletion is cell‐intrinsic. In contrast, downregulation of CD25, the α chain of the IL‐2R, did not occur in the presence of WT T cells, indicating that its expression does not depend on CD28 signals in cis.     

Interrupting CD28 costimulation before antigen rechallenge affects CD8+ T‐cell expansion and effector functions during secondary response in mice

The role of CD28‐mediated costimulation in secondary CD8⁺ T‐cell responses remains controversial. Here, we have used two tools — blocking mouse anti‐mouse CD28‐specific antibodies and inducible CD28‐deleting mice — to obtain definitive answers in mice infected with ovalbumin‐secreting Listeria monocytogenes. We report that both blockade and global deletion of CD28 reveal its requirement for full clonal expansion and effector functions such as degranulation and IFN‐γ production during the secondary immune response. In contrast, cell‐intrinsic deletion of CD28 in transferred TCR‐transgenic CD8⁺ T cells before primary infection leads to impaired clonal expansion but an increase in cells able to express effector functions in both primary and secondary responses. We suggest that the proliferation‐impaired CD8⁺ T cells respond to CD28‐dependent help from their environment by enhanced functional differentiation. Finally, we report that cell‐intrinsic deletion of CD28 after the peak of the primary response does not affect the establishment, maintenance, or recall of long‐term memory. Thus, if given sufficient time, the progeny of primed CD8⁺ T cells adapt to the absence of this costimulator.     

Differential STIM1 expression in T and B cell subsets suggests a role in determining antigen receptor signal amplitude

Ca²⁺ acts ubiquitously as a second messenger in transmembrane signal transduction. In lymphocytes, calcium mobilization is triggered by antigen and chemokine receptors, among others, and controls cell functions ranging from proliferation to migration. The primary mechanism of extracellular Ca²⁺ entry in lymphocytes is the CRAC influx. STIM1 is a crucial component of the CRAC influx mechanism in lymphocytes, acting as a sensor of low Ca²⁺ concentration in the ER and an activator of the Ca²⁺ selective channel ORAI1 in the plasma membrane. While STIM1 function has been studied extensively, little is known regarding whether it is differentially expressed and thereby affects the magnitude of calcium mobilization responses. We report here that STIM1 expression differs in murine T and B lymphocytes, and in respective subsets. For example, mature T cells express ∼4 times more STIM1 than mature B cells. Furthermore, we show that through the physiologic range of expression, STIM1 levels determine the magnitude of Ca²⁺ influx responses that follow BCR-induced intracellular store depletion. Considered in view of previous reports that differences in amplitude of lymphocyte Ca²⁺ mobilization determine alternate biological responses, these findings suggest that differential STIM1 expression may be important determinant of biological responses.     

Endothelin‐1‐induced proliferation of human endothelial cells depends on activation of K+ channels and Ca2+ influx

Aims: Endothelin‐1 (ET‐1) promotes endothelial cell growth. Endothelial cell proliferation involves the activation of Ca²⁺‐activated K⁺ channels. In this study, we investigated whether Ca²⁺‐activated K⁺ channels with big conductance (BK_Ca) contribute to endothelial cell proliferation induced by ET‐1. Methods: The patch‐clamp technique was used to analyse BK_Ca activity in endothelial cells derived from human umbilical cord veins (HUVEC). Endothelial proliferation was examined using cell counts and measuring [³H]‐thymidine incorporation. Changes of intracellular Ca²⁺ levels were examined using fura‐2 fluorescence imaging. Results: Characteristic BK_Ca were identified in cultured HUVEC. Continuous perfusion of HUVEC with 10 nmol L^?1 ET‐1 caused a significant increase of BK_Ca open‐state probability (n = 14; P < 0.05; cell‐attached patches). The ET_B‐receptor antagonist (BQ‐788, 1 μmol L^?1) blocked this effect. Stimulation with Et‐1 (10 nmol L^?1) significantly increased cell growth by 69% (n = 12; P < 0.05). In contrast, the combination of ET‐1 (10 nmol L^?1) and the highly specific BK_Ca blocker iberiotoxin (IBX; 100 nmol L^?1) did not cause a significant increase in endothelial cell growth. Ca²⁺ dependency of ET‐1‐induced proliferation was tested using the intracellular Ca²⁺‐chelator BAPTA (10 μmol L^?1). BAPTA abolished ET‐1 induced proliferation (n = 12; P < 0.01). In addition, ET‐1‐induced HUVEC growth was significantly reduced, if cells were kept in a Ca²⁺‐reduced solution (0.3 mmol L^?1), or by the application of 2 aminoethoxdiphenyl borate (100 μmol L^?1) which blocks hyperpolarization‐induced Ca²⁺ entry (n = 12; P < 0.05). Conclusion: Activation of BK_Ca by ET‐1 requires ET_B‐receptor activation and induces a capacitative Ca²⁺ influx which plays an important role in ET‐1‐mediated endothelial cell proliferation.     

Activation of CD4+ T cells by delivery of the B7 costimulatory signal on bystander antigen-presenting cells (trans-costimulation)

Increasing evidence in both murine and human systems suggests that the interaction of the T cell surface antigens CD28/CTLA4 with their ligand B7 on the antigen-presenting cells (APC) is the critical costimulatory pathway involved in the induction of maximal T cell activation and the prevention of induction of anergy. It has also been demonstrated that efficient induction of clonal expansion of normal CD4⁺ T cells requires the delivery of the T cell receptor (TCR) ligand and costimulation by the same APC. We demonstrate here that normal murine CD4⁺ T cells can be efficiently activated by soluble anti-CD3 cross-linked by fixed macrophages and by a costimulatory signal delivered by a bystander APC, B7-transfected L cells. The major factor which determined the ability of an APC to provide costimulation in “trans” was the level of cell surface B7 expression. The requirement for B7 costimulation appears to be at initial stage of TCR engagement since optimal T cell activation was only observed when TCR triggering and B7 costimulatory activity were delivered at same time by different APC. Induction of maximal proliferation of both naive CD45RB^hi and memory CD45RB^lo CD4⁺ T cells was B7 dependent and both populations of cells responded equally well to the B7 costimulation delivered in “trans”. Furthermore, trans-costimulation provided by B7 transfected L cells efficiently prevented the induction of anergy in normal murine CD4⁺ T cells induced by anti-CD3 cross-linked by fixed-resting macrophages. Addition of exogenous interleukin-2 (IL-2) and IL-7 to the primary culture in the absence of B7-transfected L cells or addition of IL-2 to the culture containing the B7 transfectant and CTLA4Ig completely prevented the induction of hyporesponsiveness. These findings raise the possibility that in certain pathological states, CD4⁺ T cells in vivo may be activated by costimulation delivered by bystander APC.